TRANSPORT PHENOMENA

IN THE NERVOUS SYSTEM

Physiological and Pathological Aspects
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
Nathan Back State University of New York at Buffalo
N. R. Di Luzio Tulane University School of Medicine
Bernard Halpern College de France and Institute of Immuno.Biology
Ephraim Katchalski The Weizmann Institute of Science
David Kritchevsky Wistar Institute
Abel Lajtha New York State Research Institute for Neurochemistry and Drug Addiction
Rodolfo Paoletti University of Milan

Recent Volumes in this Series

Volume 62
CONTROL MECHANISMS IN DEVELOPMENT: Activation, Differentiation,
and Modulation in Biological Systems
Edited by Russel H. Meints and Eric Davies • 1975
Volume 63
LIPIDS, LIPOPROTEINS, AND DRUGS
Edited by David Kritchevsky, Rodolfo Paoletti, and
William L. Holmes - 1975
Volume 64
IMMUNOLOGIC PHYLOGENY
Edited by W. H. Hildemann and A. A. Benedict· 1975

Volume 65
DILEMMAS IN DIABETES
Edited by Stewart Wolf and Beatrice Bishop Berle - 1975
Volume 66
IMMUNE REACTIVITY OF LYMPHOCYTES: Development, Expression,
and Control
Edited by Michael Feldman and Amiela Globerson • 1976
Volume 67
ATHEROSCLEROSIS DRUG DISCOVERY
Edited by Charles E. Day. 1976
Volume 68
CURRENT TRENDS IN SPHINGOLIPIDOSES AND ALLIED DISORDERS
Edited by Bruno W. Volk and Larry Schneck. 1976

Volume 69
TRANSPORT PHENOMENA IN THE NERVOUS SYSTEM: Physiological and
Pathological Aspects
Edited by Giulio Levi, Leontino Battistin, and Abel Lajtha • 1976

Volume 70
KIN INS : J>harmacodynamics and Biological Roles
Edited by F. Sicuteri, Nathan Back, and G. L. Haberland -1976

Volume 71
GANGLIOSIDE FUNCTION: Biochemical and Pharmacological Implications
Edited by Giuseppe Porcellati, Bruno Ceccarelli, and Guido Tettamanti • 1976
TRANSPORT PHENOMENA
IN THE NERVOUS SYSTEM
Physiological and Pathological Aspects

Edited by
Giulio Levi
National Research Council
Rome, Italy

Leontino Battistin
University or Padua Medical School
Padua, Italy

and
Abel Lajtha
Research Institute of Neuroehemistry
New York, New York

PLENUM PRESS • NEW YORK AND LONDON

 Library of Congress Cataloging in Publication Data
Main entry under title:
Transport phenomena in the nervous system.
(Advances in experimental medicine and biology; v. 69)
Includes indexes.
1. Neurochemistry-Congresses. 2. Biological transport-Congresses. 3. Blood-
brain barrier-Congresses. 4. Nervous system-Diseases-Congresses. I. Levi,
Giulio, 1937- II. Battistin, Leontino, 1939- III. Lajtha, Abel. IV.
International Society for Neurochemistry. V. Series. [DNLM: 1. Neurophysi-
ology-Congresses. 2. Nervous system-Pathology-Congresses. 3. Neural transmis-
sion-Congresses. WI AD559 v. 69 1975/WLl02 T774 1975)
QP356.3.T68 612'.8'042 764839
ISBN-13: 978-1-4684-3266-4 e-ISBN-13: 978-1-4684-3264-0
DOl: 10.1007/978-1-4684-3264-0

Proceedings of the Satellite Symposium of the International Society for Neuro-

chemistry held in Padua, Italy, September 9-11, 1975

© 1976 Plenum Press, New York

Softcover reprint of the hardcover 1st edition 1976
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N.Y. 10011
United Kingdom edition published by Plenum Press, London
A Division of Plenum Publishing CompanY, Ltd.
Davis House (4th Floor), 8 Scrubs Lane, Harlesden, London, NWI0 6SE, England
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from the Publisher
The editors would like to thank the following Institutions for the
generous support given for the organization of the Symposium:
Consiglio Nazionale delle Ricerche, Roma
Universita di Pad ova
Comune di Pad ova
Cassa di Risparmio di Padova e Rovigo
Ente Provinciale del Turismo di Pad ova .
Coca Cola Co., Milano
Cooper Lab., Inc., DIF A, Milano
Essex-Schering Farmaceutici, Milano
Falorni Farmaceutici, Firenze
Farmitalia Farmaceutici, Milano
Fidia Farmaceutici, Abano Terme, Pad ova
Hoechst Farmaceutici, Milano
Icpharma ICI, Milano
Italchimici Farmaceutici, Pomezia, Roma
Italseber Farmaceutici, Milano
Lepetit Farmaceutici, Milano
Marxer Farmaceutici, Torino
Merck, Sharp and Dohme, Roma
Roche Farmaceutici, Milano
Philips, Sezione Duphar, Milano
Simes Farmaceutici, Milano
Squibb Farmaceutici, Roma
Wyeth Farmaceutici, Latina
 Preface

This book presents the papers that were delivered at the

Satellite Symposium of the International Society for Neurochemistry
in Padua, September, 1975. Having such Satellite Symposia was a
new experiment for the Society, and all signs, including those
from Padua, indicate that it was a very successful experiment, which
will be an old tradition for the Society. The large international
meeting affords the opportunity for presentations from all areas,
for meeting of colleagues from various backgrounds and disciplines.
The Satellite Symposia allow people from the same area of interest
to discuss their subject in depth, and as such represent meetings
of possibly the greatest practical significance for the partici-
pants.

The Padua Symposium was no exception: all who particiDated in

it could testify to its success in exchanging of information,
learning of new approaches, and acqu~r~ng of new ideas, also the
strengthening of old friendships, forming of new friendships and
new collaborations, and exposing ideas to criticisms, suggestions,
discussions. As a sign of the interest and success of the partici-
pants I can mention that all participants have sent in their
contributions. Perhaps editors of volumes would anpreciate that
the very last came in no later than two months past the deadline.
The symposium could not have been organized at a more suitable
place than Padua -- for two days in the University, one of the
oldest and richest in traditions, and a third day at the villa
Simes under the most spacious, indeed princely circumstances. All
these aspects, and the friendly hospitality that contributed so
much to the cooperative spirit, would not have been possible with-
out the help and efforts of the hosts, the organizers, and the
participants.

I have no doubt that this meeting will belong among the most
pleasant memories and among the most productive and profitable
times of the participants.

Barriers of the brain, somewhat misnamed the "blood-brain

barrier", have been with us for a long time, have been investigated
often, and are considered in general among the most specific and
prominent properties of the brain. In many ways, in spite of
separating this organ from the rest of the organism and providing
it with special properties and functions, it is more often
misunderstood than understood and its complexity is rarely recogniz-
ed. It is more recently becoming increasingly clear, however, that
the homeostatic equilibrium, and its physiological and pathological
changes, are governed by a complex set of mechanisms residing
vii
viii PREFACE

heterogeneously in a complex set of membranes. Mechanisms residing

in the capillary endothelium are of importance - especially in
short term penetration studies of administered substances from
blood to brain; the physiological and pathological equilibria
are likely to be more influenced and governed by mechanisms residing
in neuronal, glial, nuclear,and lysosomal membranes~to name a few.
For functional importance in turn,mechanisms residing in mito-
chondrial and synaptosomal membranes are of crucial importance,
for transport out of the system those present in choroid plexus
among others have to be studied. To focus all our interest on one
membrane, or to believe that the distribution of substances in brain
is governed by the properties of a single membrane would constitute
ignoring the complexities of the nervous system - an obvious error.
The present symposium focussed its interest on the transport systems
present in these various membranes, and showed the great deal of
important information that has been obtained in the past few years
in this area. The information discussed here, beyond summarizing
our present knowledge, can serve to stimulate further advance in this
important field. Membrane phenomena are a most important and
exciting field in biology, and in few areas are they of greater
interest than in studies of neural function. As in all such
symposia, a number of potential contributors could not participate,
but the possibilities of the field and the breadth of approach is
clearly illustrated in this volume, which represents a rapidly
developing field of investigation.

The meeting would not have been possible without much help and
support. Special thanks are due to our host, Professor S. Rigotti
of the Department of Neurology and Psychiatry of the University of
Padova and also to Professor P. Pinelli of the Italian Society of
Neurology. My personal appreciation is added to those of the
participants, to myoid friends and colleagues, Dr. L. Battistin
and G. Levi, for their local arrangements and their arranging and
editing of the program. Special thanks are due also to the
University and to the Pharmaceutical firms, for their most generous
support, which made it possible for all of us to have such pleasant
surroundings for the exchange of information, such rich circumstances
for social exchange. These are hard days for people engaged in
medical research, but the three days of this symposium were a most
pleasant exception to such hardship. It was not the first or the
last symnosium on the subject, and if others will match its
cooperative and interested spirit, the field has a rosy future.

Abel Lajtha
February, 1976
 Contents

SECTION I
METABOLITE TRANSPORT
METABOLITE TRANSPORT AT CELL MEMBRANES
H.N. Christensen 3
Mediation of apparently spontaneous metabolite migrations
Each active transport has a reverse phase
Meaning of duality or multiplicity of transport systems
Evidence that exodus occurs by reversal of a weaker pump
Increased importance of discriminating distinct transport
systems
Roles of receptor sites for amino acid transport in
neurotransmission?
The place of the hydrogen ion
The future: Generation versus interconversion of
energetic gradients

POSSIBLE ROLE OF GLUTATHIONE IN TRANSPORT PROCESSES

M. Orlowski 13
Enzymatic synthesis and degradation of glutathione
The ~-glutamyl cycle
Evidence for the function of the ~-glutamyl cycle in
amino acid transport
Heritable disorders of metabolism due to deficiencies in
enzymes of the ~-glutamyl cycle

SECTI ON I I
BARRIERS IN THE LIVING BRAIN
TRANSPORT MECHANISMS IN THE CEREBROSPINAL FLUID SYSTEM
FOR REMOVAL OF ACID METABOLITES FROM DEVELOPING BRAIN
N. H. Bass and P. Lundbo rg 31
Maturation of bulk flow
Development of intracranial mechanisms for efflux of:
(a) para-aminohippuric acid (PAll)
(b) 5-hydroxyindoleacetic acid (5-HIAA)
Sink action of the cerebrospinal fluid (CSF) system

THE MORPHOLOGICAL APPROACH TO THE STUDY OF NORMAL AND

ABNOR~~L BRAIN PERMEABILITY
M.W. Brightman and R.D. Broadwell 41
Induced opening of the blood-brain barrier
tumors
hyperosmotic opening

ix
x CONTENTS

hypertensive opening
Loci where there is no barrier
permeable vessels

THE TRANSPORT OF METABOLIZABLE SUBSTANCES INTO THE

LIVING BRAIN
O.E. Pratt 55
Glucose and other energy yielding substances
glucose
ketone bodies
pyruvate and lactate
Amino acids
differences in influx of amino acids into the brain
exclusion of amino acids from the brain by inhibition
of transport mechanisms
two sites of cerebral transport
The relation of cerebral transport to various brain disorders
aminoacidurias
changes in transport which affect neurotransmitters
hormonal abnormalities affecting transport into the brain

THE SPECIFICITY OF AMINO ACID AND SUGAR CARRIERS IN THE

CAPILLARIES OF THE DOG BRAIN STUDIED IN VIVO BY RAPID
INDICATOR DILUTION
D.L. Yudilevich and F.V. Sepulveda 77
Methods and interpretation
Carrier for amino acids
Sugar carrier

POSSIBLE ROLE OF INSULIN IN THE TRANSPORT OF TYROSINE

AND TRYPTOPHAN FROM BLOOD TO BRAIN
A. Tagliamonte, M.G. DeMontis, M. Olianas,
P.L. Onali, and G.L. Gessa 89
Insulin effect on amino acid concentrations
Mechanism of the effect
Conclusion

THE INFLUENCE OF LIVER-BYPASS ON TRANSPORT AND

COMPARTMENTATION IN VIVO
J.E. Cremer 95
Consideration of the transformation of metabolizeable
molecules after they have entered the brain from the blood
Metabolic labelling patterns from various precursor substrates
the evidence for the compartmentation of metabolism within
the brain
Evidence for rapid transformation of transported substrates
Substrate uptake and metabolism in a pathological condition
affecting astrocytes
comparative studies between normal rats and animals
with a portocaval anastomosis.
 xi
CONTENTS

CERTAIN ASPECTS OF DRUG DISTRIBUTION TO BRAIN

W.H. Oldendorf 103
Transcapillary exchange
Blood-brain barrier permeability
Drug distribution to brain

PENETRATION OF PROTEINS IN THE CENTRAL NERVOUS SYSTEM

E. Levin and C.E. Tradatti 111
Penetration of proteins into the CSF
Penetration of proteins into the tissue
Routes of penetration
Increased penetration
Pathological conditions
Pharmacological studies

SECTION I I I
TRANSPORT STUDIES IN VARIOUS NERVOUS TISSUE PREPARATIONS
THE CHARACTERISTICS OF GLUCOSE TRANSPORT ACROSS THE BLOOD
BRAIN BARRIER AND ITS RELATION TO CEREBRAL GLUCOSE
METABOLISM
A.L. Betz, D.D. Gi1boe, and L.R. Drewes 133
Glucose transport kinetics under physiological conditions
Glucose transport kinetics during hypoglycemia
Glucose transport kinetics during anoxia
Mechanism of glucose transport
Comparison with glucose transport in the erythrocyte
Proposed model for glucose transport at the blood brain
interface

~mCHANISMS FOR THE PASSIVE REGULATION OF EXTRACELLULAR K+

IN THE CENTRAL NERVOUS SYSTEM: THE IMPLICATIONS OF
INVERTEBRATE STUDIES
N.J+ Abbott and Y. Pichon 151
K homeostasis
The function of astrocytes
Invertebrate studies
Vertebrate studies
glia as spatial buffers for K+
Crustacean studies

AMINO ACID TRANSPORT IN SPINAL AND SYMPATHETIC GANGLIA

P.J. Roberts 165
Structure and functions of dorsal root and superior
cervical ganglia
The neurone - glia functional unit
Uptake of putative amino acid transmitters
effect of dorsal root section
metabolic requirements
kinetic characteristics and sodium dependence
xii CONTENTS

Localization of amino acids

Drug inhibition of GABA and glutamate uptake
Amino acid release
Exchange processes
Conclusions

UPTAKE OF NEUROTRANSHITTERS AND PRECURSORS BY CLONAL CELL

LINES OF NEURAL ORIGIN
B. Haber and H.T. Hutchison 179
Transport in clonal cell lines
Uptake of Y-aminobutyric acid (GABA)
Uptake of aspartate, glutamate, glycine and taurine
Transport of choline
Transport of precursors and biogenic amines

ON THE UPTAKE MECHANISM OF CHOLINE IN NERVE CELL CULTUR~S

R. Hassare11i and P. Mandel 199
Cell cultures
Choline uptake experiments
Results
Discussion

THE UPTAKE AND RELEASE OF y-AMINOBUTYRIC ACID (GABA) BY

THE RETINA
M.J. Neal 211
Role of GABA in the retina
GABA uptake in the retina
Effect of GABA-T inhibitors on GABA uptake
Sites of GABA uptake
subcellular distribution of (3H)GABA
auto radiographic localization of GABA uptake sites
Efflux of GABA from the retina

AMINO ACID TRANSPORT IN ISOLATED NEURONS AND GLIA

A. Hamberger, B. Nystrom, A. Sellstrom and
C.T. Woiler 221
Bulk-prepared neuronal and glial cell fractions as models
for amino acid transport studies
General features of amino acid transport in neuronal and
glial cells
High-affinity uptake in relation to transmitter inactivation
Metabolic and ionic requirements for amino acid uptake
Amino acid release from isolated fractions as studied by
perfusion
Inhibitors as tools to study cell specificity of amino acid
uptake
Glutamate-glutamine compartmentation
Summary
CONTENTS xiii

TRANSPORT OF TAURINE IN THE CENTRAL NERVOUS SYSTEM

S.S. Oja, P. Kantra, and P. Lahdesmaki 237
Taurine transport in vivo
Taurine influx in vrtro--
Taurine efflux in vitro
Taurine bindinglto synaptosomal membranes

TRANSPORT OF ADENINE DERIVATIVES IN TISSUES OF THE BRAIN

H. McIlwain 253
Translocation and central effects of adenosine
Entry of adenine derivatives to tissues of the brain
adenine
adenosine
hypoxanthine
adenine mononucleotides
Output of adenine derivatives from cerebral tissues
released compounds and cyclic AMP
Intracellular movements of adenine derivatives

KINETICAL ANALYSIS OF THE UPTAKE OF GLUCOSE ANALOGS BY

RAT BRAIN CORTEX SLICES FROM NORMAL AND ISCHEMIC BRAIN
H. Lund-Andersen and C.S. Kjeldsen 265
Uptake of glucose analogues by slices from normal brain
A model of the brain slice
determination of model parameters
uptake of glucose analogues and mannitol
determination of the cellular transport by model fitting
extracellular diffusion versus membrane transport
Uptake of glucose analogues by slices from ischemic brain
determination of model parameters
uptake of glucose analogues and mannitol
comparison between the membrane transport in slices from
normal and ischemic brain
Summary and conclusions

UPTAKE AND EXCHANGE OF GABA AND GLUTAMATE IN ISOLATED NERVE

ENDINGS
G. Levi, U. Pace, and M. Raiteri 273
Synaptosomal exchange of GABA and glutamate
concentration dependence of exchange
sodium-dependence of synaptosomal exchange of GABA and
glutamate
substrate specificity of GABA exchange
parallel decrease of GABA uptake and exchange
effects of ouabain and of calcium ionophore A23l87
Concluding remarks
xiv CONTENTS

HECHANISMS OF TRANSPORT FOR THE UPTAKE AND RELEASE OF

BIOGENIC AHINES IN NERVE ENDINGS
D.F. Bogdanski 291
general characteristics of transport
Mechanisms of transport
ion-gradient hypothesis for transport, general
ion gradient hypothesis for the transport of amines
criticisms of the ion gradient hypothesis, general
criticisms of the ion gradient hypothesis, amines
recent developments in amine transport research
effect of Na+ on the kinetic constants for transport
Electrolytes and storage and mobilization of amines
Evidence for outward transport
various effects of transport inhibitors
role of transport in synaptic transmission

CHARACTERISTICS OF THE UPTAKE AND RELEASE OF GLUTAHIC ACID

IN SYNAPTOSOMES FROM RAT CEREBRAL CORTEX. EFFECTS OF
OUABAIN
G. Takagaki 307
Preparation of slices and synaptosomes
Metabolism of glutamic acid
Uptake and release of glutamic acid
Effects of ouabain on uptake

RELEASE OF BIOGENIC AMINES FRO~~ ISOLATED NERVE ENDINGS

M. Raiteri, A. Bertollini, R. del Carmine and
G. Levi 319
Superfusion of synaptosomes
Some aspects of norepinephrine release from superfused
synaptosomes
Effect of d-amphetamine on the release of biogenic amines
Relationship between structure and releasing activity of
S-phenylethylamine derivatives
effects on norepinephrine
effects on dopamine
effects on 5-hydroxytryptamine

TRANSPORT OF DOPAMINE IN DISCRETE AREAS OF THE STRIATUM

AND OF CEREBRAL CORTEX IN THE RAT
J.P. Tassin, G. Blanc, L. Stinus, B. Berger,
J. Glowinski, and A.M. Thierry 337
Demonstration of the presence of dopaminergic terminals
in the rat cerebral cortex
Reuptake process as a tool to estimate and differentiate
catecholaminergic innervation in brain structures
general characteristics
evidence for a specific 3H-DA uptake in the cerebral
cortex of the rat
CONTENTS xv

3H-Dopamine uptake on microdiscs of cerebral tissues

distribution of dopaminergic terminals in the .rat striatum
distribution of 3H-DA uptake in the cerebral cortex of the rat
localization of the cell bodies of the cortical dopaminergic
terminals

SECTION IV
FACTORS INFLUENCING TRANSPORT
ENERGETICS OF LOW AFFINITY AMINO ACID TRANSPORT INTO
BRAIN SLICES
M. Banay-Schwartz, D.N. Teller, and A. Lajtha 349
scope
rationale for studying slices
Background
other reviews
brain slices
Does active transport of amino acids require glycolysis,
phosphorylation and ATP?
evidence from bacterial studies
comparison of properties of isolated vesicles, cells and slices
Experimental dissociation of transport from glycol¥sis
How does active transport of amino acids require K or Na+
the K+ requirement is not specific
the role of Na+ is more difficult to determine: a. changing
Na+ and K+ simultaneously
b. Na+ flux relationship to amino acid uptake
summary of Na+ relationship to uptake
Dissociation of ion pump activity from uptake

POTASSIUM EFFECTS ON TRANSPORT OF AMINO ACIDS, INORGANIC

IONS AND WATER: ONTOGENETIC AND QUANTITATIVE DIFFERENCES
L. Hertz 371
Potassium effects on amino acid transport
release
uptake
Potassium effects on transport of inorganic ions and water
Potassium effects on metabolism
energy metabolism
Na+-K+-ATPase

EVIDENCE FOR A SYNTHESIS-DEPENDENT RELEASE OF GABA

R. Tapia 385
GARA-dependent inhibition
Studies on the synthesis-dependent release of GABA in vitro
Functional significance of the release of newly synthesized
GABA and other neurotransmitters
 CONTENTS

TRANSPORT OF A~1INO ACIDS AND CATECHOLAMINES IN RELATION

TO METABOLISM AND TRANSMISSION
J.S. de Be11eroche and H.F. Bradford 395
neurotransmitter uptake
neurotransmitter release
experimental approach to the study of transmitter flux
The shuttling of dopamine across the membrane of corpus
striatum synaptosomes: The differential effect of d-
amphetamine
experimental procedure
results and conclusions
The existence of a continuous membrane flux for transmitters

CHANGES IN CEREBRAL AMINO ACID TRANSPORT DURING

DEVELOPMENT
F. Piccoli 405
Developmental changes of enzyme patterns
Extracellular space and ion content
Amino acid transport

SECTION V
RELATIONSHIP OF IN VIVO AND IN VITRO STUDIES
THE USEFULNESS OF STUDIES IN VITRO FOR UNDERSTANDING
CEREBRAL METABOLITE TRANSPORT IN VIVO
A. Lajtha and M. Banay-Schwartz---- 415
Barriers --
in ----
vivo and --
in -vitro
----
Rates of cerebral protein synthesis in vivo and in vitro
Observations on -------
in vitro alterations
Substrate specificity of transport in vivo and in vitro
Aspects of transport that can be studied better in vitro
Function of the barriers
Metabolite compartmentation in vivo and in vitro
Conclusions

RELEASE OF AMINO ACIDS FROM THE SPINAL CORD IN VITRO AND

IN VIVO
--R.W.P. Cutler 435
Release of amino acids from spinal cord slices
Release of amino acids from spinal cord nerve endings
Release of amino acids from isolated spinal cord
Release of amino acids from intact spinal cord
Conclusion

THE DISTRIBUTION OF DRUGS IN THE CENTRAL NERVOUS SYSTEM

A.V. Lorenzo and R. Spector 447
Drug distribution between blood and CSF
salicylic acid
p-aminosalicylic acid
CONTENTS xvii

penicillin
gentamicin

SECTION VI
ALTERATIONS OF TRANSPORT IN PATHOLOGY
CEREBRAL PERMEABILITY PHENOMENA IN EPILEPSY
L. Battistin 465
Level and uptake in vivo
Uptake in vitro - - -
Regional uptake in vivo
Conclusions

PATHOLOGICAL ASPECTS OF BRAIN TRANSPORT PHENOMENA

M. Spatz and I. Klatzo 479
The effect of mercury on BBB
The effect of hyperosmolar perfusate on BBB
Ischemia
the effect on BBB
the effect on synaptosomes
The effect of oxygen saturation and p C02 tension on the
transport from blood to brain
In -
- vivo
- studies
In vitro studies

BRAIN DAMAGE AND ORAL INTAKE OF CERTAIN AMINO ACIDS

J . W. 01 ney 497
Excitotoxic amino acids
Cysteine neurotoxicity
Discussion

PHYSIOPATHOLOGY OF THE BLOOD-BRAIN BARRIER

M.W. Bradbury 507
Cerebral oedema and fluid movement across cerebral capillaries
Volume control of cerebral fluids
Solute movement and accumulation as volume determinants
Primary mechanisms of volume control
Conclusions

BRAIN BARRIER PATHOLOGY IN ACUTE ARTERIAL HYPERTENSION

B. Johansson 517
Acute hypertension induced by vasoactive substances
Acute hypertension induced by clamping of the thoracic aorta
Renal hypertension
The location of BBB lesions in different species
The pathophysiology of the permeability disturbance
the influence of vasoactive substances
mechanical effects of high intraluminal pressure
xviii CONTENTS

Fluorescence microscopical and ultrastructural studies of the

BBB lesions. Location and mechanisms of the vascular damage
The protective effect of structural adaptation in the resistance
vessels in essential hypertension
The relevance of animal studies on acute hypertension to the
clinical syndrome of hypertensive encephalopathy
Conclusions

AUTHOR INDEX 529

SUBJECT INDEX 531
 THE OPENING ADDRESS OF THE SYMPOSIUM

Ladies and Gentlemen:

I am very pleased and honored, as Director of the Department

of Neurology and Psychiatry of the University of Padova, to give
to all of you my best welcome and to thank you for coming here
to participate in this Symposium. Many of you came from overseas
to give us the precious contribution of your experience, and I
think that your presence here is the best reward for the tireless
efforts of Dr.Battistin, who took care of the organization of this
Symposium with great enthusiasm; I also want to express my sincere
gratitude to all Institutions and Pharmaceutical Firms that supported
our efforts.

Let me address particular appreciation to Professor Pinelli,

President of the Italian Society of Neurology, who is today with us,
as well as to Professor Merigliano, Rector of the University of
Padova, for his support, and his kindness in being your host this
evening in the Ancient Archive.

There you can admire a very insp~r~ng surrounding, where the

great tradition of this ancient University resides; the importance
of this University, even though strictly linked to our city, sur-
passes it and enters rightly into the history of culture and of
universal scientific progress.

We can say indeed that the Paduan School has always been the
heart of European Medicine, and that here was the origin of modern
medicine. In fact, during 1500, anatomical research on the corpse,
vivisection, and anatomo-comparative survey were systematically
carried out in this University. Moreover, here began the pharma-
ceutical teaching in the famous "Botanical Garden", as well as the
concept of "animal infection" and the clinical teaching at the bed
of the patient.

All these methods of research spread out in different ways to

every part of Europe, so that we can state that the teaching of
Fabrizio d'Acquapendente and Galileo certainly had a remarkable role
xix
xx s. RIGOTTI

in Harvey's discovery of blood circulation.

I could say much more about the history of medicine in Padova,

which is rich in basic events in the progress of science; I only
wish to recall, particularly for our overseas guests, the visit
that Dr.John Morgan from Philadelphia made in the second half of
1700 to Gian Battista Morgagni; that was the first contact between
American and Paduan medicine, and it is renewed today in a hearty
spirit of cooperation, leading to a warm relationship.

I am, proud to belong to this School, and I apologize for my

long description of the history of this University, which is always
open to every new technique and every trend. We are very pleased to
give our hospitality for this Symposium of the International Society
for Neurochemistry.

Neurochemistry is a relatively new branch, which only recently

acquired an autonomous position; but only a few years after its
timid appearance it caught the attention of neurologists and psych-
iatrists as well as biochemists, as is attested by several congresses
and international Symposia held in these last years, and even during
these last months.

The success of Neurochemistry, like that of other branches, for

instance Neuropharmacology, brings for us clinicians the problem of
the relationship between our branch and new disciplines. This re-
lation should not still be simple coexistence, as it was in the past,
but it should lead to real multidisciplinary research work, based on
strict cooperation; we could say that we need an osmotic process
among the various clinical and basic research branches.

I think that everybody agrees with this opinion, but it is its

realization that meets obstacles and difficulties.

Nobody doubts the soundness of Neurochemistry, and indeed this

Symposium itself proves its importance; therefore nobody doubts its
right to being an autonomous branch. Nevertheless, it is necessary
to avoid having this autonomy foster isolation, even if theoretically
it doesn't lead to it.

For Neurochemistry, such isolation would mean lack of contact

with clinical problems, and for neurologists, the impossibility to
ask your cooperation in carrying out the investigations on patho-
genesis and treatment of various diseases. In order to avoid this,
it is necessary to act on men and on structures, keeping in mind
the following points:

First of all, we must recognize the main importance of research

on brain functions, whose results, widening our knowledge on superior
OPENING ADDRESS xxi

nervous activity,represent progress not only·for medicine but also

for science in general. Therefore scientists of this branch have
the right to ask for a priority that - actually - is not always
recognized.

Secondly, we think that it is extremely important - without

passing over the fundamental research on elementary mechanisms,
which are certainly indispensable - to plan to have a closer
relationship of your branch of science with man.

Therefore researchers should more often be physicians, in the

sense that a greater number of physicians should apply themselves
to research, and also that non-medical scholars should become at
least to some extent experts in medicine; this clearly implies
a specific cultural background and a particular intellectual
approach.

In fact, we should not forget that almost all progress during

these last 10 years came either from the work of one specialist
who tried to extend his knowledge and technique towards another
branch, or from strict cooperation among researchers of different
disciplines.

From such hopes is derived the need of meetings like this, where
non-medical researchers get closer to clinical science and share its
problems and anxieties; on the other hand, physicians can, by coming
into contact with. researchers, improve their technical knowledge with
better methodological and mental understanding.

Finally, the third point regards structures - the need for a

closer link to clinical and hospital grounds, so that topography
also can support the intimate alliance among scholars of different
disciplines.

This implies, and the observation concerns mainly Italy, the

need of more flexible and open structures, and the possibility of
exchanges among researchers, both inside our country and abroad.

It is a new mentality that will rise, an intellectual approach

able to cancel bureaucratic links and to surpass traditional schemes
in order to create a new reality; that is the true multidisciplinary
research.

Only in this way can clinical science really improve and be

useful to the patient: it is sufficient to think what support was
brought to therapeutics by the studies on neurotransmitters of the
nervous system, serotonin, noradrenaline, dopamines, and GABA.

For all these reasons we will carefully listen to your communi-

cations and discussions in this Symposium, which I am sure will be
xxii S. RIGOTTI

full of interesting data, and rich in new perspectives.

Therefore I wish you good work, but I also hope that you
will find the time to visit our Padova, which with its old roads
and its various architecture shows some lovely contrasts and some
peculiar perspectives as well as some important monuments, that
incomparably testify to art and human creative genius.

With this wish I renew my friendly regards and my sincere

welcome.

Thank you.

Professor S. Rigotti

Padova, September 1975

Metabolite Transport
METABOLITE TRANSPORT AT CELL MEMBRANES

HALVOR N. CHRISTENSEN

THE UNIVERSITY OF MICHIGAN

ANN ARBOR, MICHIGAN 48104 U.S.A.

INTRODUCTION

From the assigned title I am glad to asswne that the organizers

would like to have me discuss some general iieas of metabolite trans-
port, rather than to try to summarize the findings specific to the
nervous system, the details of which most of you may know rather bet-
ter than I do. Fortunately, transport behavior seems in general to
be similar among tissues, so that the history of transport ideas and
findings is a single history and not one specific to each anatomical
or functional field, despite occasional impressions to the contrary.
I assume that the means of discrimination of distinct transport sys-
tems falls among the subjects that I might more logically discuss.
That subject continues to deserve atte~tion, because one still sees
bent Lineweaver-Burk plots treated as though they were sufficient evi-
dence for the contribution of two parallel transport systems. I feel
obliged, however, not to spend much of my time in this presentation
in explaining what my colleagues and I have been doing in past years
to codify the several amino acid transport systems, or in indicating
what the new difficulties are, or in summarizing how these codifica-
tions have so far applied to nervous tissue. Instead I want mainly
to treat a theoretical problem that has come to seem to me critically
restrictive to progress. I trust I can later help you with some use-
ful thoughts On the discrimination of the several systems, as such
comments may become appropriate during the discussion time.

MEDIATION OF APPARENTLY SPONTANEOUS METABOLITE MIGRATIONS

At a remote time in the past biologists thought metabolites just

"soak" into and out of cells. At another, not quite so remote time

3
4 H.N. CHRISTENSEN

the biochemist considered the "permeability problem" either a dread-

ful nuisance or a flexible means by which he could rationalize the
failure of his experiments. I believe one can now assert success-
fully that almost all of the passage of metabolites into and out of
cells occurs by a "determined" process. By the word determined, I
mean that the molecule of the metabolite is recognized by the cellu-
lar or organelle membrane, and that its passage is permitted or im-
pelled in one direction or the other as a result of that recognition.

We have no trouble accepting that metabolite migration is a de-

termined or mediated process when it proceeds against an electroche-
mical gradient. When the migration occurs instead in the direction
we suppose to be the spontaneous one, however, we continue to be
tempted to assume that it is just as spontaneous (and therefore ener-
gy-wasting) as it looks. We speak all too easily of simple diffusion
when it is highly improbable that most of our hydrophilic ions or
molecules finds a path open through the plasma membrane to any simple
kind of diffusion. Our metabolites are simply not the kinds of par-
ticles that move unrestrainedly through our kinds of membranes.

Fig. 1 is a picture of a model in the Museum of the History of

Science in Florence, called a Paradosso Meccanico. Perhaps you will
excuse me if I use this device to illustrate uphill transport in a
simple-minded way. We look at the inclined plane and decide intui-
tively that the rotor will spontaneously roll down the plane, just
as we decide that a manifestly downhill migration of a metabolite
will be fully spontaneous. What we have overlooked is that there is
another gradient here, one directed in the opposite sense. As a re-
sult, the spontaneous direction for the rotor to roll is actually up
the inclined plane. Just as surely a metabolite migrates in the di-
rection that represents spontaneity for the whole transport process,
and not just for the migration considered alone.

I offer this device as model for the coupled process, A + Xl ~

B + X2' in which an exergonic chemical reaction A ~ B provides
enough energy to drive the transport of X from phase 1 to phase 2.
We will let the slope of the inclined plane represent the gradient
[X]2/[X]I, against which the transport can take place; and we will
let the slope of the cones which form the axes of our rotor repre-
sent the ratio, [B]/[A]. This ratio determines how strong a gradient
[X]2/[X]1 the reaction can produce. If instead I let this cylinder
roll down the inclined plane, I illustrate the spontaneous direction
of the unhindered transport, as an isolated event. If I merely let
this conical rotor move across the leveled plane, I illustrate the
full spontaneity of the uncoupled reaction. But, once these two
gravitational forces come to apply simultaneously to the rotor all
that matters is the residual spontaneity. If we observe whether the
center of gravity of the rotor is moving upward or downward, we can
predict the spontaneous direction. The fall of the center of gravity
TRANSPORT SYSTEMS 5

Fig. 1. Photograph of model "Paradosso Meccanico," reproduced cour-

tesy of the Museum and Institute of the History of Science, Florence.

corresponds in the biological situation to the decrease of the free

energy. We need then to think of both the gradients, [X)2/[X]1 and
[B]/[A], to get the sign of the free energy change, and hence to
identify the direction of spontaneity.

EACH ACTIVE TRANSPORT HAS A REVERSE PHASE

Notice by reference to the model how easily we can go from the

condition where the chemical reaction drives the transport, to the
condition where the transport drives the chemical reaction. By put-
ting a shim under one end of the device, I can reverse the direction.
In correspondence, the downhill movement of a metabolite,which looks
so spontaneous to us, may be only slightly spontaneous because it lS
doing unsuspected work. Furthermore, small concentration changes can
make it spontaneous in the other direction.

According to all indications, it is thermodynamically impossible

to construct a purely unidirectional uphill transport process. Each
transport system must have both directions inherent in it. Just as
one of the slopes in our model can produce motion against the opposed
slope only if the latter is not too steep, so the concentration ratio
[B]/[A] of our chemical reactants can raise the gradient, [X]2/[X]1,
only to a certain magnitude. If [X]2/[X]1 is too large, the sponta-
neous direction will have the transport driving the chemical reac-
tion. In molecular events the thermodynamic limit is characteristi-
cally established by the tendency of the whole process to reverse it-
self. My subject then is to relate the problem of the energization
of transport to the important question of how exodus is mediated.
6 H.N. CHRISTENSEN

Fig. 2 illustrates the relationship in a schematic way. First

we see a scheme in which the linkage between A ~ B and Xl ~ X2 ap-
plies only to the forward direction. A backflow of X is also shown,
but it is dissociated from the chemical reaction. Such a backflow
cannot, however, be the process that sets the thermodynamic limit
that the gradient [X]2/[X]1 can approach. Instead we should see it
only as a leak. We could seal such a leak and cause the gradient to
go still higher, until the true equilibrium is reached. Unless a
true return route exists, however, we have a perpetual-motion ma-
chine. By putting heads on both ends of the two arrows (right-hand
scheme), both on the arrow showing the purely osmotic effect and on
the one showing the purely chemical effect, we indicate how the equi-
librium can be reached. I suspect many of us at one time or another
have drawn schemes like the first one, and fancied that energy trans-
fer can be restricted to one flux of the transport process. It can-
not. Therefore it seems an idle question, to which flux is the
energy applied?

MEANING OF DUALITY OR MULTIPLICITY OF TRANSPORT SYSTEMS

Let me now turn to another significant feature of metabolite

transport at the cellular membrane. This feature is the tendency of
each metabolite to be transported by what appear to be at least two
distinct agencies. Furthermore these transport systems are in some
conspicuous cases energized in two distinct ways. Each neutral amino
acid is transported into cells by a system energized at least in part
by the electrochemical gradient of Na+; but also at the same time by
another, Na+-independent system which may well be energized by ATP
cleavage. One of these systems can typically generate rather steep
gradients, say 10:1 or 50:1, but the other usually generates only mo-
dest gradients, say 2:1. In our experiments we are inclined to set
the conditions so that each of these systems is seen operating in-
wardly. We tend then, in a teleologic way, to suppose that they both
operate physiologically in that direction, one system helping the
other, or one providing a margin of safety in case of genetic failure
of the other to develop.

A B
A B

XI ~4------------------ XI'' ' L.

Fig. 2. Two schemes to show the energization of transport.
TRANSPORT SYSTEMS 7

HOW WILL TWO INDEPENDENT, UNEQUAL PUMPS "COOPERATE"?

In reality, two transport systems are unlikely to establish such

a "helping" relation with each other. What we see instead is that
the more steeply uphill system serves for net uptake, and the less
steeply uphill system for net exodus . If the equation for the latter,
more weakly uphill system is written Q + Xl + R + X2' then it will
come to operate in the net direction X2 + R + Xl + Q. A consequence
is that a total reaction A + R + B + Q can be produced by coupling
through the two transport processes. Note that we reach in this way
a generalized form of the Mitchell hypothesis.

Fig. 3 may help you visualize how two pumps working together can
establish a cyclic flow. The stronger pump is able to maintain the
tank level at the higher point, Ii the weaker pump, only at the lower
point, II. At those points as many molecules of water are moving
downward as upward through the pump used. When both pumps work to-
gether, however, net entry occurs by the first, net exodus by the se-
cond, and an intermediate level (III) is maintained. Unfortunately
the analogy of Fig. 3 is defective in that it attributes the limit of
the level attainable by each pump to its inefficiency, whereas in the
biological context we should expect the energy available to each to
be an important factor. The model would be better if it showed the
second pump driven backward, so that it operated as an electric gen-
erator, when the water in the tank stands at the indicated level.

EVIDENCE THAT EXODUS OCCURS BY REVERSAL OF A WEAKER PUMP

Incompletely interpreted evidence for such a division of fluxes

between transport systems has long been available from the observation
that the stereospecificity of exodus of amino acids (e.g. alanine)
from the Ehrlich cell is substantially greater than the stereospeci-
ficity of entry. We assign a much larger component of exodus there-

-I

-__..::.~- m

- IT

Fig. 3. A hydrostatic analog for the interaction between two paral-

lel transport systems.
8 H.N. CHRISTENSEN

fore to the more highly stereospecific system ASC. 9 Special systems

for exodus, rather than ordinary systems operating backwards, have oc-
casionally been proposed instead. Another line of evidence for our
proposal comes from the observations of the partial depolarization of
the plasma membrane of epithelial cells of the intestine 15 and of the
Triturus kidney 12, 14 when alanine or glucose is taken up. This depo-
larization begins immediately when alanine is added. It arises from
the introduction ofNa+ into the cell, along with the organic nutrient,
by a Na+-dependent transport system. An important question is whe-
ther the depolarization continues only while net inward movement of
the metabolite takes place, or whether it is maintained at the steady
state. Hoshi's experiments 12 show that the latter is the case for
the renal epithelial celli the depolarized state continues for the
whole period of observation, although alanine accumulation is quickly
complete. The implication is that although alanine enters the cell
in company with Na+ through co-transport, it must leave the cell to a
substantial extent by a Na+-independent flow.

In the past the tendency has been to suppose that alanine and
other amino acids leave the epithelial cell by simple leakage - but
this tendency represents I believe the same fallacy that the mechani-
cal analog brought out: If the migration is down the gradient, we
tend to assume that it is just as spontaneous, just as energy wasting,
as it looks. Furthermore, when the spontaneity is in fact unknown,
we should avoid terminology that suggests high spontaneity. It is in
fact clear that amino acids are pumped into the intestinal epithelial
cell in the postabsorptive state and not merely during absorption.
Clearly the amino acid nutrition of the epithelial cell must be pro-
tected by having amino acids pumped in. In the same way the cellular
levels need to be protected during absorption by restraining exodus
by having it work against a pump. A high population of Na+-indepen-
dent pumps for amino acids and sugars appears to be characteristic
of the basolateral aspects of the epithelial cells of the proximal
tubule.

You may find it a little harder, for cells not engaged in absorp-
tion or secretion, to understand why a metabolite should be brought
in by one transport system and released by another. Consider, how-
ever, how often biochemical architecture of sorts other than gradients
is built up by one route and degraded by another. Such arrangements
no doubt permit superior control and greater energetic economy than
the alternatives.

INCREASED IMPORTANCE OF DISCRIMINATING DISTINCT TRANSPORT SYSTEMS

These ideas tend if anything to increase the importance of codi-

fying the various transport systems for organic metabolites, in order
to recognize couples between such systems that balance entry and exo-
dus at the steady state. At this point I am tempted to refer somewhat
TRANSPORT SYSTEMS 9

skeptically to an example: I should like to believe that in the

cells of the cerebral cortex there are two different transport sys-
tems for the basic ~ino acids in nervous tissue, as we once proposed
for the Ehrlich cell,iCf.6,7 and just as there are at least two for
the neutral amino acids. I feel obliged, nevertheless, to serve as
a devil's advocate on the point of the presumed second general system
for basic amino acids, and to remark that I find the evidence so far
insufficient. Nevertheless, we are certainly correct to pay attention
to the important differences in the structures of the acidic, the
neutral, and the basic amino acids, and to all that we can learn from
other important structural differences in this interesting group of
metabolites. We now have to take into account that, by each producing
prompt changes in the electric polarity of the plasma membrane, two
metabolites transported by separate systems can nevertheless interact
as though they migrated bY'a single system, by showing either mutual
transport inhibition or mutual accelerative exchange. This situation
makes harder the differentiation of distinct transport systems.

ROLES OF RECEPTOR SITES FOR AMINO ACID TRANSPORT IN NEUROTRANSMISSION?

Realizing that the transfer of an amino acid across the synaptic

membrane could sharply modify the electric polarity of the membrane,
I urged in a 1970 symposium 2 exploration of the possibility that spe-
cial Na+-dependent transport receptor sites might mediate the neuro-
transmitter actions attributed to certain amino acids. Transport
sites have now been evaluated for this role, along with amino acid
receptor sites of high and low affinity not related to transport, with
results that so far appear extremely interesting even though equivo-
cal. Such possible mediating roles in neurotransmission exerted by
amino acids through their ionophoric actions in the presence of an
appropriate transport-mediating system, deserve, I think, continued
consideration. The outward as well as the inward flux of the amino
acid, or both, could be involved. Note the analogy shown by the che-
motactic response of E. coli: The binding protein associated with
transport of a nutrient appears to be essential also for the recogni-
tion of a gradient of that nutrient in the environment 10 • The high-
affinity systems may prove effective for terminating neurotransmitter
actions, as originally suggested, but I find it a little hard to un-
derstand how a recognition site is to give a signal if its affinity
is so high as to leave it fully engaged at all metabolite concentra-
tions encountered physiologically.

THE PLACE OF THE HYDROGEN ION

The greatest current excitement as to metabolite transport into

cells probably is that arising from the realization that these move-
ments are regularly electrogenic even for uncharged substances, and
that in some prominent instances they may be coupled to the flow of
10 H.N. CHRISTENSEN

the hydrogen ion rather than Na+. Co-transport of amino acids with
the hydrogen ion has now been seen in several species. The phenomenon
may follow logically from the ability of the membrane to recognize
the amino acid molecule first in one and then in another state of pro-
tonation 5 • Co-transport with H+ has, however, also emerged for sugars,
whose molecule seems unlikely to yield a hydrogen ion during the
process I6 ,17,13.

Transport by System ~ can be shown to be accelerated by increased

external hydrogen-ion concentrations, either by making the tests in
Na+-free, choline-containing media, or by using selected substrates.
In the latter case the parallel movements of H+ into the Ehrlich cell
can be shown 3 • J. Garcia-Sancho and A. Sanchez 8 have now observed in
my laboratory that the intensified uptake of glutamic acid by the same
cellon lowering the pHIl arises from the emergence of a transport of
this amino acid by System ~, at the same time that a slow uptake by
System A is largely eliminated. The new component of glutamic acid
uptake,-increasing as the pH is lowered from 7 to 4, is Na+-indepen-
dent and readily inhibited by 2-aminonorbornane-2-carboxylic acid
(Ki = O.2mM), the best model substrate for System L, and is unaffected
by the presence of ~-(methylamino)-isobutyric acid~ the most trustwor-
thy model substrate known for System!. In turn, external glutamic
acid strongly stimulates the exodus from the cell of the previously
accumulated bicyclic amino acid, whereas ~-(methylamino)-isobutyric
acid does not. The identification of this H+-stimulated component
of glutamic acid uptake was complicated by the circumstance that Sys-
tem L becomes much less selective as to its substrates at pH 4 to 5,
so that amino acids taken up almost exclusively by System! at pH 7.4
(e.g., AlB, glycine) come to enter the cell instead by System L, not
system A, at the low pH values, at the same time retaining their in-
hibitionof glutamic acid uptake, which, however, now becomes Na+-in-
dependent 8 • Thus AlB loses its place as a trustworthy model substrate
for System A. This widening of the scope of System L as the pH is
lowered und;ubtedly has unfavorable effects on the ability of the cell
to retain the amino acids ordinarily most strongly accumulated.

Because the titration curve showing the acceleration of glutamic

uptake by System L is significantly displaced upward from the titra-
tion curve of the-distal carboxyl group of glutamic acid, we suppose
that a hydrogen ion is added to bridge between that carboxyl group
and an unidentified structure at the receptor site. The dicarboxylic
amino acids should therefore provide a strong opportunity to locate
this point, whose protonation appears otherwise to enhance operation
of the system. The sidechain of aspartic acid seems too short to
bridge well to the sub-site, because this amino acid shows only one-
fifth as much uptake by this route as glutamic acid. The Ehrlich
cell lacks a transport agency seen in other biological systems, which
transports the dicarboxylic amino acids as anions, including even the
analog cysteic acid, despite the improbability that its sulfonate
group can be protonated in the physiological context.
TRANSPORT SYSTEMS 11

From the point of view of neurochemistry, these results may be

significant in bringing attention to the role of the hydrogen ion in
transport, and in supporting an earlier proposal that alkali-metal-
ion gradients may have been substituted stepwise through evolution
for a primitive but more troublesome service of hydrogen-ion gra-
dients as the major form of storage of membrane energy.

THE FUTURE: GENERATION VERSUS INTERCONVERSION OF ENERGETIC GRADIENTS

A figure published elsewhere 4 serves to pose the question, what

process represents the obligatory coupling by which electron transport
can drive oxidative phosphorylation, and vice versa, and by which
either electron transport or ATP hydrolysis can drive active trans-
port? Only one of these events needs to occur, however, for transport
to result, because the activated state can arise quite as well from
one as from the other source. In various intact cells and vesicles,
in contrast to the so-called energy-transducing membranes, we see the
simplified situation in which one or the other or these energy inputs
is excluded, so that transport arises from the other output.

What makes identification of the actual linkage to transport

difficult is that the "activated state" of the membrane may charac-
teristically involve not only the unidentified obligatory component,
but various ancillary forms of en~rgy storage that can arise reversi-
bly from the obligatory one. Where do we place gradients of ~, gra-
dients of Na+ and K+ and of metabolites, the transmembrane potential,
the formation of high-energy compounds? And which of these then is
obligatory to the flow of energy between electr0n transport and ATP
function, or between one of these and membrane transport? These an-
cillary forms of energy storage may also drive transport by main-
taining the activated state of the membrane. To what degree can we
experimentally dissociate the obligatory feature from secondary
features?

Let me propose what seems to me the most important question of

active transport: Most central is not how this or that ion or mole-
cule is caused to respond by electrophoresis to a potential gradient,
or by co-transport to a gradient of H+ or Na+. Rather the primary
question is how a molecular species is caused in the first place to
move against such gradients, and therefore to create them, thus to
generate the activated state to which electrophoresis and co-trans-
port can then be spontaneous responses.

The part of the underlying research that derives from my labora-

tory received support (Grant HD01233) from the Institute for child
Health and Human Development, National Institutes of Health, U.S.
Public Health Service. I am indebted to the collaborators named
in the references below.
12 H.N. CHRISTENSEN

REFERENCES

1. Christensen, H.N. (1964) A transport system serving for mono- and

diamino acids. Proc. Nat. Acad. Sci. U.S. 51, 337-344.
2. Christensen, H.N. (1972r-:Nature and-Roles of Receptor Sites for
Amino Acid Transport. Adv. in Biochem. Pharmacol. 4, 39-62.
3. Christensen, H.N. (1974)--On the meaning of effects of substrate
structure on biological transport. J. Bioenergetics 4, 233-263.
4. Christensen, H.N. (1975) Biological Transport, Second edition, W.
A. Benjamin, Reading, Massachusetts, U.S.A., pp. 368, 359.
5. Christensen, H.N. (1975) A Cycle of Deprotonation and Reprotona-
tion in Amino Acid Transport? Proc. Nat. Acad. Sci. U.S. 72, 23-27.
6. Christensen, H.N., and Handlogten~.E:-cI96gr- Reactions of Neu-
tral Amino Acids plus Na+ with a Cationic Amino Acid Transport System.
FEBS Letters 1, 14-17.
7. Christensen, H.N., and Liang, M. (1966) Transport of diamino
acids into the Ehrlich cell. J. Biol. Chem. 241, 5542-5551.
8. Christensen, H.N., Handlogten;-M.E., Garcia-Sancho, J., and San-
chez, A. Protonations and Deprotonations in Amino Acid Transport.
Possible Relations to Energy Transfer. Proceedings Intl. Symposium
Amino Acid Transport and Uric Acid, Innsbruck, June, 1975, in press.
g:-christensen, H.N.~iang, ~and Archer, E.G. (1967) A distinct
Na+-requiring transport system for alanine, serine, cysteine and simi-
lar amino acids. J. Biol. Chern. 242, 5237-5246.
10. Hazelbauer, G;L., and Adler,~ (1971) Role of the Galactose
Binding Protein in Chemotaxis of Escherichia coli toward Galactose.
Nature New Biol. 230, 101-104. ----
11. Heinz ~Pichler, A.G., and Pfeiffer, B. (1965) Studies on the
transport of glutamate in the Ehrlich cell - Inhibition by other amino
acids and stimulation by H-ions. Biochem. Z. 342, 542-552.
12. Hoshi, T. (1975) Electrophysiological-studies on amino acid
transport across the luminal membrane of the proximal tubule of the
Triturus kidney. I. Alanine-induced depolarization and hyperpolariza-
tion. Abstract 158, VI International Congress of Nephrology, Florence.
13. Kashket, E.R., and Wilson, T.H. (1972) Protonmotive Force in
Fermenting Streptococcus lactis 7962 in Relation to Sugar Accumulation.
Biochem. Biophys. Res. Commun. 49, 615-620.
14. Maruyama, T., and Hoshi, T. (1970) The Effect of Glucose on the
Electrical Potential Profile across the Proximal Tubule of the Newt
Kidney. Biochim. Biophys. Acta 282, 214-225.
15. Murer, H., Sigrist-Nelson, K., and Hopfer, U. (1975) On the
Mechanism of Sugar and Amino Acid Interaction in Intestinal Transport.
~. Biol. Chem. 250, in press.
16. West, I.C. (1970) Lactose Transport Coupled to Proton Movements
in Escherichia coli. Biochem. Biophys. Res. Commun. 41, 655-661.
17. West, I.C., and Mitchell, P. (1972) Proton-Coupled S-galactoside
Translocation in Non-Metabolizing Escherichia coli. J. Bioenerg. l,
445-462.
POSSIBLE ROLE OF GLUTATHIONE IN TRANSPORT PROCESSES

Marian Orlowski
Department of Pharmacology
Mount Sinai School of Medicine of the
City University of New York
New York, New York

INTRODUCTION

A discussion of the possible function of glutathione in trans-

port processes should consider two structural features of the mo-
lecule; namely the presence of a SH group and the presence of
a y-glutamyl group. Glutathione is present in all mammalian tissues
and cells. Its concentration in the kidney was reported as 2 to 4
mM 20 . Concentrations as high as 10 to 12 mM, however, have been
found in some other tissues 62 , 84. In spite of its "ubiquitous" pre-
sence the role of glutathione is not yet sufficiently understood.
Most of the studies on glutathione have centered on its function in
maintaining sulfhydryl groups of proteins in a reduced state, on its
protection of cell membranes against an oxidative stress, and on its
role in detoxification of foreign compounds. Glutathione has also
been credited with a coenzyme function in several enzymatic reac-
tions. All these functions are dependent on the presence of an in-
tact sulfhydryl group. The possible role of the y-glutamyl gro~p
of glutathione has attracted less attention. This discussion will
be limited to the enzymology of the y-glutamyl group. Some experi-
ments, however, will be reviewed in which the evidence can be taken
as supporting the involvement of either the y-glutamyl group or the
SH group in the transport of amino acids.

ENZYMATIC SYNTHESIS AND DEGRADATION OF GLUTATHIONE

Glutathione is synthesized in two separate enzymatic steps,

catalyzed in sequence by y-glutamylcysteine synthetase (reaction 1)
and glutathione synthetase (reaction 2):
1) L-Glutamate + L-cysteine + ATP ~ L-y-glutamyl-L-cysteine
+ ADP + Pi

13
14 M.ORLOWSKI

2) L-y-glutamyl-L-cysteine + glycine + ATP ~ L-y-glutamyl-L-

cysteinylglycine (GSH) + ADP + Pi
The complete synthesis of glutathione from its component amino
acids was first accomplished by Bloch and his coworkers74,
The main pathway of glutathione degradation is the reaction
catalyzed by y-glutamyl transpeptidase, This membrane-bound enzyme
catalyzes the transfer of the y-glutamyl group of glutathione to
amino acids or some peptides according to the following reaction:
3) Glutathione + amino acid (or peptide) ~ y-glutamyl amino
acid (or peptide) + cysteinylglycine
This reaction was first observed by Hanes and his coworkers30,
The enzyme seems to be identical with the activity studied earlier
by several authors, and referred to as "antiglyoxylase" or "gluta-
thionase"9,19,9 0 , In addition to catalyzing the transfer reaction
(reaction 3) the enzyme is capable of hydrolyzing the y-glutamyl
bond of glutathione (reaction 3a) and catalyzing an autotranspepti-
dation reacti9n in which the substrate itself serves as acceptor
(reaction 3b)6,23:
3a) Glutathione + H20 ~ glutamic acid + cysteinylglycine
3b) Glutathione + glutathione ~ Y-glutamylglutathione + cyste-
in;y-lglycine
Similar reactions occur with other y-glutamyl peptides or y-
glutamyl amino acids,
y-Glutamyl amino acids which are formed in the transpeptida-
tion reaction between GSH and amino acids can be converted to
pyrrolidone carboxylate (synonyms of L-pyrrolidone carboxylate
are L-5-oxoproline, L-pyroglutamate, 5-oxopyrrolidine-2-carboxylate)
and free amino acid by the cytoplasmatic enzyme, y-glutamyl cyclo-
transferase (reaction 4)17:
4) L-y-glutamyl amino acid - L-pyrrolidone carboxylate + amino
acid
L-Pyrrolidone carboxylate is converted to L-glutamate in the
kidney and other tissues50,51 ,60,69, An enzyme, L-pyrrolidone car-
boxylate hydrolase (5-oxo-L-prolinase), has been found in mammalian
tissues85 to catalyze an ATP dependent hydrolysis of L-pyrroli-
done carboxylate to L-glutamate (reaction 5):
5) L-Pyrrolidone carboxylate + ATP + 2H20 ~ L-glutamate + ADP
+ H2 0
Cysteinylglycine, which is released during the action of
y-glutamyl transpeptidase on GSH, is hydroly~ed to its component
amino acids by one or more peptidases present in tissues (reaction
6)10,41:
6) L-cysteinylglycine - L-cysteine + glycine
ROLE OF GLUTATHIONE IN TRANSPORT 15

THE y-GLUTAMYL CYCLE

The amino acid dependent degradation of GSH and the reactions

leading to its resynthesis have been integrated into a cyclic pro-
cess called the "y-glutamyl cycle"5 0 • The cycle was proposed as
an amino acid transport system in the kidney, and possibly in some
other tissues. Evidence which ~ummarized and extended this proposal
was subsequently published4~,4j.The hypothesis linking the y-gluta-
my1 cycle to amino acid transport assumes that the reaction cata-
lyzed by membrane bound y-glutamyl transpeptidase operates in the
translocation step of this transport process.
There are limitations to this hypothesis. For example some
cells have little if any y-glutamyl transpeptidase although these
cells are still capable of amino acid transport. It seems therefore
unlikely that the cycle represents a general amino acid transport
system for all amino acids in all cells. Its function is probably
limited to selected sites where y-glutamyl transpeptidase is highly
concentrated, The well documented transport of non-metabolizable
amino acids lo probably cannot be mediated by the y-glutamyl trans-
peptidase reaction, since amino acids in which the a-hydrogen is re-
placed by some other group are not substrates for the enzyme. At the
present time no single system can account for all transport phenome-
na of all amino acids described in experiments in vitro and in vivo.

y-Glu-cysH

Fig.l. The y-glutamyl cycle. 1, y-glutamylcysteine synthetase;

2, glutathione synthetase; 3, y-glutamyl transpeptidase; 4, y-glu-
tamyl cyclotransferase; 5, pyrrolidone carboxylate hydrolase;
6, cysteineglycine dipeptidase. AA = amino acid; PCA = pyrrolidone
carboxylate
16 M. ORLOWSKI

y-Glutamyl transpeptidase, the key enzyme of the y-glutamyl

cycle, which can interact with GSH and amino acids, is associated
with the insoluble particulate fractions of tissue homogenates. Pu-
rification of the enzyme can be achieved by treatment of the par-
ticles with detergents, such as deoxycholate and also by treatment
with I-butano1 23,49,75,81. The preparations obtained in this manner
are usually heterogeneous with respect to molecular rreight,l,6sedimen-
tation coefficient and migration in electrophoresis49,r5,( ,elf The
isolation of two isozymes of y-glutamyl transpeptidase was reported
from bovine and rat kidney75,79. This finding, however was not con-
firmed in later studies 82 • These isozymes may be artefacts resulting
from the fact that unrelated, membrane-derived proteins or polypep-
tide chains remain bound or aggregated with the enzyme.
y-Glutamyl tran§P~tidase is a glycoprotein containing up to
36% carbohydrate49,7),(~. The enzyme is very resistant to the action
of trypsin and other proteolytic enzymes9, and this property has
been utilized in its purification 65. The large amount of carbohyd-
rate apparently renders the sensitive peptide bonds inaccessible to
attack by proteolytic enzymes.
We have recently obtained a homogeneous preparation of the en-
zyme from sheep kidney cortex (Zelazo & Orlowski, unpublished re-
sults from the authors' laboratory). The enzyme has a molecular
weight of approximately 90,000. It is composed of two unequal sub-
units: one with a molecular weight of 27,000, the other with a mo-
lecular weight of 65,000. The enzyme shows little activity in the
absence of an added metal ion or acceptor (Table *). Sodium and po-
tassium and even more so the divalent cations Ca2 and Hg2+ strong-
ly activate the enzyme. Among the L-amino acids, the greatest acce-
leration of the reaction is obtained with methionine and glutamine.
It is of interest that glutamate and aspartate are much less effec-
tive than their corresponding amides.
Simultaneous determination of the products of the hydrolytic
and transfer reactions of the enzyme showed that none of the metals
significantly affected the formation of glutamate, and that the ac-
tivating effect of metal ions is entirely due to the acceleration
of the transfer reaction, and formation of y-glutamylglutathione
(Table 2). In the presence of acceptor amino acids, transfer of
the y-glutamyl group of glutathione to the acceptor represents ini-
tially the main reaction. Although the ratio of the transfer reac-
tion to the hydrolytic reaction in vivo is not known, and might be
dependent on pH and on the concentration of metal ions and amino
acids, it can be assumed that the degradation of glutathione depends
to a large extent on the presence of amino acids.
After the discovery of the y-glutamyl transpeptidation reaction,
Hanes considered the possibility that the reaction may function in
protein synthesis 30 • Studies in this direction, however were unsuc-
cessful. The possibility that y-glutamyl transpeptidase might func-
tion in amino acid transport was metioned by several authors 5 ,8,33.
This idea was not pursued and did not attract the attention of re-
searchers working in the field mf amino acid transport.
ROLE OF GLUTATHIONE IN TRANSPORT 17

Table 1. Activation of y-glutamyl transpeptidase by metal ions

and several acceptor amino acids*

Add i t i o n s Activity
Specific Relative

None 20 100
Na+ (150 mMj 44 220
K+ (150 roM 46 230
CaZt- ~10 mM 150 750
Mg2+ 10 roM 140 700
Mg2+ 10 roM~
+ Na+ (150 roM) 80 400
Glycine 253 1300
L-Leucine 242 1200
L-Phenylalanine 253 1300
L-Aspartate 104 520
L-Asparagine 412 2100
L-Glutamate 205 1000
L-Glutamine 709 3500
L-Methionine 784 3900
L-Arginine 429 2100
L-Lvsine 366 1800
*The reaction mixtures contained GSH (0.005M), Tris-HCl buffer
(0.08 M; pH 8.8), dithiothreitol (0.005 M), enzyme and additions in-
dicated in the table, in a final volume of 0.5 ml.Activity is in
pmoles of cysteinylglycine released per mg enzyme per min. Relative
activities are relative to those obtained in the absence of any ac-
tivators. The activating effect of amino acids (20 roM) was measured
in the presence of Mg2+ (10 mM)

Table 2. Effect of metal ions on the hydrolytic and transfer

activity of y-glutamyl transpeptidase*
Rea c t i o n produc t
Metal ion Glutamate y-Glutamyl-
glutathione
nmol nmol
None 12.2 18 \1.~~
Na+ (150 mM) 15.0 55 (3.7
K+ (150 mM~ 15.0 56 p.7)
Ca2+ (10 roM 14.8 198 13.4)
Mg2+ (10 roM) 15.3 182 (11.9)

*The reaction mixtures (no acceptor added) were the same as i n

Table 1. The products were determined by amino acid analysis. y-Glu-
tamylglutathione was determined as the S-acetamido derivative. Va-
lues in parenthesis are ratios of transfer reaction to hydrolysis.
18 M. ORLOWSKI

New interest in the physiological function of y-glutamyl trans-

peptidase was stimulated by the introduction of chromogenic substra-
tes. These substrates were used for the histochemical localization
pf the enzyme and for the rapid determination of its activity26,47,
48,56,5771. The determination of y-glutamyl transpeptidase in se-
rum is now generally recognized as one of the most sensitive tests
in the diagnosis of liver disease (for reviews see ref.37,47,66).
Histochemical studies showed that the localization of y-glutamyl
transpeptidase is consistent with the possibility of its function
in transport. The enzyme is highly concentrated in th~ brush border
of the proximal convoluted tubules of the kidneyl,2,24,47,7 0 , in the
apical portion of the cells of the intestinal epitheliurn1,27,36 , in
the choroid plexus, in the epithelium of brain capillaries 3 , 55, in
the ~~ithelial cells of the ciliary body, in the capsule of the
lens6j,6~and in several glandular epithelial ,2,24. These sites can
be expected to be highly active in amino acid transport.

EVIDENCE FOR THE FUNCTION OF THE y-GLUTAMYL CYCLE IN

AMINO ACID TRANSPORT
It was postulated that the interaction of y-glutamyl transpep-
tidase with GSH and amino acids leads to the formation of the res-
pective y-glutamyl derivatives at the cell membrane 50 • The release
of the amino acid from y-glutamyl linkage can be accomplished in se-
veral ways. y-Glutamyl amino acids are substrates for y-glutamyl
transpeptidase and can participate in further transpeptidation re-
actions with incoming amino acids, thus allowing the translocation
of several amino acids per each y-glutamyl group of GSH. Such re-
actions readily occur under experimental conditions in vitro.
A second possible amino acid release mechanism could be the hydroly-
tic function of y-glutamyl transpeptidase. Although hydrolysis is
very slow under experimental conditions it could have significance
in vivo. Finally,those y-glutamyl amino acids released into the
cytoplasm by y-glutamyl transpeptidase, could be broken down to
pyrrolidone carboxylate and free amino acid bl the cytoplasmatic en-
zyme, y-glutamyl cyclotransferase (reaction 4).
Membrane bound transpeptidase might be the site at which compe-
tition among different amino acids for a common binding site on the
enzyme occurs. The observation that an infusion in vivo of almost
any amino acid leads to a fairly generalized aminoaciduria, may re-
sult from such a competition. It is of interest that the infusion
of glutamate and aspartate is much less effective in producing ami-
noaciduria than the infusion of glutamine and asparagine 35 • This is
consistent with the finding that y-glutamyl transpeptidase is much
more active with glutamine and asparagine than with the correspon-
ding dicarboxylic acids. In addition, the fact that proline is trans-
ported by a separate transport system72 is consistent with the fin-
ding that proline is not a substrate for the transpeptidase.
Amino acid reabsorption is one of the major functions of the
ROLE OF GLUTATHIONE IN TRANSPORT 19

kidney. Kidney also contains high concentrations of y-glutamyl

transpeptidase, and the enzyme was shown to be associated with an
isolated fraction containing brush border fragment s 25. Kidney also
contains high concentrations of other enzymes of the y-glutamyl
cycle 50 -53. y-Glutamylcysteine synthetase was reported to consti-
tute approximately 2% of the soluble protein of rat kidney homoge-
nates52 • Although such high concentrations have not been found in
the mouse and rabbit kidney58 , the enzymes of the y-glutamyl cycle
are nevertheless very active in the kidney of these species.
High concentrations of y-glutamyl transpeptidase were also
found in the choroid plexus 45 ,54,83, in isolated br~in capillaries 56
in the ciliary body and the capsule of the lens 61 ,68 and in the
intestinal epithelium3 6 .
It would be expected that if GSH metabolism is associated
with the function of the kidney in amino acid transport, then its
turnover in the kidney should be higher than in any other tissue.
It is of interest in this respect that two minutes after injection
of 14c-L-glutamate, the specific activity of kidney GSH exceeded
many times the specific activity of the tripeptide in liver
brain or red blood cells 40 • A recent study showed that the turn-
over of GSH is approximately five times more rapid in mouse kidney
than in the liver?3. When L-methionine-RS-sulfoximine, an inhibitor
of y-glutamylcysteine synthetase, was administered to mice, it
caused a rapid and sharp decrease in the concentration of kidney
GSH, and a much slower decrease in the liver59 • This result is con-
sistent with a faster turnover of GSH in the kidney than in the
liver.
The functioning of the y-glutamyl cycle in vivo is indicated
by the finding of its intermediates in mammalian tissues. Studies
in our laboratory 89 as well as by others 86 , have demonstrated the
presence of considerable amounts of free pyrrolidone carboxylate
in animal tissues and body fluids under normal conditions. These
studies show that pyrrolidone carboxylate is a normal intermediate
in mammalian metabolism. In addition,y-glutamyl derivatives of va-
line, leucine, and isoleucine have been identified in human urine l 5.
Small amounts of the y-glutamyl derivatives of glutamate, glutamine,
glycine, alanine, serine, valine, and isoleucine were identified and
isolated from bovine brain?l. These findings indicate that the
y-glutamyl cycle is functioning in mammalian tissues.
Attempts have been made to obtain evidence for the functioning
of the y-glutamyl cycle in the transport of amino acids by determi-
ning the concentration of intermediates of the cycle in tissues
after amino acid loading. These experiments were based on the
assumption that administration of amino acids should induce in vivo
increased transpeptidation with GSH, formation of y-glutamyl amino
acid~and also an increase in the concentration of pyrrolidome car-
boxylate. Studies in our laboratory have shown that after adminis-
tration of several amino acids to mice, small amounts of the corres-
ponding y-glutamyl amino acids can be detected in the kidney5 8 • The
concentration of pyrrolidone carboxylate in the kidney, liver, and
20 M.ORLOWSKI

brain of mice also increased after administration of a mixture of

amino acids, the highest increase occurring in the kidney58 • Other
studies have shown that the administration of L-2-imidazolidone-4~
carboxylate, an inhibitor of L-pyrrolidone carboxylate hydrolase 8 7,
together with several amino acids, provoked a much greater accumu-
lation of pyrrolidone carboxylate in mouse tissues than the admi-
nistration of the inhibitor alone 86 • This finding was interpreted
as evidence for increased transpeptidation of amino acids with GSH
after administration of amino acids 86 • In view, however, of the ra-
ther small accumulation of pyrrolidone carboxylate after amino acid
loading experiments, it seems rather unlikely that there exists
a stoichiometric relationship according to which the transport of
each amino acid molecule results in the formation of one molecule
of pyrrolidone carboxylate.
Further suggestion of an association between kidney amino acid
transport and the y-glutamyl cycle came from studies involv~ng ma-
leate. Maleate has been long known to produce aminoaciduria4 ,jl af-
ter administration to rats. Studies have shown th~t maleate impairs
the uptake of amino acids by kidney-cortex slices67 • Transfer of
the slices into a maleate-free medium reverses the inhibition,
indicating that no covalent bond formation between maleate and cel-
lular thiol groups is involved in the mechanism of inhibition. It
was recently reported that maleate strongly stimulates the hydro-
lytic activity of y-glutamyl transpeptidase and decreases the trans-
peptidation reaction catalyzed by the enzyme I8 ,80. Thus maleate
decreases the ability of y-glutamyl transpeptidase to use amino
acids as acceptors. Meister and his coworkers pointed outthat this
action of maleate would be consistent with the proposed function of
y-glutamyl transpeptidase in amino acid transport 8D •
Hajjar and Curran29 studied the specificity of the neutral amino
acid transport system in the brush border of mucosal cells of the
rabbit ileum. They found that electron withdrawing substituents in
the ring of phenylalanine increased the affinity of the amino acid
for transport and that electron releasing groups decreased the affi-
nity. Bodnaryk has shown that y-glutamyl transpeptidase has a simi-
lar specificityll. Furthermore, the essential structural requirement
for reactivity in both systems was the presence of a carbonyl group
on the carbon adjacent to the a-carbon with the amino group.
Work showing that the translocation of phenylalanine is media-
ted by the reactions of the y-glutamyl cycle, has been described by
Bodnaryk in the housefly14 (Musca domestica). Growing larvae of the
housefly accumulate a large pool of y-glutamylphenylalanine. This di-
peptide is synthesized by transpeptidation from GSH to phenylalanine
catalyzed by y-glutamyl transpeptidase. The enzyme has been locali-
zed histochemically in the brush border of the proximal part of the
r·1alpighian tubules and in the brush border of the cells in the an-
terior and posterior portion of the midgut13. y-Glutamylphenylala-
nine provides precursors needed for the synthesis of quinones for
tanninig cuticle proteins. The puparium tanning process is accompa-
nied by a rapid, ecdysone~induced appearance of y-glutamyl trans-
ROLE OF GLUTATHIONE IN TRANSPORT 21

peptidase on the epidermal membrane at the interface bet.Teen the

epidermis and cuticle. The induced enzyme liberates phenylalanine
from its Y-glutamyl derivative and translocates the amino acid for
its subsequent conversion to tyrosine and quinones needed for the
tanning of the puparium. The liberation of phenylalanine is also
accompanied by a ten-fold increase in activity of y-glutamyl cyclo-
transferase. The newly formed enzyme (in contrast to mammalian
cyclotransferases) shows the highest activity with L-Y-glutamyl-L-
phenylalanine as the substrate12 .

HERITABLE DISORDERS OF METABOLISM DUE TO DEFICIENCIES

IN ENZYMES OF THE y-GLUTAMYL CYCLE

The significance of the y-glutamyl cycle in amino acid trans-

port could be more directly evaluated if specific inhibitors were
available to interfere with the function of one or more of the en-
zymes of the cycle. Unfortunately, no such inhibitors are yet avai-
lable. Some insight into the function of the y-glutamyl cycle has
been obtained however, from studies of patients with inborn defi-
ciencies of two separate enzymes of the cycle. Three patients have
been described with "pyroglutamic aciduria", an inborn error of me-
tabolism in which the patients excrete large amounts of pyrrolidone
carboxylate in the urine 2l ,28,34 • One of these patients, a mentally
retarded boy with a metabolic acidosis and spastic tetraparesis,
excretes 25 to 35 g of pyrrolidone carboxylate per day. Furthermore,
his excretion increased to 80 g after an intravenous infusion of
a mixture of amino acids. A massive aminoaciduria was observed du-
ring the infusion2l. It was first thought that the patient suffered
from a deficiency of pyrrolidone carboxylate hydrolase. Later stu-
dies, however, have shown that the metabolic defect results not
from failure to metabolize pyrrolidone carboxylate but rather from
its overproduction22 • Two of the three patients have been found to
have a deficiency of glutathione synthetase 88 • This apparently leads
to an increased synthesis of y-glutamylcysteine and its conversion
to pyrrolidone carboxylate. It was postulated that y-glutamylcyste-
ine can sUbstitute for glutathione in transpeptidation reactions,
and that the presence of this dipe~tide would normally prevent the
appearance of aminoaciduria42 ,43,88.
Several observations remain unexplained. Although patients46
have been previously described with hemolytic anemia and a defi-
ciency of GSH synthetase, no pyroglutamic aciduria or metabolic aci-
dosis was reported in those patients 44 • It is not known ~hether such
changes escaped detection, or they do not appear in all patients
with GSH-synthetase deficiency. Further studies are needed in order
to establish whether the deficiency of GSH-synthetase results in a
uniform metabolic disturbance. The possibility exists that the en-
zyme deficiency can be limited only to certain tissues or cells.
Supporting evidence for the functioning of the y-glutamyl cycle
 22 M. ORLOWSKI

in amino acid transport came also from the study of hereditary hemo-
lytic anemia due to a deficiency of ¥-glutamylcysteine synthetase 64
In addition to hemolytic jaundice, the patients have signs of dege-
nerative disease of the CNS and signs of spinocerebellar degenera-
tion. Examination of the urine of the patients showed a marked ami-
noaciduria resulting from an increase in the excretion of both neu-
tral and basic amino acids.

CONCLUSIONS

Further studies are needed on the function of the ¥-glutamyl

trai!lspeptidase reaction in transport. The direction of this reac-
tion is not known, and it is possible that the enzyme functions
under certain conditons in the reverse direction, to perform a sec-
retory function for amino acids. Such function would be consistent
with the localization of the enzyme in the apical portion of cells
where secretion occurs.
The possibility must also be considered that the enzyme func-
tions in the uptake of ¥-glutamyl amino acids, ¥-glutamyl peptides,
and glutamine. This would be analogous to the postulated function
of dipeptidases in the transport of dipeptides. Studies in our la-
boratory have shown that after administration to mice of the ¥-glu-
tamyl derivatives of ~-aminobutyrate, phenylalanine, and glycylgly-
cine, they rapidly accumulate in the kidney, along with metabolites
derived from them (glutamate, aspartate, glutamine, ophthalmic acid).
The need for a system for uptake of ¥-glutamyl compounds is justi-
figa by the presence of free and protein bound glutathione in plas-
ma • The free GSH in human plasma, if filtered in the glomeruli,
would present more than 0.5 umol of GSH per min to the proximal tu-
bules of the kidney for reabsorption.
Some evidence presented in this discussion may be interpreted
as either supporting the involvement of the ¥-glutamyl group or the
SH group of GSH in transport processes. These two possibilities can
explain both the aminoaciduria provoked by the administration of
maleic acid, or that observed in ¥-glutamylcysteine synthetase de-
ficiency with a low tissue GSH level. Reagents that block membrane
SH groups generally inhibit transport of amino acids7. A similar
explanation underlies aminoacidurias observed in heavy metal poiso-
ning. GSH is needed for maintaining the integrity of cell membra-
nes and this function is especially prominent in red blood cells.
Reduction of red cell GSH contents, or impairment of the enzymatic
system for the reduction of oxidized GSH renders the cell susceptible
to hemolysis, especially if subjected to oxidative stress. Experi-
mentally such a situation can be generated by the use of reagents
that oxidize intracellular GSH38,39. Diamide has been reported to
inhibit the uptake of amino acids by kidney cortex slices32. This
inhibition could be reversed by the addition of exogenous GSH. It
is of interest that although some reversal of inhibition could be
ROLE OF GLUTATHIONE IN TRANSPORT 23

obtained with dithiothreitol and mercaptoethanol these thiols were

not as effective as glutathione.
The evidence reviewed here clearly suggests the importance of
glutathione in transport phenomena. Much, however, remains to be
done before the mechanism by which glutathione affects transport
processes can be completely unraveled. It seems that both the func-
tion of the SH group and the y-glutamyl linkage of glutathione
should be considered in future studies.

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 24 M. ORLOWSKI

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ROLE OF GLUTATHIONE IN TRANSPORT 25

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26 M. ORLOWSKI

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ROLE OF GLUTATHIONE IN TRANSPORT 27

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28 M. ORLOWSKI

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 Barriers in
the Living Brain
TRANSPORT MECHANISMS IN THE CEREBROSPINAL FLUID SYSTEM FOR

REMOVAL OF ACID METABOLITES FROM DEVELOPING BRAIN

Norman H. Bass and Per Lundborg

Department of Neurology, University of Virginia
School of Medicine, Charlottesville, Virginia U.S.A.
and Department of Pharmacology
University of Goteborg, Sweden

Postnatal development of a '~lood-brain-barrier" for protecting

the central nervous system from fluctuating concentrations of
charged lipophobic molecules in blood, parallels the maturation of
unique and highly efficient transport mechanisms for the intra-
cranial removal of organic aCids 3 ,4. Prior studies have clearly
delineated anatomic barriers called "tight junctions" in brain
capillaries which prohibit ~enetration of proteins having molecular
diameters larger than 20 ~l • The permeability of this same barrier
to smaller and more polar molecules has not been determined. Recent
observations strongly suggest that homeostatic mechanisms designed
to protect the internal milieu of brain may be viewed not only as
a membranous barrier for blood-borne molecules based on their size,
lipid solubility, and ionization constant, but as a series of
selective filtration sites for carrier-mediated transport of water-
soluble, polar molecules from the intracranial cavity into the
systemic circulation 13 ,17.

The ependymal and pial linings of immature and adult brain

offer little restraint to the passage of acid metabolites from the
interstitial fluid to the CSF compartment 9 ,11 Hence, it has been
suggested that the CSF system with its markedly low concentrations
of acid metabolites relative to high levels in brain interstitial
fluid may serve as a "diffusional sink" or "quasi-lymphatic system"
for elimination of substances such as S-HIAA, from brain 12 ,lS.

Abbreviations: Para-aminohippuric Acid = PAH; S-Hydroxyindoleacetic

acid = S-HIAA; Cerebrospinal Fluid = CSF.

31
32 N.H. BASS AND P. LUNDBORG

The central nervous system of the 5 day old rat contains: blood
vessels composed of primitive epithelial elements forming barely
patent channels surrounded by loosely apposed end-feet 7 ; extra-
cellular "lakes" formed by cellular processes of numerous poorly
differentiated neurons and few undifferentiated glia 7 ; and
relatively undifferentiated secretory epithelium in the choroid
plexus 19 At 30 days of age, extracellular spaces are reduced
to small intercellular clefts by growth of numerous neural processes,
and both brain capillaries and choroid plexus show an adult appear-
ance. Since intracranial sites for carrier-mediated transport of
organic acids contain poorly differentiated cells in the 5 day old
rat, we have taken an ontogenetic approach to the question: how
does the brain, which lacks a lymphatic system of the usual type,
remove its products of amine metabolism?

If an extracellular marker, such as carboxyl-14C inulin is

infused into the CSF system of unanesthetized 5, 10 and 30 day old
rats, diffusion across the ependymal and pial surfaces into brain
is significant but extremely small with respect to CSF concentr~tions,
and there is a relatively rapid escape of the solute into blood •
This facilitated exit of inulin into blood is accomplished by the
circulatory dynamics of CSF, which is secreted primarily by the
choroid plexus 8 ,16 and reabsorbed in bulk into dural sinus blood 6 ,13.
Total CSF volumes, estimated by intrathecal infusions post-mortem
in 5, 10, and 30 day old rats were 122, 241 and 250 ~l/min,
respectively2. Values for inulin clearance derived after CSF
pressure returned to resting levels averaged 1.8 ~l/min for 10 and
30 day old rats, in relative agreement with previously reported
values of 2.2 ~l/min for anesthetized adult rats using ventriculo-
cisternal perfusion, and approximating values reported for other
animal species expressed as per cent of the total CSF volume formed
per minute 2 • In contrast, the rate of inulin clearance from the
CSF system of the 5 day old rat was 0.34 ~l/min, approximately
6-fold less than found in the 10 and 30 day old animal. Thus,
between 5 and 10 days of postnatal life, the total CSF volume and
rate of CSF formation increased from 40 to 95% of adult values,
correlating with morphologic observations showing rapid maturation
of the choroid plexus epithelium during the first two weeks of post-
natal life 19 . During this same period, brain weight increased
from 28 to 61% of adult values, suggesting that maturation of the
CSF system precedes the attainment of adult brain mass.

The rate of formation of 5-HIAA by the brain of 5 day old rats

is about 0.7l ng/min 20 , in association with CSF concentrations
of 125 ng/ml. It follows that if bulk flow were the sole mech-
anism for removal of acid metabolites from the brain of the infant
rat, CSF would have to be cleared at a rate of 5.7 ~l/min to account
for total efflux of 5-HIAA by this route. Similarly, in the 30 day
old rat, where the rate of 5-HIAA formation is about 4.2 ng/brain/min
and CSF concentrations of 5-HIAA are 126 ng/ml, complete clearance
REMOVAL OF ACID METABOLITES BY CSF IN DEVELOPMENT 33

of this acid metabolite by the CSF system would require a bulk flow
rate of 33 pI/min. It is apparent that the normal circulatory rate
for CSF in the 5 day old rat can account for elimination of not more
than 6% of the 5-HlAA formed. Moreoever, the same condition holds
for the 30 day old rat, where its facilitated rate of bulk flow has
merely kept pace with an increased number of serotonergic synapses,
and is similarly capable of eliminating only 6% of the 5-HlAA formed
per minute. Clearly, if the CSF system is to function in the removal
of products of amine metabolism from the infant and adult central
nervous system, sites for carrier-mediated transport of such organic
acids must occur in proximity to the route of bulk flow.

100

~'~.' -: "" '-: . :-:-; ::- '. :-:.-:-::-: : ..... .-.-._._.,

'.
Q
5 20
~ - .. ..... -
-- -- - +',
"

:cZ 10

.. ........
0::
o'"
:II
5
'--
:u ..........
2
!
...Z
Q S Day Old Rat

..::;
...~ --,I I~ .....,..
oQ

'----.
e
'-----.
...... 50
!
...
.
o

.....
20
~
U

0. 10

30 Day Old Rat

Figure 1. Disappearance of 3H- PAH (.... ---.l) and 14C_inulin (A--l1) from
the CSF system of 5 and 30 day old rats following a five minute de-
livery of 40 and 90 ~l volumes respectively into the spinal subarach-
noid space (----1'"). (0-._.<» denotes 3H- PAH efflux after probenecid
(250 mg/kg, subcut); (.'.0') denotes combined intrathecal infusion
of 3H- PAH and 5-HlAA.
34 N.H. BASS AND P. LUNDBORG

The ontogenesis of active transport mechanisms for acid meta-

bolites was assessed in 5 and 30 day old rats by measuring the rate
of elimination of 3H- PAR from brain and CSF after intrathecal
infusion, before and after saturation with 5-HIAA, and after
competitive inhibition with probenecid (Figure 1). PAH was chosen
as a marker substance because it shares certain properties with
5-HIAA; both organic acids have similar molecular weights, almost
identical dissociation constants, relatively low lipid solubilities,
and relative resistance to enzymatic degradation. Additionally, PAR
is commercially available as a tritium labeled compound with higher
specific activity than 5-HIAA, permitting infusion of the radio-
actively labeled acid into the CSF in concentrations below the
saturation threshold for the active transport system.

Although the CSF system of the 5 day old rat has a markedly
sluggish rate of bulk flow, PAR was eliminated from the CSF at a
rate which was l7-fold greater than that found for inulin. This
efflux rate for PAR closely approached values which could theoret-
ically account for total removal of the 5-HIAA formed by the immature
brain. Moreover, addition of high doses of unlabeled 5-HIAA to the
infusion fluid, or systemic pretreatment with probenecid, decreased
the elimination rate by approximately 50% while having no effect
on inulin efflux suggesting that: (1) PAR is eliminated from the
CSF system by a carrier-mediated transport mechanism: and (2) 5-HIAA
and PAR probably share a common transport site. Previous in vitro
studies have shown that the choroid plexus of fetal and newborn
chicks, rats, rabbits, cats and dogs possesses an active transport
system for acid-sulfonated dyes, and radiolabeled sulphate, iodide,
and PAH 5,18 Hence, it may be concluded that poorly differen-
tiated choroid epithelial cells have a carrier-mediated transport
mechanism for organic acids, whose efflux rate approaches that
required for complete removal of metabolites of biogenic amines from
immature brain.

In contrast, PAH was removed from cisternal CSF of 30 day old

rats at a rate similar to that found for elimination of inulin by
mechanisms of bulk flow. Moreover, a carrier-mediated transport site
in the cerebral ventricles could not be demonstrated, as the addition
of high doses of unlabeled 5-HIAA or systemic pretreatment with
probenecid did not influence the rate of acid efflux. This obser-
vation, at first glance, may appear to contradict prior studies in
adult animals demonstrating an "acid pump" in the choroid plexus by
in vitro techniques 10,16, and the in vivo transport of acid meta-
bolites from the caudate nucleus to artificial CSF perfusing the
ventricles 1 Although we emphasize that transfer of organic acids
to ventricular CSF and their subsequent active transport to blood via
the choroid plexus is not a major route for removal of acid meta-
bolites from the whole brain of 30 day old rats, we nevertheless
acknowledge that certain areas of adult brain situated proximal to
the ventricles may continue to use this route. However, in terms of
REMOVAL OF ACID METABOLITES BY CSF IN DEVELOPMENT 35

whole brain metabolism, the calculated e f flux rate for organic acids
from the CSF system of the 30 day old rat is too slow to constitute
the sale route for preventing their accumulation in brain. Hence,
further studies were performed to explore prior evidence suggesting
that the major route for removal of acid metabolites from adult brain
exists at transport sites presumably situated at the glia-capillary
interphase in the brain parenchyma.

Although the rate of elimination of PAH from the brain parenchyma

of 5 and 30 day old rats was rapid, no evidence for a carrier-mediated
transport route was found in the infant animal (Figure 2). Hence,
it may be postulated that organic acids are removed from the brain of
the infant rat by diffusion through its relatively large extracellular
space into ventricular CSF which serves as a "sink" for efflux of
acid metabolites by the action of carrier-mediated transport sites in
choroid plexus epithelium. The brain of the 30 day old rat, however,

40

20

10

..:c'"
z

...:z:
w

o
Z 5 Day Olel Rot

..
:c
t;
w
mnufes 60
(5
o
...
S
~
20
~
(;
...
Z
10

~
5
...~ '"
'"
2

"
30 Day Old Rat

mnutes

Figure 2. Disappearance of 3H- PAH (A- - ---A) from the brain of 5 and
30 day old rats following a five minute delivery of 40 and 90 ul
volumes, respectively into the spinal subarachnoid space (----t),
~.- . -o) denotes 3H- PAH efflux after probenecid (2~0 mg/kg, subcut);
(." ,.) denotes combined intrathecal infusion of H-PAH and 5- HIAA.
 36 N.H. BASS AND P. LUNDBORG

clearly possessed an active transport mechanism for rapid elimination

of organic acids (Figure 2). The elimination of PAR from brain to
peripheral blood was completely inhibited by large doses of 5-HIAA
and systemic injection of probenecid, supporting prior studies
demonstrating the existence of a probenecid-sensitive, active trans-
port mechanism for acid metabolites in the brain parenchyma of adult
rats 12,14 These observations and prior work showing an abundance
of capillary sites for efflux of 5-HIAA over the surface of the
cerebral hemispheres adjoining CSF pathways of the adult animal 21
explain our previous failure to demonstrate active transport sites
from values obtained by sampling CSF from the cisterna magna of the

INTRACRANIAL MECHANISMS FOR REMOVAL OF 5-HYDROXYINDOLEACETIC ACID

FROM BRAIN DURING POSTNATAL DEVELOPMENT*

5 day old rat** 130 day old rat**

% of % of
ng/min formation ng/min formation
rate rate
r
iFormation of 5-HIAA 0.72 -- 4.17 --
in whole brain***

5-HIAA elimination by 0.04 5.6 0.24 5.8

bulk flow of CSF

5-HIAA elimination by 0.67 93.1 0.69 16.5

transport sites in
the CSF system

5-HIAA elimination by not 0 3.03 72.7

transport sites in demonstrable
brain capillaries

~otal intracranial 0.71 98.6 3.96 95.0

elimination of 5-HlAA
(bulk flow + transport) I
*Based on data previously reported in detail 4

**5 day old rat: Body wgt. 13.8 g; brain wgt. 0.43 g;
30 day old rat: Body wgt. 62.0 g; brain wgt 1.39 g.

***Rate of 5-HlAA formation based on data of Tissari 20:

Value for 5 day old rat 0.1 pg/g/hr; value for 30 day old rat
assumed to be 60% of that reported for 75 day old rat
(0.3 pg/g/hr) based on brain concentrations of serotonin
REMOVAL OF ACID METABOLITES BY CSF IN DEVELOPMENT 37

30 day old rat. The appearance of carrier-mediated efflux sites in

capillaries on the surface and within the cerebral hemispheres
combined with an enhanced rate of bulk flow results in a multiplicity
of routes capable of removing acid metabolites from adult brain.

By combining values for total CSF volume and bulk flow 2 with
rates of accumulation of 5-HlAA in brain and CSF after probenecid
treatment 4 , steady-state elimination rates for 5-HlAA from intra-
cranial compartments of 5 and 30 day old rats have been calculated
(Table). The total amount of 5-HlAA eliminated per minute from the
intracranial cavity of the 5 day old rat was 0.71 ng, a value in close
agreement with the reported rate of formation of this metabolite
in the brain of infant rats 20 No evidence for carrier-mediated
efflux at brain capillaries was found, as 5-HlAA did not accumulate
in immature brain after probenecid treatment. This lack of response
of the infant brain to inhibition of active transport was not caused
by a failure of 5-HlAA formation, since a rapid decrease of 5-HlAA
has been observed after monoamine oxidase inhibition 20 .
Additionally, bulk flow of CSF eliminated 0.04 ng of 5-HlAA/min,
amounting to only 6% of its formation rate. Hence, active transport
sites in the CSF system comprised the route for removal of 93% of
the 5-HlAA formed in the brain of the infant rat, eliminating the
metabolite at a rate of 0.67 ng/min. The total amount of 5-HlAA

Figure 3. Sink action of the CSF and its proposed role in the
removal of 5-HlAA from the brain of 5 day old (A) and 30 day old
(B) rats. Note developmental changes in major (~) and minor
(---~) efflux routes associated with reduction of intercelluar
clefts between neurons (N) and glia (G) and the appearance of
mediated transport sites at brain capillaries (~). Modified from
Davson and Bradbury (Symposium, Soc. Exp. BioI. 19:349, 1965).
38 N.H. BASS AND P. LUNDBORG

eliminated per minute from the intracranial cavity of the 30 day old
rat was 3.96 ng, a value in relative agreement with the endogenous
formation of this organic acid when corrected for decreased turnover
of serotonin to 60% of values reported for adult rat brain 20.
However, in contrast to the 5 day old animal, only 23% of adult brain
5-HIAA formed per minute (0.93 ng/min) was eliminated via CSF path-
ways by the combined mechanisms of active transport and bulk flow. In
fact, the maturation of active transport sites at the glia-capillary
interphase constituted the major route for efflux of 73% of the 5-HIAA
formed in the brain of the 30 day old rat, efficiently removing the
metabolite at a minimum rate of 3.03 ng/min.

In summary, during postnatal development, unique and highly

efficient intracranial mechanisms evolve for the removal of acid
metabolites from brain interstitial fluid into the peripheral
circulation, thereby preventing an accumulation of substances, such
as 5-HIAA, an end-product of serotonin metabolism. In the 5 day old
rat, the CSF system constitutes the major route for removal of acid
metabolites (Figure 3A). The brain parenchyma with its poorly
differentiated vascular system and relatively large extracellular
space contains high concentrations of 5-HIAA in its interstitial fluid
compartment, while the CSF maintains markedly low concentrations by
means of its active transport sites in the choroid plexus epithelium
and its continuous, albeit sluggish, flow through the ventricles and
subarachnoid spaces into the dural sinuses. Since the ependymal
linings exert little restraint on the passage of substances from
brain interstitial fluid into ventricular CSF, the concentration
gradient allows for continuous diffusion of acid metabolites from
brain into blood via CSF pathways. The CSF system of the 5 day old
rat would then serve as a "sink" or permanent drain for acid meta-
bolites analogous to the lymphatic system found in all other organs
of the body.

With increasing brain maturation, relatively large extracellular

spaces are reduced in size by the growth of neural processes, and
numerous blood vessels become enveloped by astrocytic end-feet. By
30 days of age, a new site for carrier-mediated transport of acid
metabolites appears at glia-capillary interphases situated throughout
the brain parenchyma (Figure 3B). Such transport sites at brain
capillaries can cope with only 3/4 of the 5-HIAA accumulating from
the enhanced rate of serotonin metabolism. Hence, approximately 1/4
of amine metabolites continue to diffuse into CSF through the narrow
intercellular clefts of periventricular brain regions. These products
of amine metabolism which overflow into the now rapidly circulating
ventricular CSF, are swept over the cerebral hemispheres and after
diffusing across the pial surface are actively transported into the
peripheral circulation by the glia-capillary network.
 REMOVAL OF ACID METABOLITES BY CSF IN DEVELOPMENT 39

ACKNOWLEDGMENTS

This research has been supported in part by the Swedish State Medical
Research Council (no. B73-l4P-3266-03 and B73-l4X-2464-06C),
Expressens Prenatal Forskning, and a Career Development Award to
N.H. Bass from the National Institutes of Health (NS 28-155)

References:

1 Ashcroft, G.W., Dow, R.D., and Moir, A.T.B., The active trans-
port of 5-hydroxyindolylacetic acid and 3-methoxyhydroxyphenyl
acetic acid from a recirculatory perfusion system of the cerebral
ventricles of the unanesthetized dog, {. Physiol. 19
(1961) 153-160.
2 Bass, N.H. and Lundborg, P., Postnatal development of bulk flow
in the cerebrospinal fluid system of the albino rat. Brain
Res.52 (1973) 323-332.
3 Bass, N.H., and Lundborg, P., Postnatal development of mechanisms
for the elimination of organic acids from the brain and cerebro-
spinal fluid system of the rat. Brain Res.56 (1973)285-298.
4 Bass, N.H., and Lundborg, P., Mechanisms for the elimination of
5-hydroxyindole-acetic acid from brain and cerebrospinal fluid
of the rat during postnatal development, Brain Res. 77 (1974)
111-120.
5 Bierer, D.W., and Heisey, S.R., Organic anion transport in the
maturing dog choroid plexus, ~rain Res b6 (1972) 113-119.
6 Butler, A.B. Mann, J.D., and Bass, N.H., Identification of the
major site for cerebrospinal fluid efflux in the albino rat.
Anat. Rev. 181 (1975) 323.
7 Caley, D.W. and Maxwell, D.W., Development of the blood vessels
and extracellular spaces during postnatal maturation of rat
cerebral cortex. J.Comp. Neurol. 138 (1970) 31-48.
8 Cserr, H.F., Physiology of the choroid plexus. Physiol. Rev.
51 (1971) 273-311.
9 Cserr, H.F., Relationship between cerebrospinal fluid and inter-
stitial fluid of brain, Fed. Proc. 33 (1974) 2075-2078.
10 Cserr, H.F., and Van Dyke, D.H., 5-Hydroxyindoleacetic acid
accumulation by isolated choroid plexus. Amer. J. Physiol.
220 (1971) 718-723. ~- -
11 Fenstermacher, J.D., Patlak, C.S. and Blasberg, R.G., Trans-
port of material between brain extracellular fluid, brain cells,
and blood. Fed. Proc. 33 (1974) 2070-2074.
12 Meek, J.L. and Neff, H.H., Is Cerebrospinal fluid the major
avenue for the removal of 5-hydroxyindoleacetic acid from the
brain? Neuropharmacology 12 (1973) 497-499.
13 Mi1horat, T.H., The third circulation revisited, J.Neurosurg.
42 (1975) 628-645.
40 N.H. BASS AND P. LUNDBORG

14 Neff, H.H., Tozer, T.N., and Brodie, B.B., Application of

indoleacetic acid from brain to plasma, J.Pharm. Exp. Ther.
158 (1967) 214-218.
15 Oldendorf, W.H., and Davson, H., Brain extracellular space
and the sink action of cerebrospinal fluid, Arch. Neurol.
17 (1967) 214-218. --
16 Pollay, M., Transport mechanisms in the choroid plexus, Fed.
Proc. 33 (1974) 2064-2069. ---
17 Pollay, M. and Davson, H., The passage of certain substances
out of the cerebrospinal fluid. Brain 86 (1963) 137-150.
18 Robinson, R.J., Cutler, W.P., Lorenzo, A.V., and Barlow, C.E.
Development of tranport mechanisms for sulfate and iodide in
immature choroid plexus, ~. Neurochem. 15 (1968) 455-458.
19 Tennyson, V.M., and Pappas, G.D., The fine structure of the
choroid plexus: Adult and developmental stages. In A. Lajtha
and D.H. Ford (Eds.), Brain Barrier Systems, Progr. in Brain
Res. Vol. 29, Elsevier, Amsterdam 1968, pp. 63-85.
20 Tissari, A.H., Serotoninergic mechanisms in ontogenesis. In
L.O. Boreus (Ed.), Fetal Pharmacology, Raven Press, New York
(1972), pp. 237-257.
21 Wolfson, L.I., Katzman, R., and Escriva, A., Clearance of amine
metabolites from the cerebrospinal fluid: The brain as a "sink".
Neurology 24 (1974) 772-779.
THE MORPHOLOGICAL APPROACH TO THE STUDY OF NORMAL

AND ABNORMAL BRAIN PERMEABILITY

M.W. Brightman and R.D. Broadwell

Laboratory of Neuropathology and Neuroanatomical Sciences

NINCDS, National Institutes of Health
Bethesda, Maryland U.S.A.

The rich capillary bed of the central nervous system (CNS) is

impervious to protein. This generalization derives from two
observed features of the cerebral capillary wall: (a) the tight
junctions between contiguous endothelial cells comprise uninterrupted
belts that arrest the passive, intercellular movement of protein and
(b) the vesicles that bud off the endothelial cell membrane do not
appear to carry protein across the cell from blood to perivascular
tissue 19 . We would like to review briefly where and how this
blood-brain barrier (BBB) to protein and peptide is circumvented in
the normal and experimentally manipulated mammal. It will become
apparent that in some vessels which are naturally permeable or
rendered permeable experimentally, one or the other of both features
is altered: the junctions are open and no longer tight and the
endothelial plasmalemma of certain vessels is capable of vesicular
transport. A third means of bypassing the barrier must still be
considered as a strong suspicion rather than proven: fenestrae or
round windows that perforate the attenuated endothelium of capil-
laries in a few specific regions are bridged by thin diaphragms that
may be permeable to protein and peptide. A fourth bypass is one t~at
we have just come to appreciate: the very axons of the neurons
within the cerebral parenchyma. It is through the cytoplasm of these
axons that large molecules can be brought from extracerebral, peri-
pheral blood to neuronal cell bodies of the brain and spinal cord6 .

Experimental attempts to open the barrier have led to the sub-

stitution of a continuous endothelium by a fenestrated one that also
has visibly open junctions in a virally-induced tumor and to a more
subtle deformation of tight junctions in response to hyperosmotic and
hypertensive conditions. We should like to summarize briefly these

41
42 M.W. BRIGHTMAN AND R.D. BROADWELL

experimental modifications of the barrier first and then present

evidence for the "natural" gaps in the barrier.

INDUCED OPENING OF THE BLOOD-BRAIN BARRIER

Tumors

In spontaneous cerebral tumors of man 16 and in those virally-

induced in lower mammals 5, the normal cerebral capillary is
replaced by one that is permeable to protein. One feature common to
both types of tumor is a continuously open cleft between adjacent
endothelial cells. In choroid plexus papillomas induced by the
intracerebral injection of newborn animals with SV40 virus, the
endothelial cells are often separated by a cleft that is not sealed
by a belt of tight junctions. Blood-borne Evans blue-albumin and
horseradish peroxidase (HRP) rapidly escape from these vessels into
the pericapillary spaces and the clefts between tumor cells 5 .

The vessels of the choroid plexus papilloma, like those of

normal choroid plexus, are perforated in their attenuated regions by
small, round windows or fenestrae approximately 600 A in diameter.
Each fenestra is covered by a diaphragm about 50 A thick. From
counts made on micrographs of thin, plastic sections, the fenestrae
outnumber the endothelial junctions in the papillomas by about 4 to
1. Judging from the myriad fenestrae revealed in a single replica
of normal choroid plexus, however, this ratio is probably much
higher 5.

Fenestrae comprise another feature common to induced neoplasms

and, in man, some primary 16 and metastatic 12 ones as well.
The decision as to whether open junctions or fenestrae account for
the leakiness to protein of tumor vessels depends upon whether the
fenestral diaphragms are permeable to protein. If they are, then
the fenestrae would be the major pathway. Even so, the junctions do
provide a ready exit for protein and other large and small molecules
from blood to the extracellular fluid of cerebral tumors.

Hyperosmotic Opening

When hyperosmotic solutions of different substances are applied

topically to the surface of the brain or infused into an internal
carotid artery, blood-borne Evans blue that is bound to serum albumin
escapes from the affected vessels 17. The exudates appear as
randomly dispersed blue spots that form in the regions supplied by
these vessels. For urea, a concentration of about 2.2 M is the
threshold dose at which blood-borne protein escapes from the vessels.
As with other polar substances, the opening of the BBB to dye-protein
MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 43

complex is reversible whereas it becomes irreversible when non-polar

substances with relatively higher oil-water coefficients are used l7 ,

When, in addition to Evans blue, HRP is injected into a peri-

pheral vein a few minutes before flushing one carotid artery of a
rabbit with 2.5 M urea, the alteration in the BBB can be followed in
greater detail. The use of HRP as a probe molecule not only enables
one to see the distribution of escaped peroxidase in thick (50 to
200 ~) sections, but after osmication, to see the ultimate distri-
bution with the electron microscope. With such techniques, the route
of escape can be delineated and some understanding can be gained of
how hyperosmotic agents act. It is thus apparent that peroxidase
crosses some vessels, that are primarily but not exclusively capil-
laries, by an extracellular route. The evidence for this conclusion
is the penetration of HRP into extracellular pools that lie between
successive tight junctions. The pools that are farthest from either
the capillary lumen or its perivascular space are normally inacces-
sible to protein. In the urea treated specimens, protein enters all
of the pools, presumably by moving through the row of tight junctions
that have been rendered permeable 4. It is assumed that the
increased permeability results from a slight separation of contiguous
cell membranes at tight junctions due to shrinkage of the endothelial
cells.

In order to see whether structural changes were wrought by hyper-

osmotic urea, as a result of cell shrinkage or, possibly, denatur-
ation, the tight junctions of choroid plexus epithelium were examined
by freeze-fracture techniques. These tight junctions are far more
readily fractured ~ face than those of endothelium.

The freeze-fracture technique involves brief fixation in alde-

hydes, buffer rinse, cryoprotection by immersion in 20 percent
glycerol or other agents, cleaving in vacuuo, and the deposition of
a thin layer of platinum and carbonC)nto the cleaved surface. The
tissue is then brought to atmospheric pressure and digested away from
the replica. The replica is washed and dried onto copper grids for
examination in the electron microscope. The fracture splits the
membrane through its interior thereby generating two faces: the
inner half of the membrane which is viewed toward the cytoplasm of
the cell (A face) and the outer half of the membrane, viewed toward
the extracellular space (B face).

In such replicas, the tight junctions appear as narrow ridges or

strands 22 that are continuous at some places and, at others, may
be discontinuous and composed of linear rows of separate particles
on the inner half of the membrane (fig. 1). Complementary grooves,
which accommodate the strands, score the outer half of the membrane
22 belonging to the adjoined cell (fig. 2). The strands may be
mUltiple and form approximately parallel rows that may anastomose
with each other. The strands thus project across the intercellular
44 M.w. BRIGHTMAN AND R.D. BROADWELL

Figure 1. Inner half of freeze-cleaved cell membrane. Tight

junctions appear as several rows of more or less parallel, inter-
rupted strands with very few anastomotic branches. X 120,000.

Figure 2. Outer half of freeze-cleaved cell membrane. Tight

junctions appear as parallel, anastomotic grooves containing a
few particles (arrows) derived from the inner half of the conti-
guous cell's membrane. Microvilli (V) have also been cleaved.
X 100,000.
MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 45

space to form more or less continuous shelves that compartmentalize

the space. It has been proposed, with rather convincing documenta-
tion, that the degree to which a tight junction is impervious is
reflected by the number of its strands or ridges and the complexity
of their interconnections 8. According to such criteria, the mere
inspection of micrographs taken of choroid plexus epithelium suggests
that its tight junctions may be leaky. Because of the fewer rows of
strands, discontinuities in the strands, and fewer interconnecting
loops compared to those in junctions known to be impervious to ions,
the choroidal junctions appear to be less tight. The impression
gained from the replicas is supported by physiological evidence 26
and another kind of morphological observation. When lanthanum salts
are infused into blood, black deposits penetrate successive inter-
junctional pools between cells of the choroid plexus. The lanthanum
is believed to be in ionic form 7 • Even when lanthanum hydroxide,
presumably a colloid, is used as a tracer and perfused through
cerebral ventricles, it, or perhaps some of it which is ionic, can
penetrate several tight junctions between contiguous cells. Eventu-
ally, the progress of the tracer is halted by what is deduced to be
a continuous belt of tight junctions 3 . The "semi-tight" junction
that is impermeable to proteins, a heme-peptide 10 and colloidal
lanthanum can let ionic lanthanum pass. These studies show that, in
addition to the configuration of a particular junction, the size of
the probe molecule is essential for the definition of that junction.

The physical effects of hyperosmotic urea on tight junctions

was examined in the mucosal epithelium of the toad stomach where the
junctional ridges are continuous and anastomotic. When toad urinary
bladder is bathed on its mucosal surface by 0.2 M urea while its
opposite, serosal side faces isotonic fluid, the gradient causes
water to flow into the extracellular pools between successive tight
junctions. The pools are then distended. In freize-fracture, the
anastomosing ridges are themselves ballooned out 3, In our exper-
iments, no attempt was made to establish a concentration gradient of
urea. Instead, the entire stomach was immersed in 2.2 M urea for 30
to 60 minutes, fixed in urea-containing aldehydes, frozen, and frac-
tured. Our results, still preliminary, indicate that the continuous,
interconnected strands of the tight junctions in stomach epithelium
become fragmented in many places. This type of change suggests that,
in addition to cell shrinkage and a possible separation of junc-
tional strands, there might also be a denaturation of the protein
making up the strands. We have yet to determine whether these
changes, like those in cerebral vessels, are reversible.

Hypertensive Opening

When the arterial blood pressure of cats 13 or rats 21 is

raised about 90 mm Hg above the resting level by the intravenous
46 M.W. BRIGHTMAN AND R.D. BROADWELL

.-

•
"",.

. ,,(

Figure 3. Within 8 hours after an intravenous injection of 50 mg

HRP, the protein has been taken up by neurosecretory nerve terminals
in the neurohypophysis and/or median eminence and transported intra-
axonally back to the parent cell bodies in the hypothalamic supra-
optic (SON) and paraventricular (PVN) nuclei. X 30.

Figure 4. Pericytes (arrow) around a large blood vessel are

diffusely labeled by peroxidase, most of which has ~resumably
crossed that segment of the vellel wall in vesicles 5. X 560.
MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 47

infusion of aramine (metaraminol bitartrate), the BBB to protein is

opened. Evans blue-albumin complex leaves the lumen of some cortical
and subcortical vessels to form round perivascular exudates distri-
buted around segments of the vessels. When HRP is also infused
before the administration of aramine, the blue exudates that are
immediately apparent also contain brown spots that form after incuba-
tion for peroxidatic activity. Within a given cerebral region, the
number of HRP spots often outnumber the blue ones, and other regions
that appear to be intact because they are not tinted by the dye may
contain a number of brown extravasations; thus, peroxidase is a more
sensitive delineator of barrier leaks than is Evans blue.

The peroxidase technique enables counts to be made of the number

of exudates in 200 ~ thick slices of the entire brain. Only a
fraction of the cerebral vessels are affected whether the arterial
pressure rose either 90 mID Hg or 150 mID Hg above the resting level.
In the altered vessels, only segments appear to leak. Consistently,
the greatest number of extravasations in the rat occur in the
cerebrum, the next highest in the diencephalon, somewhat less in the
midbrain and least in the cerebellum and medu11a 2I This antero-
posterior decrement may reflect a diminution of intraluminal pressure
from carotid to vertebral beds.

The means by which peroxidase escapes from the affected vessels

is still unclear. One unlikely possibility is that aramine in some
way enhances vesicular transport of protein across the endothelium.
However, aramine does not appear to increase the number of vesicles
in capillary endothelium, and in arterioles and venu1es, many of the
luminal pits appear to be unlabeled by the protein. In order to
test this notion of a heightened vesicular transport, a pulse of
saline rather than aramine was quickly infused into one carotid artery
at a rate of 0.6 m1 per second over a 3 to 10 second interval I8
The results were comparable to aramine. Within two minutes, HRP
exuded from many vessels, the number decreasing in the same manner as
with aramine. Considerably more vessels, in each region of the brain,
were rendered leaky. This rapid egress of protein may more likely
be due to a transient opening of endothelial tight junctions as
initially suggested by Johansson et a1. I3 , rather than by a height-
ened vesicular transport. ----

LOCI WHERE THERE IS NO BARRIER

Permeable Vessels

Protein can circumvent the barrier to its extracellular movement

in the brain by crossing capillaries that are normally permeable to
large molecules. These permeable vessels are situated within and
48 M.W. BRIGHTMAN AND R.D. BROADWELL

Figure 5. Two hours after an intravenous injection of 50 mg HRP,

neurons within the hypothalamus dorsal to the median eminence
contain exogenous peroxidase freely dispersed throughout their
cytoplasm. Erythrocytes (E) not washed out by the perfusion lie
within adjacent blood vessels. X 750.

Figure 6. Neurons of the hypoglossal nerve nucleus contain exo-

genous peroxidase in particulate form at 12 hours after intravenous
injection. See fig. 8. X 250.
MORPHOLOGY OF ABNORMAL BRAIN PERMEABI LlTY 49

Figure 7. Just ventral to the retrogradely labeled neurons of the

ambiguus nucleus (arrow) is a more diffuse cluster of neurons, the
noradrenergic A-I group 9, also labeled retrogradely with blood-
borne peroxidase. Dark field. X 175.
50 M.W. BRIGHTMAN AND R.D. BROADWELL

outside of the eNS. Within the brain, the vessels of the choroid
plexus, as noted above, together with those supplying the circumven-
tricular organs (eVO) are fenestrated and contain more pits and
vesicles than the endothelium of the more common vessels in the eNS
20, 24. When HRP is injected intravenously, it rapidly crosses
vessles in the neurohypophysis median eminence, area postrema 20,
and other eva 6, 24. The extracellular spaces within these organs
become flooded with peroxidase. The neurohemal endings of the hypo-
thalamic neurosecretory cells avidly take up HRP from the spaces
around the fenestrated vessels in the posterior lobe of the pituitary
gland and median eminence. From these terminals, blood-borne
peroxidase is carried back to their cell bodies in the supraoptic,
paraventricular (fig. 3) and associated neurosecretory nuclei 6.
With time, the HRP flooding the extracellular clefts of the eva
spreads to adjacent areas. When high concentrations of 50 to 100 mg
of HRP (Sigma type VI or Worthington), tenfold greater than "probe"
amounts, are injected intravenously into adult mice, the areas around
the eva are completely inundated with protein within 2 hours 6. So
great is the spread from the median eminence, for example, that the
ventromedial nucleus and the dorsal and lateral areas of the hypo-
thalamus are also flooded. The inundation is intracellular as well
as extracellular. The cell bodies of neurons become homogeneously
brown, their cytoplasm having been infiltrated by HRP (fig. 5).
Protein that is freely dispersed in the cytoplasm connotes damage
to the cell membrane, since such a large molecule could only pass
directly across a membrane that is no longer semi-permeable 2.
If sufficient time (4 hours) is allowed, the protein is cleared from
the cell body by, presumably, orthograde cytoplasmic flow from soma
toward axon terminals. Between 8 and 12 hours, the HRP appears in
the cytoplasm of the neuronal soma as discrete particles: the
membrane-bounded organelles that are the recipients of protein
pinocytosed by the axon terminals (fig. 6).

Pericytes around blood vessels larger than capillaries also take

up exogenous protein (fig. 4). The incorporation is confined to only
some of the pericytes and so probably reflects the vesicular transfer
of HRP across these vessel segments. Segments of cerebral arterioles
normally transfer peroxidase in vesicles to perivascular tissue even
when one hundreth the amount used here is infused intravenously 25.
In the present experiments, the labeled pericytes are indicators of
large amounts of HRP having circulated through cerebral blood for
some hours before fixation. The diffuse labeling of these cells
indicates that either the cell membrane or the membranes of cyto-
plasmic organelles has been damaged. It would be of some interest
to determine whether the pericytes are more numerous around those
segments of arterioles that transfer protein at a particular time.

Although a small fraction of extracellular protein that has left

MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 51

Figure 8. Ventral to the area postrema (AP). the compact dorsal

motor nucleus of the vagus (X) and the nucleus of the hypoglossal
(XII) nerve are heavily labeled with HRP granules at 12 hours after
intravenous injection. Dark field. X 75.

Figure 9. When one hypoglossal nerve is ligated, the nucleus (XII)

on that side is unlabeled. Thus, blood-borne peroxidase is carried
retrogradely within axons to cell bodies in the opposite side. Dark
field. X 75.
52 M.W. BRIGHTMAN AND R.D. BROADWELL

fenestrated vessels within the eNS is pinocytosed by adjacent neuronal

somata, most of it reaches the cell body by retrograde transport from
its axonal terminals 15. Thus, adrenergic cell bodies situated deep
in the medulla and pons near the ambiguus and facial nuclei respect-
ively 9 might project to, presumably, the median eminence and
become labeled by HRP (fig. 7)6. Similarly, some perikarya in the
nucleus of the tractus solitarius also become labeled. These neurons
project to the area postrema and their axonal endings probably imbibe
HRP from this region to transfer it back to their cell bodies o.

Other neuronal cell bodies intrinsic to the eNS, including those

of cranial nerve motor nuclei III, IV, V, VI, VII, X and XII also
become labeled with HRP. Becuase the cell bodies of cranial nerve
nuclei X and XII lie close to the leaky area postrema (fig. 8), it is
conceivable that they could have directly pinocytosed some of the
extracellular peroxidase. That the cell bodies are, rather, labeled
by retrograde axonal transport is demonstrated by ligating one hypo-
glossal nerve just prior to the intravenous injection of peroxidase.
The hypoglossal nucleus on that side does not receive detectable
amounts of protein (fig. 9). The protein granules, discernible after
12 hours in the hypoglossal nucleus of the non-ligated side, must
have reached the cell bodies from their peripheral arborizations 6 •

Blood vessels outside of the eNS are the source of protein for
the cranial nerves. The capillaries of striated and cardiac muscle
are permeable to peroxidase 14. After intravascular injection, the
protein can then spread through the extracellular spaces of muscle to
synaptic clefts at myoneural junctions. From here, axonal terminals
of motor nerves can pinocytose the HRP 27 and transfer it back to
the centrally located parent cell body 15 In addition to motor
neurons, sensory and preganglionic autonomic neurons are also access-
ible to blood-borne protein 6. Thus, large molecules circulating
in peripheral, extracerebral blood can be carried intraneuronally
deep within the eNS even though these molecules, flowing in cerebral
capillaries, cannot reach the same neurons because of the capillary
barrier. Hematogenous viruses 1, proteins like nerve-growth factor
11, and presumably, some hormones and toxins may also gain entry to
the eNS from peripheral blood along these intracellular routes rather
than through the extracellular clefts within the brain. In this way,
the maintenance of certain neurons within the central n~rvous system
can be influenced by large and small substances in peripheral blood.
These substances, taken up by axon terminals, require a finite time
for retrograde transport and could trigger events in the cell body
long after they have been cleared from the blood.
MORPHOLOGY OF ABNORMAL BRAIN PERMEABILITY 53

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54 M.W. BRIGHTMAN AND R.D. BROADWELL

17. Rapoport, S.I., Hori, M., and Klatzo, I., Testing of a

hypothesis for osmotic opening of the blood-brain barrier,
American Journal of Physiology, 223 (1972) 323-331.
18. Rapoport, S.I., and Thompson, H.K., Opening of the blood-brain
barrier (BBB) by a pulse of hydrostatic pressure, Biophysical
Journal, 15 (1975) 326a (Abstracts).
19. Reese, T.S., and Karnovsky, M.J., Fine structural localization
of a blood-brain barrier for exogenous peroxidase, Journal of
Cell Biology, 34 (1967) 207-217.
20. Reese, T.S., and Brightman, M.W., Similarity in structure and
permeability to peroxidase of opithelia overlying fenestrated
cerebral capillaries, Anatomical Record, 160 (1968) 414 (Abstracts).
21. Robinson, J.S., Brightman, M.W., and Rapoport, S., Opening of
the blood-brain barrier during induced hypertension, (in
preparation).
22. Staehelin, L.A., Further observations on the fine structure of
freeze-cleaved tight junctions, Journal of Cell Science, 13
(1973) 763-786.
23. Wade, J.B., and Karnovsky, M.J., Fracture faces of osmotically
disrupted zonulae occludentes, Journal of Cell Biology, 62 (1974)
344-350.
24. Weindl, A., Neuroendocrine aspects of circumventricular organs.
In Ganong, W.F. and Martini, L. (Eds.), Frontiers of Neuroendocrin-
~, Oxford University Press, New York, 1973, pp. 3-31.
25. Westergaard, E., and Brightman, M.W., Transport of proteins
across normal cerebral arterioles, Journal of Comparative
Neurology, 152 (1973) 17-44.
26. Wright, E.M., Mechanisms of ion transport across the choroid
plexus, Journal of Physiology, 226 (1972) 545-571.
27. Zacks, S.I., and Saito, A., Uptake of exogenous horseradish
peroxidase by coated vesicles in mouse neuromuscular junctions,
Journal of Histochemistry and Cytochemistry, 17 (1969) 161-170.
THE TRANSPORT OF METABOLIZABLE SUBSTANCES

INTO THE LIVING BRAIN

O.E. Pratt

Department of Neuropathology
Institute of Psychiatry
De Crespigny Park, London SE5 8AF, England

Much knowledge about the many enzymic processes by which the

cerebral cells can change one substance into another has been de-
rived from in vitro work. However, such in vitro experiments do
not tell us the quantities of the various nutrient substances that
the living brain needs to obtain from the blood in order to function
normally. Nor do they tell us about the direction in which the var-
ious metabolic pathways act in life. In order to discover the needs
of the cerebral tissues for metabolizable substances the quantities
that enter and leave the brain during life must be studied. Over
the last decade advances in technique in the in vivo field have en-
abled the study of various aspects of cerebral metabolism to be
carried out with greater accuracy than has been possible in the
past. For example, advances have been made in the following fields:-
the rates at which substances enter the brain; the factors which
interfere with the entry of these substances; the conditions under
which supply becomes inadequate to meet fully the metabolic needs
of the brain and how far the brain, when deprived of normal substrate,
can make good the deficiency by metabolizing an alternative substance.
From these advances a common pattern emerges - that transport pro-
cesses are of great importance in controlling the levels of meta-
bolites in the brain. 43
A new technique for achieving steady concentrations of various
substances in the bloodstream of living animals (Fig. 1) has recently
been developed and should aid in the development of work on the met-
abolism of the brain in vivo. 21 , 22, 27, 53

GLUCOSE AND OTHER ENERGY YIELDING SUBSTANCES

Until quite recently glucose was believed to be the only

ss
56 O.E. PRATT

substance used by the brain to any appreciable extent for energy-

yielding metabolism. It has now been shown that under certain
conditions, e.g. fasting, the brain can utilize the ketone bodies
3-hydroxybutyrate and acetoacetate 52,40,25,26 and possibly some
glutamate (the transport of this substance into the brain will be
considered together with that of the other amino acids). For con-
venience two intermediates in the metabolism of glucose or ketone
bodies (pyruvate and lactate) will also be considered in this
section.

1. Glucose

Glucose enters the brain by a mechanism showing stereospecif-

icity and competitive inhibition. 18 ,19,38,5l,58 For the quanti-
tative study of glucose transport from the blood into the brain it
is essential to maintain steady degrees of hypo- and hyperglycaemia
over suitable periods of time. This was done by various combinations
of fasting and of controlled injections of insulin and of glucose. 2 ,28
These studies showed that in the living anaesthetized rat glucose
enters the brain by a saturable carrier-mediated process conforming
to Michaelis type kinetics (Fig. 2). The maximum transport rate was

30

PLASMA
GLUCOSE
.t' mol ml-1
10

o 25 50
MIN

Fig. 1. A steady raised level of glucose in the circulating blood

of a baboon (about 2.5 times normal) maintained by a continuous
programmed intravenous injection of glucose at a variable rate
(from Daniel, Donaldson and Pratt 22).
METABOLITE TRANSPORT IN THE LIVING BRAIN 57

found to be 2.23tO.06 ~mol glucose min-lg- l brain and the Michaelis

constant for half saturation 10.5±1.2 rnM plasma glucose 28 which
means that the carrier will be nearly 50% saturated at normal blood
glucose levels.
In order to investigate the mechanisms by which the concen-
tration of glucose within the cerebral cells is maintained within
reasonable limits the arterio-venous differences of glucose were
measured under conditions parallel to those in which influx had
been studied. It is difficult to measure glucose efflux from the
brain directly but it is possible to estimate it indirectly by
making use of the measurements of arterio-venous glucose differences.
Since the cerebral blood flow in the rat is remarkably constant 45
it is possible to calculate from the cerebral arterio-venous glucose
difference the cerebral glucose gain, which represents the difference
between influx and efflux. The glucose gained will normally (but
not necessarily) be metabolized by the brain. Some workers have
used the term "cerebral glucose uptake" but "gain" seems to be pref-
erable, not only as a more precise description but also to avoid
possible confusion with the glucose influx into the brain. At normal
concentrations of glucose in the plasma the cerebral glucose gain is

• •
.....
A A •

INFLUX RATE
jJmol min-1 9-1 brain

50 100
GLUCOSE IN ARTERIAL PLASMA jJmol ml-1

Fig. 2. The, effect of increasing the concentration of glucose in the

circulation upon its influx into the brain. • animals fasted 24
hours and given a continuous intravenous injection of insulin for 17
minutes before and during influx measurements • • controls not given
insulin (from Daniel, Love and Pratt 28).
58 D.E. PRATT

0.28 ~mol min-lg- l brain 28 which is close to the normal utilization

rate of glucose by the rat brain of 0.3 to 0.35 ~ mol min-lg- l brain. 48
Thus all the glucose gained is being metabolized. How does the influx
compare with these figures? Under comparable conditions it is 0.9
to 1.0 ~mol min-lg- l brain (Fig. 2). The large difference which ex-
ists between the influx and the gain (presumably because more glucose
is entering than is being metabolized) shows that under normal con-
ditions there is a considerable efflux of glucose from the brain
back into the blood and that the supply of glucose to the brain is
normally more than adequate to meet cerebral metabolic needs.
In severe hypoglycaemia (with the plasma glucose below about
2 mM) there is little if any difference between the cerebral influx
and the gain of glucose, showing that efflux does not occur apprec-
iably in these circumstances. 28 In this state the cerebral trans-
port mechanism is unable to extract from the blood the normal amount
of glucose that is needed so that hypoglycaemic coma develops. If
the glucose in the plasma is maintained at progressively higher levels
through the normal range and above, up to about 20 mM, the cerebral
glucose influx increases more than the gain, indicating that glucose
efflux from the brain also increases. As the plasma glucose con-

2·0

GLUCOSE GAIN
BY BRAIN
jJmol min-1g-1brain

1-0

10 20 30
GLUCOSE IN ARTERIAL PLASMA mM

Fig. 3. The glucose gain by the brain at various blood glucose con-
centrations. • insulin-treated animals; • controls not given
insulin. Each point is mean of 3 to 12 experiments. The curved
line is taken from Fig. 2 (from Daniel, Love and Pratt 28).
METABOLITE TRANSPORT IN THE LIVING BRAIN 59

centration is raised above about 20 roM the influx does not increase
much more because the transport system is almost saturated. Despite
this, the cerebral glucose gain continues to rise until it catches
up with the influx, which means that, at this stage in the experi-
ments, the glucose efflux falls sharply (Fig. 3). Under these con-
ditions, especially if insulin is also given, more glucose enters
the brain than can be oxidised and an excess must be accumulating
within the brain cells. These experimental conditions correspond
roughly with what will happen shortly after a large glucose load
(as in an intravenous glucose tolerance test) when a combination of
hyperglycaemia and hyperinsulinaemia will not endure for long so
that glucose can only accumulate in the brain cells to a limited
extent. Efflux, which occurs under normal conditions, is likely to
be an important mechanism which protects brain cells from the osmotic
effects of an excess of glucose within them.

2. Ketone Bodies

The influx of 3-hydroxybutyrate and of acetoacetate into the

brain increases more or less in direct proportion to the concen-
tration in the blood plasma, up to levels well in excess of the
value of 5 roM (a level which is commonly found in severe diabetic
ketoacidosis in man). The further finding that the cerebral gain
of each ketone body is close to its influx shows also that the brain
uses almost all of the 3-hydroxybutyrate and acetoacetate that enters
and there is little if any efflux. 25 Therefore, when the ketone
bodies in the blood are increased, as during fasting or in diabetic
ketoacidosis, there will be an increase both in their .influx into the
brain and in the rate at which they are metabolized. Under such
circumstances they appear to be used as an addition to, or as an
alternative to, glucose. When the blood levels of the ketone bodies
are very high the brain takes up more of them than it can oxidise
completely with the oxygen that it takes from the blood. 25 It can
be calculated from the influx values (Table 1) that in diabetic keto-
acidosis over 50% of the total oxygen uptake of the brain would be
required to oxidise the ketone bodies gained by the brain. It is
possible that such a metabolic load could precipitate cerebral oxygen
lack and be one factor in causing damage to the nervous system in un-
treated diabetes.

3. Pyruvate and Lactate

The influx of pyruvate and lactate into the brain is dependent

upon the blood level (in contrast to that of 3-hydroxybutyrate or of
acetoacetate). Clear evidence of saturation of the transport mech-
anism is seen. At blood levels within the normal range efflux occurs
and often exceeds inf1ux,26 so that it is clear that a proportion of
the pyruvate or lactate formed in the brain by glycolysis is not oxi-
dised in the cerebral cells but normally passes out into the systemic
circulation.
60 O.E. PRATT

TABLE 1. ENTRY OF ENERGY- YIELDING SUBSTRATES AND OF AMINO ACIDS

INTO THE RAT BRAIN IN VIVO, THE NORMAL INFLUX IS SHOWN AND THE
RATIO: INFLUX/CONCENTRATION IN PLASMA (BELOW THE LEVEL AT WHICH
THERE IS APPRECIABLE SATURATION)

Substance Influx Influx at normal

Plasma concentration plasma concentration
nmo1 min- 1g- 1brain roM- 1 nmo1 min- 1g- 1 brain
Mean ± SEM (No. of ex-
periments in parentheses)
L-1ysine (a) 24.8 ± 4.6 (9) 7.5
L-1eucine (a) 65 ± 20 (5) 6.2
L-pheny1a1anine (c) 110 ± 6.5 (11) 5.6
L-tyrosine (c) 62.6 ± 3. 1 (7) 4.9
L-histidine (c) 63.7 ± 4 (5) 4.6
L-a1anine (b) 8.7 ± 2.0 (6) 2.7
L-serine (b) 12.8 ± 2.8 (8) 2.38
L-threonine (a) 12.3 ± 4.0 (4) 2.31
L-i so leucine (a) 26 ± 3 (8) 1.89
L-valine (a) 12.8 ± 1.4 (9) 1.84
L-tryptophan (c) 135 ± 10 (5)* 1. 76
L-methionine (a) 43 ± 6 (8) 1.56
L-cysteine (b) 11.1 ± 2 (5) 1.16
L-arginine (a) 10.4 ± 1 ( 14) 1.06
L-g1utamate (b) 6.3 ± 2.6 (4) 0.98
glycine (b) 2.9 ± 0.1 (14) 0.65
L-ci tru11ine (b) 9.3 ± 3 (3) 0.57
L-proline (b) 1.9± 0.2 (7) 0.31
taurine (a) 3.0 ± 0.3 (5) 0.13
L-aspartate (a) 7 ± 1 (5) 0.14
L-dopa (d) 48 ± 3 (3)
D-g1ucose (e) 129 ± 7 (12) 972
3-hydroxybutyrate ( f) 22.0 ± 6.3 (8) 2.7
acetoacetate ( f) 37.0 ± 7.5 (5) 2.5
lactate (g) 42.8 ± 5.7 (4) 11.3
pyruvate (g) 53.8 ± 11.7 (5) 53.0

Sources: (a) Ba~os, Daniel, Moorhouse and Pratt;6 (b) Ba~os,

Daniel, Moorhouse and Pratt;8 (c) Daniel, Moorhouse and Pratt;31
(d) Ba~os, Daniel, Moorhouse, Pratt and Wilson; 9 (e) Danie1~ Love
and Pratt;28 (f) Daniel, Love, Moorhouse, Pratt and Wi1son;~5
(g) Daniel, Love, Moorhouse, Pratt and Wi1son. 26

* Free, not bound tryptophan.

METABOLITE TRANSPORT IN THE LIVING BRAIN 61

TABLE 2. SATURATION OF TRANSPORT SYSTEMS CARRYING METABOLIZABLE

SUBSTANCES INTO THE BRAIN. ESTIMATES OF THE MICHAELIS CONSTANT
FOR HALF SATURATION DERIVED FROM INFLUX MEASUREMENTS IN VIVO

Substance Blood plasma concentration at

which transport is half saturated
mM As multiple of
norma 1 leve 1

L-leucine (c) 0.9f" 0.1 9

L-phenylalanine (b) 1.6 t 0.3 25
L-histidine (b) 1.5 t 0.4 25
L-methionine (d) 2 - 4 60
L-serine (c) 3.0tO.7 16
L-tryptophan* (b) 2.2:1-3.4 160
L-isoleucine (c) 3.3±1.0 50
Lactate (g) 5.7 ± 0.9 23
Pyruvate (g) 5.S ± O.S 6
L-arginine (a) 6 :I- 2 60
L-threonine (c) 7 ± 2.6 16
L-valine (c) S :I- 1.S 56
L-lysine (a) 10 :I- 2 33
D-glucose (e) 10.5 :I- 1.2 1.3
Acetoacetate (f) > 30
3-hydroxybutyrate (f) > 30

Sources: (a) Banos, Daniel and pratt ll . (b) Daniel, Moorhouse and
pratt 3l ; (c) Daniel, Pratt and Wilson 32 (d) Crockett, Daniel and
Pratt l7 ; (e) Daniel, Love and Pratt 2S ; (f) Daniel, Love, Moorhouse,
Pratt and Wilson 25 ; (g) Daniel, Love, Moorhouse, Pratt and Wilson. 26

* Free, not bound, tryptophan.

AMINO ACIDS
It is important to understand clearly how changes in the con-
centration of an amino acid in the blood plasma of a living animal
affect its rate of transport into the brain. When the concentration
of the amino acid is within, or not far above, the normal range the
cerebral influx is for all practical purposes directly proportional
to its concentration in the plasma, or in the case of L-tryptophan,
to the fraction that is not bound to albumin. 6 ,S However, at high
concentrations in the blood the amino acid saturates the transport
carrier (Table 2) and its influx eventually approaches a limiting
62 D.E. PRATT

value. 4 ,11 For a number of amino acids an indication of the

concentration at which carrier saturation becomes important is
given in Table 2 which shows estimates of the blood concentration
at which the transport carrier is half saturated. The fact that
carrier-mediated transport is important for the supply of amino
acids from the blood into the brain does not exclude the possib-
ility of the operation of facilitated exchange mechanisms of the
kind described by Christensen, de Cespedes, Handlogten and
Ronquist. 15 It must also be remembered that the barriers at the
walls of the brain capillaries and at the surfaces of the cere-
bral cells, which prevent free diffusion of amino acids, may not
be completely impermeable and that slow diffusion as well as
transport may take place.

1. Differences in Influx of Amino Acids into the Brain

The wide difference in the rates at which amino acids are

transported into the brain in vivo, first reported by Banos,
Daniel, Moorhouse and Pratt4 ,5 has now been confirmed. 12 ,51,6,8
Comparison of rates of entry (Table 1) shows that the influx of
all the nutritionally essential amino acids into the brain is
high, which ensures that the brain normally receives an adequate
supply of these substances for protein synthesis. The other
amino acids that are found in protein are not needed in the diet
since they can be made by some organ in the body (usually the
liver) but few of them can be synthesized in the brain itself.
Table 1 shows that the influx of a group of seven amino acids is
within the range of those of the nutritionally essential group.
These seven are mainly amino acids which the brain cells cannot
make or cannot make easily. They include the semi-essential
amino acids, L-histidine and L-arginine, as well as a number of
non-essential amino acids, e.g. L-tyrosine which probably cannot
be made in cerebral tissue from L-phenylalanine at an appreciable
rate. 36 ,39 Even when the brain can make one amino acid from
another there are problems in connection with the interconversion
which can only be solved by in vivo work. For instance L-serine
and glycine are readily convertible 55 but the high entry rate of
serine (Table 1) shows that in life the cerebral cells make
glycine from serine, rather than the reverse. Thus the serine
influx into the brain must meet the combined need of the cerebral
cells for serine plus glycine: thus serine has a high cerebral
transport rate comparable with the rates of the essential amino
acids (Table 1). A similar explanation may account for the high
rate of transport of L-a1anine into the brain compared with the
lower influx of L-aspartate and L-g1utamate (Table 1) since
alanine is likely to be used by the cerebral cells as a precursor
for the other two amino acids. 3 The dibasic amino acids,
L-citru11ine and L-ornithine, which are formed in the liver but
cannot readily be made in the brain46 also have a high influx into
METABOLITE TRANSPORT IN THE LIVING BRAIN 63

the brain (Table 1) and although not used for cerebral protein
synthesis they may meet some other metabolic need.
Finally there is a third group, the amino acids which the
brain can readily synthesize, e.g. L-aspartate 35 and L-proline 57
and also amino acids which are not used for protein synthesis,
e.g. taurine. The neurotransmitter amines are not readily trans-
ported into the brain5l nor are the postulated neurotransmitter
amino acids. 59 Table 1 shows that the actual rates of transport
of aspartate, glutamate, glycine and taurine are slow. The slow
rates at which the amino acids in this third group enter the brain
are close to the entry. rates of those amino acids which are not
normally found in the body like 2-aminoisobutyrate and 2-amino-
adipate. 6 It may well be that all these amino.acids which are
not needed by the brain only cross the capillaries slowly by
diffusion even if some of them enter the brain cells by transport
systems. 13 In this group aspartate, glycine and glutamate may
be concerned in osmoregulation (Bradbury, this volume, p. 483 )
and proline and glycine have low entry rates even though present
at high concentration in the blood plasma.
On the other hand there are at least fourteen amino acids that
the cerebral cells either cannot or do not synthesize but which
they use in considerable quantities. The influx of these amino
acids (Table 1) is high enough to ensure entry at rates which are
related to the rates at which the brain uses them for protein syn-
thesis. It can be calculated from the mean rate of turnover of
cerebral protein of 0.7% hr- l , as given by Seta, Sansur and
Lajtha,54 that the rat brain uses amino acids at a rate of the
order of 75 nmol min-lg- l brain. Not all of these amino acids
are obtained directly from the blood. Some amino acids, that are
freed by the breakdown of cerebral protein, will be re-cycled,
bridging the gap between utilization and influx. Thus, the
fourteen amino acids with influx rates greater than about 1.0 nmol
min-lg- l brain provide a combined supply to the brain of 46 nmol
min-lg- l brain, i.e. less than two thirds of what the brain needs
(Table 1). These calculations suggest that the influx rates of
many of the amino acids may, on occasion, be barely adequate to
meet the needs of the brain for protein synthesis. It is signif-
icant that Lajtha44 has estimated that the half-life of many amino
acids free in the brain is less than 30 minutes.

2. Exclusion of Amino Acids from the Brain by

Inhibition of Transport Mechanisms

Competition between amino acids for entry into the brain in

vivo was first shown to occur between the dibasic amino acids,
L-arginine, L-lysine and L-ornithine. lO If one of these is present
at a high concentration in the plasma it largely prevents the others
from entering the brain but this exclusion can be overcome if the
concentration of the excluded amino acid in the circulating blood
64 D.E.PRATT

is simultaneously increased. 11 Another example of competition is

provided by the interaction between the branched chain amino acids,
L-iso1eucine, L-1eucine or L-va1ine, anyone of which if present in
the circulating blood at a high concentration (of the order of
10 roM) not only saturates its own transport system but partially
excludes the other two from entering the brain. For example, a
progressive diminution in the influx of L-1eucine into the brain
can be achieved by raising the concentration of L-va1ine in the
blood plasma to successively higher levels. However, this ex-
clusion of L-1eucine from the brain can be overcome by simultan-
eously increasing (Fig. 4) its concentration in the b1ood. 32
Another group showing saturation and cross inhibition are the
aromatic amino acids L-pheny1a1anine, L-tyrosine, L-tryptophan and
L-histidine. 31 In this group also is the aromatic amino acid
L-dihydroxypheny1a1anine which is present in the brain but not
normally in blood. It is widely used to treat Parkinsonism and,
at a blood concentration of less than 1 roM, considerably reduces
the influx into the brain of other aromatic amino acids. 9

a
2000 •• •

RECIPROCAL OF
ENTRY RATE OF
L-LEUCINE
,Pmo1-1. b'
min grain •
1000

20 40 60 80 100
-1
L-VALINE .... mol ml

Fig. 4. Inhibition of carrier-mediated transport of L-1eucine into

the brain by progressively higher concentrations of L-va1ine in the
circulation. The inhibition is largely overcome by increasing the
concentration of the L-1eucine in the circulation from 0.096 roM (a)
to 0.84 roM (b) and to 3.9 roM (c) (from Daniel, Pratt and Wi1son32 ).
METABOLITE TRANSPORT IN THE LIVING BRAIN 65

TABLE 3. EXCLUSION OF TRYPTOPHAN FROM THE BRAIN CAUSED BY RAISED

LEVELS OF OTHER NEUTRAL AMINO ACIDS IN THE CIRCULATING BLOOD*

Inhibitor Estimate of concentration

of inhibitor in the plasma
needed to halve rate of entry
of tryptophan into the brain
mM

L-phenylalanine 0.2 - 0.3

L-tyrosine 0.7 - 0.9
L-histidine 1 - 1. 5
L-leucine 1 - 1. 3
L-isoleucine 2 - 3
L-methionine 3 - 4
L-valine 2 - 4

* From Daniel, Moorhouse and Pratt 3l

When different amino acids are tested as potential inhibitors

of the influx into the brain of one particular amino acid, e.g.
tryptophan (Table 3), it is generally found that there is a wide
variation in the blood concentration at which they cause effective
inhibition. Exclusion of an amino acid from the brain is usually
most effective between related amino acids and is more likely to be
of practical importance if it takes place at low blood concentrat-
ions of the inhibitor. On the other hand the aromatic amino acids
are not only the most effective inhibitors of tryptophan transport
(Table 3) but they also inhibit leucine transport at lower plasma
concentrations than do other neutral amino acids, including even
the other two branched chain amino acids. 17

3. Two Sites of Cerebral Transport

It seems likely that there are separate sets of amino acid

transport systems in the brain, one located in the surface mem-
branes of the cerebral cells, the other at the walls of the
cerebral blood capillaries. Study of transport systems in vitro,
e.g. with brain slices, yields information only about the cell
membrane systems: the systems at the cerebral capillaries can only
be studied in the living animal. The necessarily limited duration
of experiments upon the transport of metabolizable substances into
66 D.E. PRATT

the brain in vivo makes it difficult to study each of the systems

separately in the way that has been done for sodium transport. 47
Our experiments have usually been kept short, one minute or less
(to minimise metabolic changes), but when experiments are contin-
ued for 30 minutes or more (Fig. 5) there are no obvious discon-
tinuities in the curves obtained, such as might be expected if the
extracellular space was filled first and then the substances
entered the cells more slowly. Failure to observe any such dis-
continuities for glucose or various amino acids, with or without
carrier saturation and with or without inhibitors, provides evi-
dence that the rates of transport across capillaries are not
widely different from those into cells. That these two systems
for the transport of amino acids into the brain are essentially
similar was suggested by Lajtha42 and has been confirmed by com-
parison of recent work in vivo with previous work in vitro. 50 ,16
Thus both systems are essentially carrier-mediated so that trans-
port across the capillaries, like that into the cells, can be
impeded by competition between closely related groups of amino
acids for the saturable transport carriers.

Ratio of radioactivity:

-1
.uCi g brain
-1
f..lCi ml plasma

Min
Fig. 5. Exchange of radioactivity while constant specific activity
of l4C histidine is maintained in the circulating blood (from
Daniel, Love, Moorhouse and Pratt 24 ).
METABOLITE TRANSPORT IN THE LIVING BRAIN 67

THE RELATION OF CEREBRAL TRANSPORT TO VARIOUS BRAIN DISORDERS

1. Aminoacidurias

The immature brain, which is rapidly making new protein, re-

quires relatively more amino acids from the bloodstream than the
brain of the adult. 5 If it does not get what it needs cerebral
development and function will be adversely affected. 32 Examples
of cerebral damage are seen in many of the aminoacidurias in which
the concentration of one or more amino acids in the blood is con-
siderably raised. 56 That damage to the developing brain is due
directly to the amino acid imbalance is illustrated in maternal
phenylketonuria where the incidence of mental retardation in the
metabolically normal foetus is almost 100%.41,34 The foetal
brain must suffer damage from the phenylalanine which has passed
through the placenta into the foetal blood and is present there in
abnormally high concentration. The cerebral damage is not necess-
arily due to a direct effect of the phenylalanine upon the brain
but may be caused indirectly because the high level of phenyl-
alanine in the foetal blood interferes with the transport of other
amino acids into the brain, excluding some from entering in suf-
ficient quantity. A defect of brain development is likely to be
caused by a shortage in the brain of one of the excluded amino
acids, especially if any of them are essential for the proper
metabolism of nerve cells and thus for the normal functioning of
the brain. It is worth noting that the amino acids which have
been shown to inhibit brain transport systems (Table 5) include
phenylalanine, the branched chain amino acids and lysine. 9 ,11,3l
Various of these are found in high concentration in the blood in
some metabolic defects associated with mental retardation. The
generally accepted treatment of the aminoacidurias consists in
restricting the intake of that amino acid that is present in the
blood of the patient at a raised level, in an attempt to reduce
its concentration to the normal. The work described above
suggests that the amino acid whose metabolism is defective acts
in vivo as a competitive inhibitor and that the damage to the
brain in a patient with an aminoaciduria may be due to exclusion
from it of other amino acids, whose transport systems are blocked
by the competitive inhibitor. Since such exclusion can be over-
come by increasing the concentration of the excluded amino acid
in the circulation, we have suggested that treatment might be
improved by feeding supplements of any excluded amino acids.?

2. Changes in Transport which affect Neurotransmitters

With varying degrees of certainty some ten or so substances

have been established as neurotransmitters in the brain. 1 9a
68 D.E. PRATT

All of these, except acetylcholine, are either amino acids or are

made in the brain from amino acid precursors. The quantities of
amino acids used in neurotransmitter metabolism are minute com-
pared to the requirements of the brain for protein metabolism. 14
However, is so far as the supply of amino acids to the brain
determines the availability of free amino acids within the brain
it will also influence the levels of the neurotransmitters. There
are changes in the levels of various monoamine neurotransmitters
in the brain in a number of neuropsychiatric disorders, including
Parkinsonism, Huntington's chorea, hepatic encephalopathy and that
associated with perinatal malnutrition.
The monoamine neurotransmitters which are synthesized from the
aromatic amino acids L-tyrosine and L-tryptophan are an important
group. The influx into the brain of these two amino acids is
largely determined by their concentration in the blood plasma
(within the normal range) but can be reduced sharply by competitive
inhibition. Thus, they can be excluded from the brain by other
neutral amino acids, especially other aromatic amino acids, if
these are present at high concentration in the blood. 3l In par-
ticular when the concentration of L-phenylalanine in the blood is
raised somewhat above normal it will largely exclude tryptophan
(the precursor of serotonin) from the brain (Table 3) and thus
probably reduce the production of serotonin in the brain. On
the other hand since the transport of L-tryptophan into the brain
is a very active process (Table 1) working against a rather low
(15 - 20 ~M) concentration of the amino acid in the plasma (the
major part normally being bound to plasma albumin) any rise in the
blood level of tryptophan, or anything which prevents it being
bound to albumin, may produce a relatively large increase in the
supply of the amino acid to the brain, raising its concentration
in the brain cells and causing over-production of serotonin.
Different hypotheses have been put forward to explain how such an
over-production occurs and could cause encephalopathy in acute
hepatic failure. 49 ,20,23 The question is how the various changes
in blood amino acids which have been reported in acute hepatic
failure affect the influx of tryptophan into the brain. Influx
will be increased only slightly by the removal of inhibition due
to the fall in the levels of the circulating branched chain amino
acids but this increase will be more than offset by greater inhib-
ition due to the raised level of phenylalanine in the circulation. 23
There may, however, be other more important factors, for instance
an increase in the concentration of free tryptophan in the plasma,
which work in the opposite direction.

3. Hormonal Abnormalities Affecting Transport into the Brain

Relatively little attention has been given to the potentially

important field of hormonal control of brain transport mechanisms
and much of the work has been concerned with one hormone, insulin.
METABOLITE TRANSPORT IN THE LIVING BRAIN 69

It has only recently become clear that insulin has an effect upon
the exchange of glucose between the brain and the blood.
The ability to control independently the insulin and glucose
levels in the blood has made it possible to separate the direct
effects of insulin upon cerebral transport or metabolism from
those due to changes in blood glucose. If the blood glucose is
raised to high levels the cerebral transport system becomes sat-
urated thus limiting the exchange of glucose between the blood
and brain. The increased influx in hyperglycaemia leads to
increased cerebral glucose gain. 28 This continues until a rise
in intracerebral glucose causes a comparable increase in efflux
which establishes a new equilibrium.
The response to hyperglycaemia is different when the blood
insulin is also raised to a high level. 28 The glucose influx
into the brain is not altered but the cerebral gain of glucose is
raised. Even at normal blood glucose levels insulin causes a
significant increase in gain which is associated with a reduction
in the efflux of glucose. Fig. 3 shows that as the blood glucose
is raised above normal in insulin-treated animals the cerebral
glucose gain rises faster than the influx until the efflux falls
to zero i.e. all the glucose transported into the brain is being
metabolized or retained. In these insulin-treated, hyperglycaemic
animals the rate of glucose gain is several times normal and is
sustained for 20 minutes or more. 28 This changed response to
hyperglycaemia as a result of insulin is likely to be due either
to a block of glucose efflux or to an increase in the rate of
cerebral glucose metabolism. Some of the extra glucose which is
gained by the brain when both insulin and glucose levels in the
blood are high may be oxidised to compensate for the lack of
ketone bodies which are removed from the bloodstream by the sys-
temic action of insulin. However, in hyperglycaemia (above about
20 mM plasma glucose) the cerebral oxygen uptake is insufficient
to oxidise all the glucose that is gained. In all probability
much of the excess glucose is being used to make glycogen, non-
essential amino acids or lipids. 29 This surprising effect of
insulin upon the brain merits further study.

Thyroid hormones. Another example of an endocrine influence upon

cerebral transport mechanisms is that due to thyroid hormones
(Table 4). The stimulation by thyroid hormones of the influx into
the brain of L-Ieucine, and probably also of other amino acids 30
is likely to be important in ensuring an adequate supply of these
amino acids to the brain for protein synthesis. The finding that
thyroid hormones play a part in amino acid transport into the brain
may help to explain why the reduction in protein synthesis in vivo
in the brains of thyroidectomised animals 37 is not observed in
vitro. l Lack of thyroid hormones will be another factor which
may prevent the brain obtaining the amino acids that it needs,
providing one possible explanation for the mental retardation
associated with thyroid deficiency.
 ~

TABLE 4. THE EFFECT OF THYROIDECTOMY UPON THE INFLUX OF SCME ESSENTIAL AMINO ACIDS INTO THE BRAIN
AND UPON THEIR INCORPORATION INTO CEREBRAL PROTEIN (Means ± S.E; No. of experiments in parentheses)

Amino Thyroidectomized group Control group

acid
Influx Influx/amino Percentage of Influx Influx/amino Percentage of
acid concen- amino acid acid concen- amino acid
tration in entering brain tration in entering brain
plasma that becomes plasma that becomes
(nmol min- 1 protein bound (nmol min- l protein bound
(nmo1 min -1 g-l brain (runol min- 1 g-l blain
g-l brain) nW l ) g-l brain) nM- )

L-leucine 5.65±0.77(4)* 40.7 r S.5(4)* 1O.8±3.1(4)** 9.95 r O.75(3) 7S.6 r S.7(3) 21. ]±0.4(3)
L-valine 1.5a±O.22(S)* 10.4±1.5(S)* 3.1±0.9(S)** 2.94±0.44(2) 16.2±2.S(2) 1l.5±0.8(2)
L-lysine 2.26±0.41(4) 12.1±2.2(4) 2.9±0.2(4) 7.4G±1.4 (9) 24.8±4.6(9)

Significantly different from control *(P < O.OS, t-test), **(P < 0.01, t-test)
o
(From Daniel, Love and Pratt 30 ) m
"tI
::0
»
=I
METABOLITE TRANSPORT IN THE LIVING BRAIN 71

In conclusion work in vivo has shown that metabolizable sub-

stances are transported into the brain largely by carrier-mediated
processes at rates bro~dly corresponding to the needs of the brain
for these s~bstances. Thus, because the need of the brain for
glucose is high, its influx is of an order of magnitude larger
than that of other nutrients such as amino acids. Of the amino
acids that the cerebral cells need for the synthesis of protein
some fourteen are not made by the brain and these enter from the
blood at fairly high rates. At the other extreme are those amino
acids which can readily be synthesized by cerebral cells and whose
influx is low.
As well as these specific differences in the activity of the
transport systems for various substances, the rate at which any
particular metabolizable substance enters the brain will depend
upon its concentration in the blood plasma and upon the level of
inhibition which it encounters from competing substances in the
circulation and often upon the levels in the blood of certain
hormones.
The transport systems may sometimes become unable to meet the
cerebral needs, e.g. in severe hypoglycaemia the transport system
cannot extract sufficient glucose from the blood to supply an
adequate quantity to the brain and in thyroid deficiency the
influx of certain amino acids is inadequate for protein synthesis.
An excess of certain nutrients in the blood may also lead to
trouble, e.g. in hyperphenylalaninaemia certain other amino acids
are excluded from entering the brain. Transport processes are
clearly of fundamental importance in controlling the levels of
metabolizable substances in the brain and thus of transmitter and
protein synthesis.

I would like to thank my colleagues, especially P.M. Daniel,

E.R. Love and S.R. Moorhouse, for help in preparing the manuscript.
The work was assisted by grants from the Research Fund of the
Bethlem Royal and Maudsley Hospitals, the Medical Research Council,
the British Diabetic Association and the National Fund for Research
int 0 Crippling Diseases.

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3 Balazs, R., Control of glutamate metabolism. The effect of
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METABOLITE TRANSPORT IN THE LIVING BRAIN 73

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74 D.E. PRATT

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 METABOLITE TRANSPORT IN THE LIVING BRAIN 75

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52 Owen, O.E., Morgan, A.P., Kemp, H.B., Sullivan, J.M., Herrera,
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53 Pratt, O.E., An electronically controlled syringe drive for
giving an injection at a variable rate according to a preset
programme. Journal of Physiology, 237 (1974) 5-6P.
54 Seta, K., Sansur, M., and Lajtha, A., The rate of incorporation
of amino acids into brain protein during infusion in the rat.
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55 Shank, R.P., and Aprison, M.H., The metabolism in vivo of
glycine and serine in eight areas of the rat central nervous
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56 Stanbury, J.B., Wyngaarden, J.B., and Fredrickson, D.S., The
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57 Strecker, H.J., Biochemistry of selected amino acids. In A.
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58 Yudi1evich, D.L., and De Rose, N., Blood-brain transfer of
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THE SPECIFICITY OF AMINO ACID AND SUGAR CARRIERS IN THE CAPILLARIES

OF THE DOG BRAIN STUDIED IN VIVO BY RAPID INDICATOR DILUTION

D,L, Yudilevich and F,V, Sepulveda~<

Department of Physiology, Queen Elizabeth College

University of London, Campden Hill Road, London, W8 7AH

INTRODUCTION

It is well established that some metabolites are transported

from blood to brain by carrier systems which presumably are located
in the capillary endothelium. In the dog the original evidence of
carrier mechanisms was derived from Crone's and our own experiments
on intact animals in which the unidirectional flux of sugar 6 ,25 and
amino acids 26 was studied by means of the first passage multiple
tracer dilution technique 4 , With David Gilboe, when he came to my
laboratory in Chile some years ago, we applied these techniques
to his isolated dog brain preparation in order to measure kinetic
constants for the glucose carrier 3 , He reports elsewhere in this
Symposium the results he and his collaborators obtained when extending
these methods to other molecules and experimental conditions. I
shall concentrate here on demonstrating that the glucose carrier of
the brain is of a type similar to that of the erythrocyte and in
characterising the neutral amino acid carrier demonstrated by
Yudilevich, de Rose and S~pulveda26. I also wish to give what I
think are convincing arguments for the localisation of these carriers
in the capillary endothelium. It is important, I believe, to
emphasize that similar indicator dilution experiments could be made
in humans lO , particularly since recently glucose and other
metabolites have been labelled with C-ll, a short-life radioactive
isotope of carbon l9 ,

* From the Departement de Chirurgie Experimentale, HSpital

Cantonal Universitaire, Lausanne.

77
78 D.L. YUDILEVICH AND F.V. SEPULVEDA

METHODS AND INTERPRETATION

Measurement of the unidirectional flux of a substance was made

in mongrel dogs anaesthetised with 30 mg/Kg (i.v.) of sodium
pentobarbital (Nembutal) using techniques described by Crone 6 •
A mixture of radioactive tracers was injected through a catheter
introduced into the lumen of the carotid artery via the cranial
thyroid branch. Venous blood was obtained from the sagittal sinus.
Each experiment consisted of a rapid (about 1 sec) intracarotid
injection of about O.lml of a solution containing two tracers,
which was followed by collection of 10-25 equal venous samples in

r--l
"0
0
0
:a
E
'" 0.1
..
d
..........
on
0
0
c..
...
u
~

a.
L-...J

G.Ol
3 5 7 9
seconds
1.0

,--,
z
...
..
~
-......
J:;
<l.

:x:
""
I
-' 0.0
L-.-J
20 40 60 110 100
Percent total area un der 22Na

Ii I i , , , , If
1 2 3 4 5 6 769
seconds

Fig. 1. Above: typical "control" simultaneous dilution curves.

Below: plot of the 3H/22Na concentration ratio (R) versus the
accumulated area up to each sample, of the reference tracer 22Na •
From about 10 to 80% of the total area, R is relatively constant:
at about 80%, R increases. The maximal fractional extraction of
L-(3H)phenylalanine is computed from E = 1 - R(10-80), in which
R(10-80) is the average ratio value in the 10-80% interval.
L-(3H)phenylalanine backflux becomes significant 5 secs after
tracer appearance.
 SPECIFICITY OF CAPILLARY TRANSPORT 79

ten to twenty seconds. The injectate solution contained the 3H-

or 14C-1abe11ed test molecule and also 22Na whi~h we showed pre-
viously is an intravascular reference substance 5. Up to eight
dilution runs were performed on each dog with a resting interval of
about 10 minutes between runs 21 The "control" injectates were
prepared by diluting the tracers in solutions of either NaC1 0.9g%
or mannitol (0.1, 1. OM) or sucrose (3M). In the "experimental"
run the test competitor for the transport of the labelled substance
was added to the injectate at concentrations of 0.1, 1.0 and 3.0M.
It was estimated that the concentration in brain capillaries was
about 10 times lower. The maximal fractional extraction E is
computed from the tracer concentration ratio as explained in the
legend to Figure 1.

Unidirectional flux from blood to brain is the product of E,

plasma flow and capillary plasma chemical concentration of the
substrate. However, based on the simple measurement of E,
competition of various molecules for a given carrier can be tested
in vivo since the other two parameters can be assumed to remain
constant. E decreases if inhibition of the transport of the
labelled substance has occurred. For the analysis of the relative
effectiveness of competitors a percentage inhibition, I, was
calculated as I = (1 - Eexp/Econtrol) X 100.

CARRIER FOR AMINO ACIDS

The high fractional loss of some amino acids in the single

passage through the brain circulation is seen from E values for
various labelled amino acids summarised in Table 1. Only L-valine,
L-leucine, cycloleucine, L-tyrosine, L-phenylalanine and L-histidine
are extracted in significant amounts. In these experiments no amino
acid carrier was added to the injectate. The injectate concentration
is dependent on the specific activity of the labelled amino acids.
We have previously reported examples of self- and cross-inhibition
in experiments in which carrier amino acids were added to the
injectate at O.lM. This led Yudilevich, de Rose and S~pulveda to
the suggestion of a common carrier for all the long-chain neutral
amino acids and that this was the only amino acid carrier present in
brain capillaries 26 • It was also shown that glucose did not inhibit
amino acid transport 26 • To further characterise the amino acid
carrier we can analyse the cross inhibition experiments shown in
Table 2. The carrier concentration in the injectate was always O.lM.
L-methionine greatly inhibited the transport of L-leucine and to a
lesser but significant extent that of L-tyrosine. L-leucine inhibited
the transport of cycloleucine and L-tyrosine. No significant
inhibitory effect of L-arginine on L-histidine was observed and nor
did L-alanine have an effect on L-phenylalanine transport.
80 D.L. YUDILEVICH AND F.V. SEPULVEDA

TABLE 1 FRACTIONAL EXTRACTION OF AMINO ACIDS

Amino acid Concentration Average of fractional

(mM) extractions ± S.D.
(No. of experiments)

Neutral
(14C)Glycine (0.041) -0.03 ± 0.04(3)
L-(14C)Alanine (0.059) -0.02 ± 0.04(5)
L-(14C)Serine
(14C)GABA
(0.053)
(4.357)
0.02 $
0.04(4)
-0.09 - 0.07(6)
L- (14C )Valine (0.033) 0.17 ± 0.05(6)
L-(14C)Glutamine (1.343 ) 0.01 ± 0.05(4)
£ (1.300) 0.33 ± 0.05(21)
!
L- 3H )Leucine
(1 C)Cyc10leucine 0.17 0.02(3)
L-(3H)Tyrosine (0.016) 0.47 - 0.04(5)
L-(3H)Phenylalanine (0.014) 0.39 ± 0.07(8)
L-(3H)Histidine (0.003 ) 0.26 ± 0.06(2)

Acidic
L-(14C)Aspartic acid (0.041) -0.08 +
- 0.08(4)
L-(3H)Glutamic acid (0.034) 0.03 ± 0.06(4)

Basic
L-(3H)Lysine
L-(14C)Arginine
(0.016)
(0.027)
0.03 !
0.07(5)
0.06 - 0.06(9)

In conclusion it appears that in the dog there is no evidence

for a basic amino acid carrier but that a carrier for long-chain
neutral amino acids is clearly demonstrated. Histidine, which in
other tissues shares a basic amino acid carrier, is only transported

TABLE 2 CROSS INHIBITION OF AMINO ACIDS

TRACER/INHIBITOR % INHIBITION
(individual dogs)
(14C)Cyc101eucine/Leucine 100; 67; 100
(3H)Tyrosine/Leucine 100; 54; 27
(3H)Tyrosine/Methionine 25; 21; 13
(3 H)Leucine/Methionine 74; 74; 72
(3H)Histidine/Arginine 3', 9', 12; 16
(3H)Pheny1a1anine/Alanine 4; 7; 3 ,. 0
SPECIFICITY OF CAPILLARY TRANSPORT 81

by the neutral amino acid carrier. However, at plasma pH about 96%

of the total number of histidine molecules have a net charge equal
to zero, so it resembles more a neutral amino acid than a basic one.
Since all of the amino acids transported are included in the L-system
of Oxender and Christensen 15 we propose that the carrier in brain
capillaries is similar to that found in~lich ascites cells or in
red cells 24 • In the rat a carrier for the basic amino acids arginine
and lysine has been found in addition to the L-carrier system by
01dendorf 14 and by Banos, Daniel and Pratt 2 • Even though further
work remains to be done particularly since the techniques have
different sensitivities, I wish to propose that there is a species
difference which accounts for the findings in the rat and in the dog.

SUGAR CARRIER

One of the experiments designed to establish relative order

of affinity for the glucose carrier of a number of hexoses and
pentoses is illustrated in Figure 2. In these experiments up to
8 successive first passage tracer dilution runs with D-(3H)glucose
were made in each dog. Control injectates (D-mannitol or NaCl
0.9g %) were alternated with experimental runs made with injectates
containing potentially competitive sugars (0.1, 1.OM). It can be
seen that D- and L-arabinose produced no significant change in the
extraction of glucose. D-galactose reduced the extraction at 1M
and it was only slightly effective at O.lM. Phlorizin at 0.07M
almost totally inhibited transport of glucose. Further experiments
(Figure 3) showed that 3-0-methyl-D-glucose is a more potent
inhibitor than D-galactose and that L-arabinose, D-ribose and
D-arabinose inhibit in decreasing order. In dog 11, D-mannose
D-xylose and D-fucose, all inhibit more than 50% whereas D-lyxose
inhibits only 16%.

Table 3 describes inhibitions of D-glucose transport showing

a higher affinity for L- than for D-arabinose. Phlorizin (7mM)
is a much more potent inhibitor than D-galactose (10 or 10OmM).
Of special interest is the finding that phloretin at equimolar
concentration is a more potent inhibitor than phlorizin.

Experiments suggested to us by John Pappenheimer were

performed with 3-0-methyl-D-(14C)glucose. In Table 4 it can be
seen that the extraction of this molecule was reduced to zero by
D-glucose as well as by the cold derivative.

It may be concluded that in the capillaries of the dog brain,

the specificity of the sugar carrier depends on the conformation of
the transported sugars in the same way as sugar transport in the
red cell 12 • The carrier in the intestinal epithelium is more
specific; for example mannose does not inhibit glucose transportS.
82 D.L. YUDI LEVICH AND F.V. SEPULVEDA

.1
1.0

0.5
~;~
L-Arab

5 5
l' , i;" , 0

... 0
0
~

... e
-..
N
Z

'E:::
N ci
..
N
.1
'"u0 0'" i
.= 0
<.!) 0. ~ c..
.....
O-Gal
...z u
.02
1M
ili'i" ,
1 5 Q.
1 1 5

G
I
0 ...
z
N
N

'0E=
0.5~

1 5
.1

.02
1
m
Phlor

5
NaCI

r"T'T"TTT'I
1 5
seconds seconds

Fig. 2. Right: 22Na dilution curves successively obtained in a

dog. D-C 3H)glucose concentrations are not drawn. Left: Ratio
curves identified by the same symbols used in 22Na dilution curves.

The fact that phloretin is a more potent competitor than phlorizin

also suggests the carrier resembles that of the erythrocyte 23 • Hence
the blood-brain barrier system should also be ~a-independent as
opposed to the ~a-dependent carrier in the intestine and kidney22.
Very recently pardridge and Oldendorf 17 confirmed +Na-independence
in the rat blood-brain barrier and also demonstrated that phloretin
is about 25 times more potent an inhibitor than phlorizin.

In order to detect a Na-K pump in the brain capillary

endothelium the extraction of labelled ouabain was measured.
SPECIFICITY OF CAPI LLARY TRANSPORT 83

TABLE 3 '70 INHIB IT ION OF D-GLUCOSE TRANSPORT

Dog No. L-Arabinose D-Arabinose
lOOmM 100mM
4 ~ --u-
5 0 10
6 0 0
7 40 6
300mM 300mM
10 ~ --u-
12 53 14
Phlorizin D-Galactose
7mM lOmM lOOmM
4 lOa 14
5 73 29 52
6 67 21 55
Phloretin Phlorizin
lmM lmM
l3 3-9- 1-6-
14 92 34

I-
oa:
S!~
CI)
DOG 10 DOG 11 z
<t
a:
I-
w ::i
CI)
0 u.i
0 U (/j
0 0 :::J +1
0 C")
0 ....
0 0...1
0.... .c 0 II)
It)<!)
'0
0
8....
C") I/)
U ~ 0
~
0
....
0 0
c
I 0
....
0 ....
0 )(
>-
z
aI 0
C")
C
II) II)
I/)
..J
0
i=
E
(J
.cIII c::
I/)
.Q 0 C
I
ED
I
~
cu
(!) «
... ~
cu >-
)(
(J
~
u. ::I
0 ~
I I I I I I

*
C") C ..J C C C
0

Fig. 3. Inhibition of D-glucose transport by other sugars.

Concentrations (mM) are 1/10 that of the injectate, i.e. the
estimated capillary concentration.
84 DoL. YUDILEVICH AND FoVo SEPULVEDA

TABLE 4 3-0-METHYL-D-(14C)GLUCOSE EXTRACTION

Control Carrier D-Glucose Carrier 3-0-M-

100mM D-Glucose 100mM
4 dogs 3 dogs

0.31 + 0.06 0.03 :: 0.05 -0.01 +

- 0.06

From Table 5 it is seen that no significant extraction was found

at any ouabain concentration. This indicates the absence of
Na-K pump at the interface where glucose and amino acid carriers
are located. In the heart Duran and Yudilevich detected with
identical methods ouabain binding to myocardium 7 • The finding in
brain correlates with the absence of action of this drug on glucose
transport 17 •

The present characterisation of the glucose carrier in the dog

may be compared with results in other species. In the rabbit
using the indicator dilution technique Agnew and Crone l attempted
a similar study, However, as they stated, "the differences in
transport rate of the various sugars tested were so small that it
would be unjustified to draw any conclusions with regard to this
aspect". The reason for this may have been the large amounts of
cold substances used and the fact that glucose was always present
in the injectate at a relatively high concentration. This study
was further complicated by the finding of a large (65 to 85%)
extraction of mannitol which moves by simple diffusion and is not
extracted in brain capillaries 25 •

Measuring rapid uptake of tracer in the rat brain Oldendorf

found a similar order of affinities but molecules in the lower
range were not tested 14 • In other work on rodents ll ,18 and in the

TABLE 5 FRACTIONAL EXTRACTION OF 3H-OUABAIN

ouabain 4 x 10-8 10-6 10-5 10-4
capillary
Dog No. (mM)

FC-l -0.01
FC-2 -0.04
FC-3 0.03
FC-13 0.00 0.00 -0.03
FC-14 0.02 -0.02 -0.02
 SPECIFICITY OF CAPILLARY TRANSPORT 85

cat 8 ,20 the results may be more strongly influenced by parenchymal

cell uptake and metabolism, for their methods used longer uptake
times. This may well account for the differences with our results.
Thus Eidelberg et al found no effect of phlorizin on arabinose
transport 8 • Furthermore, they found that D-arabinose was transported
at a higher rate than L-arabinose 8 • Le Fevre and Peters found that
D-galactose and D-xylose do not share the glucose-mannose carrierll.
Neame has reviewed the literature on transport of monosaccharides
from blood to brain 13 • However, he tabulated indiscriminately in
vivo and in vitro experiments which may explain his conclusion that
entry of glucose-into brain is facilitated by a carrier system
apparently unlike that of the erythroctye and somewhat similar to
that found in intestine and kidney.

Our data bear an important relation to the anatomical location

of the blood-brain barrier and particularly with the location of the
glucose and amino acid carriers we have described. The endothelial
cells and glial cells have been proposed as alternative sites.
Following the arguments made by pappenheimer l6 a "critical
experimental test in favour of the capillary endothelium would be
the finding that the analogues of glucose cause an instantaneous
reduction of glucose extraction". Our data show that this happens.
Furthermore, the relatively large molecule of phlorizin, mol wt 436,
is a potent inhibitor and it is reasonable to assume that this
molecule could not pass through the tight junctions of the brain
capillary. Thus the site of action must be on the luminal face of
the endothelial cells. Identical reasoning led Kleinzeller and
McAvoy9 to propose the anti-luminal face of the cells for the
location of glucose carriers in the kidney tubule. We hope that
current work with polyphloretin (mol wt above 1000) will further
substantiate our conclusion.

Acknowledgement This work was started at the Faculty of Sciences

of the University of Chile and was concluded in Great Britain.
D.Y. would like to thank the Medical Research Council, The Department
of Physiology at University College, London, and especially Hugh
Davson for their kind support. We are also grateful for secretarial
assistance from Miss Lesley Bridge, for technical assistance from
Mrs. Lynda Clarke and for unending collaboration from Mr. David Barry.

REFERENCES

1. Agnew, W.F. and Crone, C. Permeability of brain capillaries to

hexoses and pentoses in the rabbit. Acta Physiol.Scand.
70 (1967) 168-175.

2. Ba~os, G., Daniel, P.M. and Pratt, O.E. Saturation of a shared

86 D.L. YUDILEVICH AND F.V. SEPULVEDA

mechanism which transports L-Arginine and L-Lysine into the

brain of the living rat. J.Physiol. 236 (1974) 29-41.

3. Betz, A.L., Gilboe, D.D., Yudilevich, D.L. and Drewes, L.R.

Kinetics of unidirectional glucose transport into the isolated
dog brain. Am.J.Physiol. 225 (1973) 586-592.

4. Chinard, F.P., Vosburgh, C.J. and Enns, T. Transcapillary

exchange of water and other substances in organs of the dog.
Am.J.Physiol. 183 (1955) 221-234.

5. Crane, R.K. Intestinal absorption of sugars. Physiol.Rev. 40

(1960) 789-825.

6. Crone, C. The permeability of brain capillaries to

non-electrolytes. Acta Physiol.Scand. 64 (1965) 407-417.

7. Duran, W.N. and Yudilevich, D.L. Capillary and cellular barriers

to ouabain transport in the heart. Microvasc.Res. 7 (1974)
84-88.

8. Eidelberg, E., Fishman, J. and Hams, M.L. Penet!ation of sugars

across the blood-brain barrier. J.Physiol. 191 (1967) 47-51.

9. Kleinzeller, A. and McAvoy, E.M. Sugar transport across the

peritubular face of renal cells of the flounder. J.Gen.Physiol.
62 (1973) 169-184.

10. Lassen, N.A., Trap-Jensen, J., Alexander, S.C., Olsen, J. and

Paulson, O.P. Blood-brain barrier studies in man using the
double-indicator method. Am.J.Physio1. 220 (1971) 1627-1633.

II. Le Fevre, p.G. and Peters, A.A. Evidence of mediated transfer

of monosaccharides from blood to brain in rodents. J.Neurochem.
13 (1966) 35-46.

12. Le Fevre, p.G. and Marshall, J.K. Conformational specificity in

a biological sugar transport system. Am.J.Physiol. 194 (1958)
333-337.

13. Neame, K.D. Transport of monosaccharides, amines, and certain

other metabolites. In A. Lajtha (Ed) Handbook of Neurochemistry,
Plenum Press, New York, Vol. 4, 329-359.

14. Oldendorf, W.H. Brain uptake of radiolabelled amino acids,

amines and hexoses after arterial injection. Am.J.Physiol.
221 (1971) 1629-1639.

15. Oxender, D.L. and Christensen, H.N. Distinct mediating systems

for the transport of neutral amino acids by the Ehrlich cell.
SPECIFICITY OF CAPILLARY TRANSPORT 87

J.Biol.Chem. 238 (1963) 3686-3699.

16. Pappenheimer, J.R. Discussion in C. Crone and N.A. Lassen (Eds)

Capillary Permeability. Proc.Alfred Benzon Symp., Munksgaard,
Copenhagen, 1970, pp 477-479.

17. Pardridge, W.M. and Oldendorf, W.H. Kinetics of blood-brain

barrier transport of hexoses. Biochim.Biophys.Acta 382
(1975) 377-392.

18. Park, C.R., Johnson, L.H., Wright, J.H. and Batsel, H. Effect
of insulin on transport of several hexoses and pentoses into
cells of muscle and brain. Am.J.Physiol. 191 (1957) 13-18.

19. Raichle, M.E., Larson, K.B., Phelps, M.E., Grubb, R.L., Welsh,
M.J. and Ter-Pogossian, M.M. In vivo measurement of brain
glucose transport and metabolism employing glucose-llC.
Am.J.Physiol. 228 (1975) 1936-1948.

20. Sacks, J. and Bakshy, S. Insulin and tissue distribution of

pentose in nephrectomised cats. Am.J.Physiol. 189 (1957)
339-342.

21. Slpulveda, F.V. and Yudilevich, D.L. The speciHcity of glucose

and amino acid carriers in the capillaries of the dog brain.
J.Physiol. 250 (1975) 2l-23P.

22. Stein, W.D. The movement of molecules across cell membranes.

Academic Press, London, 1967.

23. Wilbrandt, W. and Rosenberg, T. The concept of carrier transport

and its corollaries in pharmacology. Pharmacol.Rev. 13 (1961)
109-183.

24. Winter, C.G. and Christensen, H.N. Migration of amino acids

across the membrane of the human erythrocyte. J.Biol.Chem.
239 (1964) 872-878.

25. Yudilevich, D.L. and de Rose, N. Blood-brain transfer of glucose

and other molecules measured by rapid indicator dilution.
Am.J.Physiol. 220 (1971) 841-846.

26. Yudilevich, D.L., de Rose, N. and S~pulveda, F.V. Facilitated

transport of amino acids through the blood-brain barrier of
the dog studied in a single capillary circulation. Brain
Research 44 (1972) 569-578.
POSSIBLE ROLE OF INSULIN IN THE TRANSPORT OF TYROSINE

AND TRYPTOPHAN FROM BLOOD TO BRAIN

A. Tagliamonte, M.G. DeMontis, M. Olianas,

P.L. Onali, and G.L. Gessa

Institute of Pharmacology, University of Cagliari

Cagliari, Italy

The penetration of amino acids into the brain and into other
tissues is mediated by energy-requiring systems l - 3 , These systems
are not specific for each amino acid, but distinguish amino acids
into three groups: neutral, acidic, and basic 4 - 6 , The rate of
entry of anyone amino acid into the brain is controlled by the
ratio between its concentration in plasma and the concentration
of all other amino acids belonging to the same groupS-7.

Since tryptophan is the only amino acid present in plasma bound

to a protein, albumin8 , the question arises as to whether it is
only its free fraction or rather its total plasma concentration that
competes with the other neutral amino acids for brain penetration,

The issue has its physiological implications, since the rate of

synthesis of brain serotonin is, in most cases, proportional to the
concentration of tryptophan in the central nervous system (CNS)9,10.

The main evidence that free plasma tryptophan has a physiological

role in controlling brain tryptophan levels is the following:
a) drugs that displace tryptophan from its binding to serum albumin
decrease total serum tryptophan concentration while increasing free
serum tryytoyhan, brain tryptophan level, and brain serotonin
synthesis 1, 2; b) treatments that increase free fatty acids in
plasma have the same effect 13 ,14 in that free fatty acids compete
with tryptophan for its binding to serum albumin 14 ; c) the increase
in brain tryptophan concentration following the administration of
L-tryptophan parallels the increase in free serum tryptophan and not
that of total serum tryptophan 15 •

On the other hand, the hypothesis that !2!!l, and not free,

89
90 A. TAGLIAMONTE ET AL.

tryptophan concentration in plasma controls brain tryptophan

concentration relies mostly on the fact that the administration of
insulin or the consumption of a carbohydrate diet (which causes the
release of endogenous insulin) increases the concentration of
tryptophan in the rat brain 16 • Since the above treatments decrease
the plasma levels of most amino acids, including the free fraction
of tryptophan, the accumulation of this amino acid in the brain has
been attributed to an increase in the ratio of total plasma tryptophan
to other neutral amino acids 17 •

The present report shows that insulin no~ only increases the
brain level of tryptophan but also increases the level of another
neutral amino acid, namely tyrosine, from blood to brain, though
decreasing its plasma concentration. This might suggest that
insulin directly controls the entry of amino acids into the brain.

MATERIALS AND METHODS

Experiments were carried out with male Sprague-Dawley rats

(Charles River), weighing 180-200 g. They were housed at 2l o C,
four to a cage, exposed to light from 9:00 to 21:00 and had free
access to water and food. The latter was removed 20 hours before
treatments, which were given at 10:00.

Animals were stunned with a blow on the head, their blood was
rapidly collected from the abdominal aorta and allowed to clot at
+4oC, and the serum was frozen at -200C until analyzed.

Brains were rapidly removed, frozen in dry ice, and stored at

-20 0 C until analyzed. Serum was processed for free tryptophan
determination according to Stefanini 18 and for total tryptophan
and tyrosine as described by Costa et al. 19 • Brain tyrosine and
tryptophan concentrations were determined as already described lO
Values were analyzed statistically by the two-tailed Student's
test 20 •

RESULTS

Table 1 shows the effects of a single dose of insulin on the

concentrations of t~ptophan and tyrosine, both in serum and in
brain. As expected 16 , insulin increased total serum tryptophan
and brain tryptophan levels, while it decreased significantly the
free fraction of this amino acid in serum. In addition, insulin
decreased the concentration of serum tyrosine at all three times
studied. However, to our surprise, the hormone did not lower brain
tyrosine, but actually increased its concentration to a maximum of
143% 90 minutes after treatment.

Table 2 shows that the oral administration of glucose produced

the same pattern of changes in the concentration of tryptophan and
tyrosine in serum and brain, as obtained following exogenous insulin.
ROLE OF INSULIN IN TYROSINE AND TRYPTOPHAN TRANSPORT 91

TABLE I
Effect of Insulin Administration on the Concentrations of Tyrosine
and Tryptophan in Serum and in Brain.

Min. after TrXEtoEhan Txrosine

insulin Serum Brain Serum Brain
administration (~/ml) (j-Lg/g) (~/ml) (~/g)
(no. of animals) free total

0 (36) 3.26 18.5 4.46 14.3 12.3

30 (18) 2.54* 20.0 5.58** 11.8*** 14.4***
60 (18) 2.15* 23.0*** 6.21* 10.5** 15.5*
90 (18) 2.08* 22.9*** 6.56* 10.3** 17.6*

Each value is the mean ± S.E. of experiments reported in

parentheses. Insulin was injected S.C. at the dose of 2 I.U.
* P ~ 0.001 in respect to control values.
** p~ 0.01 in respect to control values.
*** p ~ 0.05 in respect to control values.

TABLE 2
Effect of Oral Glucose Administration on the Concentrations of
Tyrosine and Tryptophan in Serum and in Brain.

Min. after Tryptophan Txrosine

glucose Serum Brain Serum Brain
administration (~/ml) (j-Lg/g) (j-Lg/ml) (j-Lg/g)
(no. of animals) free total

o (36) 3.26 18.5 4.46 14.3 12.3

30 (18) 2.14* 18.8 5.13*** 12. 6*~\-* 15.3**
60 (18) 2.19* 20.1 6.89* 11.3** 18.5*
90 (19) 2.04* 20.4 5.09*** 13.1 16.3**

Each value is the mean ± S.E. of experiments reported in

parenehteses. Glucose was administered by gavage at the
dose of 2.5 g/Kg in 5% H2 0.
* p < 0.001, in respect to control values
** p <" 0.01 in respect to control values
*** p -< 0.05 in respect to control values
92 A. TAGLIAMONTE ET AL.

DISCUSSION

The mechanism by which insulin or glucose administration

increases the level of brain tyrosine, as well as that of brain
tryptophan, might be an increased amino acid transport from blood
to brain. The enhanced transport of tyrosine might be explained by a
smaller decrease than that of the other neutral amino acids, which
compete with tyrosine for the same transport into the brain. This
possibility is supported by the results of Yamada and Swendseid 2l ,
indicating that insulin does, indeed, reduce the plasma levels of
valine and isoleucine to a much greater extent than that of tyrosine.
As reported above, a similar mechanism has been suggested to explain
the insulin-induced rise in the level of brain tryptophan l7 .

An alternative possibility might be that insulin directly

stimulates the transport of tyrOSine and tryptophan from bloo~2to
brain, as it does for other amino acids into striated muscles •

Similar arguments can be extended to tryptophan. Insulin might

stimulate its transport from blood to brain either directly, or
2
indirectly by enhancing the ratio of f ee serum tryptophan to the
sum of the other competing amino acids 3. However, two mechanisms
are probably operating to decrease free serum tryptophan: one is
an enhanced penetration and retention by tissues, including the
brain, the other is an increased binding to serum albumin. The
latter effect is the consequence of the insulin-induced decrease in
serum non-esterified fatty acids which compete with tryptophan for
the same binding onto serum albumin 14.

Our theory is that as tryptophan binds to albumin, its avail-

ability for tissues is limited; this would explain why, following
insulin administration, bound tryptophan accumulates in serum, while
free tryptophan falls in the same way as do other unbound amino acids.

This view is opposite to that proposed by Wurtman, who considers

the physiological significance of bound tryptophan to be that of
remaining in serum and, therefore, being available for the brain,
while the other amino acids that compete with it for brain uptake
are declining 24 • Among the problems to be Clarified is that of the
physiological significance 0.£ the increased turnover of brain
tyrOSine, with respect to the synthesis of brain proteins and/or
catecholamines. It is pertinent, at this point, to mention that
recent results from our laboratory25 and from Wurtman's26 suggest
that tyrosine concentration in the brain plays an important role in
the syntheSiS of dopamine.
SUMMARY
The administration of insulin or the ingestion of glucose
increases the concentration of tryptophan and tyrosine in the
brain. This increase is associated with a parallel decrease in the
ROLE OF INSULIN IN TYROSINE AND TRYPTOPHAN TRANSPORT 93

concentration of free tryptophan and tyrosine in serum. The results

suggest that insulin enhances the transport of tyrosine and trypto-
phan from blood to brain. The results, moreover, suggest that
exogenous or endogenous insulin enhances the transport of tyrosine
and tryptophan from blood to brain.

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94 A. TAGLIAMONTE ET AL.

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amino acid concentrations on the synthesis of brain monoamines,
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leucine on amino acid levels in plasma and tissue of normal
and diabetic rats, Am. J. Physiol., 215 (1968) 1324.
22. Manchester, K.L., The control by insulin of amino acid
accumulation in muscle, Biochem. J., 117 (1970) 457.
23. Biggio, G., Fadda, F., Fanni, P., Tagliamonte, A., and Gessa,
G.L., Rapid depletion of serum tryptophan, brain tryptophan,
serotonin and 5-hydroxyindoleacetic acid by a tryptophan-free
diet, Life Sci., 14 (1974) 1321.
24. Wurtman, R.J., In, P. Seeman, G.M. Brown (Eds.), Frontiers in
Neurology and Neurosciences Research, First Int. Symposium
of the Neuroscience lnst., Toronto, 1974.
25. Tagliamonte, A., Tag1iamonte, P., Corsini, G.U., Mereu, G.P.,
and Gessa, G.L., Decreased conversion of tyrosine to catechol-
amines in the brain of rats treated with p-ch1oropheny1a1anine,
.]. Pharm. Pharmac., 25 (1973) 101.
26. Wurtman, R.I., Larin, F., Mostafapour, S., and Fernstrom,
Brain catechol synthesis: control by brain tyrOSine concentrat-
ion, Science, 185 (1974) 183.
THE INFLUENCE OF LIVER-BYPASS ON TRANSPORT

AND COMPARTMENTATION IN VIVO

Jill E. Cremer

M.R.C. Toxicology Unit

Medical Research Council Laboratories
Carshalton, Surrey, U.K.

In this paper consideration will be given to the transport of

metabolizeable molecules and their transformation once they have
entered the brain from the blood. Few studies on uptake processes
have also followed the rapidity with which some substances can be
transformed by established enzymic pathways. Furthermore, even
substrates undergoing certain metabolic sequences in common, such
as the formation and oxidation of acetyl-CoA, appear to do so in
a non-uniform manner. This may indicate a "directed" movement of
small molecules to specific sites for metabolism. This could be
into particular types of cells or into discrete parts of the same
cell type.

Metabolic Labelling Patterns from Various Precursor Substrates

A schematic outline of the theoretical fate of l4C atoms

originating in different precursors and becoming incorporated into
acetyl-CoA is shown in Figure 1.

l4C becomes incorporated not only into the individual

carboxylic acids of the cycle but also into several closely assoc-
iated amino acids·. If all the labelled precursors fed into common
metabolic pools of acetyl-CoA then the distribution of l4C in
subsequent metabolites would be expected to be the same. That
this is not so in the intact brain has long been known and two
typical patterns of metabolite labelling are shown in Figure 2.

The most notable difference between them is in the amount of

l4C incorporated into glutamine relative to glutamate. The high

95
96 J.E. CREMER

[2-14(:] Glucose [1-14(:1 Butyrate [1 J4c] Octanoate

CH3.
1
*co. COOH
l,+c~
CH3. CH2. : CH2. *COSCoA
+CO~CH3 " .. ~
*COSCoA ~-----.....

~
*Aspartic ~*GI utamic-tGlutamine
acid ~ cycle j" acid
~
Figure 1. Schematic outline of the labelling of
metabolites during the oxidative metabolism of substrates
specifically labelled with l4c.

(2 - t4C) glucose (1- t4C) butyrate

~
~ 10·0
>0
:s E a·o "
, ... --- .... .... ,
.~~ ~
....

,
"
......
.......... GLN

,:
E S-O '
~ Q.

.2 ~ )( 4·0
:t:1C)
U
CD '0 2-0
Q. -
Cf)

Time after intravenous injection, min

Figure 2. Adult, 200 g, rats were injected with either

10 f.LCi or (2_l4C) glucose or 15 f.LGi of sodium (1_14C)
butyrate in 0.2 ml of 0.9% NaCl into a lateral tail
vein. At selected time intervals after injection the
supratentorial regions of the forebrain were removed by
the rapid freeze-blowing technique ll .
LIVER-BYPASS, TRANSPORT, AND COMPARTMENTATION 97

ratio for the specific radioactivity of glutamine relative to

glutamate found with many precursors at early times after injection,
deviates from the precursor-product relationship expected of a
homogeneous (single component) system. It is evidence of multi-
compartment metabolism (see reviews l ,2).

The amount of a substance g~v~ng this very high labelling

ratio, for example butyrate, that is metabolized by the brain is
likely to be very small in comparison with the main substrate,
glucose 3 • This does not appear to be the reason for marked
differences in metabolite labelling patterns as can be seen when
data for 3-hydroxy(3- l4 C) butyrate and (1_14C)butyrate are
compared as in Table 1.

TABLE 1. Comparison of the glutamine/ylutamate labelling ratios

following the injection of various ( 4C)-labelled substrates.

GLN/GLU sp. radioactivity ratio

Time after injection:- 3 min. 10 min.

Substrate injected

(2_l 4 C)Glucose 14 0.35 0.54

D-(-)-3-Hydroxy-(3- C)butyrate 0.34 0.60
(1_14C)Acetate 2.5 1.9
(1_14C)Butyrate 4.2 3.8
(1_14C)Octanoate 4.3 3.8

Labelled substrates were injected into a tail vein of 200 g

rats. Each of the two amino acids was isolated from the
brain and the specific radioactivity determined as d.p.m./~ol.

In fed adul~ rats the concentration of ketone-bodies in plasma

and brain is low , and therefore the net rate of 3-hydroxybutyrate
oxidation by the brain will be small. Interestingly, the relative
specific radioactivity ratios of the amino acids found after 3-
hydroxy-(3- l4 C)butyrate were closely similar to those observed after
(2_l4C)glucose and were quite distinct from those found after
(1_14C)butyrate (Table 1). The enzymes required for the initial
steps of conversion of 3-hydroxybutyrate to acetoacetyl-CoA are
different from those required for converting butyrate. Differences
in labelling of subsequent metabolites might be due, at least in
part, to these enzymes being present in different cells or regions
of a cell.

Evidence for Rapid Transformation of Transported Substrates

It has been noted 3 that in the brain of a rat injected intra-

venously with (1_14C)butyrate, in less than 1 minute over 80% of
98 J.E. CREMER

TABLE 2. The rapid metabolism of (1_14C)octanoate by rat brain.

d.p.m.!g of brain
Time after Total % of total
injection radioactivity Glutamate Glutamine counts in
(seconds) GLU + GLN

13 42,400 8,200 1,700 23

20 52,700 13,400 6,200 37
58 71 ,400 16,000 28,900 63

Adult, 200 g, rats were injected with 10 ~Ci of sodium

(1_14C)octanoate into a lateral tail vein and at the
times indicated brains were removed by the freeze-blowing
technique ll •

the total radioactivity is present in the amino acid fraction.

This shows the efficiency with which butyrate molecules are
transformed within the brain. To account for such efficiency
one might suggest that butyrate is not evenly distributed through-
out brain tissue but is taken up preferentially by those cells
able to metabolize it •• This suggestion does not apply only to
butyrate. Another fatty acid of longer chain length, octanoate,
has been studied at eveq earlier times after intravenous injection
and shown to be rapidly transformed (Table 2).

Substrate Uptake and Metabolism in a Pathological Condition Affecting

Astrocytes.

Following from this idea consideration has been given as to

where this rapid metabolism might occur. A possibility seemed to
be astroglia and to test this a neuropathological condition was
chosen in which astroglia were known to undergo ultrastructural
alteration. In rats given a portocaval anastomosis the astrocytes
throughout the brain show a marked watery swelling during the
first few weeks after the operation 6 . No changes are observed in
other types of cells during this stage.

Comparative metabolic studies have been made between control

and QPioated rats injected with various 14C labelled precursors
3,4,1, • With (14C)glucose only minor differences have been
ob~erved in the rate of utilization 3 but with (14C)acetate and
(14C)butyrate there is a marked reduction in the total radio-
activity in the brains of operated animals 3 ,4,10 (Table 3). This
is reflected in the reduced amount of label incorporated into
metabolites, including amino acids (Table 3). However, with
octanoate there is no reduction in the amount of label found in
the brains of the operated rats (Fig. 3). In control animals the
pattern of labelling of brain metabolites by octanoate is virtually
identical to that found with butyrate.
LlVER·BYPASS, TRANSPORT, AND COMPARTMENTATION 99

TABLE 3. Radioactivity in the brains of control rats and rats with

a portocaval anastomosis after an injection of (1-14C)acetate or
(1_14C)butyrate.

10 -3 x d.p.m./g of brain
14 ~1_14C2But~rate
(1- C2Acetate

Animals Time after Total Glu GIn Total Glu GIn

injection radio- radio-
(min) activity activity

Control 2 26 7 7 67 19 30
Operated 2 4 1 0.7 33 10 11

Control 5 36 9 8.5 103 36 35

Operated 5 16 3.5 4.7 42 8 28

(1_14C)Acetate (20 ~Ci/lOO g body wt) was given sub-

cutaneously4. (1_14C)Butyrate (5 ~Ci/lOO g body wt)
was given intravenously (lateral tail vein)3,4. Operated
animals were rats which had been given a portocaval
anastomosis 3 weeks before the metabolic study.

Control rats p)
or rats 3 weeks after a portocaval-anastomosis (e)

[IJ4C] octanoate injection [1_ 14C 1 butyrate injection

120 • •
100 #Oe
0
0 0 0 0
0
80 0
00
0
60 0
0

40
•• • •
20 •
0
Minutes after intravenous injection

Figure 3
100 J.E. CREMER

TABLE 4. Uptake of straight-chain monocarboxylic acids by rat brain.

Compound Brain Uptake Index*

Acetate 14
Butyrate 46
Octanoate 95

*Values are from Oldendorf 8 • The uptake of a l4C_

labelled acid is expressed as the percentage of the
uptake of tritiated water.

The discrepancy between the findings for octanoate comparea

with those for acetate and butyrate might be due to differences
in the lipid solubility of these compounds which would influence
their transport from blood to brain ce11s. Some evidence for
this comes from the work of Oldendorf 8 ,9 who has shown that in
normal rats octanoate enters the brain readily, whereas acetate
and butyrate have a restricted entry as judged from the brain
uptake index (BUI) given in Table 4.
The increase in BUI with chain length parallels increasing
lipid solubility of the acids. However, as pointed out by Oldendorf
8,9, the uptake of the shorter acids is probably greater than would
be expected if lipid-water affinity was the only determining factor.
Saturation kinetics and competitive inhibition studies have indicat-
ed the presence of a monocarboxylic acid carrier system associated
with the blood brain barrier 9 . Such a carrier would enhance the
uptake of the more water soluble shorter chain acids in normal
animals and might be a site for inactivation in pathological condit-
ions.

Some preliminary studies have been made in portocaval anastomosed

rats using the carotid artery injection technique 9 with tritiated
water as the reference substance. The BUI found with (U-14C)acetate,
at a concentration of 0.15 mM, was 16.72 + 1.73 (S.D.) and 6.88 +
1.18 for control and operated rats, respe~tively (G. Sarna, unpublish-
ed observations). Results with (14C)butyrate were equivocal and
more experiments are needed. If there is a reduction in the BUT
this is likely to be less marked than for acetate. With (U-14C)-
glucose, for both groups of animals, the BUI averaged 23. These
uptake data, showing a gross impairment of acetate and no effect on
glucose, are similar to the earlier results from metabolic studies
3,4,10.

The influence that metabolism may have on the uptake index is

not well understood and might be greater on substances with an
apparently restricted entry than on those that are more highly lipid
 LlVER·BYPASS, TRANSPORT, AND COMPARTMENTATION 101

soluble. In the intact brain, to distinguish between an effect on

transport per ~ or on subsequent transformation of metabolizeable
molecules is not easy. The experimental results reported here have
only highlighted the problem rather than given any clear-cut answers.

Acknowledgements

I would like to thank Mr. G. Sarna, Department of Physiology,

King's College, London for permission to quote his recent data; and
Professor J.B. Cavanagh, Institute of Neurology, London for his help
and collaboration with several of the other experiments previously
published.

References

1. Berl, S., and Clarke, D.D., Compartmentation of amino acid

metabolism. In A.Lajtha (Ed.), Handbook of Neurochemistry, Vol. 2,
Plenum Press, New York-London, 1969, pp. 447-472.
2. Berl, S., Biochemical consequences of compartmentation of
glutamate and associated metabolites. In R. Balazs and J.E.
Cremer (Eds.), Metabolic Compartmentation in the Brain, Macmillan,
London-New York, 1973, pp. 3-17.
3. Cremer, J.E., Heath, D.F., Teal, H.M., Woods, M.S., and Cavanagh,
J.B., Some dynamic aspects of brain metabolism in rats given a
po~tocaval anastomosis, Neuropathology and Applied Neurobiology,
3 (1975) 293-311.
4. Cremer, J.E., Heath, D.F., Patel, A.J., Balazs, R., and Cavanagh,
J.B., An experimental model of CNS changes associated with chronic
liver disease. Portocaval anastomosis in the brain. In S. Berl
and D.D. Clarke (Eds.), Metabolic Compartmentation and Neuro-
transmission, Plenum Press, New York-London (in press).
5. Hawkins, R.A., Williamson, D.H., and Krebs, H.A., Ketone-body
utilization by adult and suckling rat brain in vivo, Biochemical
Journal, 122 (1971) 13-18.
6. Kyu, M.H., and Cavanagh, J.B., Some effects of portocaval
anastomosis in the male rat, British J. Exp. Path. 51 (1970)
217-227.
7. Matheson, D.F., Cavanagh, J.B., and Woods, M.S., Effect of porto-
caval anastomosis on the incorporation of (U_1 4 C)glucose into
protein, cholesterol and lipid of rat brain, J. Neurol. Sci.,
23 (1975) 433-443.
8. Oldendorf, W.H., Blood brain barrier permeability to lactate,
European Neurology, 6 (1972) 49-55.
9. Oldendorf, W.H., Carrier mediated blood-brain barrier transport
of short-chain monocarboxylic organic acids, Amer. J. Physiol.
224 (1973) 1450-1453.
10. Patel, A.J., Balazs, R., Kyu, M.H., and Cavanagh, J.B., Effects
of portocaval anastomosis on the metabolism of (1-14C)acetate and
on metabolic compartmentation in rat brain, Biochem. J ., 127
(1972) 85P.
102 J.E. CREMER

11. Veech, R.L., Harris, R.L., Veloso, D., and Veech, E.H.,
Freeze-blowing: a new technique for the study of brain in
vivo, Journal of Neurochemistry, 20 (1973) 183-188.
CERTAIN ASPECTS OF DRUG DISTRIBUTION TO BRAIN

William H. Oldendorf

Brentwood Hospital, Veterans Administration

Los Angeles, California 90073
UCLA School of Medicine, Los Angeles, California 90024

INTRODUCTION

The wall of the general capillary (tissues other than brain)

is freely permeable to all small molecules but the brain capillary
wall, the blood~brain barrier (BBB), is impermeable to many small
molecules but permeable to certain others. The fundamental differ-
ence between these quite different capillary permeability character-
istics is diagrammed in Fig.l. In general capillaries, blood plas-
ma solutes exchange with the more stationary pericapillary extra-
cellular fluid (ECF) largely by diffusion through clefts between the
cells making up the capillary wall. This exchange is, accordingly,
via extracellular pathways.

In brain, the cells making up the capillary wall are fused to-
gether, thus closing the intercellular cleft and obliterating this
non-specific route of exchange. Exchanges between plasma and brain
ECF must take place through the brain capillary and are trans-cell-
ular. Undoubtedly, such trans-cellular exchanges take place through
the general capillary wall but the extracellular route is so effici-
ent that it obscures any transcellular exchanges.

The permeability of general capillaries is only crudely selec-

tive, restricting molecules greater than 20,000-40.,000 MW but indis-
criminately passing all smaller molecules 1. The BBB exchanges
only certain classes of solutes and this selectivity is based on
specific molecular characteristics th~t determine whether they can
enter and traverse the capillary cell .

That the BBB must be permeable to certain substances could be

predicted because some drugs given intravenously have an instantan-
103
104 W.H.OLDENDORF

eous effect on brain and certainly the BBB must be permeable to

metabolic substrates such as glucose. These sUbstances are able tQ
enter and traverse the brain capillary cell.

INTER-CELWLAR TRANS-CELWLAR
EXCHANGE EXCHANGE

GENERAL BRAIN
CAPILLARY CAPILLARY

Fig.l. Indicating the apparent fundamental difference between non-

neural (general) and brain capillA.ries. In the general capillary
exchange of small molecules takes place extracellularly between the
cells of the capillary wall. In brain these intercellular clefts
are closed (tight junctions) leaving the only pathway of exchange
through the cells making up the capillary wall. Such trans-cellular
exchange probably also takes place in general capillaries but its
measurement is made difficult by the efficient non-specific pathway
between the cells.

The capillary cell is made up of an inner and outer bimolecular

membrane and a thin layer of interposed cytoplasm. These membranes
and the cytoplasm are all potential obstacles to successful traver-
sal of the BBB. A plasma molecule must be able to detach itself
from plasma molecular components, enter the inner membrane, detach
itself from this membrane and enter the cytoplasm. It must survive
the effects of the many cytoplasmic enzymes, diffuse to the outer
membrane, enter and penetrate the outer membrane, probably in much
the manner it penetrates the inner membrane. A drug encounters,
therefore, both membranous and enzymatic barriers on its way from
plasma to brain tissue cells.

The key elements of BBB permeability are shown diagrammatically

in Fig.2. The inner capillary cell membrane is the blood-brain inter-
face and is shown in the 'legend diagrammed in Fig.2. The four im-
portant classes of molecules present at the interface are plasma
water, plasma proteins, membrane lipid and membrane carrier proteins.
BRAIN PERMEABILITY TO DRUGS 105
BLOOD - BRAIN

•
INTERFACE

tV
ENZYMATICALLY
PLASMA DRUG CARRER
PROTEIN....... MOL~ PROTEIN

~,
---. SUBSTRATE

-
MOLECULE

CARRIER
PLASMA7'f'" " •• "PROTEIN
WATER
o
ENom.~JAL
PLASMA
CYTOPLASM
t
INNER
t
OUTER
MEMBRANE t.£t.tlRANE
LIPID LIPID

Fig.2. At the blood-brain interface, entry into the capillary cell

is governed by the plasma drug or metabolic substrate's relative
affinity for the four relevant classes of molecules present. These
are: plasma water, plasma proteins, membrane lipid and membrane
carrier protein. The affinity for plasma proteins and membrane car-
rier proteins cannot easily be predicted but the affinity for plasma
protein can be measured empirically by in-vitro dialysis. Affinity
for membrane carrier proteins cannot easily be measured even empiri-
cally so this area is quite unknown (thus the dashed arrow from
drug to carrier). When a solute is ionized the charge site renders
the molecule highly polar and this firmly anchors the molecule in
water and this minimizes entry into the membrane lipid. Polarity of
neutral molecules is largely determined by their hydrogen-bonding
capacity and this is a very important characteristic determining
BBB permeability. It may be the neutral species of drugs largely
ionized at body pH which is likely to penetrate the BBB.

The relative affinity of the molecules for the plasma compon-

ents and the membrane components determines the rate of transition
from plasma into membrane. The relative affinity for plasma water
can be fairly easily predicted considering the interface as a
simple water-lipid two phase system. To a large extent partitioning
of a drug into these plasma and membrane compartments can be predic-
ted from an in vitro lipid-water partition coefficient (PC) measure-
ment. It is the relative affinity for these two phases (lipid vs.
water) which is important and not the absolute solubility in either.

The affinity of a drug for the plasma and carrier proteins is

less easily predicted and must be determined empirically. There
probably are no useful rules predicting ability of plasma proteins
to bind drugs nor is the affinity of drugs for membrane carrier
106 W.H.OLDENDORF

proteins understood. The carrier proteins presumably allow the

entry into the membrane of very polar metabolic substrates, such as
glucose, which otherwise would remain anchored in the plasma water
and very rarely have the kinetic energy required to enter the lipid.

DRUG DISTRIBUTION TO BRAIN

A bewildering array of physico-chemical factors govern the
appearance of a drug in the blood plasma and its ultimate delivery
to the brain ECF. For simplicity, it will be assumed here that a
drug is injected intravenously and appears immediately in the venous
blood plasma. A drug so introduced first passes through the lung
capillary bed where it comes into partial equilibrium with various
lung tissue compartments (Fig.3).

When we studied lung, brain and plasma levels of various lipid-

soluble drugs following intravenous injection we encountered a con-
siderable sequestration and retention in lung of some very lipid sol-
uble drugs. For example, LSD (octanol/water PC=180) exhibited the
tissue concentrations shown in Fig.4. The same measurements for nic-
otine (olive oil/water pc=o.4) and ethanol pc=o.o4 are also shown.

The anatomic location of this seemingly large lung lipid

compartment is uncertain but it may, to a significant degree, con-
sist of alveolar surfactant. With very high partition coefficient

DISTRIBUTION OF 1.\1. DRUGS

TO LJ..NG AND BRAIN

Fig.3. After intravenous injection a drug is immediately brought

into at least partial equilibrium with lung lipid and water. The
drug remaining in blood then is distributed to various organs ap-
proximately in proportion to the fraction of cardiac output going
to that organ. If there is sufficient BBB permeability to the drug,
this flow-limited distribution also applies in brain.
BRAIN PERMEABILITY TO DRUGS 107

drugs, sufficient sequestration in lung may occur to substantially

reduce the amount remaining in blood to be distributed to brain and
other organs (Fig.4). It is possible that the lung may have spe-
cific drug affinites giving it unexpectedly large distribution
spaces for certain drugs.

\, 1.-____________ _

~ ----------_____ LSD (LUNG) ~

"t---N.. --L
r--=-----:'-::::I-~~_~~~-----~ (LUNG) ~

:--
~ ~N NICOTIE (BRAW) PCO.4 -------------------E

~
~ OHMCL_
E
LSD (BRAIN)

124 8 16 64
TIME IN (MIN) AFTER I.V. IN.JECTION

Fig.4. The absolute concentration of various lip~d-soluble drugs in

rat-lung and brain after intravenous injection. H-water is also
shown in brain. Very lipid-soluble LSD (pc=180) is highly seques-
tered in lung. The tissue concentration is expressed as a per-cent
of mean body concentration and allows easy conceptualization of the
amounts of various drugs retained in various tissues. Substantially
less LSD (pc=18o) distributes to brain than does nicotine (pc=o.4)
or eth nol (pc=o.o4), probably because so much of the LSD is seques-
tered and retained in the lung during its first passage after i.v.
injection.

The drug molecules survlvlng lung passage are then distributed

to the various organs and, when tracer studies are performed, most
of the tracer delivered to each of the organs other than brain comes
sufficiently toward equilibrium with the local ECF that it largely
remains in the tissue. The relative amount delivered to the various
tissues is nearly proportional to the fraction of cardiac output dis-
tributing to the organ. If the BBB is sufficiently permeable to the
drug, this flow-limited distribution also holds for brain.

We have measured BBB permeability to a number of drugs by

injecting some radiolabeled drug into the rat carotid artery and
108 W.H.OLDENDORF

measuring the percentage left in brain following a single microcir-

culatory passage 3-5. The brain uptake is measured relative to a
highly diffusible tracer, 3H-water (THO) injected simultaneously.
The THO is assumed to be completely removed by brain (actually only
85-90%) and the up-take of the drug is expressed as a percentage of
the THO. When the lipid/water PC of a number of drugs is plotted
vs. the % clearance, the curve shown in Fig.5 is generated.

••
NlCO-.• IM"UMINt
"HANOL
".eMU
N~~~N'~____________________
e __ •
100 •
•
..
.0 .~
O,FFI4NI
e.8_NlETMYL_

.
HEROIN

•
METHADONE

.
2 30

•
'HENOIAlIITAl
•
DUNnN

ASCOIIiC ACD
• MOI.'HIHI
MnHO~.EJlA!~nL5AUC'uc • ______ ~T~OD_ '.!.C!!I~~ ~E!.. ___________ _
2 ----l"iCm- - - - - - - - - - - -

.-
(noSH: • • IENZYLPINICLUN
AI.IWOSIDE $-IODO·2·DfOXYUIIDINE

10 100

'"OLIVE
.001 .10 1.0
Oil
....RTITION COfFfICENT
H 20

Fig.5. Plotting % clearance of various drugs during a single brain

circulatory passage vs. olive oil/water partition coefficients.
When the coefficient exceeds about 0.02 substantially complete clear-
ance is observed.

These data suggest that most drugs having a partition coeffi-

cient greater than about 0.03 will undergo substantially complete
clearance during a single brain passage. Accordingly, if one were
designing drugs to enter brain there would seem little reason to
greatly exceed this threshold. It would seem desirable in such drug
design to avoid very high partition coefficients. Not only do such
drugs tend to be retained in lung but they quickly wash out of brain
redist~ibuting to other tissue compartments. Very lipid-soluble
drugs will largely redistribute to depot and other fat compartments
BRAIN PERMEABI L1TY TO DRUGS 109

where they may reside for very long intervals. This rapid washout
and redistribution of very lipid-soluble barbiturates (such as thio- 6
pental) has been shown to be the basis of their brief central action.

Ethanol probably has an ideal partition-coefficient (0.04) since

this is adequate to allow its complete clearance by brain but low
enough that little accumulation in fat occurs. This, together with
its rapid metabolism, probably explains its rather short duration of
action. Such short-action is probably desirable in a drug taken
socially for its desirable mental effects since it is much less
likely to interfere with the user's overall life-pattern than is a
drug such as tetrahydrocannabinol which has a partition coefficient
of about 6000, as a result of which it is retained in the body for
very long periods 7 .

REFERENCES

1. Landis, E.M., and Pappenheimer,J.R.,Exchange of substances

through capillary walls. In Handbook of Physiology, Sect.2.
Circulation. Vol II. (P.Dow,exec. ed., W.F.Hamilton,Sect.Ed.)
American Physiological Soc., Washington, D.C. 1963,pp.961-1034.
2. Oldendorf,W.H., Blood brain barrier permeability to drugs.
Ann. Rev. of Pharm.,14 (1974) 239-248.
3. Oldendorf,W.H., Measurement of brain uptake of radiolabeled sub-
stances using a tritiated water internal standard. Brain Re-
search, 24 (1970) 372-376.
4. Oldendorf,W.H., Brain uptake of radiolabeled amino acids,amines,
and hexoses after arterial injection. Amer. J. Physiol. ,221
(1971) 1629-1639.
5. Oldendorf,W.H., Lipid solubility and drug penetration of the
blood brain barrier. Proc.Soc.Exp.Biol.Med.,147 (1974) 813-
816.
6. Gold~tein,A., and Aronow, L., The duration of action of thiopen-
tal and pentobarbital. J. Pharm.Exp.Ther. , 128 (1960) 1-11.
7. Kreuz,D.S., and Axelrod, J., Delta-9-tetrahydrocannabinol:
localization in body fat. Science 179 (1973) 391-393.
PENETRATION OF PROTEINS IN THE CENTRAL NERVOUS SYSTEM

Emanuel Levin* and Carlos E. Tradatti

Instituto Nacional de Farmacologia y Bromatologia

Cas eros 2161 and
Instituto Antartico Argentino
Buenos Aires, Argentina

INTRODUCTION

The subject of penetration of protein derivatives and other

macro~olecules into the CNS has no extensive literature; until
recently it was assumed that such entry was practically negligible.
The well known barrier phenomena for substances of lower molecular
weight and less ~ructural complexity suggested that exogenous
proteins should normally be excluded from the CNS.

Primarily, interest was centered on the increased CSF protein

concentrations in pathological states in which knowledge of quant-
itative and qualitative variations might help the diagnosis, and
orognosis, of CNS diseases. Studies related to development of edema
in the nervous parenchyma mention the increased extravasation and
movement of plasma proteins in different parts of the brain.

The possibility of increasing the CNS permeability to oroteins

and other macromolecules, pharmacologically, has had limited
development despite its potential importance. If we consider that
the highest exoressions of central nervous activity such as learning
and memory may be intimately related to specific proteins, we might
soeculate on the impact of introducing "memory molecules" in the
brain or of administering nucleic acids to modify behavior.

In the treatment of CNS oathology, we can envisage globulins

and other antibody derivatives neutralizing toxins and noxious

* Established Investiga~or of the ~Consejo Nacional de

Investigaciones Cientificas y Tecnicas, Argentina.

111
112 E. LEVIN AND C.E. TRADATTI

antigens in the intimacy of the nervous system structures. This

possibility of therapeutic use of macromolecules with biological
activity in the eNS requires deeper knowledge of the barrier
mechanisms that regulate their meager passage into the brain.
Then, it should be possible to modify the barrier transitent1y
without disrupting the delicate and efficient homeostasis that
protects the functioning of the eNS.

In our laboratory work related to this subject utilized as

a model the Fab fraction of an immunoglobulin which has approximate
molecular weight of 50,000, and thus is 1/3 or 1/4 the size of
the corresponding globulin. Fab was obtained by mild proteolysis
of the globulin, followed by separation on DEAE-Sephadex gels.
Fab retains the antibody properties of the parent globulin. Here
we intercalate parts of our work pertinent to the matter.

PENETRATION OF PROTEINS INTO THE CSF

Assuming that all CSF proteins were derived from blood, their
plasma liquor ratio would be approximately 200, showing on the one
hand the low permeability for these compounds, and on the other
hand the presence of regulatory mechanisms capable of maintaining
a steady protein concentration in CSF. We know very little about
these mechanisms apart from a presumably passive efflux by bulk
flow of the CSF.

One way to ascertain the orlgln of CSF proteins is by electro-

phoresis. This assumes that protein fractions from blood with simil-
ar mobilities to those in nervous tissue and CSF indicate a common
orlgln. However, this single parameter is not sufficient. For
example, CSF prealbumin exhibits peptidase activity that may cleave
some proteins coming from blood, thereby altering their electric
mobility. The neuraminidase activity of. the CSF can also liberate
neuraminic acid from glycoproteins entering the CSF, thus/changing
their electrophoretic polarity 19. Therefore, an immunological
identification of the different fractions confirmed the predomin-
ance of plasma proteins as the source of the CSF protein fractions.
A contribution from nervous tissue is also present although to a
much smaller degree.

In pathological states with some degree of trauma, specific

derivatives from the parenchyma become more evident. Quite often
these are accompanied by altered vascular permeability which obscures
the origin of the fractions that increase. For instance, in multiple
sclerosis there is an increase in CSF immunoglobulins~,t9 and r,
partly from blood and partly from the eNS. Furthermore, it is a
selective enhancement particularly for the fractions passing from
blood.
PROTEIN UPTAKE BY THE eNS 113

Proteins with smaller size enter the eNS more easily than
bigger ones, again with selective differences. Serous-transferrin,
type II (mol.wt.88,000) is found in normal eSF, but transferrin
type I of the same size is not - it has been found only in abnormal
eSF when the total protein content rises above 100 mg percent 19.
Albumin,~;-glycoprotein, transferrin and some lipoproteins, all
wi th mol. wt. below 200,000 are present normally in eSF. In contras t,
fibrinogen,~glycoprotein, and certain lipoproteins (mol.wts.above
200,000) are excluded or only traces are present.

New techniques, such as electroimmunodiffusion, isoelectric

focusing, isotachophoresis, among others, are able to detect
and characterize minute protein fractions that were unsuspected
only a few years ago, e.g,,~/-Bntitrypsin, tau-protein,~2-hapto­
globin, are among more than 40 species already demonstrated.
Reviews of this field can be found in references 8, l8a and 29.

With respect to quantitative aspects of protein penetration

into eSF, Hochwald et al. 12 , working with cats, established an
influx of radioiodinated serum albumin (RISA) of 1.3 ~g/min
(approximately 2 mg/day) from blood to a ventriculo-cisternal
perfusate. The albumin specific activity equilibrated with that
of blood in 20-24 hours. Treatment with acetazolamide, which
inhibits eSF production by choroid plexus, indicated that approx-
imately 50 percent of the albumin entered through the choroid while
the remainder entered by extrachoroidal pathways, e.g. tissue
capillaries and pial vessels. By perfusing an isolated area of
the craniql subarachnoid space in monkeys, Matsen and West 20 deter-
mined that the albumin flux was 0.8 fl8' min-I. cm- 2 from blood to
the perfusate, in experiments lasting 9 hours. They concluded that
for albumin,pial vessels have a permeability similar to choroid
capillaries. The influx of albumin from blood was 0.65 to 0.89 ~g.
min- l 13 in subarachnoid segments. In patients!with normal eSF
protein levels the turnover of parenteral RISA in the eSF was 17
mg in 24 hours 5 When the CSF albumin was increased, the influx
was proportional to the eSF protein concentration: in a case exhib-
iting a total protein of 100 mg/IOO ml CSF, over 2 g albumin ex-
changed daily between the eSF and blood. Equilibrium of the eSF
and plasma specific activities was reached between 24 and 72 hours
after the isotope injection.

Differences in protein concentration in cisternal, cranial

and spinal fluids can be attributed to a balan~e of influx and
efflux in each region and to enrichment by proteins coming from
the neural parenchyma (permeating from the eNS capillaries) as the
eSF travels from the ventricles towards the venous sinuses.
Permeability coefficients were calculated for the cat, for the
three fluid spaces, with quite different values among them 6
ventriculo-cisternal, 0.30; cranial-subarachnoid, 0.42; spinal-
114 E. LEVIN AND C.E. TRADATTI

subarachnoid, 0.08 ~g min.cm 2 It should be noted that the pro-

tein concentration in the spinal subarachnoid space is higher than
in the other two compartments. This is due to local protein
accumulation because of the sluggish circulation of the fluid at
this level against a low, but constant, protein influx.

To summarize, normal CSF proteins mainly arise from blood;

which implies passage of protein derivatives through the capillary
wall. The rate of transport is low and has been measured with
isotopically labeled proteins. Choroid, tissue, and pial vessels
are the nexi of protein transport.

PENETRATION OF PROTEINS INTO THE TISSUE

Studies on penetration of macromolecules into normal nervous

parenchyma were mainly qualitative since the poor passage of these
compounds hindered precise measurements: radioautographic and
histochemical methods showed the limited spread of various proteins
introduced by blood, CSF,or directly in the brain. More recently,
new extranervous routes are utilized, injecting markers in muscle,
eye, tooth, etc., while looking for the rate and mechanism of
movement inside the nerve and neuron.

How do macromolecules cross the boundaries, the endothelia of

blood vessels, t~e nerve endings,and the limiting membranes of the
CNS? Once in the nervous system, depending on the place of entry,
the movement of the compound varies. If the molecule traversed the
endothelium of the brain capillaries, do the basal membrane and
astrocytic feet play a restrictive role in the displacement of the
protein? If the molecule was introduced into or reached the CSF,
did its movement from the liquor to the intimity of the parenchyma
follow a simple diffusional pattern or was it distorted by binding,
pinocytosis or attack by proteolytic enzymes? Only part of the
answers are known and most of these are incompletely demonstrated.

Routes of penetration:

a) From blood: Electron microscopic studies show the morphol-

ogical barriers to the penetration of macromolecules from blood.
Using marker proteins it is possible to see the two ways, inter
and intracellular, by which these molecules could cross the cap-
illary endothelium.

Tight junctions between vascular endothelial cells in the

nervous parenchyma and in the pial membrane 3 oppose inter-
cellular progress of proteins. In places where the vessels do not
present such junctions, e.g., choroid plexi and "leaky" areas such
as the median eminence, protein passage is confined to these
PROTEIN UPTAKE BY THE eNS 115

parenchyma

Fig.l. Diagram showing intercellular pathways in the movement of

proteins coming from blood or CSF. Tissue cells (squares) or
vascular endothelial cells (flat rectangles) touch each other,
are tight junctions interrupting the movement of macromolecules.
Shaded tracings represent the vessels' lumens. Non-barrier
regions, such as the area postrema, median eminence, etc., are
represented at the right side of the diagram.

particular zones by the intimate apposition of epithelial cells of

the plexus and ependymal cells of non-barrier regions (Fig.l).
Intracellularly, pinocytosis by engulfing vesicles
appears the main mechanism of transfer of macromolecules through
the capillary endothelial cells. Such filled vesicles can travel
as single bodies or coalesce to form cisterns and "channels"
bridging the luminal and basal surfaces of the cell. The picture
of intracellular channels carrying proteins is better seen in
elasmobranch fishes 6. In mammalian brain, the agranular reticul-
um might play such role. Morphological evidence of proteins reach-
ing the cell soma by axonal transport suggests this possibility.
The amount of material that can be transferred by these intra-
cellular routes is difficult to evaluate. Morphologically, the
cerebral capillary endothelia have fewer vesicles than those of
extranervous tissues. This may be an additional factor contributing
to the barrier to proteins in the CNS. On the other hand, endo-
116 E. LEVIN AND C.E. TRADATTI

thelial cells of some cerebral arterioles are richer in vesicles

and cisterns, perhaps as a way of circumventing (at this level) the
blockade of the intercellular junctions 3l • (The morphological ap-
proach to this subject is more extensively treated in another chap-
ter of the book.)

With reference to quantitative studies, after 9 hours of i.v.

injection of l3II-albumin (RISA) in monkeys 20 , passage into
brain tissue expressed as TIM· 100 (T=tissue; M=plasma), was 0.6
for cortex (grey matter) and 0.1 to 0.25 for subcortical samples
(mixture of grey and white matter, with a predominance of the
latter). According to the serum specific activity, the amount
entering the tissue was between 40 and 250 ~g in the experimental
period. After 1 hour of blood injection, Lorenzo, et al. 18
obtained values of 0.15 for caudate nucleus, 0.17 for thalamus
and 0.23 for colliculi.

In our study with radio.iodinated serum albumin (RISA) and

l3l I - Fab we obtained similar values, varying within 0.2 to 0.5
(TIM· 100), up to 6 hours after injection of the isotopes.
Regional differences were not significant, although there was
a tendency to higher values in the thalamus and caudate nucleus.

b) From CSF. Ventriculo-cisternal and cranial subarachnoid

perfusions are the usual methods used to study the passage of
proteins from CSF to the underlying tissue. Protein markers
show again how the tight junctions between endothelial cells
stop the extracellular movement of macromolecules on their way
into the capillary lumen after ventricular injection. It is also
possible to see intracellular vesicles and cisterns carrying
proteins to the luminal side of endothelial cells when the marker
has penetrated the basement membrane 3

The extent of penetration could be better evaluated in

quantitative studies by current perfusion methods of the CSF
spaces, if one measured thereafter the concentration of the
marker proteins in successive slices at increasing distance from
the perfused surface. We have placed a ~lastic ring on the open
cranial subarachnoid space of rabbits 1 forming a reservoir
where the labeled proteins, l3lI - Fab or RISA, dissolved in artific-
ial CSF, were in contact with the pial surface. After 3 hours,
0.5 mm thick slices from a block of tissue under the reservoir
were obtained with microtome and the radioactivity was measured.
The penetration into the tissue, expressed as TIM· 100 of the total
radioactivity in the medium, varied from 12, for the first underlying
slice, to 2.5 for the deeper, fourth slice. But, after homogenizing
the tissue with TCA, most of the radioactivity was recovered in the
TCA supernatant. Thus, proteolytic or at least I-splitting activity
was very pronounced when the proteins penetrated the brain by this
route.
PROTEIN UPTAKE BY THE eNS 117

Table 1. Percent of TCA soluble radioactivity found

in several brain regions after introduction of
l3lI-Fab and RISA by blood and by CSF.

Introduction by blood Introduction by CSF

Tissue region Fab RISA Tissue slice Fab RISA

Cortex 5.5 4.5 First 41.5 27.0

White matter 29.3 16.0 Second 70.2 53.2

Thalamus 14.1 10.2 Third 74.2 65.5

Caudate nuc. 11.1 5.5 Fourth 91.6 84.8

Tissue samples in both experimental conditions are not the same,

except for Cortex and First slice. 100 percent radioactivity
corresponds to total homogenate.

When penetration of these proteins was measured from blood

after various pharmacological treatments, the proportion of TCA
soluble radioactivity was much lower. Appropriate controls were
run, i.e., the same pharmacological treatment at the beginning of
the "CSF experiments" and in the "blood experiments", killing the
animals 3 hours after the pharmacological treatment.

We proposed a functional differentiation in the extracellular

space of the brain according to the route of introduction of extra-
cellular markers 16 • The present results agree with this inter-
pretation. Proteins corning from CSF are exposed to more of the
splitting enzymes on their way to the depth of the parenchyma.
When the markers are provided from blood, most of the exogenous
protein could be stopped at the level of the basement membrane,
with a lower splitting activity. Subsequently, proteins permeating
the basement membrane could pass to the astrocyte feet and only
part would have intercellular paths in common with the first route.
References to quantitative penetration of labeled proteins from
CSF to the surrounding nervous 8arenchyma do not refer to the state
of the label in the tissue 4, 2 .

The profile of penetration was different for total and for TCA
insoluble radioactivity, and thus, it was possible to determine the
diffusion coefficient (D) for Fab and for RISA in the tissue.

Both proteins have the same rate of diffusion in living brain,

which is lower than in agar and also is lower than that of other
extracellular markers 17 (Fig. 2).
 118 E. LEVIN AND C.E. TRADATTI

.7 I
I
I
RISA ,, Fab
E ,,
I I

,,
I I
:)
.4 I

"0
I
,,
I

,
CI>
I
E I
I

...
I \

,
u
I
C
0 I ' ...
u I
.......... .1 I
CI>
u
-;;
I
,, tissue
I
tissue
u
C
.....
0
u

.01~ __ ~~ ____ ~____ ____

~ ~

5 10 5
distance from medium surface in mm

Fig.2. Diffusion patterns for Fab and for RISA in brain tissue
and in agar. -1
Diffusion coefficients in cm- 2 .sec .10- 7 Fab:Dtissue = 1.27
Dagar = 6.48 - RISA:Dtissue = 1.20; Dagar = 18.8; Dtissue for
inulin is 12.0 and for sulfate is 52.1.
Dotted line = total radioactivity (protein bound + TCA soluble),
deviating from diffusional patterns.

c) From extranervous tissues. Recent research indicates

the possibility of neuronal and synaptic endings being an entry
route for exogenous macromolecules. Although it has been known
for some time that neurotropic viruses can travel via peripheral
nerves in to central nuclei, it was accepted that their entry
was due to an abnormal permeability or damage of the terminal
nerve membranes by the same noxious agent 15. It is now
demonstr2ble that this process of macromolecular penetration
occurs under normal conditions and plays an important role in
the regulation of neural functions, e.g., it is proposed as one
of the mechanisms of interaction, controlling the local homeo-
stasis between the internal tissue milieu and the activity of the
neuron. Phenomena such as regulation of nerve growth according
to the size of the area to be innervated, chromatolysis and
regeneration after nerve lesions, glial reactivity, retrograde
transynaptic changes, and cybernetic regulation of the synthesis
PROTEIN UPTAKE BY THE eNS 119

of hormones and enzymes in the neuronal soma, are thought to be

influenced by movement of such informational proteins and peptides.

The classic concept of informational messages in the eNS is

that the transmitter molecule attaches to a specific receptor
(resulting in metabolic changes and a firing of a nerve impulse)
and produces an electrical event rapidly propagated by the neuronal
chain. Now, a new concept emerges - an extranervous macromolecular
transmitter that does not become attached to a receptor, but travels,
carrying the message "personally" to the proper cellular structure
in the neuronal soma. This trophic message is not rapid. If the
nerve impulse flies, these trophic messengers walk (slow axonal
transport) or run (fast axonal transport).

Different types of proteins, some with biological activities,

others utilized only as markers, have been examined using local
injection in the tissue mass where the nerve endings terminate.
After various periods of time, the protein is sought along the
nerve tract and in the cell soma, usually quite distant from the
place of injection. This is a retrograde transport as opposed to
the orthograde trophic flow from the soma to the nerve ending.

Peroxidase is one of the preferential markers for tracing the

routes of progression and the fate of the injected material.
Neurotubules inside the nerve can be seen filled with the tracer,
and in the cell body it has been shown in vesicles and in the
agranular reticulum 21 . Lysosomes appear to participate in the
degradation of the protein derivatives and it is assumed that this
is a sort of regulatory mechanism to destroy the foreign protein
once it has acomplished its mission 22

Selectivity in the retrograde axonal transport has been

demonstrated in sympathetic ganglia. Preferential entry of the
nerve growth factor (NGF) over other proteins of higher and lower
molecular weight, indicates the importance of structural config-
uration in the process 10 • Quantitative determinations showed
that this route can provide more NGF than is normally found in the
ganglion. In animals with an axotomy that interrupts the possible
progression through the nerve, subcutaneous injection of NGF re-
sulted in 50 percent inhibition of tyroxine hydroxylase, an enzyme
induced by NGF. The biological importance of the nerve route is
then evident 23 .

Not all nerve endings exhibit this permeation of e~ogenous

macromolecules through their terminal membranes. The appearance
of moveable proteins in sensory nerves is not a general feature.
In one lot of rats, only 60 percent showed peroxidase uptake by
sensory nerve terminals of the optic tract 28 . There was,
additionally, some species specificity for rats and fishes;
120 E. LEVIN AND C.E. TRADATTI

peroxidase uptake was not demonstrable in birds and snakes. No

clear explanation was advanced for these negative results.

Other substances and metabolites besides protein have access

to the CNS by this route in normal and pathological conditions.
Further studies should disclose the biological role and possible
pharmacological use of this new transport boundary in the nervous
system.

INCREASED PENETRATION

Pathological conditions. In many pathological states of the

CNS there is abnormal permeability to macromolecules (proteins).
In general, when certain types of edema, called vasogenic by Klatzo,
are present, increased protein concentration in the tissue and
or in the CSF is one indicator of the alterations in the vascular
barrier to these compounds.

We refer above to those pathological conditions wherein the

vascular endothelium is morphologically continuous, but has an
abnormal functional permeability. In contrast, stab wounds,
necrosis or any other cause of vascular rupture, besides being
foci of blood extravasation, are surrounded by an area of increased
passage of proteins, altered electrolyte composition, collection of
fluid, etc. Transitorily, the normal regulatory mechanisms are
unbalanced, breaking the homeostasis of fluid exchanges between
blood and nervous compartments. However, the integrity of the
membranes that act as barriers or exchangers is preserved, thu~
permitting recovery after varying times of the transport balance
and of the functional normality of the tissue.

There is an extensive literature about protein variations

during pathological states in the CNS. We shall examine only
certain aspects related to the possible mechanisms of transport,
stressing that the modified physico-chemical environment is an
important determinant of the altered state of the proteins in
the tissue.

Variations in the protein and matrix hydrations, displace-

ment of charges in the membrane proteins, new interactions
between surface free radicals, hydrophobic forces and other
physico-chemical parameters are profoundly influenced by the
colloidal change resulting from the higher water concentration
in the edematous tissue. The binding of the exogenous proteins
entering the tissue is altered (probably loosened), enhancing the
mobility and displacement of the incoming molecules.

One of the singular morphologic features of the edematous

parenchyma, as revealed by electron microscopy, is the watery
PROTEIN UPTAKE BY THE eNS 121

appearance of the astrocyte cytoplasm, from which we infer that

water and extracellular ions have ~ained access to the interior
of these cells in contrast to the unmodified neurons. The
presence of foreign proteins inside glial cells in cases of exper-
imental edema is established. Brightman 2 observed the presence
of ferritin- and peroxidase-filled vesicles and free ferritin
granules in swollen astrocytes. The perivascular basement membrane
did not impede the movement of the marker. This is in contrast to
normal conditions wherein the basal membrane is postulated to be
a potential barrier for the ingress of exogenous proteins. Very
probably, the binding properties of the basal membrane are modified
by the swelling, allowing the displacement of protein molecules.
The enlarged astrocytic foot, now filled with a watery milieu with
an ionic content of unusual nature, could also change the spatial
configuration and charges of its membrane lipoproteins, thus.l per-
mitting an increased protein passage.

In a study on protein penetration in the brain after seizures

induced by metrazol 18 ,passage of RISA from blood was greatly
increased (20-fold in the thalamus). The unbound I-radioactivity
(TCA soluble) was 24 to 30 per cent of the total radioactivity in
comparison with 5 to 9 percent in normal animals. Convulsions
were not prevented by administration of an anticholinergic agens
but RISA penetration was lower and the unbound radioactivity
represented only 7 to 16 per cent of the total. (It is tempting
to speculate on the involvement of acetylcholine (Ach) regarding
permeation of proteins in the CNS. As a neurotransmitter, an
excess of Ach in pathological conditions could alter the equilibrium
of the Na:K balance and the intra and extracellular ionic medium,
causing an increased permeability also for macromolecules. Alter-
natively, besides its neurotransmitter function, the polar con-
figuration of the Ach molecule might interact with radicals of the
membrane and of the entering molecule, varying their surface
charges and configuration. Changes in the activity of proteinases,
altered mobility and modified binding could facilitate macromolecular
passage as result of these interactions).

Increased protein penetration from blood into the nervous

parenchyma is a well establisherl feature in experimentally-induced
brain edema. Cutler, et al; 7 studied the intracellular location
of the incomin~ protein. After subcellular fractionation, 18 per
cent of the total bound radioactivity from an edematous brain
tissue homogenate was found in the nuclear sediment. When the
increased protein penetration was due to hypercapnia 1.6 per cent
of the bound radioactivity was present in the nuclear fraction.
The difference was accounted for by the microsomal sediment, which
in the edematous tissue retained 30 per cent of the total radio-
activity vs. 14 percent in that of the C02-treated animals. Perhaps
hypercapnia, influencing the acid-base balance inside the cell,
122 E. LEVIN AND C.E. TRADATTI

changes the protein attachment. Or, perhaps after edema and the
appearance of new protein species, there may be new interactions
and binding with the entering molecule. It is known that during
brain edema new proteins appear from proteolysis, metabolism,
and from removal of the macromolecules 27 • When new fractions
are formed, even nonspecific binding of proteins could be very
strong and might be fundamental to the state of the molecules in
a biological medium.

The molecular basis of abnormal penetration and movement of

proteins in the CNS remains unexplored. It is a challenging and
rewarding field for research.

Pharmacological studies. The transitory breakdown of

brain barriers by drugs has been one of the objectives for intro-
ducing different substances into the CNS. Thus, besides behavioral
changes, it might be possible to modify physiological responses;
to inactivate viruses and other noxious agents in the nervous paren-
chyma; to attack benign and malignant tumors in the brain, etc.

The main problem is to find a way by which to increase

permeation through the barriers which does not have serious sequ-
elae. To a certain extent, alteration in the blood and CSF pressures.
EEG, mild inflammatory phenomena, and other minor consequences
could appear as side effects of the treatment, so long as the
normal homeostasis can be restored in a reasonable time.

The difficulty is greater when the substances to be introduced

are macromolecules, because the barriers to these compounds are
more restrictive than for others. If we look for a moderate in-
crease in permeability, to study, for instance, the routes of entry
of proteins, perhaps it would be possible by mild procedures to de-
monstrate histologically what differences occur in relation to
normal conditions. But, if one's interest is to transfer proteins
with biological activity to produce an effect that requires a thresh-
old dose, then it is necessary to approach limits of injury, such
that lesions to the vessels, tissue edema, and more severe inflamm-
atory reactions are very likely to develop.

Cerebral vasodilator drugs were among the first agents tried.

Histamine, physostigmine, papaverine, and serotonin, were examined,
not only for any ability to increase protein penetration,but also
for their effects on the passage of smaller molecules. With res-
pect to proteins the results were contradictory and extravasation
of proteins was observed when structural alterations of the vessels
did occur. Most positive results were qualitative, without precise
measurement of the degree of penetration.

More detailed studies were performed with C02' a well known

vasodilator of the cerebral vasculature. Animals breathed CO 2-
PROTEIN UPTAKE BY THE eNS 123

containing air or mixtures of C02-02 at varying concentrations.

When the proportion of C02 was above ten percent, increased pene-
tration of protein molecules was observed. Vasodilatation and
acidification were the two effects first investigated to clarify
the mechanism of action of C02. Cutler et al. 7 demonstrated that
the increased permeability was not related to either of these
effects. Increased passage of RISA, up to 20 times normal values
(thalamus and colliculus)was obtained in regions where no vaso-
dilatation was present. On the contrary, the correlation of
penetration with the degree of vasodilatation was inverse in
experiments employing inhalation of 25 percent of C02 for one hour.
The effect of acidosis was investigated by parenteral infusion of
0.1 N HCl for one hour after injection of RISA. Blood pH was re-
duced to 7.1 or less and pC02 was maintained at normal levels. No
increase in protein penetration was observed. The effect of C02
on protein penetration was readily reversible and could not be
observed 10 min after discontinuing the hypercapnia. Penetration.
of RISA into CSF under hypercapnia was reported to be a very rapid
response to C02 inhalation. In cats, RISA influx from blood to a
ventricular perfusate rose from 1.0 to 11.4 ~g/min and descended
when the C02 was replaced by airll. The mechanism of action of the
C02 on protein penetration in the CNS remains to be explained.

The use of hypertonic solutions is another procedure. Several

substances were tried, and generally, those with lower lipid sol-
ubility (being more restrained in their passage to the brain) showed
greater osmotic effects, favoring protein penetration 24 . Lipid sol-
uble substances, being more permeable, acted more as agents of injury
on the cell membrane. The threshold osmolarity for "opening" the
barrier varied slightly depending on the substance. The range f0r
several radiological contrast media and for NaCl was between 0.8
and 1.5 osmolal, when these were injected in the internal carotid 26
For urea and lactamide the threshold was at 2 molal concentration 25
In all cases measurement of positive results were semiquantitative.
Apparently, passage was due to shrinkage of the nerve and endothel-
ial cells, widening of intercellular spaces, and separation of the
tight junctions in the endothelium of capillaries and arterioles.
Peroxidase was seen as a"string of sausages" that formed pools
between successive tight junctions. Our impression is that the
separations are transitory and that progression of the material is
worm-like, moved by impulses from successive openings and closings
of the junctions. However, not all regions where peroxidase was
extravasated showed this picture, nor were there signs of shrunken
cells 1, 30

The objective of our study on Fab was to obtain a considerable

increase in penetration without producing brain damage for possible
therapeutic applications. We decided empirically that at least one
third of the extracellular space should be occupied by the protein
124 E. LEVIN AND C.E. TRADATTI

for the procedure to be considered as potentially effective. If we

accept that 20 percent of the tissue volume is extracellular space,
our goal was to produce Fab values in the nervous tissue 6 percent
above the Fab plasma levels, when the protein was introduced by
blood. 131I-human serum albumin (RISA) was used in the preliminary
trials and also for comparison as an inert protein of a similar
molecular weight. Damage to the tissue, the other obstacle to
be overcome, was judged in a first approximation by macroscopic
inspection of the brain and in cases considered as normals, by optic
microscopic examination. Findings of microhemorrhages, mild peri-
vascular edema, cellular distortion, pyknotic nuclei, or any other
sign of microscopic tissue injury determined the classification of
an experiment as "tissue damage positive (+)".

Hypercapnia, induced by inhalation of C02, 20 percens + 02,

80 percent, for 2 hours, did not increase RISA penetration above
the 6 percent positive threshold we were looking for. The use of
NaCl and urea above the threshold osmolarities proposed by Rapoport
caused the expected increased penetration in rabbit brain, but also
produced macroscopic signs of tissue damage.

The combination of hypertonic solutions at concentrations below

those producing "osmotic opening" combined with other active agents
gave better results. We introduced the osmotic agents through a
cannula placed caudally in the external carotid. Thus, the solut-
ion reached the internal carotid without interrupting the blood
flow to the brain. Infusion for 5 min at 1 ml/min was allowed for
the osmotic action. Five min after infusion the animal was decap-
itated and samples of brain tissue were taken for radioactivity
counting. We applied corrections for unbound radioactivity and
residual blood volume. Four brain areas were compared for regional
selectivity of penetration (Table 2), and we measured radioactivity
of a homogenate of the entire hemisphere.

The results of trials with hypertonic solutions near the thresh-

old for obtaining positive results are summarized in Table 3.

The main differences between this and other studies that utilized
hypertonic solutions, are: a) The combination of an "osmotic" solute
with another agent active in transport processes; b) blood supply
to the brain was maintained during the infusion; c) quantitative
measurement of the protein penetration; d) the addition of a lipid
soluble substance (DMSO) favored the protein penetration; and e) we
obtained frankly positive results, i.e., up to 20 times control
values, without macro- or micro-evidence of tissue damage.

Because circulation is maintained during the infusion, the

real osmolarity acting on the brain vascular bed is lower than that
in the solution. During three treatments with NaCl + DMSO (4 percent
PROTEIN UPTAKE BY THE eNS 125

+ 4 percent), osmolarity = 1.87, giving positive results, when

blood samples were taken from the internal carotid (which contains
a mixture of arterial blood and the infusate) the osmolarities in
situ were 0.49, 0.49, and 0.42.

Tightness of binding between the entering molecule and the

tissue was studied by the method of King 14. Three degrees of
binding are: a) Total binding measuring the amount of protein
retained in the tissue after 5 rapid washings. b) Firmly bound
protein: that remaining in the tissue after 2 days incubation in
cold saline solution. c) Urea-resistant binding: that remaining
in the tissue after 2 hours incubation of the tissue containing
firmly bound protein in 8 M urea.

Table 2: Results of a typical positive experiment.

Male rabbit, 2.3 Kg - iv injection of l3lr-Fab.

After 20 min, intracarotid infusion (blood flow
maintained) of NaCl 4 percent + DMSO
4 percent, 1 ml/min for 5 min. Maximal arterial
pressure at 3 min = 154 mm Hg.

Tissue Tissue Hemisphere Hemisphere

sample damage infused side noninfused
side

Cortex 12.3 1.6

White matter 2.2 0.9

Thalamus 13.9 1.1

Caudate nucleus 3.6 1.2

Homogenate 7.6 1.3

Figures represent (tissue/plasma radioactivity) X 100.

To evaluate the results of a single infusion, values for cortex,
thalamus and caudate nucleus were averaged: 9.9. Normal range:
0.2 to 0.5.
126 E. LEVIN AND C.E. TRADATTI

Table 3. Effect of carotid hipertonic infusions on the

penetration of 3Ir-Fab in the brain

TIM
N Agents Conc.* Osmol. more than Tissue
6 percent damage
(Positive
penetration)

(4) NaCl 6 1.83 1 +

(3) NaCl 5 1.44 o
• [(5) NaCl + C02 6 + 20 1.84 2 +
(2) NaCl + C02 5 + 20 1.44 o +
NaCl + C02 6 1.83 3 +
A[(5)
(3) NaCl + C02 5 1.44 3 +
o (2) DMSO 4 0.57 o
(2) DMSO 15 2.20 o
(1) NaCl + DMSO 6 + 5 1 +
o
(6) NaCl + DMSO 5 + + 2.14 4 +
(7) NaCl + DMSO 4 + 4 1.87 5

(3) NaCl + DMSO 3 + 3 1.38 o

(1) Urea 18 2.81 1 +
(2) Urea 12 1.89 o
(5) Urea + DMSO 12 + 5 2.38 1

(2) Urea + DMSO 9 + 5 2.04 o

Number of positive animals (TIM column), from the total number
assayed (N).
* Two values in the column correspond to the concentrations of
first and second agent, respectively. Solids = g percent. C02=vol.per
cent
• Animals received 30 min C02 inhalation;and during the last 5 min
of this inhalation were treated with hypertonic infusion.
~ The NaCl solution was gassed with C02 before infusion which pro-
duced pH 5.0 - 5.2 0 Dimethylsulfoxide
PROTEIN UPTAKE BY THE eNS 127

The results are presented in Table 4. Considering that "in

vivo" the proteins were in contact with the tissue for only a few
minutes vs. 1 hour during incubation, we conclude that macromole-
cules in the extra and intracellular sap are not free but are
attached with a certain firmness to the parenchymal structures.
Some degree of regional specificity can also be appreciated. Urea-
resistant Fab binding in vivo was stronger than that for RISA.

It was important to establish whether the binding did modify

the biological properties of the protein. We did not observe this.
For example, in one experiment, the antibody titer of the Fab was
the same before and after 1 hour "in vitro" incubation with cortical
slices.

Our experiments are in progress. Some data presented here show

the complexity of the problems that must be solved to gain a better
understanding of the mechanisms that govern the penetration and
movement of macromolecules in the nervous system. Then, it should
be possible to drive proteins with biological activity towards
specific CNS targets, in normal and pathological states, to improve
or restore the activity of this most highly evolved biological
system.

Table 4. Firmness of binding of Fab and RISA with brain tissue.

FIRMLY BOUND * UREA-RESISTANT ~

Tissue Fab RISA Fab RISA
sample in in in in in in in in
vivo vitro vivo vitro vivo vitro vivo vitro

Cortex 39 60 23 58 62 75 20 80

White matter 42 55 31 70 81 83 12 86

Thalamus 40 53 52 71 35 65 27 82

Caudate nuc. 37 46 51 73 57 100 35 86

In vivo: Intracarotid hypertonic infusion of NaCl 4 per cent +

DMSO 4 percent for 5 min. Average ryrain TIM: 8.1 percent for Fab
and 6.7 for RISA.
In vitro: Incubation of tissue samples with l3l I - Fab or RISA for
1 hour.
Each value is the average of 2 experiments.
* Values represent per cent of the total bound radioactivity.
~ Values represent per cent of the firmly bound radioactivity.
128 E. LEVIN AND C.E. TRADATTI

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PROTEIN UPTAKE BY THE eNS 129

18. Lorenzo, A.V., Shirahige, I., Liang, M., and Barlow, C.F.,
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 Transport Studies in
Various Nervous Tissue
Preparations
THE CHARACTERISTICS OF GLUCOSE TRANSPORT ACROSS THE BLOOD BRAIN

BARRIER AND ITS RELATION TO CEREBRAL GLUCOSE METABOLISM

A.L. Betz, D.D. Gilboe, and L.R. Drewes

Departments of Neurosurgery and Physiology

University of Wisconsin, }1adison, Wisconsin 53706

INTRODUCTION

Normal neuronal function requires the presence of a suitable

ionic and metabolic environment. It is well documented that in
cerebral tissue, the interface between blood and brain is select-
ively permeable to plasma solutes. Therefore, this so-called
"blood brain barrier" (BBB) plays a major role in maintaining
the milieu interieur of the brain. A complete description of
the barrier's function must consider the relationship between
transport and metabolism. The barrier could serve as a primary
regulator of cerebral metabolism by controlling the entry rate
of substrates into the brain and thus becoming the rate limiting
step. On the other hand, the BBB may serve a more passive role
by maintaining the cellular concentration of substrates whose
rates of metabolism are regulated by other mechanisms. This is
particularly relevant to glucose because most of the energy
produced by the brain is derived from glucose metabolism.
According to current theory, the aYstomic site of the BBB is
the cerebral capillary endothelial cell . Such a hypothesis is
consistent with the observati~g that the barrier has many of the
properties of a cell membrane . A solute can cross a cell
membrane either by simple diffusion or by specific carrier-mediated
transport systems; however, only the latter process would permit
appreciable quantities of a polar solute such as glucose to enter
the brain.
In the study of solute transport across the BBB, the integrity
of the BBB must be preserved; thus, it is necessary to use intact
organs. This is best accomplished by using experimental systems
such as whole animals or isolated, perfused brain preparations
which maintain a normal relationship between capillaries and brain

133
134 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES

cells. The many studies using brain slices are probably more
relevant to transport across brain cell membranes than to trans-
port across the BBB.
Quantification of the rate of transport requires knowledge of
the blood flow rate and solute concentration. With few exceptions,
these values are unknown when whole animals are used; therefore,
studies made with the entire animal are generally qualitative
rather than quantitative. Interpretation of data obtained in
studies using intact animals is further complicated by the hormon-
al and metabolic influences of extracerebral tissues.
The ability to measure and control the perfusate composition
and flow rate in the isolated perfused canine brain combined with
the ability to study unidirectional solute extraction using the
indirttor dilution technique has allowed us to quantify the
rate of unidirectional glucose transport across the BBB. Thus,
the effects of both physiologic conditions and pharmacologic
agents on the kinetics of glucose transport across the BBB can be
studied.

GLUCOSE TRANSPORT KINETICS UNDER PHYSIOLOGIC CONDITIONS

It is well established that glucose enters the brain by a

transport mechanism.. This is appaIin17fI§m3~t~5i~~ ~2ich show
that glucose uptake ~s a saturable ' , , , , , stereo-
specific ~2035s~Owhich can be competitively inhibited by glucose
analogues ' , 12C~~nter-transport between glucose and 3-0-
methyl-D-glucose ' is additional evidence that glucose uptake
by the brain is carrier-mediated.
Transport is similar in many respects to an enzymatic re-
action. Thus, analysis of these transport data is simplified by
assuming that the solute in blood, the carrier, and the solute
inside the cell are analogous to the substrate, enzyme, and
product, respectively.
When the rate of unidirectional transport has been determined
over a range of blood glucose concentrations, the results can be
analyzed using conventional enzyme kinetic techniques. The
apparent K and V for transport are calculated (Fig. 1) by
fitting th~ data ~8Xthe Michaelis-Menten equation
V S
v = max
K + S
m

Kinetic constants for cerebral glucose transport have been

determined in a variety of species including mice, rats, rabbits,
dogs, and sheep. It appears that the apparent Km is general1.;T
hetween 5.5 and 8.7 mM, while the apparent Vroax ranges from 0.7 to
2.8 ~mole/gm of brain per min2,11,7,8,9,15,2~,42.
The K for an enzymatic reaction provides a means by which the
degree of Waturation of the catalytic sites on the enzyme can be
estimated. For substrate concentrations much lower than the K ,
m
GLUCOSE TRANSPORT AND METABOLISM 135

1.60 • •
•

c • •
E 120
"e •
•
"..
0>

'"
'0
e~ 0.80
~

0.40
0.10 0.20 0.30 0.40 0.50 0.60
-!,.(mM'-'
A

10 20 30 40 50
A (mM)

Figure 1. The rate of unidirectional glucose flux is plotted

versus average capillary glucose concentration. The line was
obtained by fitting the data directly to the Michaelis-Menten
equation. A Lineweaver-Burke plot (insert) of the same data is
shown. The direct fit is more valid because it permits use of
weighting factors.

the rate of catalysis is a first-order function of the substrate

concentration. When the substrate concentrations are much higher
than the K , the enzyme is nearly saturated and the catalytic rate
is indepen~ent of the substrate concentration (zero-order kine-
tics). For substrate concentrations near the K , the reaction
rate is a mixed first-order and zero-order func¥ion of the sub-
strate concentration. Under physiologic conditions, the apparent
K for unidirectional glucose transport into the isolated, perfused
dNg brain is about 8.3 roM 11 • Since, in the intact animal, the
blood glucose concentration is normally maintained between 4 and 6
roM, it is unlikely that saturation of the BBB glucose carrier
would ever occur. Furthermore, the rate of unidirectional glucose
transport i§t~9brain is about 3 times the rate of cerebral glucose
utilization' . Therefore, the transport of glucose across the
BBB is probably not a rate-limiting step in cerebral metabolism
under physiologic conditions.
Glucose transport could become rate-limiting when the blood
glucose concentration is very low (hypoglycemia) or when there is
an increase in the rate 'Of glucose utilization by the brain (e.g.
during anoxia).
136 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES

GLUCOSE TRM~SPORT KINETICS DURING HYPOGLYCEMIA

Hypoglycemia produces distinct neurologic symptoms which are

probably the result of altered cerebral metabolism. As the blood
glucose concentration is lowered, the steady-state glucose concen-
tration in the brain extracellular space would be expected to
decrease until the rate of glucose utilization exceeds the rate of
transport from blood to brain. At this point, the brain glucose
concentration will be near zero and cerebral metabolism will be
greatly impaired. If one assumes that the metabolic sequirement
for glucose remains constant at 0.26 ~mole/gm per min and that
the apparent kinetic constants for glucose transport into the 11
canine brain are K = 8.26 mM and V = 1.75 ~mole/gm per min
the blood glucose ~oncentration whi~xwould permit a rate of uni-
directional glucose transport equal to the rate of glucose utiliza-
tion is 1.44 mM (26 mg/l00ml).
Neurologic symptoms of hypoglycemia reportedly occur when the
blood glucose concentration falls in the range of 2.22 to 2.78 mM.
The symptoms are probably the result of a decrease in the rate of
glucose transport from the extracellular space into the neuron when
brain glucose levels are low. It is likely that the kinetics of
glucose transport at z8e neuronal cell membrane are diff ent Z7
from those at the BBB . The studies of Gilboe and Betz ,predict
that the net flux of glucose will fall below 0.26 ~mole/gm per min
(at a blood flow rate of 68 mi/IOO gm per min) when the arterial
glucose concentration is under 2.85 mM. Thus, it is the overall
rate of glucose transport from blood to brain extracellular space
to brain cell that determines the amount of glucose available for
metabolism. In order for the BBB glucose transport system to
maintain the normal brain glucose levels in the presence of low
blood glucose concentrations, it would be necessary to have a lower
K or higher V
m max
GLUCOSE TRANSPORT KINETICS DURING ANOXIA

A second situation in which BBB glucose transport can become

rate-limiting occurs during anoxia or ischemia when the utilization
of glucose is increased. We have studied the changes in glucose
transport and metabolism which result when the isolated canine
brain is perfused with blood having a nO~91 glucose concentration
but an arterial P0 2 of less than 10 mmHg , . During the first
minute following initiation of anoxic perfusion, the rate of glyco-
lysis ~~creases 5-fold as indicated by the rate of lactate accumu-
lation . At the same time, the rate of unidirectional glucose
transport remains constant; however, the net uptake of glucose more
than doubles (Fig. 2). Thus, the rate of glucose utilization
exceeds the capacity for glucose to be transported across the BBB
at a normal arterial glucose concentration. The result is that the
whole brain glucose concentration falls rapidly (Fig. 3) to less
GLUCOSE TRANSPORT AND METABOLISM 137

"2
·e....
E
!?
,,
.,
II>
0.40
,
~
~
0.30
'y.-- --t- -i---i
? 0.20

0.10

o 5 10 15 20 25 30 35 40 45 50 55 60
Minutes of Anoxia

Figure 2. The mean unidirectional (closed circles) and net (open

circles) glucose flux (± SE) into the isolated canine brain are
plotted versus time after initiation of anoxia.

123456 10 15 20 25 30
Minutes of Anoxia

Figure 3. Cerebral glucose uptake (closed circles) and cerebral

glucose content (open circles) are plotted versus time after
initiation of anoxia. The data are shown as percent of control +
SE. Normal cerebral glucose uptake is 0.26 + 0.03 )JTIlole/g per min
while normal cerebral glucose content is 1.72 + 0.14 )JIDole/g.
138 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES

than 24% of normal a~§er 10 min of anoxia and to near zero follow-
ing 30 min of anoxia . These data suggest that the brain could
survive longer periods of anoxia if the blood glucose concentra-
tion were elevated. Prolonged post-decapitation EEG activity and
maintenance of a more normal metabolic state ~tS been observed in
mice made hyperglycemic prior to decapitation .
The entry of glucose into the brain during anoxia becomes
g
even more inadequate as the result of subsequen ~?anges in
properties of the BBB glucose transport system ' . After 2
minutes of anoxia, the rate of unidirectional glucose transport
begins to decline and after 15 minutes it levels off at a rate
that is approximately half that of the control value (Fig. 2).
These data indicate that the BBB glucose transport mechanism is
altered as a consequence of cerebral anoxia. That this is not due
to a general change in BBB permsability is indicated by t?5 fact
that both diffusion of fructose and transport of leucine are
unchanged by anoxia. There are several possible explanations for
this phenomenon: a) an inhibitor of glucose transport may be
produced during anoxia, b) cerebral blood flow may be substantia-
ly redistributed during anoxia, c) the facilitated diffusion --
transport system may be modified as a result of the decrease in
brain glucose levels, d) glucose transport across the BBB may be
directly coupled to metabolic energy, or e) glucose transport
across the BBB may be indirectly ~ouple$ to metabolic energy
through cotransport with ions (Na or H). Each of these possibi-
lities is discussed.

MECHANISM OF GLUCOSE TRANSPORT

We have no data to support or refute the proposal that an

inhibitor of glucose transport is produced during anoxia. A
kinetic analysis of the changes in glucose transport during anoxia
would be useful in deciding the issue; howeve~ we have not attempt-
ed to obtain such data due to technical problems.
There does appear t24be a slight redistribution of cerebral
blood flow during anoxia ; however, it is unlikely that this
redistribution is of sufficient magnitude to explain the observed
changes in glucose transport. Furthermore, cerebral blood flow
changes which do occur take place during the first 5 minutes
following the onset of anoxia. Changes in cerebral blood flow
would be expected to affect all BBB transport and the unidirect- 10
ional transport of L-Ieucine appears not to be altered by anoxia
Facilitated diffusion of glucose has been extensively studied
in the human erythrocyte. It was reported that the kinetics of
transport are altered by the concentration of glucot~ ~~ the side
of the membrane toward which transport is occurring , . In
particular, an increase in the glucose concentration inside the
red cell causes an increase in the rate of transport from the
outside to the inside of the cell. This phenomenon has been
called accelerative exchange diffusion and is usually explained
GLUCOSE TRANSPORT AND METABOLISM 139

using a mobile-carrier model for facilitated diffusion in which 37

the loaded carrier has greater mobility than the unloaded carrier .
Since brain glucose levels decrease during anoxia, the
decreased rate of glucose transport may be due to a decrease in
the accelerative exchange diffusion of glucose across the BBB. In
order to examine the importance of accelerative exchange diffusion
of glucose between blood and brain, the kinetics of glucose trans-
port wgre determined at each of five different brain glucose
levels. The existence of accelerative exchange diffusion was
indicated by an increase in the apparent V for transport as the
brain glucose level increased (Fig. 4). AT~fiough the data predict
a decrease in glucose transport during anoxia, the predicted

43.9±0.6mM
2.0
C 26.3±0.2mM
·E 16.8.:tO.3mM
......
E 1.5 6.11±0.!7mM
~
~'"
0
E 1.0
<.
~
0.5

20 40 60 80 100
A (mM)

Figure 4. Effects of plasma glucose concentration on cerebral

glucose transport kinetics. Curves were fitted to indicator
dilution data followfng equilibration of isolated brain with
various levels of plasma glucose. The glucose concentration with
which the brains were equilibrated is indicated to the right of
each line.

decrease (5%) is less than the observed decrease (50%). Therefore,

the BBB glucose transport system cannot be described by either a
simple symmetric mobile-carrier model for facilitated diffusion or
by an asymmetric model with unequal carrier mobilities. Both
models were also rejected as explanations of the facilitated5d~a­
fusion system for glucose transport in the human erythrocyte' .
Neither model can explain the large decrease in the V that is
max
140 A.L. BETZ. D.O. GILBOE. AND L.R. DREWES

observed when the rate of glucose transport into erythrocytes

con~aining no glucose is compared to the rate of transport into
cell~ which are equilibrated with the external glucose concentra-
tion. This finding is analogous to our observation of an un-
expectedly large decrease in the rate of glucose transport into
brain during anoxia when brain glucose levels are low.
The fundamental question as to whether or not energy is
required for BBB glucose transport remains unanswered. Although
it has generally been assumed that transpori5ff9m4~l~2d to brain
occurs by a facilitated diffusion mechanism ' , , , there is
little evidence to support this hypothesis. The usual argument in
favor of a facilitated diffusion mechanism is based on the obser-
vation that whole brain glucose t,vels are lower than the cor-
responding plasma glucose levels • This finding has been inter-
preted to mean that cerebral glucose transport does not occur
against a concentration gradient. 22he brain glucose level in the
isolated dog brain is 1.72 ~mole/g • It is not known how glucose
is compartmented within the brain, but it has been su§gested that
free glucose exists only in brain extracellular space and4~hat
this space comprises approximately 18% of the brain weight
This results in a calculated glucose concentration of 9.55 ~mole/ml
of extracellular water which is nearly twice the normal plasma
glucose concentration (5.55 ~mole/ml). Thus, it is conceivable
that the initial transport step is into a compartment with a high
glucose level and that brain glucose transport takes place against
a concentration gradient. Even if glucose transport were observed
to occur down a concentration gradient, an energy dependent process
could be involved in glucose translocation.
If the decrease in glucose transport during anoxia is due to
an energy dependent process, then it is difficult to explain why
the rate of transport does not continue to decrease after 15 min
2
of anoxia. Brain ATP levels decrease from 23% of normal a §er 10
min of anoxia to about 5% of normal after 30 min of anoxia . It
is possible that the glucose transport process is somehow closely
coupled to the small amount of energy that is generated during
anoxia, perhaps by a systj, similar to the bacterial PEP-dependent
phospho transferase system • Our observations could also be
explained by an alternative model which invo4ge~7two carriers, one
that is energy dependent and one that is not ' • However, if
two carriers were present, the transport data could not be des-
cribed by simple Michaelis-Menten kinetics and the resulting
double reciprocal plots would be nonlinear. There is no sign of
nonlinearity in anY4~f the control data that we have generated.
A recent study has shown that the number of mitochondria in
brain capillary endothelial cells is approximately five times that
of capillary endothelial cells from medialis muscle. Since the
capacity to catalyze oxidative phosphorylation is believed to
reside exclusively in the mitochondria, it would appear that
cerebral capillary cells have an enhanced ability to generate
A.L. BETZ, D.O. GILBOE, AND L.R. DREWES 141

energy. This additional energy may be necessary to support active

metabolite transport across the BBB.
Although primary (non-coupled) active transport of sugars
occurs in other organisms, no such system has as yet been a3mon-
strated in any mammalian species. Pardridge and Oldendorf have
recently shown that t¥e rate of cerebral glucose extraction is not
altered by reduced Na (10 or 70 mEq/l) or the presence of ouabain
(lor 10 rnM). Therefore, i~ is unlikely that glucose transport
into brain is coupled to Na transport as it is in intestine and
kidney.
A model for solute transport that has thus far been demonst-
rated only in nonmammalian systems, but which deserves conside3~­
tion here, is the chemiosmotic model as formulated by Mitchell .
In this theory it is postulated that solutes transported across
cell membranes are accompanied by protons. Electrochemical balance
is maintained by extruding these protons from the cell with energy
derived from electron transport, ATP hydrolysis or membrane poten-
tials (ion gradients). An interesting feat~oe of this proposal
which is supported by experimental evidence is that transport
should be low in cells in which the internal pH is lower than the
external pH. This is precisely the situation during cerebral
anoxia when the rate of unidirectional transport falls by 50% and
the tissue pH decreases to about 6.8, presumably due to lactic
acid formation. Although the chemiosmotic theory has, to our
knowledge, never been invoked to describe BBB transport, it is a
hypothesis that should be considered and tested.
In summary, several lines of inferential evidence indicate
that BBB glucose transport may be energy dependent, but there is
little conclusive evidence to support this theory.

COMPARISON WITH GLUCOSE TRANSPORT IN THE ERYTHROCYTE

From the foregoing discussion, it appears that glucose trans-

port across the BBB may occur by a mechanism which resembles the
facilitated diffusion system in the human erythrocyte. This
speculation led us to investigate other possible points of resemb-
lance between these two systems. Frequent reference is made in
the literature to the similarity between glucose transport in the
brain and in the red cell, but this comparison is based largely on
the presupposition that BBB glucose transport occurs by a facili-
tated diffusion mechanism. Other ~ore direct ex~d~~ce indicates
~hat b~t~ tYtnfgoY7 ~~s§Ims are Na -independent ' and insulin
lnsensltlve ' , , ,
Several investigators have studied the structure-ac~i~~t~5
relationships for glucose transport into the erythrocyte' , .
Less comprehensive studies of D-glucose transport from blood to
~ra~n.have.shown tht~ ~5v~Oal structural analogues of glucose also
lnhlblt thlS system ' , .
By kinetic analysis, 3-0-methyl-D-gl~cose was shown to be a
competitive inhibitor with a Ki of 1.8 rnM. If it is assumed that
142 A.L. BETZ. D.O. GILBOE. AND L.R. DREWES

Table 1
Effectiveness of Various Monosaccharides as Inhibitors
of Glucose Transport in Brain and Erythrocyte

Percent normal brain

glucose transport in Order of inhibitor
presence of inhibiror effectiveness in
erythrocyte
Inhibitor LeFevre Barnett Kahlenberg
et al et al
a-D-glucose 53
a,i3-D-glucose 58 2 2 1
2-deoxy-D-glucose 61 1 1 2
3-0-methyl-D-glucose 62 3 4
i3-D-glucose 63
D-galactose 67 4 8 5
D-mannose 69 3 4 3
1,5-anhydro-D-glucitol 77 7 9
D-xylose 80 5 6 6
D-fucose 85 8 5 7
5-thio-D-glucose 86 8
D-ribose 88 7 12
L-arabinose 90 6 9 10
i-inositol 91
L-fucose 94 9 11
sorbitol 97
L-glucose 100 10 13
D-fructose 101
0.9% NaCl (control) 100

Unidirectional glucose flux was determined in the presence of

the compounds listed. The average capillary glucose concentration
for this study was 4.94 + SE 0.06 mM. All inhibitor concentrations
were identical. For further details see Betz et al. 1975c.

other glucose analogues are also competitive inhibitors, then the

capacity of an analogue to inhibit glucose transport is directly
related to its affinity for the glucose carrier.
Eighteen glucose analogues (Table 1) were tested for their
ability tg inhibit glucose transport into the isolated, perfused
dog brain. Several preliminary conclusions about the structural
features required for the binding of a sugar to the glucose carrier
can be drawn. Inhibition of glucose transport by 1,5-anhydro-D-
glucitol and i-inositol and lack of inhibition by sorbitol suggest
that glucose is normally transported in the pyranose ring confor-
mation. It appears that no single hydroxyl group is absolutely
required for binding since elimination or epimerization at any
single asymmetric carbon does not abolish the ability of the
analogue to inhibit glucose transport. The hydroxyl on C-1
GLUCOSE TRANSPORT AND METABOLISM 143

probably is involved in binding since elimination (1,5-anhydro-D-

glucitol) or anomerization (a vs S) at this position decreases the
inhibitory potency. The hydroxyl on C-2 is not involved in bind-
ing since there is no change in the rate of transport when it is
eliminated (2-deoxy-D-glucose). However, epimerization from the
equatorial to the axial conformation (D-mannose) decreases binding
probably due to steric hindrance. Either addition of a methyl
group to the hydroxyl on C-3 (3-0-methyl-D-glucose) or epimeriza-
tion at C-4 (D-galactose) results in a decrease in the ability to
inhibit transport, but we cannot determine whether this is due to
steric hindrance or to interference with a hydrogen bond which is
normally present. The hydroxyl on C-6 is probably involved in
binding since elimination of the hydroxymethyl group on C-5 of
glucose (D-xylose) reduces inhibitory potency. In addition,
elimination of either the hydroxyl on C-6 of D-galactose (D-
fucose) or of the hydroxymethyl group on C-5 of D-galactose (L-
arabinose) causes a further decrease in the ability to inhibit
glucose transport. Finally, replacement of the oxygen of the
glucopyranose ring with an hydroxymethyl group (i-inositol) or
with sulfur (5-thio-D-glucose) results in compounds with low
affinity for the glucose carrier. This low affinity is probably
due to steric hindrance and/or absence of hydrogen bonding to the
substi tuted groups. Thus, the ring oxygen of D-glu'cose is re-
quired for optimal binding of glucose to the carrier.
These results resembLe studies of structure activity relation-
ships using the human erythrocyte (Table 1). However, one dif-
ference in the results is that the BBB glucose transport system
appears to have slightly higher affinity for the a-D-glu~ose
anomer while the erythrocyte system prefers the S-anomer .
Inhibition of glucose transport by the phenol, phloretin, and
its glucoside, phlorizin, has been studied extensively in several
tissues. Significant differences have been reported for inhib-
itory activities in erythrocytes and in kidney and intestine. It
was found that, in the erythrocyte, both compounds are competitive
inhibitors of glucose transport and ph~~r~§in is effective at
lower concentrations than is phlorizin ' . In t~e kidney and
intestine where glucose transport is coupled to Na transport,
phlorizinli~lseveral orders of magnitude more effective than
phloretin' . Furthermore, phlorizin is fully competitiv Z1 in
hamster small intestine, while phloretin is noncompetitive
In the isolated, perfused dog brain, both compounds are
competitive inhibitors of glucose transport across the BBB;
however, phloretin (apparent Ki = 0.29 ± .04 mM) is partially
competitive and inhibits at lower cogcentrations than does phlor-
izin (apparent Ki = 1.11 ± 0.12 mM) . In these studies no
conversion of phlorizin to phloretin was detected in the per-
fusate. Thus, the pattern of inhibition by these two compounds is
similar to the pattern observed in human erythrocytes but dissimi-
lar to the pattern observed in the intestine and kidney.
Cytochalasin B is a fungal metabolite that effects a wide
144 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES

variety of cellular processes. Recently, cytochalasin B was found

to be a potent noncompetitivel~n~~b~5or (Ki about 0.5 ~M) of
erythrocyte glucose transport ' , . Glucose transport from
blood to brain is also inhibited by cytochalasin B (unpublished
results). Available evidence indicates that the inhibition is
noncompetitive and the Ki, based on the whole blood cytocholasin B
level, is approximately 25 ~M. Inhibition is completely reversi-
ble within 4 minutes after exposure to cytochalasin B. This
similarity of inhibition patterns is additional supportive evi-
dence for the mechanistic and structural similarities between the
glucose transport systems in the erythrocyte and in the BBB.

PROPOSED MODEL FOR GLUCOSE TRANSPORT AT THE BLOOD BRAIN INTERFACE

A sketch of the proposed glucose binding site at the blood

brain interface has been constructed based on data from the study
of blood brain glucose trans po 48
inhibition and current concepts
of membrane structure (Fig. 5) . Membranes are composed mainly

outs ide inside

phenol bind ing site

hypothet ico l
cytocho losin 8
binding si te

Figure 5. Proposed model of the glucose transport site in the

cerebral capillary endothelium showing: A) the lipid bilayer and
the carrier protein and B) the cleft with sites for hydrogen
bonding of the Cl chair configuration of glucose, an adjacent
phenol binding site and a cytochalasin B binding site. Although a
cytochalasin B binding site is shown, its acutal location is
unknown.
GLUCOSE TRANSPORT AND METABOLISM 145

of lipid bilayers together with integral and peripheral proteins.

Membrane proteins which participate in transport are presumed to
be integral proteins that span the lipid bilayer (Fig. SA). The
glucose carrier is visualized as a membrane protein with a char-
acteristic binding site which is capable of specifically binding
D-glucose. Once the glucose molecule is bound, translocation to
the opposite side of the membrane can occur by a configurational
change in the membrane protein or by another suitable release
mechanism. It should be emphasized that this model is designed
only to depict the integration of carrier and binding site in the
membrane and not to imply the mechanism by which translocation
occurs.
At the glucose binding site (Fig. 5) the conformational
requirements resemblI, 4
in some wa s 33 those for the erythrocyte as
proposed by Alvarado , and others' . Evidence indicates that
the chair conformation of the a-D-glucose pyranose ring is the
preferred sugar configuration for binding to the glucose carrier.
The hydroxyls on carbons 1,3,4, and 6 as well as the ring oxygen
contribute significantly to the interaction between the solute and
protein. This interaction likely occurs via hydrogen bonding to
neighboring groups such as the imidazole of histidine, the E amino
of lysine, the B-hydroxyl of serine, the guanidinium of arginine
or the sulfhydryl of cysteins. Based on the inhibitory patterns
of phlorizin and phloretin, a hydrophobic binding site for phenols
probably exists in close proximity to the glucose binding site.
Cytochalasin B, which is bound at a more distant site, reduces the
apparent V but does not alter glucose binding. This is a
characteri~~tc of noncompetitive inhibition.

SUMMARY

The evidence suggests that glucose transport across the

blood brain barrier (BBB) in the dog is normally not a rate-
limiting step in cerebral metabolism; however, transport may
become rate-limiting under conditions of extreme hypoglycemia or
anoxia. Studies on the mechanism of glucose transport from
blood to brain do not at this time permit us to distinguish
between active transport and facilitated diffusion; however, a
decrease in the rate of unidirectional transport during anoxia
suggests that an energy-dependent process may be involved. In
spite of this evidence, glucose transport across the BBB is
similar to the facilitated diffusion of glucose into the red
cell in terms of the structural requirements of the glucose
molecule, the pattern of inhibition by phlorizin,+phloretin and
cytochalasin B, and the lack of sensitivity to Na or insulin.

ACKNOWLEDGEMENT

This research was supported by Grant NS05961 from the National

Institute of Neurological Diseases a nd Stroke.
146 A.L. BETZ, D.O. GILBOE, AND L.R. DREWES

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MECHANISMS FOR THE PASSIVE REGULATION OF EXTRACELLULAR K+ IN THE

CENTRAL NERVOUS SYSTEM: THE IMPLICATIONS OF INVERTEBRATE STUDIES

N. Joan Abbott and Y. Pichon*

Department of Physiology
King's College, Strand, London WC2R 2LS, U.K.
and
*Laboratoire de Neurobiologie Cellulaire
CNRS, 91190 Gif-Sur-Yvette, France

INTRODUCTION: K+ HOMEOSTASIS

Homeostasis of the brain extracellular K concentration is a

necessary prerequisite for neuronal functioning and synaptic
integration 3 ,7. There is considerable evidence for such regulation
in vertebrate brain, and when the interstitial K concentration is
maintained constant in the face of chronically altered K levels in
blood, an active, energy-dependent mechanism is suggested 7 • However,
it is not known to what extent the brain can maintain a degree of
internal homeostasis by purely passive 'K buffering' systems. In
this paper we shall examine the evidence for active and passive
regulation of K in the brain microenvironment and discuss some
results from a crustacean preparation which indicate that passive
K buffering may occur. Possible involvement of glia, and alternative
mechanisms, will be discussed. As astrocytes have been suggested
as a major site for K buffering in the vertebrate brain, evidence
for a homeostatic function of astrocytes will be considered.

THE FUNCTION OF ASTROCYTES

Neuroglial cells, which may make up 50% of the volume of

central nervous tissues, and outnumber neurones by 10 to 1, are
the source of considerable frustration to neurophysiologists. While
speculations about their functions 13 ,14 have existed since their
earliest histological recognition, very few of the proposed functions
have been amenable to experimental testing, and the role of astro-
cytes, in particular, is still far from clear.

151
152 N.J. ABBOTT AND Y. PICHON

INVERTEBRATE STUDIES

A significant advance was the investigation of an invertebrate

preparation A the segmental ganglion of the leech, by Kuffler and
co-workersl~,14. Aided by the large size and ready identification
of the glia in this preparation, accurate information about the
membrane properties could be obtained by electrophysiological
techniques, and the metabolic properties of the cells could be
studied. It was possible to show that the glial cells were
characterized by large resting potentials, high internal K concen-
tration, electrical coupling between adjacent cells, and a membrane
that behaved as a more or less perfect K electrode. Subsequently,
it was shown that stimulation of neurones caused depolarization of
adjacent glia 2 , due to potassium liberated during the neuronal
action potential.

VERTEBRATE STUDIES: GLIA AS SPATIAL BUFFER FOR K+

The electrophysiological technique was extended to amphibian

glia, studied in the optic nerve of the mudpuppy (Necturus), and
the frog 6 ,lS,2l, and very similar results were found. On the basis
of these studies, Kuffler and co-workers proposed a mechanism by
which the glia might act as 'spatial buffers' in the distribution
within the nervous system of K produced by localized regions of
neuronal activi ty13,2l. The necessary geometry is shown in Fig. 1.
If K accumulates in the cleft adjacent to surface A of the glial
cell, this membrane will tend to depolarize, and a small quantity
of K will enter the cell. If the glial cell is assumed to be
coupled to others electrotonically14, the membranes of the glial
network, B, distant from regions of neuronal activity, will face
extracellular clefts with normal K levels, and therefore will not
be depolarized. The resulting difference in potential between
the two extremities of the glial intracellular compartment will
cause K ions to diffuse into the glia adjacent to active neurones
and out in inactive regions, causing a circulation of current back
through the extracellular space. Thus, as long as the glial
membranes are depolarized asymmetrically, the glial compartment
can act as a K buffer, removing K from regions of high activity,
and distributing it at a distance, where it will be diluted by
the bulk interstitial fluid. This is proposed as one possible
mechanism by which local accumulation of extracellular K to
levels which would significantly interfere with neuronal activity,
is reduced. The neurones themselves are rather insensitive to K
over the range of K fluctuations normally encountered in vivo 14 ,
so the action potential mechanism is unlikely to be se~ously
affected, but synaptic regions are much more sensitive 3 •

Evidence that such a spatial buffer might be acting is

surprisingly difficult to produce, even in lower forms. In
mammalian brain, identified glia do not generally appear to behave
REGULATION OF EXTRACELLULAR K+ 153

Figure 1. Mechanism for 'spatial buffering' of K by glia

proposed by Kuffler and co-workers. Glial cells (g) are
electrotonically coupled by gap junctions (gj). a: active axon.

as perfect K electrodes 8,22 If other ions than K significantly

contribute to membrane conductance, or if an electrogenic component
is present 4 ,23,28 the ability of the glial membrane to buffer
extracellular K levels might be limited.

Indirect support for a passive glial buffer comes from the

study of Trachtenburg and Pollen 29 , who measured membrane properties
of mammalian glia in vivo, and claimed that the short time constant,
low membrane specific~istance and large length constant of the
(astrocytic) glia made it particularly suitable for removing K
passively and conveying it intracellularly to a distance. In
Necturus the potentials produced by the glial cells are capable of
initiating extracellular current flow 2l ; this would only be expected
if appreciable intracellular K redistribution was occurring. The
MHller cells (large astrocytes) of Necturus retina depolarize in
response to light, with a latency and time course consistent with
an effect mediated via K liberated from distal retinal neurones 19
The resulting radial current flow through the retina could only be
explained if some portion of the large MHller cell acted as source,
and another part acted as sink for the current, as required by the
Kuffler K redistribution hypothesis.

The recent introduction of K-selective microelectrodes 3l has

greatly improved the resolution and reliability of methods for
studying K regulation in the central nervous system. With this
technique it is possible to show that the interstitial steady state
K activity is equal to that of CSF, but can be elevated by electrical
or natural stimulation, from the normal £!. 3mM, up to 9 mM12 •
Under abnormal or pathological conditions such as anoxia, epileptic
seizure~ or spreading depression, the activity may rise as high as
60 mM27 ,30. When stimulation is halted in normal preparations, a
154 N.J. ABBOTT AND Y. PICHON

frequent finding is that the extracellular K level recovers not

simply to resting level, but to less !h!ll the resting level 12 .
This underswing cannot be explained by any purely passive mechanism,
and thus must be due to active removal of K from the extracellular
space. Possible sites for such removal are the choroid plexus and
brain capillaries 7 , glia 9 ,10,24 and neurones 12 ,23, While evidence
can be presented for the ability of each of these sites to remove
K from the extracellular space, the relative contributions of each,
and the respective time courses of the removal processes, have not
been established.

An active compo~ent to K removal is clearly present during

periods of heightened activity such as found with elyctrical
stimulation, and in regions of high synaptic density 2. However,
it is not clear to what extent less active regions of the mammalian
nervous system and the brains of lower forms rely on energy-dependent
K removal processes. It is interesting that in the amphibian optic
nerve, the recovery of the glial membrane potential following maximal
stimulation of the nerve was found to be slower at low temperatures,
with a QlO calculated as 1.56 ± 0.17 5 , compared with an expected Q 0
for metabolic-dependent processes of at least 2 26. It was concluled
that passive diffusional processes were responsible for redistribution
of the extracellular K. As the 'spatial buffering' by glia is likely
to be a simple diffusional process, it could presumably contribute
to such a redistribution, but it could not be distinguished in this
study from K dissipation via the extracellular clefts. A possible
contradictory observation is that NADH levels fall when amphibian
optic nerve glia is stimulated by raised K levels 20 ; while this
could be due to a non-specific increase in glial metabolism induced
by elevated K, it could also indicate the presence of a K-stimulated
Na-K-ATPase, which could be involved in active removal of extra-
cellular K 17. Thus extracellular diffusion, passive glial K
buffering, and active K uptake may all contribute to K redistribution
in the amphibian system.

In the leech ganglion, an energy-dependent electrogenic process

has been shown to be involved in the hyperpolarization of the neuronal
membrane following activity3. There is no information about the
contribution of active vs. passive mechanisms to K redistribution
in this species, although early work suggested that passive diffusion
via the extracellular clefts could explain the observed time course
of K entry and efflux 14 • Hertz has shown that the K-stimulated
increase in oxygen uptake, which is correlated with activation of
a Na-K-ATPase in other neural tissues, is absent in leech ganglia
(L. Hertz, pers. comm.). It might then be expected that active
processes would be relatively unimportant in this preparation.

In a very elegant study in cat spinal cord, Kriz et al~12

showed that an under swing of the interstitial K concentration after
activity was observed only when the K-sensitive microelectrode was
REGULATION OF EXTRACELLULAR K+ 155

in the deeper parts of the cord, close to synaptic regions, although

large positive K alterations could be detected more peripherally.
They interpreted the association of the underswing with neurones as
evidence that active mechanisms for K removal are located on neuronal
membranes, and not associated with the glia, which is presumably
present at all depths in the cord. An alternative possibility is
that glial Na-K-ATPase activity is not uniformly present throughout
the spinal cord, but is concentrated in regions of neuronal density;
Hertz noted lO that K-stimulated oxygen uptake, presumed to be glial,
was greater in the cortex than in the spinal cord. This could
indicate that in regions of the spinal cord with low synaptic density,
passive mechanisms for K redistribution, including glial K buffering,
predominate. There is certainly evidence that the glia may contri-
bute to sustained potentials recorded in spinal cord 25 , implying
that circulation of current due to glial K distribution is present.
However, in more active neuronal and synaptic regions such as
cerebral cortex, there are indications that the time course of K
dispersion after local accumulation in the extracellular space, is
not compatible with passive entry into the glia lS •

Thus, although the idea of a passive glial K buffer has been

espoused by many workers, there is still little evidence to
support such a buffer as a physiological reality. The difficulty
arises because it is generally impossible to distinguish passive K
redistribution attributable to diffusion along extracellular clefts
from diffusion through cell membranes, even in conditions where
active mechanisms are thought to be unimportant. For this reason,
some recent observations in a crustacean central nervous system may
be interesting because they provide direct evidence for a passive
K buffering system, which could be due to glial K redistribution.

CRUSTACEAN STUDIES

The circumoesophageal connectives of the crayfish (Procambarus

clarkii) central nervous system, are convenient preparations for
application of an electrophysiological technique to the study of
ion movements, as they contain giant axons whose membrane potential
and action potential response to bathing K and Na concentrations is
known l • The time course of response of the membrane potential
when the solution bathing the intact connective is switched from
normal Ringer to a test solution containing altered K or Na levels,
can be used to calculate the half-times for K and Na entry and
efflux from the axon surface l •

Values obtained in intact preparations, and in preparations

from which the layer of outer glia, the perineurium, has been
removed (desheathed) are shown in Table 1. It is seen that K reaches
the giant axon very slowly, but K removal is rapid; the time course
of Na entry and efflux are faster, but here there is a slight
156 N.J. ABBOTT AND Y. PICHON

asymmetry in the opposite direction. Desheathing speeds up both

entry and efflux of K, especially the former.

TABLE 1

Half-times (TO. 5 ) for K+ and Na+ movements between bathing

medium and extra-axonal space of crayfish central nervous
system connective.

_____________________________
Preparation Ion T,0.5 ______________
(mins) n __

Intact K Entry 142.5 + 9.6 113

Efflux 6.0 + 0.4 113

Desheathed K Entry 4.1 ± 0.7 5

Efflux 1.5 ± 0.1 5

Intact Na Entry 2.9 + 0.9 3

Efflux 9.3 + 3.0 3

There are several models of the system which could explain

the observations:
iL A peripheral diffusion barrier (e.g. zonulae occludentes
between outer perineurial glial cells) impedes ionic movements,
and associated Na/K transport systems acting to pump Na in and
K out across the perineurial epithelium, cause the asymmetry
between K entry and efflux, Na entry and efflux.
21 No tight diffusion barrier is present, but a restricted
extracellular cleft and long diffusion pathway produce the
same net result, sufficiently impeding ionic movements so
that an active Na/K pump can control ionic entry and efflux,
as in a).
£1 Active mechanisms are unimportant, but passive mechanisms
are present which act as K buffers or reservoirs, slowing the
build-up of K in the extra-axonal space during entry, and
speeding K removal from the axon surface during efflux.

Electronmicroscopic investigation of the connective 16 suggests

that zonulae occludentes are probably absent in the outer perineurial
zone, a finding consistent with the observation that peroxidase entry
via an extracellular pathway, while slow, is demonstrable ll • The
extracellular clefts in the perineurium and other other layers of
glia are, however, tortuous, and frequently restricted by gap
junctions. Deeper in the connective, large extracellular spaces
are present, which appear not to be artefactual, and contain dense
deposits of extracellular material. Gap junctions between glia
REGULATION OF EXTRACELLULAR K+ 157

are frequent in outer layers, and are occasionally seen in deeper

zcnes. 16

These observations suggest that model a) above is ruled out,

but are compatible with models b) and c), the main difference
between these models being that active mechanisms are necessary for
b) but not for c). The effect of specific inhibitors of Na-K-ATPase,
strophanthidin and ethacrynic acid, on the time course of K movements,
was therefore investigated. If an active process is responsible for
the slow K entry (and rapid K efflux), inhibitors should speed entry
and slow efflux. Fig. 2 shows that neither strophanthidin (0.2 mM)
nor ethacrynic acid (0.3 mM) had any marked effect on rate or extent
of K entry and efflux, showing that active mechanisms are unlikely
to be important in controlling K distribution. Interestingly, 3 M
urea, which is known to uncouple occluding junctions by causing cell
shrinkage, did result in a greater rate of K entry and efflux.
Thus of the models suggested above, only c) appears able to explain
observed K and Na movements in the crayfish connective. Possible
types of passive system envisaged are:
(i) ion reservoir on extracellular matrix, likely to
be mucopolysaccharide 16 ;
(ii) intracellular ion binding with high affinity for K;
(iii) passive K buffer due to glia.

At first sight, it would seem difficult to explain the difference

in behaviour of Na and K without supposing that a system is present
which can distinguish between these cations - a property unlikely
for an extracellular ion-exchange matrix (i). However, the very
slow K entry could be due to the need for K to displace Na ions
from the matrix before reaching the axon, and fast K efflux could
be due to rapid exchange of K from the axon surface with residual
Na on the matrix (Fig. 3a). Na-free experiments from which Na +
kinetics were calculated, were performed with a medium in which Tris
was substituted for Na+; the relatively large size of the former
cation could lead to its effective exclusion from a dense extra-
cellular matrix, causing the partial retention of Na in the matrix,
even under Na-free conditions (Fig. 3b). This could explain the
apparent slowness of Na efflux, and rapidity of Na re-entry to the
axon surface on return to normal Ringer. Thus although the kinetics
of such a theoretical system need to be investigated in detail, a
passive extracellular reservoir would appear to be able to account
for the observed differences between Na and K movements.

As cell membranes, particularly glial, are readily able to

distinguish between Na and K ions, an intracellular reservoir due
to cation binding (ii), which could be selective for K, is
theoretically possible. Such a system could explain slow K entry
(adsorbed onto intracellular sites, possibly in exchange for Na)
and fast K efflux from the axon surface; Na entry and efflux would
158 N.J. ABBOTT AND Y. PICHON

a 13

mM --control
.... .... ethlcrynlc acid
- - - strophanthldln
It
_.- 3 M urea

2 8 10 14 18
min

b entry efflux
0 0

In ( K,.!C..)
\ ",
' .,,,,,".,,".".
1Io·!C..
\ " ';;''.;,; ''
-(}02 \
\
-1 , "
' ', .' .....
"'.

\ " "-,
"............
\
i
i -2
"
,'. ' "- "-
,.

"'-.,
-()-Q4
\ "-
"-
"
"'i \
-(}08
\
-3
"'\,
\
\
\
0 2 2 e 10
min '"
Figure 2a. Effect of 2 min 100 mM K Ringer pulse (horizontal
bar) on K concentration at the axon surface (K)a' in control and
after treatment with inhibitors or urea. S04 replaced Cl in the
K-Ringer. Results with KCl-Ringer were comparable.

Figure 2b. Curves from Fig. 2a replotted semilogarithmically,

to show the effect of inhibitors on rate (slope of each line) of K
entry and efflux.
REGULATION OF EXTRACELLULAR K+ 159

Medium
(a)

(b)

Tris
CI

Figure 3. Proposed influence of negatively-charged extra-

cellular matrix within the eNS on distribution and movement
of ions under various experimental conditions: (a) Replace-
ment of NaCl-Ringer by KCl-Ringer, and return to NaCl-Ringer;
(b) Replacement of NaCl-Ringer by Tris Cl-Ringer, and return
to NaCl-Ringer. pn: perineurium, outer layer of glia.

be unaffected by binding, as Na would be largely confined to the

extracellular space. However, rates of Na movement into and out
of the system might be expected to be more symmetrical than observed,
unless an extracellular matrix, or restriction of the extracellular
space, causes discrimination between Tris and Na. The properties
or location of the hypothetical intracellular binding sites are
unknown, and might be difficult to establish experimentally.

A third possibility is that the glia acts as a passive 'spatial

buffer' for K, slowing the build-up of K in the extra-axonal space
during entry, and speeding K removal from the axon surface during
efflux. Na, being extracellular, would not be buffered in the
same way, so would show relatively unimpeded entry and efflux
kinetics. Fig. 4 shows how this system might function. When
external K concentration is raised, the tortuous extracellular cleft
and gap junctions of the perineurium slow K entry to the extra-
cellular space; meanwhile K at the outer perineurium membrane A will
160 N.J. ABBOTT AND Y. PICHON

Figure 4. Mechanism for redistribution of K within the CNS by glia

acting as 'spatial buffer'. Electrotonic coupling between glia is
assumed. pn: perineurium; g: glia; a: axon.

60
mV

40

20

O+---f-"""T""__l""t'"rT'I'r---r-""""'"
20

Figure 5. Dependence of transient 'trans-perineurial' potential (mV)

on K concentration in the external medium (K). Results with KCl.
K2S0 4 results were comparable. Dotted line: gheoretical Nernst
relation.
REGULATION OF EXTRACELLULAR K.J. 161

cause depolarization. Because of the geometry of the glial network,

electrotonically coupled by gap junctions, K will tend to enter the
glia at A, and leave elsewhere in the system, e.g. B. In the
isolated preparation used in these experiments, only a small portion
of the connective surface was exposed to the test solution l , allow-
ing translocation of K laterally via the glia, and eventual dilution
by the bulk extracellular fluid in regions outside the test compart-
ment. The net result is that build-up of K at the axon surface C
is slower than would be expected if K movement was confined to the
extracellular cleft. On return to normal Ringer, the situation
is reversed, and B is now relatively depolarized compared with A.
K will tend to move in at B, out at A, so will be relatively rapidly
removed from the regions adjacent to the axons. Na entry and efflux
are assumed to be predominantly via the extracellular cleft. De-
sheathing (which removes the perineurial glial layer) or treatment
with 3M urea, disrupts the geometry of the spatial buffer compart-
ment, and allows rapid penetration of K into the extracellular
space, and hence to the axon surface.

The behaviour of such a spatial buffer depends on:

(i) asymmetry of depolarization of glial membranes, due to
diffusional restriction in the system so that not all points
on the glial membranes depolarize simultaneously;
(ii) selectivity of the glial membrane for K. An appreciable
permeability to other ions such as Na, Cl, would reduce the
efficiency of the K distribution system.

The anatomy of the glial system, and the partial diffusional

restriction provided by the perineurium would seem to ensure that
condition (i) is met. The selectivity (ii) of the outer perineurial
membrane can be estimated by studying the transient potential
generated across the perineurium by sudden changes in external
potassium concentration l • For a perfect K electrode, the transient
potential should show a dependence on log (KJ with a slope of 58 mV
(Nernst). Fig. 5 shows results obtained withOKCl in a series of
experiments. While the average slope is ca. 52 mV, preparations
have been obtained which respond to 100 ~K with the predicted
Nernst potential, suggesting that in undamaged preparations the
membrane behaves as a more or less perfect K electrode. Preliminary
experiments suggest that the response is little affected by the
substitution of S04 for Cl in the test solution. These observations
confirm the finding in other groups, that glial membrane shows a
high selectivity for K, and suggest that the second requirement ii)
above, is met in the crustacean system.

CONCLUSION

It can be concluded that the crayfish central nervous system

shows evidence of passive mechanisms for regulating K distribution,
 162 N.J. ABBOTT AND Y. PICHON

owing to intracellular or extracellular buffering systems. 'Spatial

buffering' of K by the glial network, as suggested by Kuff1er and
co-workers 21 is one possibility, but intracellular or extracellular
ion-binding need also to be considered. It is interesting that the
crayfish system appears not only potentially able to buffer K
changes produced by neuronal activity, but also those caused by
fluctuations in ionic concentrations in blood, at least over short
periods. Such a mechanism may thus represent a rudimentary form
of blood-brain barrier, as well as a primitive method for homeostasis
of the brain microenvironment.

This work was supported by a Research Grant from the Science

Research Council, and travel grants from the Royal Society and
the We11come Trust.

REFERENCES

1. Abbott, N.J., Moreton, R.B., and Pichon, Y., E1ectrophysio10gica1

analysis of potassium and sodium movements in crustacean nervous
system, J. expo Bio1., 63 (1975) 85-115.
2. Baylor, D.A., and Nicholls, J.G., Changes in extracellular
potassium concentration produced by neuronal activity in the
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3. Baylor, D.A., and Nicholls, J.G., After-effects of nerve impulses
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5. Bracho, H., and Orkand, R.K., Neuron-g1ia interaction: dependence
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REGULATION OF EXTRACELLULAR K+ 163

11. Kristensson, K., Str8mberg,E., Elofsson, R., and Olsson, Y.,

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a potassium mediated effect of neuronal activity on the glial
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cells. Ergebn, Physio1., 57 (1966) 1-90.
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properties of glial cells in the central nervous system of
Amphibia. J. Neurophysiol., 29 (1966) 768-787.
16. Lane, N.J., and Abbott, N.J., The organization of the nervous
system in the crayfish Procambarus clarkii with emphasis on the
blood-brain interface, Cell and Tissue Res., 156 (1975) 173-187.
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change during hippocampal electrical stimulation, J. Neuro~
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alterations by potassium levels comparable to those during neural
activity, Brain Res., 55 (1973) 467-471.
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nerve impulses on the membrane potential of glial cells in the
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788-806.
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cortex to increased extracellular potassium, Brain Res., 38
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23. Ransom, B.R., and Goldring, S., Slow hyperpolarization in cells
presumed to be glia in cerebral cortex of cat, J. Neurophysiol.,
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Academic Press, New York and London, 1967.
164 N.J. ABBOTT AND Y. PICHON

27. Sugaya, E., Takato, M., and Noda, Y., Neuronal and glial
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Vyskoc~l, F., Kr~z, N., an
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AMINO ACID TRANSPORT IN SPINAL AND SYMPATHETIC GANGLIA

Peter J. Roberts

Department of Physiology and Biochemistry

University of Southampton
Southampton, S09 3TU England

I. INTRODUCTION

It has long been considered that, in common with monoamine

neurotransmitters, the postsynaptic actions of the amino acids
glycine, GABA, taurine, glutamate, aspartate, and proline are term-
inated by their rapid removal from the vicinity of the receptors by
specific transport processes associated with the presynaptic nerve
terminals. In tissue slices or synaptosomes, these transport pro-
cesses possess a prominent high affinity component, which is
absolutely dependent on the presence of extracellular Na+. Although
other, non-transmitter amino acids appear also to be transported by
systems of both low and high affinities 8 , their high-affinity
components do not share the same requirement for sodium; neither are
these systems localized preferentially in those areas of the eNS
utilizing these substances as transmitters. Evidence from non-
equilibrium density gradient centrifugation studies 8 and electron
microscopic autoradiography18 has demonstrated that accumulation is
primarily into the nerve terminal regions of tissue preparations.
However, it has been argued on the basis of the compartmentation of
amino acid metabolism 6 ,7 that the glial cell is likely to be of
major importance in transmitter inactivation and, in vivo, the auto-
radiographic evidence 20 ,26 supports this suggestio~ ~umber of
preparations, including physically separated glial elements (see
Haber, this book) and the isolated retina (see Neal, this book),
have been classically utilized to demonstrate that glial cells will
specifically accumulate a variety of neurotransmitters.

Two other extremely useful preparations for the study of

neuronal and glial function are the sensory, dorsal root ganglion
165
166 P.J. ROBERTS

and the autonomic, superior cervical ganglion.

II. STRUCTURE AND FUNCTIONS

A. Sensory Ganglia. The isolated, but structurally intact,

dorsal root ganglion is highly suitable for studying the transport
of amino acids and for the elucidation of the glia-neurone relation-
ship. The chief attributes of this system are:
1. Dorsal root ganglia are morphologically discrete and easily
dissected from the animal in adequate quantities.
2. The ganglion possesses a relatively simple structure:
(a) There is little evidence for the existence of
synapses within the ganglion.
(b) The neuronal cell bodies are morphologically identical,
and all probably utilize the same sensory transmitter
substance.
(c) Each of the neuronal cell bodies is surrounded by a
number of satellite glial cells, providing a sheath around
the neurone.
(d) Each neurone and its satellite glial cells are divided
from others by connective tissue; hence each neurone and
its glia form a functional and structural unit.

B. Superior Cervical Ganglia. This sympathetic ganglion differs

from dorsal root ganglia in several major respects: preganglionic
fibres release acetylcholine onto postganglionic cell bodies, which
utilize noradrenaline as a transmitter substance. A functional role
of catecholamines within the ganglion possibly associated with
inhibitory dopaminergic interneurones~l has often been suggested,
and following release, dopamine may activate cyclic AMP formation
in the glia 17 •

Morphology and size of the neurones in the superior cervical

ganglion exhibit considerable variation as compared with the dorsal
root ganglion. However, the cell bodies are nearly always closely
ensheathed by satellite glial cells, as in the dorsal root ganglion,
and Schwann cells are abundant around the many unmyelinated fibres.
The cells which comprise the neural satellites have long been
considered as the peripheral counterparts of CNS oligodendrocytes,
although their morphology is rather dissimilar. It appears unlikely
that astrocytes occur within the ganglion.

III. THE NEURONE - GLIAL FUNCTIONAL UNIT IN SUPERIOR CERVICAL, AND

DORSAL ROOT GANGLIA

It has been suggested 16 that the glial cell package which envelops
synaptic regions in the brain and spinal cord, and also the cell
bodies of ganglia, is present to regulate the substances reaching
the neuronal membrane; such substances could include neurotrans-
mitters, K+, and blood-borne, neuroactive materials. The release
AMINO ACID TRANSPORT IN GANGLIA 167

of neuroactive materials from glia might exert a modulatory role

on neuronal excitability. Alternatively, the glia might serve as
a reservoir for, say, 5-carbon units which could be translocated
to the neurones for replenishing depleted transmitter stores.
Classically, the glia has been presumed to have a trophic function,
supporting the neurones and performing specialized functions such
as myelination of the axons.

IV. UPTAKE OF PUTATIVE AMINO ACID TRANSMITTERS

A. Tissue: Medium Ratios. The connective tissue sheath around

the ganglion provides an effective barrier to the uptake of amino
acids. For example, comparison of deshea5hed dorsal root ganglia
with ganglia where the sheath was intact 3 showed that the rate of
uptake of 3.5~ l4C-glutamate was markedly less in the latter
preparation. The rate of accumulation of all amino acids, investigat-
ed in both superior cervical lO and dorsal root ganglia preparations
30,33, was linear for several hours and relatively slow as compared
with slices and synaptosomes. However, the uptake seen with GABA,
glutamate, and glutamine was substantially greater than that of
other amino acids, such as glycine, a-alanine, and lysine (Table 1).

TABLE 1
UPTAKE OF AMINO ACIDS INTO RAT DORSAL ROOT GANGLIA
Amino Acid Tissue: Medium Ratio
0.5 hr 4 hr

GABA 4.63 ± 0.19 26.84 ± 0.31

GABA + 10~ AOAA 8.01 ± 0.64 92.36 ± 0.98
Glutamate 7.97± 0.54 70.30 ± 0.66
Glutamine 5.89 + 0.69 60.22 ± 0.73
Lysine 2.01± 0.54 22.57 ± 0.80
Glycine 1.84 ± 0.72 12.98 ± 0.83
AOAA = amino oxyacetic acid.

Ganglia were isolated and incubated in pairs for 30 min at 37 0 C

in a.5ml Krebs bicarbonate medium containing labelled amino acid
(0.1 ~). Incubations were stopped by removing the ganglia, which,
after blotting and weighing, were solubilized with 400 ~l 'Protosol'
and assayed for total radioactivity. T: M values + S.E.M. of 6
observations (values corrected for extracellular (i~ulin) space of
0.481 ml/g tissue at 30 min - not significantly increased at 4 hr).

In the absence of AOAA, the tissue: medium ratio attained by

4 hr for 3H-GABA was approximately 27 as compared with 92 in the
presence of the GABA transaminase inhibitor. A similar enhancement
of GABA uptake was seen in ganglia from AOAA - treated rats 34 • Thus,
the apparently low T : M value found in the absence of AOAA may be
168 P.J. ROBERTS

attributable to leakage of metabolites into the medium. Similarly,

glutamate was rapidly metabolized following accumulation (after a
30 min incubation, only 35% of the original radioactivity was still
in glutamate). Hence, where there is a likelihood of the formation
of labelled products which are not retained by the tissue, measure-
ment of total radioactivity accumulated inevitably underestimates
uptake. Although glutamine was taken up to a significant extent,
metabolism was slight, with 75% of the activity remaining as
glutamine after 30 min.

Experiments with superior cervical ganglia incubated with 0.4 ~

GABA in the presence of AOAA have similarly demonstrated a substantial
uptake, yielding T:M ratios of 60-70 after 4-6 hr incubation. More
than 90% of the tissue radioactivity was attributable to unchanged
GABAlO •

B. Effect of Dorsal Root Section. Transection of the efferent

dorsal root was found to produce no significant change in 3H-GABA
uptake into ganglia removed 1 month after the lesion33 • Neither
was the uptake of GABA or glutamate affected following dorsal root
section central to the ganglion. Similarly, preganglionic denervat-
ion of the superior cervical ganglion did not reduce GABA uptake,
indicating that there was no preferential uptake into presynaptic
elements.

c. Metabolic Requirements for Uptake. The uptake of GABA,

glutamate, and glutamine (the 3 amino acids most aVidly accumulated
by dorsal root ganglia) at a concentration of 1 ~ was markedly
inhibited by reducing the temperature of incubation: at OoC uptake
was"< 5% of that at 37 0 C for each of the amino acids. Metabolic
inhibitors such as ouabain, 2 ,4-dinitrophenol , p-hydroxymercuribenzo-
ate, and p-chloromercuriphenyl-sulphonate (500 ~) were also effective
in reducing rate of uptake. These data are therefore consistent
with there being an active, energy-dependent uptake process for
each of the amino acids.

D. Kinetic Characteristics of Amino Acid Uptake. The kinetics

of the uptake of several amino acids into dorsal root and superior
cervical ganglia have been investigated lO ,30,34. Least squares
analysis of data obtained with concentrations of labelled GABA,
glutamate, and glutamine in the range 0.1-1000 ~ indicated that
two distinct uptake systems could be resolved. One system W~! of
low affinity with a relatively high capacity (~100 nmol/min g-l
for each amino acid) and apparent Km's of approximately 1000 ~;
this was probably analogous to the typical low affinity systems
found in other nervous tissues 9 . The other system was one of high
affinity (Km'S of 50 ~ or less) and of low capacity (Table II).

E. Na+ Dependence of High Affinity Uptake. Absolute dependence

on the presence of external sodium ions for high affinity uptake
AMINO ACID TRANSPORT IN GANGLIA 169

into synaptosomes has been cited 8 as an important criterion for

establishing a substance's candidacy as a neurotransmitter at a
particular site. Dorsal root ganglia incubated with each of the
amino acids in Table II showed a great sensitivity to Na+ in the
medium. When the sodium content of the Krebs medium was replaced
by 50 mM Tris buffer (pH 7.4) and 230 mM sucrose, uptake was
inhibited by more than 80%.
TABLE II
KINETIC CHARACTERISTICS OF GANGLIONIC AMINO ACID ACCUMULATION
-1 -1
Amino Acid Tissue K (~) V (nmol/min g )
m max
GABA SCG l 7.0 0.20
DRG 9.7 2.68
Glutamate DRG 20.6 5.90
Glutamine DRG 50.1 11. 73

lSuperior cervical ganglia data from Bowery and Brown 1972 10 ;

dorsal root ganglia were incubated with labelled amino acid
for 20 min, as described in the legend to Table I, and total
accumulated radioactivity was determined.

Thus in many respects the properties of GABA and glutamate

uptake into sensory and sympathetic ganglia closely resemble those
described in brain slices and synaptosomes; in every case uptake
possesses a high affinity component, which exhibits saturation
kinetics and is sodium-dependent. The uptake of glutamine into
ganglia is somewhat anomalous, since synaptosomes accumulate
glutamine by a low affinity, high capacity system only29 and, follow-
ing uptake, rapidly convert the glutamine to glutamate. A high
affinity, sodium-dependent uptake system for glutamine has also
been detected in spinal nerve roots 29 and in rat brain slices 3 •
The function of this system is obscure, since glutamine is without
effect on the firing of neurones. However, it is feasible that the
high affinity 'uptake' is an artifact derived from homoexchange of
extracellular labelled glutamine with the large glutamine pool within
the satellite glial cells. Preliminary autoradiographic data
(unpublished) support the possibility of homoexchange.

The essential difference between the high affinity uptake systems

for GABA and glutamate in ganglia and those in nerve endings is the
rate of accumulation; the very low Vmax values found in ganglia may
indicate a sparsity of uptake sites.

V. LOCALIZATION OF AMINO ACIDS

A. Autoradiographic Analysis. Following incubation of dorsal

root ganglia with 5~ 3H-GABA or 1 ~ 3H-glutamate, light and EM -
autoradiography33 demonstrated an exclusive localization of radio-
170 P.J. ROBERTS

activity over the satellite glial cells. With GABA, the presence
of AOAA in the medium greatly enhanced the silver grain density.
Similar 3results were obtained with desheathed superior cervical
ganglia 6; there was heavy labelling over the satellite cells and
especially over the Schwann cells around unmyelinated fibres.
Incubation of dorsal root ganglia 34 with a high (300 ~) concentrat-
ion of 3H-GABA, i.e. with the low affinity uptake system predominat-
ing, a large number of silver grains was observed over the neuronal
cell bodies and connective tissue sheath in addition to over the
glia. When ganglia were incubated with 3H-GABA in the absence of
sodium, the extent of labelling was minimal, with no selective
localization. Investigations with tritiated glycine, a-alanine,and
leucine under the same conditions as for GABA, revealed an equal
localization over the neuronal cell bodies and satellite glial cells.

B. Compartmentation analysis. There is extensive evidence in

favour of compartmentation of glutamate (and hence of two of its
products, GABA and glutamine) within nervous tissue l ,6,7, and
studies with radiolabelled precursors indicated that the major
part of glutamine derived from glutamate, acetate, propionate, and
butyrate lies in the glia. On the other hand, glucose or pyruvate
label a large pool of glutamate that is presumed to lie within the
neurones and which is not associated with glutamine synthesis to
any degree.

When the incorporation of radioactivity into several amino acids

of dorsal root ganglia was investigated by incubation with uniformly
labelled 14C-glutamate, pyruvate, or acetate, a steady increase in
incorporation was observed over a period of 80 min. However, the
pattern of labelling was not the same for the three precursors
(Table III).

The results demonstrated that both 14C-glutamate and 14C-acetate

were incorporated into a small (probably glial) pool of glutamate,
which was rapidly converted to glutamine, yielding specific radio-
activities relative to glutamate of approximately 3. Glutamate and
acetate were more effective precursors for GABA than was pyruvate.
Pyruvate, on the other hand, was a far less effective precursor for
glutamine, whose R.S.A. remained lower than unity even at 60 min.
Thus pyruvate appeared to be incorporated extensively into a large
(neuronal) glutamate pool. Autoradiographic support for these
conclusions has recently been forthcoming 23 in that with glucose,
radioactivity was localized to a greater extent over the neuronal
cell bodies, while with acetate the labelling was predominantly
over the satellite glial cells. Hence the autoradiographic data
obtained with 3H-GABA and 3H-glutamate, concur with the biochemical
evidence in firmly ascribing an exclusively glial localization of
the uptake sites.
AMINO ACID TRANSPORT IN GANGLIA 171

TABLE III
DISTRIBUTION OF AMINO ACID RADIOACTIVITY DERIVED FROM l4C-GLUTAMATE
(5 ~), l4C-PYRUVATE (450 ~), AND l4C-ACETATE (80 ~).
l4 C_glu l4 C_pyr 14C-ac
Amino Acid Endogenous
conc. -1
(IJIIlO 1. g ) S.A. R.S.A. S.A. R.S.A. S.A. R.S.A.

Aspartate 0.80 2427 0.40 2402 1.11 1659 0.54

Glutamate 2.22 6064 (1.0) 2164 (1.0) 3073 (1.0)
Glutamine 1.34 18708 3.08 1926 0.89 8727 2.84
Alanine 0.64 1102 0.18 1829 0.85 1076 0.35
GABA 0.04 6731 1.11 1385 0.64 2090 0.68
Incubations were for 30 min.
S.A. = specific radioactivity of each amino acid (dpm/nmol)
R.S.A. = specific radioactivity of each amino acid relative to
the absolute specific radioactivity of glu in that tissue, taken
arbitrarily as = 1.0. Data from Roberts and Keen 30 •

VI. DRUG INHIBITION STUDIES

A large number of compounds have been screened in several

investigations into the structural requirements for the high affinity
uptake of GABA5,19 and glutamate 2 ,3 in rat cortical slices. Since
specific inhibitors would be of value in elucidating the physiological
role of these amino acids, or possibly to be utilized as 'false
transmitters', it would be useful to distinguish between the glial
and neuronal uptake processes, if indeed there were any qualitative
differences.

A. Uptake of GABA. GABA uptake into neither dorsal root nor

superior cervical ganglia was inhibited by a large molar excess of
glycine, glutamate, or glutamine; although labelled taurine and
~-a1anine were accumulated to ari extent comparable to that of 3H-
GABA by superior cervical gang1ia lO • In general, most compounds
were equipotent inhibitors of 3H-GABA uptake into both ganglia and
cortical slices. GABA receptor blockers such as bicucu1line and
picrotoxin were inactive, while guanidino propionic acid, 8-amino-
va1eric acid, 2-fluoro GABA and 3-hydroxy GABA were approximately
equally effective in ganglia and slices. Two compounds in particular,
however, ~-a1anine and L-2,4-diaminobutyric acid (DAB), showed
markedly different inhibition properties in both systems (Table IV).
~-A1anine was essentially without effect on the uptake of GABA by
small slices, where the accumulation occurs primarily into nerve
terminals. It was, however, a potent competitive antagonist of the
uptake into glial cells of ganglia. In contrast, DAB was a potent
(non competitive) inhibitor of3H-GABA uptake into nerve terminals,
but was rather less effective as an inhibitor of the glial uptake.
172 P.J. ROBERTS

TABLE IV
IC sO VALUES FOR THE INHIBITION OF GABA UPTAKE INTO CORTICAL SLICES
AND GANGLIA.

Compound IC501__________~_
Cortex Ganglia
700-J~
L-2,4-diaminobutyric acid 50~'~
l20-J~
!3-alanine
-J~ from Schon and Kelly34 19
** from Iversen and Johnston
IC sO value is the concn. of inhibitor required to produce a 50%
inhibition of the uptake of 0.5 ~ 3H-GABA after a 10 min incubation .
Tissues pre incubated with the inhibitor for 10 min.

3 H-!3-alanine itself was accumulated exclusively by glial

elements of both ganglia and cortical slices 35 with almost identical
kinetic systems. Uptake was enhanced by AOAA, since !3-alanine is a
substrate for GABA transaminase, and the process was strongly sodium·-
dependent. GABA, which is a more effective substrate for the glial
uptake system, competitively antagonized the accumulation of f3-alanine
(Ki = 36 ~ in slices). These results show that a high affinity
glial uptake coexists with the GABA uptake system (of greater
capacity) of nerve terminals. Thus it would seem that in studies
where the time of incubation is very short, the uptake of GABA
measured essentially represents neuronal accumulation. However, the
situation might be rather different in longer incubations, even when
using synaptosomal preparations, where there may be Significant
contamination with glial fragments ('gliosomes,)12. Since DAB is
selective for the neuronal uptake and is itself accumulated by
cortical slices 14 , it may prove to be a valuable tool as a selective
marker for the localization of GABA operated inhibitory synapses.

B. Uptake of Glutamate. Since it would be of value to distinguish

between the relative roles of glia and nerve endings in the inactivation
of synaptically released glutamate, a study similar to that carried
out with GABA was undertaken with glutamate 31 • In general, it was
found that the uptake of 14C-glutamate into synaptosomes was slightly
more sensitive to inhibition than was its glial uptake, but no
sufficiently selective inhibitor was found which might have enabled
an evaluation to be made of the roles of the 2 cell types. The
results did, however, indicate that glutamate uptake sites on both
the glia of dorsal root ganglia and synaptosomes from cerebral cortex
are structurally different from the postsynaptic receptors. Thus,
excitants such as kainate and N-methyl-D-aspartate were inactive.
DL-aspartic acid f3-hydroxamate, a weak excitant, was found to be one
of the strongest known uptake inhibitors.
AMINO ACID TRANSPORT IN GANGLIA 173

VII. AMINO ACID RELEASE

An important criterion for a neurotransmitter is the demonstrat-

ion of its release from nerve terminals during depolarization. Amino
acid neurotransmitters, subsequent to their accumulation into nervous
tissue by sodium-dependent, high affinity pr~cesses, can be selectiv-
ely released in a Ca 2 +-dependent manner by K induced (40 mM)
depolarization 8 • Several investigations have therefore been made
of the factors iDfluencing the efflux of amino acids from glia of
ganglia lO ,22,24,27.

A. Superior Cervical Ganglia. Accumulated 3H-GABA exhibited

an efflux pattern from super fused superior cervical ganglia similar
to that seen in cortex slices: a rapid phase followed after IS-20
min by a slow steady efflux. The rate of release was increased by
~~~Si~~et~:l!:S~o:~:n~~:t!~:;l:;~~e: ~;St~!m;:e!~~~:a~~ :!~~a!~:l~
which depolarizes the ganglionic neurones, indicating that the
release is from the glial cells since they are not depolarized by
cholinomimetics. Electrical stimulation of the preganglionic trunk
produced a variable release depending upon the stimulus parameters,
and was found to occur solely from the nerve trunk. A consistent
increase in release was observed during high-frequency stimulation
of long duration, across the ganglion body. The release following
electrical stimulation was not blocked when transmission was
prevented by using a medium containing no Ca 2+ or 10-30 mM Mg2+.

B. Dorsal Root Ganglia. (i) Exogenous amino acids.

Depolarizing concentrations of K+ have been found to evoke a release
of several pre-accumulated labelled amino acids 27 • In the presence
of SO mM potassium, the release of GABA was increased 2.2 times, that
of glutamate 1.8 times, and that of glycine and glutamine 1.3 times
(the results for GABA and glutamate being significant at P,O.Ol).
Only for 3H-GABA could an evoked release be seen at a concentration
of less than 30 mM of K+.

Virtually all stimulus-secretion coupling mechanisms have been

shown to be calcium-dependent; therefore it was interesting to find
that removal, or replacement, of Ca 2+ with Mg2+, or+the addition of
the chelating agents EDTA and EGTA, inhibited the K evoked release
of 3H-GABA and l4C-glutamate. In addition, the divalent cation
ionophore A23l87 (19 ~), which stimulates the release of several
neurotransmitters and neurohormones, by transferring Ca 2+ across the
cell membrane lS , increased the release of GABA 2.4 times, and that
of glutamate, 1.6 times. However, in several other respects the
nature of the calcium dependence differed from that seen in other
systems. The drugs, D600 and diphenylhydantoin, which block Ca 2+
influx during depolarization, were without effect on K+ - evoked
GABA release Z2 • Raising the calcium concentration to 10 mM was
ineffective, as was the introduction of lanthanum ions (S mM), which
174 P.J. ROBERTS

inhibit the movement of calcium across membranes. Hence it seems that

although calcium ions are required for the release of GABA and
glutamate from the glia of dorsal root ganglia, the release system
may be different from that found in other neurosecretory systems.
Similar findings have recently been reported for the release of
3H-GABA from glial cells of the posterior pituitary25

(ii) Endogenous amino acids. In order to compare the release

of radiolabelled exogenous amino acids with endogenous amino acids,
dorsal root ganglia were incubated in Krebs medium and then transferr.-
ed for a short period to one containing 50 mM K+, and the free amino
acids in the medium were determined as their 3H-dansyl derivatives 27
Of the amino acids detected, the only ones whose rates were increased
by K+ were glutamine (1.5 times) and glycine (1.6) times. Neither
the release of glutamate nor that of GABA (which in any case was
very low) was increased by K+, while the release of aspartate was
depressed.

The reason for the observed lack of correlation between the

release af exogenous and endogenous amino acids may be explicable
largely in terms of the compartmentation of amino acid pools. Labell-
ed glutamate and GABA are taken up into the small (glial) pool and
then presumably released from this site by K+. In the case of their
endogenous counterparts, where especially for glutamate, there is a
large neuronal pool, a small release from the glial cells might well
be masked by continuous release from the large pool. The increased
release seen with glutamine is compatible with the idea that glutamate
taken up by the glia is converted to glutamine, which is later
released and returned to the neurone for subsequent hydrolysis back
to glutamate 6 ,7.

VIII. HOMO- AND HETERO- EXCHANGE OF GABA BY DORSAL ROOT GANGLIA

Recent evidence (see Levi et al., this book) has indicated that
homoexchange is a major mechanism by which radioactive GABA is trans-
ported into synaptosomes. This process is strongly sodium-dependent,
and occurs even at very low concentrations of GABA in the external
medium, and hence might merely simulate a high-affinity uptake system.
To test whether homoexchange occurs in dorsal root ganglia, ganglia
were finely sliced and incubated with 3H-GABA (0.8 ~) followed by
superfusion for 30 min (unpublished). When varying concentrations
of unlabelled GABA were added to the superfusion medium, homoexchange
occurred, with a detectable increase in release of 3H-GABA occurring
at a concentration of 5 ~ unlabelled GABA, and a maximal release at
approximately l500~. The heteroexchange of 3H-GABA with DAB and
~-alanine (both 100 ~) was also investigated: ~-alanine was approx-
imately three times as effective as DAB as substrate for the carrier
system. Several other substances including O-aminovalerate and 3-
hydroxy GABA were also able to heteroexchange.
AMINO ACID TRANSPORT IN GANGLIA 175

IX CONCLUSIONS

The glial cells of both dorsal root ganglia and superior cervical
ganglia behave very similarly to isolated glia, cultured glial tumor
cells, and nerve endings in their ability to accumulate neuroactive
amino acids by sodium-dependent, high-affinity systems. Hence, it
can be proposed that the function of the glial cells in the dorsal
root ganglion and superior cervical ganglion is to regulate the levels
of neurotransmitters and other neuroactive materials in the extra-
cellular space and to form a protective barrier against substances
entering from the blood supply; however, it has never been suggested
that either glutamate or GABA playa transmitter role in ganglia,
and their neurones are insensitive to glutamate. GABA will depolarize
neurones of both sensory12 and superior cervical ganglia ll , and this
can be antagonized by the GABA - receptor antagonists, bicuculline
and picrotoxin13 • Close intra-arterial injection of GABA readily
depolarizes ganglionic neurones; hence the glial cells would not seem
to be particularly efficient at these sites in a 'protective' role.
What then are the functions of the uptake systems found in the satelli-
te glia and Schwann cells? It seems quite feasible that the uptake
sites are mere vestiges of a property common to all glial cells, but
which are active only in the central nervous system in areas where
GABA or glutamate are neurotransmitters. Such a concept is supported
by the finding that Sc~wann cells of both dorsal and ventral spinal
roots will accumulate 4C-glutamate in a similar manner28. An
alternative proposal 4 for the function of the glial uptake systems
is that the glial cells are the site of the 'GABA-shunt' in nervous
tissue.

The significance of the finding that accumulated GABA and gl¥ta-

mate were released in a calcium-dependent manner in response to K
depolarization is not clear, although receptors for GABA do exist in
the ganglia. The levels of GABA in superior cervical ganglia and
dorsal root ganglia are extremely low, and it seems unlikely that
the satellite glial cells could effectively playa modulator role
in releasing sufficient GABA during the build up of extracellular K+
in intense nervous activity to produce an inhibitory effect under
physiological conditions. Although electrical stimulation will evoke
a release of exogenous GABA, it is not calcium-dependent, unlike the
release caused by elevated K+. This must cast doubt on the proposal
that the GABA release during electrical (and hence neuronal) activity
occurs indirectly via the potassium release from the neurones.

Despite the many unanswered questions, there can be no doubt

that glial cells are intimately concerned with neurotransmitter
function, and ganglia provide a simple, glia-enriched preparation for
investigating their function and relationship to neurones.
 176 P~.ROBERTS

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UPTAKE OF NEUROTRANSMITTERS AND PRECURSORS

BY CLONAL CELL LINES OF NEURAL ORIGIN

B. Haber and H.T. Hutchison

Division of Comparative Neurobiology, The Marine
Biomedical Institute, Department of Neurology and
Department of Human Biological Chemistry and Genetics
University of Texas Medical Branch
Galveston, Texas 77550, U.S.A.

INTRODUCTION

Intercellular communication is a fundamental process under-

lying behavior. The importance of communication between neurons
in determining behavior is undisputed, but recent evidence sug-
gests that communication between glia and neurons and between
glia and other cell types may be of significance in the modula-
tion of behavior. It is well established that the principal mode
of inactivation of neurotransmitters, other than acetylcholine,
is by reuptake into presynaptic terminals l . However, the close
spatial relationships, of neurons and glia, and the glial ensheath-
ment ofaxons and synapses, suggest that glia, as well as neurons,
may participate in the uptake of synaptically released neuro-
transmitters 2 ,3. In so far as synaptic transmission depends in
part on levels of the transmitter in the cleft, rapid removal of
transmitter, either by neurons or glia, will influence or modu-
late efficacy of transmission. To demonstrate a significant glial
role in the modulation of synaptic transmission, it is first
necessary to show that glial cells possess mechanisms for the
accumulation of transmitter which are comparable in substrate
affinity to the pumps present in the specific nerve endings using
that transmitter.

Many workers have demonstrated that various brain prepara-

tions often possess two transport systems for putative neurotrans-
mitters. One system is apparently shared with structurally related
compounds and is usually a low-affinity system (Km on the order of
I millimolar); the other is often a high-affinity system (Km in the

179
180 B. HABER AND H.T. HUTCHISON

micromolar range) and is thought to be involved in transmitter

reuptake. Some evidence has been obtained using synaptosomal pre-
parations which suggests that only terminals normally using a given
transmitter are capable of accumulating that transmitter 4 ,5,6,7.
A variety of neural preparations has been used to characterize
transport processes for neurotransmitters and precursors. These
have been synaptosomes and brain slices; and to a lesser extent
bulk isolated neuronal perikarya and glia 3 • Synaptosomes and
brain slices do not permit an accurate assessment of the glial vs
neuronal contribution to the uptake process; bulk isolated prepara-
tions may suffer from varying degrees of membrane damage due to the
isolation procedure, and loss of processes. Such considerations
have prompted the use of clonal cell lines of neural origin as
model systems for the study of neuronal and glial transport func-
tions in tissue culture 8 ,9,lO,11,12,13,14,15,16,17.

Transformed lines of neuronal and glial origin have the advan-

tage that (1) they may be readily cloned and, therefore, represent
a single, genetically pure cell type, (2) biochemical studies may
be performed directly in the culture vessels, and (3) the prepara-
tion and manipulation of large numbers of cultures is relatively
easier to accomplish than are fractions prepared by bulk isolation
procedures. Though we do not know how extensive the changes in
gene expression in transformed cell lines may be, these cells pos-
sess many properties which indicate their utility as models of
neurons and glia.

TRANSPORT IN CLONAL CELL LINES

The neuronal properties of the murine C-l300 neuroblastoma

cell lines have been amply documented, as have the glial proper-
ties of the astrocytoma cell lines derived from n-nitrosoethyl
urea (NEU) induced rat tumors 18 ,19,20,21,22,23a,b,24. Experiments
done in the authors' laboratories have used primarily subclones
of the C6 astrocytoma (supplied by J. de Vellis, UCLA), subclones
of the C-l300 neuroblastoma (supplied by J. de Vellis and H.
Hirshman) and several clonal lines from rat NEU induced tumors
(supplied and characterized by D. Shubert)25. The RN22, described
as a peripheral neurinoma 26 , was supplied by S. Pfeiffer.

The kinetics of transport in clonal cell lines are analyzed

by hyperbolic least-squares regression techniques, as previously
described in detail 27 • The fitted equation for a two-carrier
mediated transport system is

v
=
S
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 181

and for a one carrier mediated transport system and diffusion is

v +D
s
Where Km and Vmax are Michaelis constants and maximum velocities,
respectively, and D is a diffusion constant. The velocities, V,
have units of picomoles of substrate taken up per milligram of
cell protein per minute of incubation; the substrate concentra-
tions, S are expressed in micromolar units. The data are plotted
as vIs vs V, a modified Hofstee 28 plot. A transport system obey-
ing Michaelis-Menton kinetics (a "carrier mediated" transport sys-
tem) yields a straight line on a plot of VIS versus V, whereas a
two-carrier mediated system for the same substrate, or a single
carrier mediated system and diffusion, yield hyperbolas. Using
the aforementioned method, kinetic data from our laboratory are
obtained using statistically weighted fits 21 and are presented with
standard errors.

UPTAKE OF y-AMINOBUTYRIC ACID (GABA)

The uptake of GABA by astrocytoma cell lines is shown in

Fig. 1. It can be seen that GABA uptake is mediated by more than
one transport system.

The Michaelis constant for the C6 astrocytoma high-affinity

system is 0.22 ~M, whereas the second component represents a non-
saturable mechanism, presumably diffusion. The high-affinity sys-
tem for GABA uptake is Na+ dependent, and the Km is in good agree-
ment with that found for bulk isolated glia and for synaptosomes 3 •
Bulk isolated glia concentrated GABA far more effectively than
neuronal perikarya 3 , but less effectively than isolated synapto-
somes 29 .

Glioma lines have been shown by others lO to take up GABA in

a Na+ dependent manner. That uptake was shown to consist of
saturable and nonsaturable component 10 , and was antagonized by
taurine, ~-alanine, low temperature and the GABA antagonist,
bicuculline. Autoradiographic evidence has confirmed the uptake
of GABA by glial cells in cerebellar cultures 30 , satellite glia
of dorsal root ganglia 31 , MUller cells of the retina 32 , connec-
tive tissue cells, probably glia, at the lobster neuromuscular
junction33 , and gliocyte cells of the pineal. 34
182 B. HABER AND H.T. HUTCHISON

10 20
V (pmoles/mg-min)

Fig. 1: C6 ast~ocytoma ce~~s we~e ~abe~ed fo~ 2 min with

1 ~Ci [3H] GABA at tota~ GABA concent~ations of 0.6-
20~. Each point ~ep~esents the mean of 3 determina-
tions. The so~id ~ine is a ~east-square8 curve fit
fo~ one c~~e~-mediated t~anspo~t system and diffu-
sion. The bottom c~e ~ep~esents the uptake in the
absence of Na+. 9

Accumulation of GABA by neuroblastoma cells was studied in

detail in clone NB4l. As in the C6 astrocytoma, the transport is
mediated by a high-affinity, Na+ dependent system, with a Km of
0.15 ~M and a second, non-saturable component 9 . In both cell
types, the low-affinity or diffusional component is unaffected
by omission of Na+ from the incubation medium. The accumulation
of GABA by the C6, but not by the neuroblastoma cells, is inhi-
bited by norepinephrine (NE), singly or in combination with
theophylline, a p~osphodiesterase inhibitor; the velocity of
GABA transport decreases with increasing age in culture and with
cell density (Haber, et al., unpublished). These results suggest
that the glial intracellular accumulation of GABA and perhaps of
other amino acids may be regulated by intracellular levels of
cyclic AMP. Similarily, the glial accumulation of NE is density
dependent and decreases with increasing cell densi ty35. The
transport of aminoisobutyric acid and cycloleucine by cultured
glial cells is stimulated by norepinephrine and dibutyryl cyclic
AMP; the time course of the stimulatory effects by NE corresponds
well with the rise and fall of intracellular cyclic AMP in these
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 183

cells 36 • Thus, the glial norepinephrine sensitive adenyl cyclase

may serve as a coupling mechanism between neurons and glia; such
coupling may be the activation of glial phosphorylase and glycogen
breakdown or alterations of glial permeabilities to both neuro-
transmitters and ions.

GABA uptake mechanism in the neuroblastoma cells differ from

that of C6 cells, as shown in Figure 2A and B.

A B
0.04
0.04
-
0.03 0.03

3-
E
f
'ii
'ii
~
0.02 i
,g.
0.02
,g.
>
>
0.01 0.01

10 20 5 10 15 20
minutes minutes

Fig. 2: GABA uptake in C6 astrocytoma cel:ls (A) and neuro-

blastoma (B) in the presence-and absence· of 1 mM amino-
oxyacetic acid (AOAA) (from Ref. 9).

Aminooxyacetic acid (AOAA), a carbonyl trapping agent, is an

inhibitor of pyridoxal dependent enzymes. It effectively inhibits
both the synthesis of GABA by glutamic acid decarboxylase (GAD) in
vitr0 37 and the metabolism of GABA by y-aminobutyric acid trans---
aminase (GABA-T) both in vitro and in viv0 38 • AOAA, at 10-4 M,
markedly inhibits both the respiration and the synthesis of amino
acids by transamination reactions from [ 14 CJ glucose and [ 14 CJ
GABA in brain cortex slices 37 . (Equivalent concentrations of AOAA
do not inhibit either the uptake of GABA by brain cortex slices or
the uptake of glycine by Ehrlich ascites cells.)
184 B. HABER AND H.T. HUTCHISON

The effects of AOAA on GABA uptake in C6 cells is shown in

Fig. 2A. It can be seen that, in these cells, there is a 5-fold
inhibition of uptake in the presence of AOAA. The specificity
of this inhibition was tested by examining the effects of AOAA on
glutamic acid accumulation. Glutamic acid is a possible excita-
tory amino acid neurotransmitter and is taken up by C6 astrocytoma
cells by a high-affinity uptake system with a Km of 7 ~M. Concen-
trations of AOAA that markedly inhibit GABA uptake have no effect
on the accumulation of glutamate by these cells. Similarly, the
uptake of glycine is unaffected by AOAA in either cell line.
These findings suggest that the inhibition of GABA uptake is not
related to general non-specific metabolic effects of AOAA such as
have been shown for brain cortex slices 37 •

In marked contrast, Fig. 2B shows that equimolar concentra-

tions of AOAA do not significantly alter the accumulation of GABA
by NB41 neuroblastoma. These somewhat unexpected findings prompted
us to examine GABA uptake in mouse brain synaptosomes. However,
as in NB41 neuroblastoma cells, AOAA does not inhibit the uptake
of GABA in synaptosomes. These findings with clonal cell lines
used as model systems suggest that in glia, but not in neurons,
GABA accumulation by the high-affinity system is coupled to its
metabolism. Neurons on the other hand, may possess a mechanism,
possibly vesicular, for the storage of GABA which is independent
of its metabolism. Thus, glial GABA accumulation may have a meta-
bolic significance, a suggestion in accord with observations of
greater GABA-T activities in bulk isolated glia than in neuronal
perikarya (Hamberger, this volume). Whether the uptake of GABA
by clonal cell lines of bulk isolated glia 3 ,29 is mediated by
high-affinity systems or also in part by homoexchange 39 is at
present unknown. The possible role of homo exchange mechanisms
in transport phenomena in the CNS is discussed in detail by Levi
and Raiteri in this volume. Taken together, the evidence suggests
that glial accumulation may represent a significant process for
the removal of neuronally released GABA, and a modulation of inhi-
bitory transmission at gabaminergic synapses.

UPTAKE OF ASPARTATE, GLUTAMATE, GLYCINE AND TAURINE

The above amino acids are suspected transmitter substances

in the mammalian CNS; their accumulation by clonal cell lines has
not been studied in great detail. Glutamate and aspartate are
taken up rapidly by glioma cells, and these cells have the ability
to synthesize GABA and S-alanine, respectivelylO. Glutamate is
accumulated by several subclones of the C6 astrocytoma by a high-
affinity system with a Km of 7 ~M; this process is not inhibited
by AOAA at concentrations found to block GABA uptake 9 • Similarly,
glycine is also taken up by both astrocytoma and neuroblastoma
cells by a high-affinity system qualitatively similar to that seen
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 185

in synaptosomes; the uptake of both glutamate and glycine is also

mediated by a second nonsaturable system which has not been well
characterized to date (Haber, et al., unpublished). Taurine uptake
in glioma cells consists of two Na+ dependent components; a rapid,
saturable component (Km 10-17 ~M) and a nonsaturable component 10 •
This uptake is antagonized by 8-alanine, but not by GABA, suggest-
ing that glial cells in culture take up the two putative neuro-
transmitters (GABA, taurine) via separate carrier mechanisms.
Electron microscopic radioautography has confirmed the glial up-
take of taurine in brain slices -
in-vitr0 40
- - • The data are consis-
tent with the suggestion that in the brain the untransformed
counterparts of glial tumor cells have the capacity to control the
extracellular concentrations of potentially neuroactive amino
acids lO •

TRANSPORT OF CHOLINE

Acetylcholine (ACh) differs from other neurotransmitters in

that it is inactivated by extracellular hydrolysis rather than by
reuptake into presynaptic nerve endings and into glia. Further-
more, the central nervous system is incapable of de novo synthesis
of choline41 ,42. Thus, for the synthesis of Ach, cholinergic
synapses must depend upon the uptake either of exogenous choline,
or of that formed by hydrolysis of ACh. Evidence that choline
may be rate limitin~ for ACh synthesis was first shown in frog
sympathetic ganglia 1 in which repetitive stimulation fails in
the absence of exogenous choline. The suggestion that reuptake
of choline is important to cholinergic function has led to a num-
ber of investigations of the transport of choline into brain
slices 43 ,44,45 and synaptosomes 46 ,47. These studies have shown
that both slices and synaptosomes accumulate choline via a high-
affinity, saturable mechanism and by a second, less well charac-
terized component of lower affinity. It has been suggested that,
at cholinergic synapses, the reuptake of choline by the high-
affinity system is tightly coupled to the synthesis of ACh 43 ,46.

The transport of choline in clonal cell lines has been studied

in a number of laboratories 8 ,11,12,13,16 and by us in the neuronal
(NB41 and N2AG) and glial (C6 and RN22) cell lines 15 ,48,49. The
several subclones of the murine C-1300 neuroblastoma can be charac-
terized as noradrenergic or cholinergic on the basis of their
relative ability to synthesize norepinephrine and acetylcholine,
respectively; the high-affinity uptake of choline does not differ
significantly in cholinergic vs. noradrenergic clonal lines 8 .
Therefore, the suggestion that the high-affinity transport for
choline in cholinergic synapses is tightly coupled to acetylcholine
synthesis 43 ,46 is not demonstrable in a tumor cell line used as a
model of a cholinergic neuron.
186 B. HABER AND H.T. HUTCHISON

In Figure 3 is shown the kinetic analysis of choline trans-

port by NB41 neuroblastoma cells in culture.

30 Km Vmax D
Ai4.2 303 10.8
. t3D tao !.6
Kml Vmax Kmz Vmax

B.
"ii5 201 2,605 34,733
t5.1 !60 !I,022 !12,744
20
V
S

10
- ---------+-
B
A

5000 10000
V

Fig. 3:
A. Graphic description of a singZe high-affinity
transport system pZus a diffusionaZ component.
B. Graphic description of a carrier-mediated trans-
port system, consisting of two components of
high and ZOW affinities.
Inset gives numericaZ vaZues for the kinetic parameters des-
cribed by curves A and B. Each point represents the mean
of five determinations; th~ bar, the standard error of the
mean (from Ref. 49).

It is clear from Figure 3 that more than one mechanism for

choline transport is operative in these cells. The solid line
(A) represents a computer fit of these points by a model consist-
ing of a single carrier-mediated transport system and a second
component, which is diffusion. The dashed line (B) represents
a double carrier-mediated transport system model fit to the same
data points. Either curve fits the data well and the kinetic
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 187

parameters obtained from these fits are shown in the inset to Fig.
3. Either fit yields an estimated Km for the high-affinity cho-
line transport system in the range of 10-14 ~M. The low-affinity
component is less well characterized by these data,but if it is a
saturable system, it has a Km greater than 1 roM and is not satu-
rated at 10 roM, the highest concentration used in this series of
experiments. These data are included to graphically illustrate the
possibility of multiple possible fits for double-affinity trans-
port systems; that even rigorous statistical evaluation of data
yields Km values which should be assigned a range rather than an
absolute numerical value. Furthermore, it is difficult, if not
impossible, to unequivocally decide whether a low-affinity system
is in fact carrier-mediated or represents diffusion. These con-
siderations apply equally to transport studies of other neuro-
transmitters and precursors.

The Na+ requirement for choline transport differs in neuronal

and glial cell lines; the high-affinity system has an absolute
requirement for Na+ in the former, whereas such transport is stimu-
lated in glial cell lines by omission of Na+. Figure 4 illustrates
the substrate dependence of choline transport in the C6 astrocytoma
cell line; the kinetic parameters of choline transport in the pre-
sence, and in the absence, of sodium in these cells are listed in
the inset to Figure 4A. The Km value of 13.8 ~M for the high-affinity
system in quite similar to that shown for the NB4l neuroblastoma
cells in Figure 3. Choline uptake at low substrate concentrations
in the absence of sodium is shown by the dashed line (B) in Figure
4A. The striking acceleration of choline transport is only seen
at low substrate concentrations. The stimulation of choline trans-
port in the absence of sodium apparently results from a decrease
in the Km of the high-affinity choline transport system. It should
be pointed out, however, that a variety of closely related curves
may be fitted to these points; thus, we do not reject the possi-
bility that the difference may be a result of a higher Vmax rather
than a lower Km. The stimulation of choline uptake decreases with
increasing Na+ concentration; at 50 roM Na+ it is reduced to half
that seen in the absence of Na+'

Inspection of the values in Figure 4B for the neuroblastoma

shows that the velocity of choline uptake by the high-affinity
system is reduced three-fold in the absence of sodium, whereas the
diffusion system is only slightly affected. These results agree
with similar studies in synaptosomes 46 and in brain slices 43 , but
differ from the recent report 16 that choline transport in similar
neuroblastoma cell lines is sodium independent. We have found a
partial sodium dependence of choline transport at low substrate
concentrations in several neuronal (N2AG and B50) and in some, but
not all, of the non-neural cell lines which we have examined.
188 B. HABER AND H.T. HUTCHISON

A B
15

I(tI'II Vrncu _ C_
. No° Il7!131 7B!n7 4~! 0 4
~ 10
- No" 1t1 4,! 1.0
S 26~24

,,
\
'---------------------------------------

100 200 1000 zooo 4000 3Z1OO JIj:;OO

v

Fig. 4: Curves A and B describe the kinetics of choline

transport in the presence and absence of Na+, respec-
tively, in Fig. 4A and 4B. Inset gives numerical
values for the kinetic parameters described by curves
A and B for Figure 4A and 4B. Each point represents
the mean of four determinations; the bar, the standard
error of the mean (from Ref. 49).

The high-affinity system for choline thus differs with

respect to its Na+ requirement in neuronal and glial cell lines;
the physiological significance of this difference is difficult
to determine. This differential Na+ requirement in these two cell
types is analogous to the sodium dependence of the transport of
the biogenic amines and their precursors into rat brain synapto-
somes: the uptake of norepinephrine (NE) and serotonin(5-HT) is
Na+ dependent, but the uptake of tyrosine and tryptophan is not 50 •
It has been suggested that the influx of Na+ during the depola-
rization of presynaptic terminals results in a transitory decrease
in extracellular Na+, and an increase in K+. Such transitory
changes in extracellular monovalent cations might trigger the
influx of choline into glial cells, whereas during repolarization
the external Na+ would increase, and thus promote neuronal reup-
take of choline for the resynthesis of ACh. The role of divalent
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 189

cations, such as Ca++ is presently not understood; however, some

evidence with bulk isolated glia and GABA transport suggests a
role for Ca++ in the coupling of transport to physiological neural-
glial interactions 29 •

The high-affinity transport of choline is inhibited by low

(10- 4 M) concentrations of hemicholinium-3 (RC-3) in both neuronal
and glial cell lines 49 , as well as by cholinesterase inhibitors
of the physostigmine type. The inhibition by cholinesterase
inhibitors is observed at relatively high concentrations (10- 3 M),
and is similar to that seen in synaptosomes. Thus, these findings
suggest the possibility that the clinical effectiveness of these
drugs in the treatment of such disorders as myasthenia gravis may
be limited by their inhibition of choline transport. The presence
of high-affinity transport systems for choline is not unique to
cell types of the nervous system. The presence of such transport
systems in glial cells, coupled with the observations of the quan-
tal release of ACh from Schwann cells at the denervated neuro-
muscular junction 51 , and the presence of some choline acetyl trans-
ferase activity (CAT) in glial tumor lines (Raber, et al., unpub-
lished) is suggestive of a glial role in cholinergic transmission.
Taken together, the data indicate that clonal lines of tumor
origin have utility in defining some aspects of the neuronal and
glial roles in cholinergic function.

TRANSPORT OF PRECURSORS AND BIOGENIC AMINES

Tyrosine transport has been studied in a noradrenergic clone

of neuroblastoma, NlE-l5, which has high levels of tyrosine
hydroxylase. The transport is saturable, and exhibits marked
exchange properties 52 • It has no apparent ionic or energy
requirements, but is sensitive to sulfhydryl reagents and low
temperature. Accumulation of tyrosine by this clone is inhibited
by molecules structurally analogous to tyrosine, such as trypto-
phan, dihydroxyphenylalanine and phenylalanine, as well as by
leucine, isoleucine and valine 52 • Transport of tyrosine is,
therefore, thought to occur in this cell line by diffusi0n and
by the leucine (L) preffering transport system 53 •

The transport of biogenic amines by clonal cell lines of

neural origin has not been extensively studied to date 54 ,55,56.
Bulk isolated glia do take up norepinephrine (NE), serotonin (5RT)
and dopamine; however, these transport processes have not been
characterized 3 • The presence of biogenic amines in cultured mouse
neuroblastoma (clone NB-2a) is demonstrable by fluoresence histo-
chemistry, as is synthesis of DA, NE, 5RT, tyrawine (TY) and octo-
pamine by these cells from C14 precursors 57 . Astrocytoma cells
also contain endogenous biogenic amines in concentrations lower
than those found in neuroblastoma cells. The amine levels are
190 B. HABER AND H.T. HUTCHISON

significantly different in the two astrocytoma clones examined 57 .

Astrocytoma cells in situ have been shown to accumulate cate-

--- 58
cholamines from the extracellular environment , and cultured
astrocytoma cells contain significant activities of both monoamine
oxidase (~~O) and catechol-o-methyltransferase (COMT)59 The
MAO activity in several clonal lines of astrocytoma has been
characterized by us on the basis of substrate specificity and
inhibition by chlorgylline and deprenyl: the ~~O is primarily
type A, though one clone was found to have type B MAO. In con-
trast to the astrocytoma, several murine and human clonal neuro-
blastomas contain only type A MAO (Haber, et al., unpublished).

5HT and NE are taken up by a high-affinity transport system

in both C6 and RN22 cell lines, and a second component of lower
affinity, which may represent diffusion27,54,55. The Km of the
high-affinity system for 5HT is less than 1 ~M for RN22 neurinoma
cells, and roughly 2 ]JH for the C6 astrocytoma. For NE, the high-
affinity system is in the range of 2-3 ~M for all glial lines
tested, and this K is approximately equal to that found for
synaptosomes. Themprecise kinetics of DA uptake have not yet
been determined: however, DA is taken up by glial cell lines in
a manner analogous to NE and 5HT. The accumulation of 5HT is
rapid and concentrative: tissue/medium ratios of better than 100:1
are seen following five minutes of incubation with micromolar con-
centrations of the amine. Competition experiments done to date,
suggest that 5HT is taken up by glial cells via a carrier system
different from that which transports NE and DA. The phenylethyl-
amine derivatives such as PMPEA are accumulated by a third carrier
system, which has some overlap with the NElDA carrier 56 The
high-affinity system for 5HT in glial cell lines has an absolute
Na+ dependence5~,55; whereas the accumulation of NE and of the
phenylethylamine derivatives is independent of Na+.

In direct contrast to the cell lines of glial origin, the

neuroblastoma cells do not appear to take up biogenic amines by
a high-affinity system. The velocities of biogenic amine uptake
by neuroblastoma are so low as to preclude calculation of kinetic
constants, degree of saturability or extent of the Na+ dependence
of the transport process. The presence of high-affinity transport
systems for 5HT, NE, and DA suggest that uptake into glial cells
may effectively modulate monoaminergic transmission. Verification
of this suggestion will depend in part on the radioautographic
glial localization of monoamines in situ. This may prove diffi-
cult to accomplish, as glial cells possibly have little storage
capacity for monoamines, but do have significant degradative capa-
city, in view of the high levels of MAO and COMT found in cultured
astrocytoma. It is suggested that glial cells may in fact repre-
sent the primary sites of extraneuronal metabolism of biogenic
 NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 191

TABLE I

Effect of Inhibitors on the Uptake of Serotonin and

Norepinephrine by Clonal Astrocytoma Cells in Culture

% INHIBITION
INHIBITOR Cone. (M) NE 5HT

Chlorimipramine 10- 5 N.D. 50

Chlorimipramine 10-4 N.D. 71
Lilly 11014 10-6 N.C. N.C.
Lilly 11014 10- 5 N.C. 56
Lilly 11014 10- 4 N.C. 65
5-0H Dopamine 10- 3 N.C. 78
6-0H Dopamine 10- 3 96 52
Paramethoxyphenyl-
ethylamine (PMPEA) 10- 3 N.C. 50
2,3 Dimethoxyphenyl-
ethylamine (DMPEA) 10- 3 10 80
Phenylethylamine (PEA) 10- 3 N.C. 53
a-Ethyl tryptamine 10- 3 N.C. 67
5-Methoxytryptamine 10- 3 N.C. N.C.

C6 astrocytoma cells were labeled with micromolar concen-

trations of H3 NE or H3 5HT, in the presence of inhibitors
at stated concentrations. Results are expressed as % inhi-
bition of the velocity observed in a control medium. An
MAO inhibitor was added to the incubation medium.
N.D. = not done N.C. = no change
(Haber, et al., unpublished).

amines, a suggestion supported by the immunochemical localization

of COMT to postsynaptic sites and to glia (Crevelling, personal
communication).

The effects of a variety of inhibitors have been examined on

the accumulation of 5HT and NE by the C6 astrocytoma cells (Table
I). Chlorimipramine at 10- 4 M inhibits 70% of 5HT accumulation.
Lilly compound 11014 has been reported as a selective inhibitor
of 5HT transport in synaptosomes 60 ; it is evident that the selec-
tivity is also seen for 5HT transport by cultured cells of glial
origin. At 10- 4 M Lilly 11014 inhibits glial 5HT transport by
65%, with no effect on NE accumulation. a-ethyl tryptamine
inhibits glial 5HT uptake, with no effect on the NE transport
system. In synaptosomes, ring substitution (5-methoxytrypt-
amine) does not inhibit 5HT transport, whereas side chain (a-ethyl)
substitution has profound effects. It is apparent that the effects
of ring vs side chain substitution of the indole nucleus affects
192 B. HABER AND H.T. HUTCHISON

5HT accumulation in a similar manner in both isolated brain synap-

tosomes and clonal tumor cells of glial orlgln. 6-0H dopamine is
neurotoxic to noradrenergic terminals: at high concentrations
(10- 3 M) it more drastically inhibits the uptake of NE than that
of 5HT by astrocytoma cells. Conversely, 5-0H dopamine, which
affects the uptake of both 5HT and NE in synaptosomes, is more
selective for the glial 5HT transport process. The 5HT analog,
6-0H tryptamine, effectively abolished the glial high-affinity
transport for serotonin, with no inhibition of catecholamine up-
take. The phenylethylamine (PEA) derivatives, paramethoxyphenyl-
ethylamine (PMPEA) and 2-3-dimethoxyphenylethylamine (2,3 DMPEA)
in vitro preferentially inhibit the accumulation of 5HT by mouse
brain synaptosomes, and to a far lesser extent than that of NE
and DA61. PMPEA, which itself is taken up by astrocytoma cells
in culture, also promotes release of glial H3 5HT. In vivo PMPEA
depletes 5HT and NE, both centrally and peripherallyOT.~e in
vitro selectivity of the phenylethylamines for 5HT transport is
qualitatively similar in both synaptosomes and cultured glia.

The above observations suggest that the accumulation of bio-

genic amines by both glia and synaptosomes can be manipulated
by pharmacological agents in a qualitatively similar manner; there-
fore, it is fair to state that studies of amine transport and
release in morphologically complex neuronal preparations, such as
slices and synaptosomes may have a significant glial contribution.

Clonal tumor cell lines of neuronal and glial origin

serve as useful model systems in delineating the possible glial
role in the modulation of transmission, and may provide useful
insights into neuronal-glial interactions in vivo.

ACKNOWLEDGMENTS

Supported in part by DHEW grants NS 11255, NS 11354, Welch

Grant H-S04 and a grant from the Muscular Dystrophy Association
of America. The collaboration of R.L. Suddith, Ph.D., is grate-
fully acknowledged.
NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 193

REFERENCES

1. Axelrod, J., The metabolism, storage and release of catechol-

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2. Curtis, R.D., and Johnston,G.A.R., Amino acid transmitters.

In A. Lajtha (Ed), Handbook of Neurochemistry, Vol. 4,
Plenum Press, New York and London, 1970.

3. Henn, F.A., and Hamberger, A., Glial cell function: Uptake of

transmitter substances, Proc. Natl. Acad. Sci., 68 (1971)
2686-2690.

4. Iversen, L.L. and Johnston, G.A.R., GABA uptake in rat cen-

tral nervous system: Comparison of uptake in slices and
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194 B. HABER AND H.T. HUTCHISON

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NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 195

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31. Schon, F., and Kelly, J.S., Autoradiographic localization of

[3H] GABA, [3H] glutamate over satellite glial cells, Brain
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of 3H-GABA in rat retina, Nature, 235 (1972) 217-218.

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of y-aminobutyric (GABA) uptake in lobster nerve-muscle pre-
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196 B. HABER AND H.T. HUTCHISON

35. Vernadakis, A., Accumulation of H3 norepinephrine in C6 glial

cells in culture, The Physiologist, 18 (1975) 3.

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glutaric acid transaminase in vivo and in vitro by U-7524
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acid at nerve endings can simulate high-affinity uptake.
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~raphy of rat hypothalamus after in vitro incubation with
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43. Kuhar, M.J., Sethy, V.H., Roth, R.H., and Aghajanian, G.K.,
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NEUROTRANSMITTER AND PRECURSOR TRANSPORT BY CELL LINES 197

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55. Haber, B., Werrbach, K., Risk, M. and Hutchison, H.T., Uptake
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56. Vance, C., Ashkenazi, R. and Haber, B., Uptake of paramethoxy-

pheny1ethy1amine (PMPEA) by isolated synaptosomes in vitro.
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58. Bondareff, W. and Narotzky, R., Uptake of exogenous norepine-

phrine from corpus callosum by neurons of the cingu1ate cortex,
Exp. Neuro1., 34 (1972) 309-315.

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methyl transferase and monoamine oxidase activity in cultured
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198 B. HABER AND H.T. HUTCHISON

60. Wong, D.T., Horng, J.F., Bymaster, F.P., Hauser, K.L., and
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phenolpropylamine, Life Sciences, 15 (1974) 471-479.

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biogenic amine uptake by sympathomimetic amines, Proc.
Soc. for Neuroscience, 29 (1974) 123.

62. Ashkenazi, R., and Haber, B., Mode of action of a sympatho-

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1 (1975) 163-168.
ON THE UPTAKE MECHANISM OF CHOLINE IN NERVE CELL

CULTURES

MASSARELLI, R. & MANDEL, P.

Centre de Neurochimie du C.N.R.S •

.11, rue Humann, 67085 - STRASBOURG, FRANCE

INTRODUCTION

Much evidence points to the possibility that the rate

limiting step in acetylcholine synthesis is bound to a mass ac-
tion effect of its substrates: choline and acetyl-CoA 1-3. The
availability of choline and acetyl-CoA thus is of crucial impor-
tance for the synthesis of the neurotransmitter.

Choline seemingly is not synthesized in the nervous tis-

sue, not at least, from a direct methylation of enthanolamine.
4-8 • Recently some authors have observed an arterio-venous dif-
ference in the concentrations of free choline in the rat 9-10.
This finding has led to the hypothesis that choline might be syn-
thesized in rat brain from methylation of lipid bound ethanolami-
ne 9-11.However, Spanner et al. 12 have not found such arterio-
venous difference in choline concentrations in the rabbit. The
mechanism of choline uptake in nerve cells remains then a basic
phenomenon for the availability of this compound for acetylcholine
synthesis.
13-14
Choline crosses the membranes of blood cells of
cells in brain slices and of synaptosomes 17-20 via a process
which is sodium-dependent and which seems to be energy-dependent.
In 1972 Yamamura & Snyder 21, using a large range of choline con-
centrations, found two apparent Km's for the uptake of choline
into synaptosomes. Further research has shown that the high affi-
nity mechanism, or low apparent Km system~ geems to be responsi-
ble for the synthesis of acetylcholine 22 2 .
We have approached the study of the mechanism of choline

199
200 R. MASSARELLI AND P. MANDEL

uptake in using a simple model of nervous tissue: nerve cell cul-

tures, whose application in studies on elementary biochemical me-
chanisms has already been shown 27-28. We have studied clones of
mouse neuroblastoma CI300 as the simplest model of an homogeneous
cell population and primary cultures from dissociated chick embryo
cerebral hemispheres as a more complex non-tumoral model.

MATERIAL & MET30D8

Cell Cultures
Neuroblastoma clones: Cholinergic clones 821 and 820
and inactive clone NI8 were a generous gift of Dr. M. Nirenberg;
astroblasts from hamster (clone NN) were obtained from North Ame-
rican Biological Inc. and glioblastoma clone C6 and neuroblastoma
clone CCL 131 neuro 2a from American Type Culture Collection. The
cells were stored in liquid nitrogen and thawed when needed. The
cultivation was performed in Falcon plastic flasks (75 cnf) with
Dulbecco's medium (Gibco) containing 10 % foetal calf serum in a
100 % humidified atmosphere of 5 % C02/95 % Air at 37 0 C. The
cultures were replicated in Falcon plastic Petri dishes (28 cm2 )
and used at confluency.
Chick embryo cultures : The cerebral hemispheres of em-
bryos at various ages were dissociated through a nylon sieve (48~)
and seeded in Falcon plastic Petri dishes. The cultures were incu-
bated in Eagle's modified Dulbecco's medium containing 20 % fetal
calf serum in a 100 % humidified atmosphere of 5 % C02/95 % Air
at 37 0 C. Cultures from 8 day old embryos in culture for 7-10 days
show a uniform2~ayer of glial-like cells on top of which neurons
are dispersed -)0. The presence of synaptic junctions has been
shown in cultures of this age. Cultures from 14 day old embryos in
culture for 7 days present the same morphological aspect 29-30;
however, when these cultures are kept for longer periods in cultu-
re (14-21 days) the neurons will degenerate and only differentia-
ting glial cells will be left 30.

Choline Uptake Experiments

The attached cells from neuroblastoma clones or from
chick embryo cultures were washed 3 times with physiological sali-
ne \ 0.147 M NaCl, kept at 37 0 C) and a measured amount of Krebs-
Ringer phosphate (usually 4 ml) pH 7.2 was added to each Pet~i
dish. After 15 min. of equilibration, radioactive choline([1 C] Me
choline, sp. act.: 60 M Ci/mmole. Amersham) was added. At the end
of the incubation, the medium was discarded and the cultures were
washed 3 times with 0.147 M NaCl. The remaining traces of water
were rapidly evaporated at 37 0 C. Two ml of concentrated formic
acid was added to the dried Petri dishes and the digestion of the
cells was controlled by light microscope observation. 0.5 ml of the
acid was then transferred into scintillation counting vials and
UPTAKE OF CHOLINE IN CELL CULTURES 201

10 ml Omnifluor (New England Nuclear) was added. The radioactivity

was then measured with an Intertechnique SL 30 scintillation spec-
tometer.
RESULTS

When neuroblastoma clones were incubated with 1.5 ~M of

[14C]-'Me choline the uptake as function of time was linear for va-
rious periods of time, depending upon the clone under study (Fig.
I). A similar result was obtained with chick embryo cultures whe-
re a saturation plateau was reached earlier in more mature cells
(Fig. 2). Experiments performed in the presence of metabolic inhi-
bitors have shown that the uptake of choline in nerve cell cultu-
res is slightly energy-dependent,while in synaptosomes contradic-
tory results were obtained (Table I). However, the concentrations
of inhibitors used in these studies were generally very elevated
(Table I).

50 100
MIN

Fig. I. (left) : Uptake of choline in neuroblastoma clones as

function of time. S20, S21a, S21b : cholinergic clones; NI8 and
131 (CCL 131 neuro 2a): inactive clones. Ie: total cellular radio-
activity, 1m: total radioactivity in the medium. (Ie/1m) x 10- 3
cpm.

Fig. 2. (right): Uptake of choline in chick brain cells. Embryos

of 8 days (E8), 12 days (EI2), and 14 days (EI4). The cultures we-
re one week old.
202 R. MASSARELLI AND P. MANDEL

CN DNP Refe-
Ouabain 4°C
rences
Cultures
S21 (ImM) 83% --- (O.lmM) 65% 10% (32)
NI8 88% --- 75% 8% (32)
E8c7
EI4c7
--- ( ImM) 61%
--- ---
81%
90%
13%
12%
(33)
(33)
S~naE-
tosomes
(4mM) 98% (0.2mM)96% (0.2mM) 74% --- (23)
( ImM) 27% (0. 2mM) 40% (O.lmM) 55% 22% (22)
(0.lmM)73%

Table I. Inhibition of choline uptake in nerve cell cultures and

synaptosomes by metabolic inhibitors and ouabain. Results expres-
sed as percentage of control. eN: cyanide; DNP:3,4 dinitrophenol;
S21: Neuroblastoma cholinergic clone; NI8 neuroblastoma inactive
clone; E8c7: chick embryo brain cell cultures: mixed neuronal-
glial population from 8-day old embryos (E)in culture (c) for 7
days; E14c7: 14-day old embryos in culture for 7 days.

When cell cultures were incubated with various concentra-

tions of substrate a complex kinetics could be revealed. The Line-
weaver-Burk plot gave two straight lines with apparent Km of about
10- 6 and 10-5M 34 . Further experiments have shown that the two
lines represented the tangents to a curve (Fig. 3) and that all
cell lines under study presented this type of uptake kinetics re-
gardless of their origin 34-37. Table 2 shows a summary of the
apparent Km values which have been obtained in synaptosomes and
cell structures.
Several inhibitors have been tested on the kinetics of
the uptake of choline. Results obtained after incubation of cell
cultures with hemicholinium-3 (Fig. 4) have shown a competitive
inhibition of the low apparent Km or high affinity mechanism.Si-
milar results were obtained in synaptosomes 24-25 . No effect was
observed in incubating neuroblastoma clones NI8 and 821 with d-tu-
bocurarine 39 . A competitive inhibition of the low apparent Km
was observed when clone NI8 was incubated with 10-6M of BW 284c51,
a selective inhibitor of acetylcholinesterase (EC 3.1.1.7.) (Fig.
5 ). However, at higher concentrations (10-4M) this compound com-
pletely blocked the high affinity component (Fig.S) .
UPTAKE OF CHOLINE IN CELL CULTURES 203

Choline KmHA KmLA Refe-

uM x 1O-6M x: 1O-5M rences
Nerve cell
cultures
NIB (2-150) 2.5 3.6 (34)
821 (0.1-10) O. II B.I (34)
NN (2-150) 1.5 5.4 (35)
C6 (2-160) 0.65 2.4 (35)
NIE 115 (x) 1.4 --- (37)
826 (x) 3.5 --- (37)
C6 (x) 2.0 --- (37)
BB2 (x) 3.3 --- (37)
EBCIO (2-150) 2.0 1.0 (36)
EI4C7 (2-150) 3.2 1.0 (36)
EI4C21 (0. I-I) 0.5 6.2 (36)

Homo~enates

corpus
striatum (0.5-100) I. 43 9.3 (22)
cerebral
cortex (0.5-100) 3.0 3.3
cerebellum (0.5-100) --- 4. I

8ynapto-
somes
--
(0.5-100) 2.0 2.S (29)
(2.0- SO) 4.B 4.0 (23)
(2. 0-100) 3.5 --- (24)

Table 2: High affinity (HA) and low affinity (LA) Km's of choli-
ne uptake in nerve cell cultures and synaptosomes. NIS: neuroblas-
toma inactive clone; 821, 826: neuroblastoma cholinergic clones;
NIE 115: neuroblastoma adrenergic clone; NN: normal astroblasts
from hamster; C6: rat glioblastoma; BB2: fibroblasts 37 ; EBCIO:
B day-old chick embryos in culture for 10 days; E14C7: 14 day-old
embryos in culture for 7 days; E14C21: 14 day-old embryos in cul-
ture for 21 days. x = not specified.
204 R. MASSARELLI AND P. MANDEL

e e
I

..1
y " .1.
y "

.1 .5 1.0 .1 I
.-1..
S

Fig. 3. (left) : Kinetics of the uptake of choline in chick em-

bryo nerve cell cultures. 8 day old embryos in culture for 7 days:
mixed neuronal-glial population. S = ~M, v = nmoles uptaken/h/mg
protein.
Fig. 4. (right) : Inhibition of choline uptake by hemicholinium-3
in chick embryo nerve cell cultures. Eight day-old embryo cultures
in culture for 10 days: mixed neuronal glial population. The con-
centration of hemicholinium-3 was of 10-4M.

DISCUSSION

Since choline does not seem to be synthesized in the

nervous tissue, its uptake in nerve cells is of primary importance
for the metabolism of phospholipids and of the neurotransmitter
acetylcholine.
We have observed that the uptake of choline as a func-
tion of time can be represented by a linear function, and a satu-
ration plateau is attained following cellular maturation. More
mature cell lines like clones S20 and S21, which were in culture
longer (60-80 replications) than the other clones (30-40 rep li-
cations),and the cultures from 14 day old chick embryo could reach
a saturation plateau faster than the undifferentiated cell types.
An earlier saturation plateau of choline would then correspond to
changes in the size of choline pools caused by cellular differen-
tiation or aging.
It has been shown that the uptake of choline in nerve
cells or in synaptosomes is sodium-dependent, but only slightly
energy-dependent. Moreover the high concentrations of metabolic in-
hibitors used for these studies (Table 1 does not support a great
UPTAKE OF CHOLINE IN CELL CULTURES 205

171

J
BW284 C51
-6 .,.,P
10 M
s
y

-30 -10 0 10 30 50
s
Fig. 5 Effect of a selective inhibitor of acetylcholinesterase
on the kinetics of choline uptake in clone N18.

importance of energy in the transport of choline. A QIO of only

2.12 found in neuroblastoma clone NI8 39 would indeed suggest
that in this type of culture the uptake of choline is mediated by
a facilitated diffusion-like process (similar results have been
obtained by Haeffner 40 on the uptake of choline in Ehrlich he-
patoma cell cultures).
It has been suggested that the so-called high affinity
uptake of choline may be rate limiting in the synthesis of acetyl-
choline. Other experiments have suggested a correlation between
cholinergic areas of the brain and choline high affinity uptake
41 and some authors have drawn a correlation between high affini-
ty uptake of choline and cholinergic neurons, claiminc the possi-
bility of using such a mechanism as neuronal marker 42 . However,
it has been shown that a complex kinetics can be found for the
uptake of choline in many cell types. The high affinity uptake of
choline can be found in cholinergic as well as inactive or glial
cell lines, whether of tumoral origin or from primary cultures of
normal origin. It should be pointed out, as has been done in this
meeting by Prof. Christensen, that the representation of a trans-
port phenomenon by a curve in a Lineweaver-Burk plot usually im-
plies conformational changes caused by increasing concentrations
of substrate, and rarely to the existence of two separate mecha-
nisms of transport. It is our opinion that such a simple kinetic
206 R.MASSARELLIANDP.MANDEL

approach is not sufficient for a correct study of the transport

of choline into nerve cells.
We have, however, tried to influence the uptake of cho-
line using some pharmacologic~l agents. It has been found that
after incubation of the cultures with hemicholinium-3 the uptake
is reduced, while d-tubocurarine and ouabain had little effect or
none. A specific inhibitor of acetylcholinesterase: BW 284 c51 at
10-4M blocks completely the high affinity component, presumably in
shifting the affinity of the carrier for choline. However, no cor-
relation has been found between choline uptake and external ace-
tylcholinesterase activity in clones S21, NI8 and NN 43.
Conversely it was shown that a certain correlation
exists between choline uptake and the amount of sialic acid groups
releasable by neuraminidase at the surface of the cellular membra-
ne 44 . We are presently investigating whether these negatively
charged groups can influence the uptake of positively charged cho-
line non-specifically or whether a glycoprotein is partly respon-
sible for the transport of choline inside nerve cells.

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UPTAKE OF CHOLINE IN CELL CULTURES 207

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208 R. MASSARELLI AND P. MANDEL

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UPTAKE OF CHOLINE IN CELL CULTURES 209

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THE UPTAKE AND RELEASE OF y-AMINOBUTYRIC ACID (GABA) BY THE RETINA

M.J. Neal

Department of Pharmacology, The School of Pharmacy

29/39 Brunswick Square, London WClN lAX

INTRODUCTION

The vertebrate retina originates embryologically as an outgrowth

of the forebrain. Only about 15% of the tissue is specialized for
photoreception and the remainder is representative of CNS grey
matter. The retina possesses several experimental advantages for
neurochemical and neuropharmacological studies. In particular, it
can be isolated in vitro, intact, without any preliminary slicing
or chopping procedures:- In addition, it can be stimulated physio-
logically with flashes of light and the response of the tissue can
be monitored by recording the electroretinogram. The organization
of the retina is relatively well understood and its well defined
layered structure facilitates the correlation of neurochemical and
physiological findings with anatomical structure.

The present study concerns the uptake and release of the putative
synaptic transmitter substance y-aminobutyric acid (GABA) by the
retina. It is reasonable to assume that the retina utilizes the
same transmitter substances as the rest of the CNS and so the study
of retinal transmitters, in addition to providing information on
visual physiology, may also provide clues to understanding central
synaptic mechanisms in general.

ROLE OF GABA IN THE RETINA

As in other areas of the eNS, retinal cells are inhibited by

GABA and there is much evidence to suggest that th§ amino acid is
concerned with inhibitory mechanisms in the retina. The retinae
of all the vertebrate species so far studied contain GABA. The
concentration of GABA in the whole retina varies between species

211
212 M.J. NEAL

but is usually about 2 ~oles/g wet weight. Intraretinal localizat-

ion studies of GABA and GAD activity suggest that the amino acid
may be concentrated in amacrine and horizontal cells 7 and isolated
fusiform axons from goldfish horizontal cells are capable of
synthesizing GABA12. These studies are consistent with the inhibit-
ory function of these cells. The effects of light and dark on the
GABA system in the retina have recently been reviewed 8;but briefly
it may be said that the adaptational state of the retina does not
produce any apparent change in GAD or GABA-T activity, or GABA
levels in the rat or chicken retina, but in the goldfish and frog,
GABA levels fall in dark adapted retinae. It is not known whether
GABA has any important metabolic role in the retina.

GABA UPTAKE IN THE RETINA

The uptake of GABA by the isolated retina was first shown

using rat tissue 5,6,23 We chose this species because the rat
retina is a convenient weight (10 mg) for uptake studies. However,
we have found subsequently that the characteristics of GABA uptake
into the rabbit retina are essentially the same as those for the

24
22
(S)
20 (4)

18
16
.2 14
C;
a:: 12 0-<> [I4 C]-GABA
E (4) lO-8 M)
.~ 10
"0 .--e L3 H]-Mannitol
-''""
~

:J
III
8
6
(lO-7 M)

III
;:: 4
2 (8) (8) (8)
__ ..... ____ .... _____ - - - - - I

5 10 15 20 25 30 35 40 45 50
Incubation Time (min)

Fig. 1. Uptake ofI14C)GABA by rabbit retina. Small portions of

retina (weight approximately 10 mg) were incubated at 37 0 C in Krebs
Ringer bicarbonate medium containingt 14 C)GABA (4 x la- 8M). ,After
the incubation, the retinae were recovered, washed and dissolved
in Soluene TmlOO. The radioactivity was then estimated by liquid
scint illation spectrometry. The accumulation of [3HJmannitol was
estimated by incubating retinae withL3 H]mannitol (10-7 M). Figures
in brackets indicate the number of determinations at each time;
vertical bars indicate S.E.M. (Atterwill and Neal, unpublished
results) •
UPTAKE AND RELEASE OF GABA IN RETINA 213

rat. The uptake of [l4CJGABA by the rabbit retina is illustrated

in Fig. 1.

The retinal uptake process for GABA was found to be markably

similar to that described previously for small 'mini-slices' of rat
cerebral cortex lO . The uptake of[3HJGABA by the retina is tempera-
ture sensitive, sodium dependent and inhibited by anoxia, dinitro-
phenol and ouabain. As in other areas of the CNS, GABA transport
in the retina is saturable, but as illustrated in Fig. 2, when
the kinetic data were plotted in a linearizing form of the Michaelis-
Menton equation (l/v against 1/s) a curve was obtained from which
two straight lines could be extracted. These data suggest that the
uptake of GABA by the retina is mediated either by two transport
processes 2 or by one process which exhibits cooperativity. On the
assumption that GABA uptake is mediated by two transport processes,
the kinetic data (Fig. 2) were used to obtain estimates of the
kinetic parameters. The parameters so obtained were refined by
non-linear regression analysis (Numerical Algorithms Group Library
Programme E04FBF) in which the data was fitted direct to a series
of models including (a) a single Michaelis-Menton equation (b)
a double Michaelis-Menton equation and (c) a single Michaelis-
Menton equation + diffusion; weighting was applied as suggested by
Ottaway2Z on the basis of constant relative error (White and Neal,
in preparation). Inspection of the residual sums of squares and
the sequential distribution of residual errors indicated that a
transport system with two components (model b) accorded best with
the data, and gave the following kinetic parameters: High affinity
process, apparent Km = 0.42 ~; Vmax = 0.775 (nmoles/min)/g wet
weight. Low affinity process, Km = 171 ~, Vmax = 129 (nmoles/min)/g
wet weight.

The retinal transport process for GABA is capable of producing

a net increase in the total GABA content of the tissue 6 • Thus,
when rat retinae were incubated for 30 min in medium containing
[3H]GABA (0.5 rnM), the total GABA content of the tissue was approx-
imately three times higher than that of retinae incubated for the
same time in GABA-free medium. It is not possible from this
experiment to decide whether the high or low affinity (or both)
transport process for GABA is responsible for this net uptake, since
with an external GABA concentration of 005 rnM, the high and low
affinity processes would be expected to contribute to the GABA
uptake. Some' of the [3HJGABA must enter the ret ina by exchanging
with a part of the endogenous GABA store because the uptake of[3H]
GABA was higher than the net increase in tissue GABA content 6 .

The specificity of the high affinity uptake process for GABA

in the rat retina is very similar to that described previously in
rat cortical slices lO The uptake ofl3HJGABA (10-8M) by the retina
was unaffected by a large molar excess of related amino acids such
as glycine, alanine, histidine, proline, aspartate, glutamate, or
214 M.J. NEAL
100

80

60

40
.!!

800 ~ 20
S

....
600 .
~
c
c
0

-20
..1 x 10-6 ""
u
V 400 -40

-60

-80
I
-100
400 600 800 1000
..1.10"3 I nl1 10nl1 100nl1 111" 10,,11 100,.,.. ImH IOml1
5 AOAA concentration

Fig. 2 Fig. 3

Fig. 2. Kinetic analysis of the effect of GABA concentration

on the accumulation of l4C GABA by rabbit retina. Estimates of
the initial velocity (V) of GABA uptake were obtained by incubat-
ing small portions of rabbit retina at 37 0 C in Krebs Ringer
brcarbonate medium for 5 min and subsequently measuring the radio-
activity accumulated by the tissue. The results are expressed in
the reciprocal form of the Michaelis-Menton equation. Each value
is the mean of at least 8 experiments. V = Velocity of GABA
uptake (10- 9 moles~min~g wet weight). S = GABA concentration (10-6M).
(Atterwill and Neal, unpublished results.)

Fig. 3. Upper panel: Effect of different concentrations of

AOAA on the accumulation of 3H GABA by rat retinae incubated at
37 0 C for 60 min in the presence of 3 H GABA (50 ~M). The retinae
were preincubated with AOAA for 15 min at 37 0 C before the addition
of 3 H GABA. The results are plotted as the percentage increase of
3H GABA accumulation above the accumulation of GABA obtained in the
absence of AOAA. Lower panel: Effect of AOAA on the activity of
GABA-T in retinal homogenates prepared from retinae which had
previously been incubated with AOAA for 15 min at 37 oC.. Each result
is the mean of at least six experiments. The vertical bars = S.E.M.
(From Neal and Starr, 1973 21 .)

2-aminobutryate. The retinal uptake of [3 HJGABA was inhibited 50%

by L-2,4,diaminobutryate (DABA) 58 ~M; S-guanidinopropionate, 63 ~M;
DL-y-amino-S-hydroxybutryate, 80 ~M; and S-alanine, 390 ~M. This
UPTAKE AND RELEASE OF GABA IN RETINA 215

pattern of specificity in the rat retina is unusual in that it

represents a glial uptake process (see later) for GABA which is
more sensitive to inhibition by DABA than by ~-alanine. In other
areas of the CNS, and in the peripheral nervous system, GABA
transport into neurones is more sensitive to inhibition by DABA
and glial uptake is more sensitive to inhibition by ~-alanine9.

EFFECT OF INHIBITORS OF GABA-AMINOTRANSFERASE (GABA-T) ON THE

ACCUMULATION OF[3H]GABA BY THE RETINA

The accumulation of(3 H]GABA by rat retina and rat cortical

slices was found to be greater at 2SoC than at 37°C. This was
thought to be due to the[3H]GABA in the tissue being more rapidly
metabolized at the higher temperature, followed by the loss from
the tissue of labelled metabolites. This hypothesis was tested in
the rat retina by studying the accumulation of[3HJGABA following
inhibition ~f GABA-T, the enzyme responsible for its metabolic
degradation 1.

In the presence of the GABA-T inhibitor, aminooxyacetic acid

(AOAA) (10 ~), the accumulation oB[3H]GABA by the retina was
doubled after 60 min incubations, although over shorter periods
it had no effect and in the retina, AOAA has no significant effect
on the initial velocity of GABA uptake except at high concentrations
(10 mM).

As illustrated in Fig. 3, the increased accumulation ofeIi1

GABA by the retina in the presence of AOAA appears to be directly
related to the inhibition of GABA-T. Thus, the maximum potentiation
of[3H]GABA accumulation did not occur until the retinal GABA-T
activity was essentially 100% inhibited. This suggestion is support-
ed by the fact that other GABA-T inhibitors also increasedL3H]GABA
accumulation by retina and this increase paralleled the degree of
GABA-T inhibition.

SITES OF GABA UPTAKE

Subcellular Distribution ofCH)GABA. The subcellular distribut-

ion of(3HJGABA accumulated by the retina was studied l ,18 in the
rabbit because, at the time these experiments were undertaken,
autoradiographic studies seemed to indicate that GABA uptake into
the rabbit retina was largely neuronal, but also because the larger
size of retina was necessary to readily obtain the necessary amount
of tissue. However, subsequent studies revealed that(3H]GABA uptake
in the rabbit retina is both neuronal and glial.

Rabbit retinae were incubated with(3 H1GABA and homogenized in

0.32M sucrose. The homogenates were then subjected to differential
and density gradient centrifugation techniques 18 • It was found that
only about 20-30% of the endogenous and{!HJGABA was present in a
216 M~.NEAL

particulate form. Of this particle bound GABA, about half was

present in the crude mitochondrial fraction (P2) and half in the
crude nuclear fraction (PI). This result was in striking contrast
to similar work involving the subcellular fractionation of cortical
slices, where it was found that only about 10% of the[3H]GABA and
endogenous GABA was in Pl but 50% was in P2 19. This difference
in the distribution of particle bound GABA between cortex and
retina may be due to the presence in the retinal Pl fraction of
large pinched-off photoreceptor terminals 14 ,18. These terminals
themselves are not thought to be GABAergic, but they possess
invaginations into which horizontal cell and bipolar cell processes
project. The terminals of these cells are frequently observed in
the invaginations of the pinched-off photoreceptor terminals, and
since the horizontal cells are thought to be GABAergic 8 this
'trapping' of small horizontal cell nerve terminals in the larger
photoreceptor terminals might be responsible for the relatively
large porportion of GABA (and GAD) found in the PI pellet.

When the crude mitochondrial fraction (P2) from retina was

subjected to density gradient centrifugation on continuous or
discontinuous sucrose gradients, more than 65% of the[!HJGABA was
recovered in a particulate form. Using continuous sucrose gradients
it was found that the particles accumulating[3H]GABA had a median
equilibrium density of 1.2 to 1.3 M sucrose. Exposure of the P2
pellet to osmotic shock before density gradient centrifugation
reduced the particle bound[3H1GABA by more than 50%. Experiments
using four-step, discontinuous, sucrose gradients revealed that the
(3HJGABA was accumulated predominantly in particles which had the
same position on the gradients as most of the GAD activity. As in
the retina GAD is believed to be localized largely in neurones; the
present results are consistent with[3H1GABA being accumulated in
GABAergic synaptosomes. However, this conclusion must be very
tentative since E.M. studies of retinal subcellular fractions have
revealed that MHller cell processes may also be pinched-off during
the homogenization process to produce 'gliasomes,l8, and the position
of these particles (if any) on sucrose gradients has not yet been
determined.

Autoradiographic Localization of GABA Uptake Sites. The sites

of uptake of GABA in the retina have been extensively studied by
autoradiography. The results of these studies have been somewhat
confusing, and the sites of GABA uptake in the retina seem to vary
considerably with different species and also with the technique
used to label the tissue. In general, the evidence available at
present suggests that non-mammalian retinae may take up labelled
GABA predominantly into neurones, whereas mammalian retinae tend
to accumulate GABA in the neuroglial MHller cells. However, the
picture in mammalian retinae is not quite clear-cut, and in some
species neuronal uptake as well as glial uptake is seen if the
labelling is accomplished by intravitreal injection in ~.
UPTAKE AND RELEASE OF GABA IN RETINA 217

In one of the earliest autoradiographical studies of GABA

localization Ehinger and Falck4 found that in the rabbit retina
[3H]GABA was accum~lated in the inner synaptic layer, some ganglion
cells and in some cells situated in the inner part of the inner
nuclear layer, which they tentatively identified as amacrines.
This pattern of labelling was essentially the same when the retina
was labelled by intravitreal injection or by incubation of the
isolated retina, therefore the polar distribution of theI3HJr,ABA
could not be due merely to a concentration gradient. The number
of cells in the inner nuclear layer taking up GABA was smaller
than the total number of amacrines, indicating that the cells
taking up GABA must be a subgroup of amacrines. The uptake of
eH]GABA by ganglion cells was variable but could not have been due
to intraretinal cells since after chronic sectioning of the optic
nerve no ganglion cells were seen to take up GABA. A neuronal
uptake of [3HJGABA was also reported in the goldfish retina both
in ~ and following intravitreal injections 13 . The GABA was
accumulated predominantly in horizontal cells and in cells with
the position of amacrines. A much greater uptake of GABA was
observed in horizontal cells of light stimulated retina. A
predominantly neuronal uptake of[3HJGABA has also been reported
in the frog 24 , chicken 15 , and pigeon 15 retina in vitro.

A different pattern of labelling was found in the isolated

rat retina by Neal and Iversen 20 • In this species the retinal
uptake of (:J:i]GABA appeared to be predominantly into the neuroglial
MUller cells. The failure to demonstrate neuronal uptake in this
retina was not due to a rapid metabolism of GABA in neurones with
a subsequent loss of labelled metabolites, since in the presence
of AOAA the same pattern of uptake was observed. Glial uptake by
the isolated rat retina was confirmed by Bruun and Ehinger 3 and
Marshall and Voaden 16 •

The factors influencing the sites of uptake of GABA have been

somewhat clarified by Bruun and Ehinger 3 who found that both the
species of animals and the method of labelling were important.
Thus,[3H)GABA applied by intravitreal injection into eyes of rats,
guinea pigs, cats, and monkeys was preferentially accumulated by
amacrines, but in vitro labelling resulted in glial uptake except
in cats and gui~a-pigs, where labelled amacrines were still
observed. In rats and monkeys glial uptake of[3 H]GABA often disguis-
ed amacrine uptake even in vitro. In a recent species comparison
of GABA uptake in mammalian retinae labelled in vitro, Marshall
and Voaden 16 found a predominantly glial uptake in the cat, baboon,
guinea-pig, goat, and rabbit retina, although in the rabbit retina,
amacrine cell uptake was also observed.

In contrast to GABA, glycine always seems to be.taken by

retinal neurones, whatever the species and both in vivo and in ~.
218 M.J. NEAL

Similarly glutamate is consistently taken up by glial cells in

the retina. It is not known why the pattern of GABA uptake shows
such wide species variation.

EFFLUX OF GABA FROM THE RETINA

There have been several reports on the release of(3HJGABA

from the superfused retina in ~, but as far as I am aware
none from the retina in ~.

The spontaneous resting release of l3H] GABA from the isolated

rat retina is low in the presence of AOAA6,25, less than 10% of
thel3H1GABA accumulated during the loading incubation being releas-
ed during a subsequent superfusion of 1 hr. If the retinal GABA-T
is not inhibited, then (3Ii) GABA is rapidly metabolized by the tissue
and 90% of the radioactivity in the retina is lost within 30 min:
Of this released radioactivity, less than 22% is present as[3H)
GABA; th~ remainder, which is not retained on Amberlite CG120 resin
in the H form is probably tritriated H206. Since the glial
Mtiller cells which accumulate [3H) GABA in the rat retina are also
the major site of GABA-T activity in the retina, this rapid
metabolism of accumulated [3H] GABA is perhaps not unexpected.

The efflux of[3H]GABA from the rat retina in the presence of

AOAA is increased by depolarizing stimuli such as high potassium
concentrations and electrical field stimulation but not by stimulat-
ion with a flashing light 17 ,25. The failure of light stimulation
to increase the release of(3H)GABA from glial cells is not surpris-
ing, but similar experiments with the frog retina, which takes up
3H GABA into horizontal and amacrine cells, also failed to demon-
strate a light-evoked release of[3HJGABAll. As in the rat retina,
the efflux of 3H GABA from the superfused frog retina was increased
by electrical stimulation, and by 40 roM potassium. The increase
in[3HJGABA efflux produced by electrical stimulation was not calcium
dependent, but the response to 40 roM potassium was almost abolished
by the absence of calcium in the medium. This calcium dependence
of the potassium evoked[3~GABA release, but apparent non-dependence
of the electrically evoked release is identical to previous studies
on the release of[3H] GABA from cortical slices.

Recently we have studied the release of endogenous GABA, and

other amino acids, from the isolated rat retina but have so far
failed to detect any increase in the release of GABA following
photic stimulation of the tissue. These results are in agreement
with those using the isolated fowl retina, where Pasantes-Morales
et al. 22 also found that photic stimulation did not increase the
release of[3HJGABA or endogenous GABA, although the same stimulation
produced clear increases in the efflux of both labelled and
endogenous taurine.
 UPTAKE AND RELEASE OF GABA IN RETINA 219

ACKNOWLEDGEMENTS

I am grateful to the M.R.C., the University of London Central

Research Fund" and the S.K.F. Foundation for grants.

REFERENCES

1. Atterwill, C.K., and Neal, M.J., The subcellular distribution

of l4C-y-aminobutyric acid, GABA, and 3H-dopamine in the
rabbit retina, Brit. J. Pharmacol., 48 (1973) 355-356.
2. Bond, P.A., The uptake of y-[3HJaminobutyric acid by slices
from various regions of rat brain and the effect of lithium,
J. Neurochem., 20 (1973) 511-517.
3. Bruun, A., and Ehinger, B., Uptake of certain possible neuro-
transmitters into retinal neurones of some mammals, Exp. Eye
Res., 19 (1974) 435-447.
4. Ehinger, B., and Falck, B., Autoradiography of some suspected
neurotransmitter substances: GABA, glycine, glutamic acid,
histamine, dopamine, and L-DOPA, Brain Res., 33 (1971) 157-172.
5. Goodchild, M., and Neal, M.J., Uptake of 3H-gamma-aminobutyric
acid (GABA) by rat retina, J. Physiol., 210 (1970) l82-l83P.
6. Goodchild, M., and Neal, M.J., The uptake of 3H-gamma-amino-
butyric acid by the retina, Brit. J. Pharmacol., 47 (1973)
529-542.
7. Graham, L.T., Intraretinal distribution of GABA content and
GAD activity, Brain Res., 36 (1972) 476-479.
8. Graham, L.T., Comparative aspects of neurotransmitters in the
retina. In H. Davson and L.T. Graham (Eds.), The Eye, Vol. 6,
Comparative Physiology, Academic Press, New York, 1974, pp.
283-342.
9. Iversen, L.L., and Kelly, J.S., Uptake and metabolism of y-
aminobutyric acid by neurones and glial cells, Biochem. Pharmacol.
24 (1975) 933-938.
10. Iversen, L.L., and Neal, M.J., The uptake of 3H- GABA by slices
of rat cerebral cortex, J. Neurochem., 15 (1968) 1141-1149.
11. Kennedy, A.J., and Voaden, M.J., Factors affecting the spontaneous
release of 3H -y-aminobutyric acid from the frog retina in
vitro, J. Neurochem., 22 (1974) 63-71. --
12. Lam, D.M.K., Biosynthesis of y-aminobutyric acid by isolated
axons of cone horizontal cells in the goldfish retina, Nature,
254 (1975) 345-347.
13. Lam, D.M.K., and Steinman, L., The uptake of y- l 3H]aminobutyric
acid in the goldfish retina, Proc. Natn. Acad. Sci. U.S.A.,
68 (1971) 2777-2781.
14. Marshall, J., Medford, P.A., and Voaden, M.J., Subcellular
fractionation of the rabbit retina: the isolation of synaptic
pedicles and inner segments of photoreceptor cells, Exp. Eye
Res., 14 (1974) 559-569.
220 M~.NEAL

15. Marshall, J., and Voaden, M.J., An autoradiographic study of

the cells accumulating 3H-y-aminobutyric acid in the isolated
retinas of pigeons and chickens, Invest. Qphthal., 13 (1974)
602-607.
16. Marshall, J., and Voaden, M.J., Autoradiographic identification
of the cells accumulating 3H-y-aminobutyric acid in mammalian
retinae: A species comparison, Vis. Res., 15 (1975) 459-461.
17. Neal, M.J., (unpublished results).
18. Neal, M.J., and Atterwill, C.K., Isolation of photoreceptor
and conventional nerve terminals by subcellular fractionation
of rabbit retina, Nature, 251 (1974) 331-333.
19. Neal, M.J., and Iversen, L.L., Subcellular distribution of
endogenous and(3H)y-aminobutyric acid in rat cerebral cortex,
J. Neurochem., 16 (1969) 1245-1252.
20. Neal, M.J., and Iversen, L.L., Autoradiographic localization
of 3H-GABA in rat retina, Nature, New Biol., 235 (1972) 217-218.
21. Neal, M.J., and Starr, M.S., Effect of inhibitors of y-amino-
butyrate aminotransferase on the accumulation ofL3Hj-y-amino-
butyric acid by the retina, Brit. J. Pharmacol., 47 (1973) 543-
555.
22. Pasantes-Morales, H., Klethi, J., Urban, P.F., and Mandel, P.,
The effect of electrical stimulation, light and amino acids
on the efflux of 35S-Taurine from the retina of domestic fowl,
Exp. Brain Res., 19 (1974) 131-141. 14
23. Starr, M.S., and Voaden, M.J., The uptake of C-y-aminobutyric
acid by the isolated retina of the rat, Vision Res., 12 (1972)
549-557.
24. Voaden, M.J., Marshall, J., and Murani, N., The uptake of
3H- y- aminobutyric acid and 3H-glycine by the isolated retina
of the frog, Brain Res., 67 (1974) 115-132.
25. Voaden, M.J., and Starr, M.S., The efflux of radioactive GABA
from rat retina in~, Vision Res., 12 (1972) 559-566.
AMINO ACID TRANSPORT IN ISOLATED

NEURONS AND GLIA

A. Hamberger, B. Nystrom, A. Sellstrom, and C.T. Woiler

Institute of Neurobiology
University of Goteborg
Fack, S-400 33
Goteborg, Sweden

INTRODUCTION

Glial cells, particularly astroglia, are thought to play an

essential role in regulating the levels of many com~onents in the
extracellular space of the central nervous system 5. Changes in
extracellular potassium concentration which accompany neuronal cell
activity for example, have been shown to trigger a number of
activities, such as uptake of potassium chloride and water into
astroglia during simultaneous increase in glial cell oxygen con-
sumption 22 • Thus a tight coupling of neuronal firing with glial
supportive activity has been suggested on the basis of work done
in the fields of ultrastructure, neurophysiology and biochemistry.
Closely connected with water and ion fluxes is the transport of
amino acids into and within the central nervous system. The
microdistribution of amino acids may well influence the composition
of brain proteins. A different aspect concerns the so-called
transmitter amino acids, namely, y-aminobutyric acid (GABA),
glutamate, aspartate, glycine, taurine, etc., in which case amino
acid transport may influence more directly neuronal electrical
activity. Virtually all interest to date has been directed toward

221
222 A. HAMBERGER ET AL.

nerve-endings as regulators of extracellular transmitter amino

acid levels through reuptake in analogy with amine transmitter
systems 26, 28, 29. But during the last few years increasing
evidence has appeared through widely different approaches that
astroglial cells may be important regulators of transmitter amino
acid uptake, release and metabolism 11,19,24,25,27.

BULK-PREPARED NEURONAL AND GLIAL CELL FRACTIONS

AS MODELS FOR AMINO ACID TRANSPORT STUDIES

There exists at present histochemical and autoradiographical

methods for studying discrete neuronal and glial cells which do not
require their physical separationl,lO, but the technical difficulties
are immense in the case of small,water-soluble, metabolically labile
molecules such as amino acids. The study of comparomentation of
amino acid transport and metabolism requires in most cases methods
which allow a separate study of the different cell-types present in
the brain. Single neuronal and glial cells can be separated by hand-
dissection 23 , 37, but the yield of tissue is too small for suitable
transport studies. Large amounts of neuronal and glial cells can be
obtained through tissue-culture of glioma or neuroblastoma cell
lines 12, 14, 50. The advantage of high viability of tissue-cultured
material, however, must be considered in relatiQnship to the problem
of malignant transformation. Selectively altered nervous tissue where
glia are made to predominate by cutting of specific fiber pathways
9, 41, or the use of tissue-models containing glia but devoid of
nerve-endings such as dorsal root ganglion, represent additional
ways to approach the problem 4, 44, 45, 48, 49. In our laboratory
we have preferred a direct approach to compartmentation by studying
the properties of bulk-isolated fractions of astrocytes and neuronal
perikarya from normal cerebral cortex in parallel with nerve endings
17. Currently employed procedures for the separation of neurons and
glial cells in bulk have been reviewed in a series of recent publi-
cations 43, 46, 51. The methods yield neuronal perikarya shorn of
their dendrites and astroglial cells which retain most of their pro-
cesses. The possibility of obtaining meaningful data depends on the
degree of integrity, purity and homogeneity of the fractions. Both
cell types appear to undergo unavoidable losses in cytoplasmic con-
stituents such as small molecules and soluble enzymes during the
separation procedure, but maintenance of characteristics such as
Na+K+-ATPase activity, active amino acid and cation transport, oxygen
consumption, and protein synthesis indicate that the cells have fully
AMINO ACID TRANSPORT IN NEURONS AND GLiA 223

retained their membrane and organelle-associated properties.

The purity of the fractions is high and certainly sufficient
to show specific properties of each cell type. The main disadvantages
other than loss of soluble constituents are (1) loss of dendrites in
the case of the neuronal fraction and (2) heterogeneity with respect
to each cell type. With respect to heterogeneity, efforts to approach
the problem via E.M. autoradiography combined with quantitative data
on neuronal and glial fractions have been hampered by the fact that
over 90 % of the amino acid radioactivity is washed out during fix-
ation, in contrast to the case in slice work 26

GENERAL FEATURES OF AMINO ACID TRANSPORT IN NEURONAL AND

GLIAL CELLS

Transport capac1t1es for amino acids by neuronal and glial

cells are given in Table 1. The index for amino acid concentrative
capacity used is tissue/medium ratio of radioactive amino acid after
a short pulse of exposure to label. The glial cells are more potent
concentrators than the neuronal perikarya for all amino acids 16.
This is particularly so for the transmitter amino acids glutamate,
aspartate and GABA. In these cases synaptosomes are even better amino
acids concentrators than the glial cells 19, 20. Glycine is not a
transmitter in cerebral cortex but only in spinal cord 57 • It may
be that the low transport in neuronal perikarya is due to the loss
of dendrites. For comparison, uptake of the amine transmitters sero-
tonine and norepinephrine into glial cells is of small importance,
whereas synaptosomal uptake is higher. It is possible that the trans-
mitter amino acid/amine differences represent genuine differences in
cellular uptake mechanisms for transmitter inactivation.
The high glial transport compared to neurons has been confirmed
in experiments where slices were exposed to label and then fraction-
ated rather than incubating the fractions themselves 18. Furthermore,
the transport enzyme, Na+K+-ATPase, was 2-3 times higher in purified
glial plasma membrane than in neuronal plasma membrane 15, 21, a
result which has been confirmed in the whole isolated cell fractions
using a different method 38. It ought to be noted, however, that
amino acid (and potassium) uptake in neuronal perikarya of the same
magnitude as in glial cells was found by Nagata et ale 40, using a
similar method for bulk-isolation of cells. This high amino acid
uptake in neuronal perikarya was paralleled by a high level of endo-
genous amino acids which did not differ much from the level in the
glia. In our system, the glial level of endogenous amino acids ex-
ceeds that of neuronal perikarya by 4-5 times 56. A number of studies
employing autoradiography to localize amino acid uptake also support
the finding that glial cells are more active concentrators than
neuronal perikarya 11, 24, 25.
224 A. HAMBERGER ET AL.

TABLE 1. UPTAKE OF AMINO ACIDS AND TRANSMITTERS INTO FRACTIONS

OF NEURONAL PERIKARYA, GLIAL CELLS AND NERVE ENDINGS
mM Neurons Glia Synapt-
osomes
L-(U_ 14 C)Phenylalanine 1.0 1.4 1.4
0.1 1.4 2.4
L-(U- 14 C)Valine 1.0 1.4 1.5
0.1 1.4 5.1
(1_14 C)-a-Amino- 1.0 1.4 2.5
isobutyric acid 0.1 1.4 5.1
L_(U_ 14 C) Threonine 1.0 2.3 5.7
0.1 2.3 11.4
L-(U_ 14 C)Alanine 1.0 1.5 3.7
0.25 2.3 7.9
14) _
L- ( U- C LeUC1ne 0.2 1.2 2.8
0.1 1.7 3.5
14) _
L- ( U- C Tyros1ne 1.0 1.2 3.0
0.1 2.2 9.6
14) _
L- ( U- C Lys1ne 0.2 1.3 3.6
0.1 2.1 4.9
14) _
L- ( U- C Ser1ne 1.0 2.1 6.7
0.1 2.6 16.0
L-(U- 14 C)Proline 0.2 2.1 7.9
0.1 2.5 9.9

(1_14 C)-y-Amino- 1.3 1.3 3.5

butyric acid 0.0001 8 53 165
14) _
L- ( U- C Glyc1ne 1.0 1.3 4.2
0.1 1.6 7.9
L- ( U- 14) . acid
C Aspart1c 1.0 1.6 7.0
0.01 8.0 22.8 77 .0
L-(U_ 14 C)G1utamic acid 1.0 1.8 15.6
0.01 5.4 32.0 100.8
CIJ
H
Q)
Serotonin 0.001 3 3 10
Q)
+J
+J
0.0005 3 3 10
C
-,-I
.,-1
S
0.0002 4 3 28
~ C\j~ Norepinephrine 0.001 3 3 7
H
+J Dopamine 0.0005 3 4
Uptake figures refer to: Free radioactivity in total pellet water
/Free radioactivity in the medium at the end of incubation. Incuba-
tion conditions: 37 0 , 5-30 min.
Data collected from published papers 16, 19, 61.
AMINO ACID TRANSPORT IN NEURONS AND GLiA 225

HIGH-AFFINITY UPTAKE IN RELATION TO TRANSMITTER INACTIVATION

Iversen and Kravitz 28 first demonstrated a high-affinity GABA

uptake system to be present in the crustacean neuro-muscular junc-
tion, while a specific localization of GABA uptake into mammalian
nerve-terminals was simultaneously shown by Iversen and Snyder 30.
In general, high-affinity sodiu~5dependent uptake of GABA, glutamate,
aspartate and glycine (~<5 xlO M), has been considered to be a
necessary criterion for a transmitter function of the amino acids
5, 35, 36. In synaptosomes, the non-transmitter amino acids such as
leucine, alanine, etc. show only low-affinity kinetics (Km>5x10- 4M)
whereas the transmitters show both kinetics components, so that high-
affinity uptake presumably represents a specific transmitter-termin-
ation process located, until recently, at least in nerve-terminals
by Snyder's group 35, 36, 60.
High-affinity uptake of labelled GABA and glutamate, however,
has also been unequivocally demonstrated in our gaboratory , using
bulk-isolated glial cells from cerebral cortex 1 , as well as for
glial cells in tissue-culture 20,50, and for dorsal root ganglia
which contain no nerve-endings 44. In contrast to the dual kinetic
behavior of glutamate, glutamine, which is not neuroactive and which
is a major metabolite of glutamate in the brain, shows only a single
low or intermediary Km in both glia (1.2 x 10-4M) and synaptosomes
(1.4 x 10-4M) 61, which is more typical of the single-affinity kine-
tics of non-transmitter amino acids. This has already been shown for
nerve-endings in earlier studies 3, 45. On the other hand, dorsal root
ganglia which presumably represent a glial model, did exhibit a high-
affinity Km with respect to glutamine in one of these studies 45. The
implications for glutamate/glutamine compartmentation are discussed
more extensively in a later section (vide infra).
Autoradiographic analysis of GABA uptake in synaptosomes gives
substantial support to the idea of a specific localization of the
high-affinity uptake system to unique populations of GABAnergic ter-
minals 26. Unique profiles of labelled amino acids on sucrose gradi-
ents have also been resolved from each other by "incomplete density
gradient centrifugation" by Snyder and co-workers with respect to
glutamate/aspartate vs. GABA in cerebral cortex and glycine in spinal
cord 57, 60. In our own autoradiographic studies, the uptake of GABA
into glial cells shows an even distribution into the total population
of astrocytes rather than any specific accumulation in discrete sub-
populations of astroglia. Nevertheless, a glial transmitter inacti-
vation role, at least for the amino acids, is a good probability in
view of the anatomical association of the intertwining processes of
glial cells at synaptic sites 13

METABOLIC AND IONIC REQUIREMENTS FOR AMINO ACID UPTAKE

Most amino acid uptake systems are active, i.e. against a con-
centration gradient. Accordingly, uptake of either transmitter or
226 A. HAMBERGER ET AL.

non-transmitter amino acids in neurons and glia would be expected to

be energy-dependent, i.e. abolished at 00 and inhibited by metabolic
uncouplers such as dinitrophenol. This, indeed, is what has been
found for GABA 19. Inhibition by ouabain 19, however, implies linking
of the transport system to Na+/K+-ATPase which combined with the
evidence for absolute sodium-dependence 53 indicates that glia and
neuronal perikarya, like synaptosomes, possess high-affinity uptake
systems driven by the sodium and potassium gradients. A mutually de-
pendent binding of sodium and amino acid to a carrier on the outside
of the membrane has been proposed as the main mechanism for GABA
transport 32. Binding would occur at high external sodium concentra-
tions while release to the inside of the cell would occur at low
intracellular concentrations of sodium. The role of potassium in
amino acid transport is perhaps more difficult to explain: maximal
uptake is obtained at approximately 5 rnM, the extracellular K+-con-
centration, and falls off on either side of this peak, but never to
zero as in the case of sodium 53. We have shown that glial accumula-
tion of K+ greatly exceeds neuronal, in parallel with amino acid
uptake 15. Conceivably, amino acid and potassium could be co-trans-
ported by th~ high-affinity carrier via displacement of sodium.
The Ca+ requirement for synaptic release has by now become a
sought-after criterion for demonstrating central neurotransmission
in vitro 31 • Of related importance then are the opposite effects
Ca++ exerts on the GABA re-uptake process in glia and synaptosomes 53 .
It has been found that lowering Ca++ in the external medium from
1.5 - 0 rnM stimulates glial uptake of GABA. However, interestingly,
its effect on synaptosomal uptake is the reverse. The stimulation of
glial uptake of GABA by withdrawal of Ca++ is paralleled by the sti-
mulating effect of Ca++-withdrawal on potassium accumulation 15. This
suggests a mechanism by which the uptake of Ca++ into the nerve-
endings during synaptic release of GABA could trigger glial uptake of
GABA and potassium while inhibiting re-uptake of the amino acid into
the nerve terminal. In this way the translocation of amino acid from
the nerve-terminal to the glial cell and post-synaptic site would be
promoted during synaptic activity. A unidirectional flux of GABA from
the synaptosomal to the glial compart~ent is also implied by the
predominant localization of glutamate decarboxylase (GAD), the enzyme
of GABA synthesis, in the nerve-terminals 47, 56, in contrast to GABA-
transaminase, the enzyme of GABA degradation, which is much more
active in the glial compartment 56. Thus there appears to be meta-
bolic data in support of a glial transmitter-inactivation role.

AMINO ACID RELEASE FROM ISOLATED FRACTIONS AS STUDIED BY

PERFUSION

In addition to possessing high-affinity uptake systems presum-

ably subserving inactivation roles for putative amino acid transmit-
ters, isolated glia and neuronal perikarya also exhibit the ability
AMINO ACID TRANSPORT IN NEURONS AND GLiA 227

Fig.l - Superfusion system. Preloaded cell fractions are centrifuged

to form a pellet on a filter paper supported by a perforated steel
disc and inserted in the chamber. The fluid reservoir above is filled
by pipetting 10 ml portions from an oxygen-gassed bottle. The fluid
is drawn down by a pump to vials in a fraction collector. The upper
pump and tubing are inserted for applying short pulses of media of
different composition. The system is used in a thermostatically reg-
ulated room at 37 oC.

to release labelled amino acid in response to high K+ in the medium.

This is best studied by means of a perfusion system (Fig.l)54. A
typical experiment involves preloading cells with labelled amino
acid,placing them in the perfusion chamber, and collecting fractions
into scintillation vials while altering the composition of the medi-
um after 15 minutes of spontaneous efflux. The effect of a three
minute exposure to 15 mM KCl on release of 3H-GABA from glial cells
is shown in Fig. 2 55. A similar pattern is exhibited by neuronal
perikarya and synaptosomes. Neither NaCl or choline chloride applied
at the same concentration led to release. The release phenomenon im-
plies that amino acid transport is bidirectional, involving an eff-
lux as well as an uptake component which appear to be the reverse of
each other, in that conditions which inhibit uptake are similar to
228 A. HAMBERGER ET AL.

120
T

~ 80
Q.
U

,.."
..
. ............ ;
I \

"..... ..........
\
40
~ .
,/

5 15 min

Fig.2 - Wash-out curve after GABA pre-loading and K+-stimulated

release. Each point represents the radioactivity per 1 min fraction
(1.2 - 1.5 ml). The peak represents the response to a pulse of
medium containing 15 rnM KCl.

those leading to high efflux rates and vice versa. Thus, ouabain and
low sodium stimulate GABA efflux from pre-loaded fractions 34 , where-
as they inhibit uptake. Of the greatest interest is that K+-stimul-
ated release of amino acids from glial cells could imply a physi-
ologically important mechanism of modulation at the synaptic site;
i.e. neuroactive amino acids released from the glial compartment in
response to high K+ during pre-synaptic activity could compete with
synaptically released amino acid for occupation of post-synaptic
receptors.
In our hands, K+-stimulated glial release of GABA failed to
show the "classical" Ca++-dependence shown for synaptosomes;
failure to exhibit calcium-dependence 55wou ld be expected of a non-
vesicle-mediated phenomenon. Neuronal perikarya, on the other hand,
showed calcium-dependence for release, like synaptosomes, and here
we are not dealing with vesicles.

INHIBITORS AS TOOLS TO STUDY CELL SPECIFICITY OF AMINO ACID

UPTAKE
· . . ++
Ot h er than t h e 1nterest1ng and 1mportant case of Ca ,there
did not appear to be any major differences in the characteristics of
uptake and release in glia compared to synaptosomes. Therefore,
cell-specific differences have been sought by means of inhibitor
studies. After having tested large series of GABA analogues, Iversen
AMINO ACID TRANSPORT IN NEURONS AND GLiA 229

80000

60000

40000

20000

GLIAL CElLS
SYNAPTOSOMES

o+---~-----r----~--~-----r----'
o 10 15 20 25 30
MIN

Fig.3 - Stimulation of 3H-GABA release from glia and synaptosomes

by perfusion with lO-3M S-alanine. Aliquots of glial cells and syn-
aptosomes were incubated for 5 min in medium containing 3H- GABA and
the suspensions were then centrifuged onto a filter paper, followed
by superfusion (cf. Fig. 1). At the arrow the perfusion medium con-
tained lO-3M S-alanine. 3H-GABA release was stimulated 25% for
synaptosomes and 45% for glial cells.

and collaborators found that while being a poor inhibitor of GABA

uptake in dorsal root ganglion used as a glial model, L-2,4-diamino-
butyric acid (DABA)was a potent inhibitor of GABA uptake in cortical
slices used as a synaptosomal model 27, 49. In contrast, S-alanine
inhibited glial GABA uptake in dorsal root ganglia but not in corti-
cal slices. These findings have been confirmed by autoradiography49.
In our own hands DABA inhibited GABA uptake equall~ well both in
bulk-isolated fractions of glia and synaptosomes 5 . The most recent
studies from our laboratory involving exchange-type release experi-
ments have, however, been in agreement with the dorsal root ganglion
studies of Iversen, at least with respect to S-alanine. Thus, cells
preloaded with 3H-GABA show a stimulated release of radioactivity
230 A. HAMBERGER ET AL.

during perfusion with unlabelled GABA. We have shown this homo-

exchange to be even more effective in glial cells 54 than in synapt-
osomes 34 • When glial fractions and synaptosomes preloaded with
3H- GABA were perfused with S-alanine, the glia again released GABA
at a considerably greater rate than synaptosomes (Fig.3). The
results suggest that the glial pool of GABA which is exchange-
releasable by GABA and GABA analogues is larger than that of synapto-'
somes. This, in turn, may imply that synaptosomal GABA may be comp-
artmented into separate bound and unbound pools, since a particul-
ate-bound pool of GABA would not be releasable by exchange. This
suggestion is also supported by our previous finding that glial
release of GABA is not calcium-dependent in contrast to synapto-
somal release, since calcium-dependence implies release from a bound,
presumably vesicle-attached, transmitter pool.

GLUTAMATE-GLUTAMINE COMPARTMENTATION

Glutamine in the brain is rapidly synthesized from a small pool

of glutamate sequestered from the larger metabolic pool 7. Most in-
vestigators agree that"the glial compartment corresponds to the
small glutamate pool 2, but are not so certain as to whether a
transmitter compartment in synaptosomes also corresponds to the
small glutamate pool. Very little glutamine synthetase is recovered
in nerve-endings 47 , while the specific activity of glutamine is very
low in synaptosomes compared to slices following incubation with
labelled glutamate 8. However, it can be argued that glutamine
synthetase is washed out, since it has a dual soluble-microsomal
localization 52. Our own studies confirm this dual subcellular
localization even when the enzyme is assayed properly by a method
which inactivates interfering ATP-ase activity 6, 42. The difficulty
in assessing neuronal/glial/synaptosomal differe~ces in glutamine
synthetase activity does not apply to glutaminase, which is a mito-
chondrial enzyme 47. Fig. 4 shows that glutaminase activity is
greatest in synaptosomes and least in glial cells, especially when
phosphate is present in the medium. Glutaminase in brain is a phos-
phate-dependentenzyme 33, 58. If glia are the main site of glutamine
syrtthesis, then this implies synaptosomal degradation of glutamine,
release as transmitter glutamate, uptake by glial cells, and re-
synthesis to glutamine. Translocation of glial glutamine back to
the nerve-ending compartment does not require a high-affinity
uptake process for glutamine. Our own studies indicate that
glutamine is transported into synaptosomes by a low-affinity process
(Km 1.4 x lO-4M) 61, in confirmation of the results of other invest-
igators 3, 45. Single-affinity kinetics with a Km of lO-4M, how-
ever, is also shown by glia. Since a high glutamine/glutamate ratio
in the extracellular compartment is presumably necessary as a means
of controlling the excitability of the neuronal membrane, it is not
surprising that the affinities of both glial and synaptosomal mem-
brane are lO-fold lower for glutamine than for glutamate.
AMINO ACID TRANSPORT IN NEURONS AND GLiA 231

300
o Ring.r

~ • II - • 5mM PO. buffer

2 ••

.c 150

'e.·
.........

'"
-Z..
·
~
E
10 •

U
•
·
'0
E

50

Neuron. Crud. "'lap.

Fig.4 - Formation of glutamate from glutamine by neuronal, glial and

synaptosomal fractions. Suspensions were incubated with 5xlO- 4 M
(U)-14C-L-glutamine in a Krebs-Ringer medium for 30 min at 37 0 • In-
cubations were terminated by addition of ethanol to a final concen-
tration of 70%. Aliquots of supernatants were spotted for paper chro-
matography in n-butanol:acetic acid: water (60:15:25 v/v). After
nin hydrin development radioactivity was measured by liquid scin-
tillation using cellosolve-toluene (1:2) as solvent.

SUMMARY

Our efforts have been directed towards characterizing aml.no

acid uptake, metabolism and release in bulk-isolated glia and neur-
onal perikarya studied in parallel with nerve-endings, especially
as it concerns the transmitter amino acids and the participation of
glia in the clearing of the synaptic space during impulse conduction.
232 A. HAMBERGER ET AI..

A possible neuromodulator role for the glia at the synapse is also

suggested by K+-stimulated release. Our most definitive conclusions
have been based so far on studies with GABA, although we are also
beginning to accumulate data for glutamate related to glutamate-
glutamine compartmentation. Glta preferentially accumulate potassium
and amino acids compared to neuronal perikarya, have higher Na+/K+-
ATPase activity, possess high-affinity, sodium-dependent uptake sys-
tems for GABA and glutamate similar to the ones in synaptosomes,
and release amino acid in response to a potassium pulse by a calcium-
independent process. Low neuronal uptake could be due to loss of
dendrites. Unidirectional GABA-flux from the synaptosomal to glial
compartment is suppor~d by high GAD in nerve endings compared to
high GABA-T in glia.
Glutamine may be a transmitter glutamate-precursor in nerve-
endings since glutaminase activity is high in nerve-endings, but
low in glia where glutamine is presumably made. Glutamine uptake in
both glia and synaptosomes obeys low-affinity kinetics in contrast
to glutamate, consistent with the inability of glutamine to excite
the neuronal membrane. The studies with GABA, which are considerably
more extensive, are supported by related work using glia in tissue-
culture and autoradiography. There appears to be a suggested diff~
erence in the behavior of amines which were poorly taken up by the
glial system. Glia, synaptosomes and neuronal perikarya, in general,
behaved similarly with respect to requirements for uptake and rel-
ease, except in the case of Ca++, which exerted opposite effects on
glial and synaptosomal uptake of GABA.
We believe that work along these lines tends to firmly estab-
lish a direct role for glial cells as modulators of neuronal excit-
ability and represents a convergence between transmitter amino acid
neuropharmacology and cellular biochemistry. This' not only deepens
and enlarges the vocabulary of synaptic biochemistry but also un-
doubtedly will have major clinical applications in the fields of
epilepsy and behavior.

ACKNOWLEDGEMENTS

C.T.W. is a recipient of a National Research Service Award

from NINDS; NIH Fellowship it. 1 F 22 NS 02592-01. This work was
supported by a grant from the Swedish Medical Research Council
(grant No. B75-l2X-164-llC) and by a grant from Ollie and E10f
Ericssons Stiftelse.

REFERENCES

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AMINO ACID TRANSPORT IN NEURONS AND GLiA 233

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TRANSPORT OF TAURINE IN THE CENTRAL NERVOUS SYSTEM

S.S. OJA, P. KONTRO and P. LAHDESMAKI

Institute of Biomedical Sciences, University of

Tampere, and Department of Biochemistry,

University of Oulu, Finland

INTRODUCTION

Taurine, 2-aminoethanesulphonic acid, is present in brain

tissue in millimolar concentrations 44 ; however, taurine levels
show considerable differences in different mammalian species 38
and at different developmental stages 37 . The physiological role
of taurine, apart from its conjugation with bile acids, is vir-
tually obscure. However, more and more attention has been paid to
its possible function in electrically excitable tissues, such as
brain, retina, heart or muscle. Taurine inhibits the spontaneous
firing of some central synapses with a potency equal to that of
y-aminobutyric acid 8 ,17 and affects the excitability of cardiac
muscle 46 ,58. Some authors are therefore inclined to consider
taurine one of the inhibitory neurotransmitters lO ,29,42.
Taurine can be synthesized in the organism through several
metabolic pathways20, but only two of them may have quantitative
importance in mammals. 1) Decarboxylation of cysteine sulphinic
acid to hypotaurine, which is subsequently oxidized to taur-
ine 9 ,14,43. Cysteine sulphinate decarboxylase (E.C. 4.1.1.29) has
been partially purified and is relatively active in brain tis-
sue 2l , but oxidation of hypo taurine has been demonstrated only in
a non-satisfactory way in the liver and not at all in the brain13 ,
56. The yield of taurine in the brain through this metabolic route
appears scanty36. 2) An alternative pathway involves de novo syn-
thesis of taurine from inorganic sulphate via an active inter-
mediate, 3'-phosphoadenosine-5'-phosphosulphate, the most active
sulphate acceptor being serine 33 ,48,57. The latter pathway is

237
238 S.S. OJA. P. KONTRO. AND P. LAHDESMAKI

well documented in liver tissue, but not in brain. The breakdown

of taurine through oxidative deamination to isethionic (2-hy-
droxyethanesulphonic) acid, proceeds slowly in all tissues 43 •
Only a negligible fraction of radioactivity was detected in brain
or plasma as isethionic acid or inorganic sulphate several hours
after parenteral administration of a tracer dose of 1 35 SItaurine
(Oja, Lehtinen and Lahdesmaki, unpublished).
If we consider the postulated role of taurine at CNS syn-
apses, the transport of taurine across cell membranes is a cen-
tral object of interest, in particular as the metabolic rates of
taurine appear to be low. Efflux from presynaptic terminals on or-
thodromic stimulation supplies taurine molecules into the synap-
tic clefts, whereas influx into presynaptic neurons may replenish
the intraneuronal supply. Furthermore, in the absence of fast
breakdown reactions, the termination of a possible synaptic action
of taurine could only be effected by an efficient reuptake across
presynaptic or postsynaptic membranes. .

TAURINE TRANSPORT IN VIVO

Only a few studies have been undertaken to elucidate the tis-

sue/plasma exchange of taurine in vivo. Minato et al. 32 injected
rats intravenously with 1 35 SItaurine and found that kidney, liver
and small intestine accumulated radioactivity very fast, while the
uptake of 35S was very slow in the brain. Spaeth and Schneider 54
followed the decay of radioactivity from tissue I 35SItaurine pools
and concluded that the half-life of tissue taurine in heart,
muscle and brain must be significantly longer than in the other
tissues studied. We have computed the rates of plasma to tissue
transfer of taurine for a number of tissues in 7-day-old and adult
mice (Table 1). It appears that the plasma to tissue exchange of
taurine is lowest in the brain, while taurine penetrates very
fast for instance from plasma to liver (and also to kidney). In
all tissues studied the exchange of taurine was faster in adult
than in 7-day-old mice. There was no distinct correlation between
the tissue levels of taurine and the rate of taurine transfer from
plasma to tissues.
Practically nothing is known of the exchange of taurine be-
tween brain and cerebrospinal fluid, even if Collins 5 has shown
that intracisternally applied I 35SItaurine accumulates in the rat
brain. Collins did not find either any correlation between the
taurine levels and the rate of disappearance of 135 SItaurine from
eight different anatomical areas of the brain. We thus conclude
that the magnitudes of transport rates of taurine between plasma
and tissues do not primarily determine the endogenous tissue levels
of taurine. The tissue concentrations of taurine are generally many
times higher than the plasma concentrations 20 • Intracellular bind-
ing or electrochemical forces may decisively help in maintaining
TAURINE TRANSPORT IN THE eNS 239

Table 1. The relative magnitudes of exchange rates of taurine

between plasma and tissues in 7-day-old and adult mice

Tissue 7-day-old mice Adult mice

Brain 1 2
Femoral muscle 3 5
Heart 10 80
Liver no 130

The exchange rates were estimated with the aid of a digital

com~uter by fitting the alterations in the specific radioactivity
of 135SItaurine in plasma and tissues after pulse labeling to a
simplified multicompartment model representing the living organ-
ism. Six experimental series, ten mice in each. The average
transport rate from plasma to brain in 7-day-old mice (1.4
nmol/min/g tissue) is taken as unit (Oja, Lehtinen and Lahdes-
maki, unpublished).

such concentration gradients. It is also striking that the plasma

to tissue exchange of taurine seems to be very slow in those tis-
sues in which a physiological role in the regulation of electri-
cal excitability has been attributed to taurine.

TAURINE INFLUX IN VITRO

A more detailed analysis of amino acid transport requires

experiments in vitro, since the experimental conditions can be
more easily modified according to needs. Taurine transport has
often been studied in vitro with brain slices, synaptosome prep-
arations or isolated retinae. In a few investigations glial tumour
cells or synaptic vesicles have been employed. A very slow ex-
change between the incubation media and intracellular spaces is
characteristic for taurine transport also in vitro 35 . At least two
taurine transport systems exist in nervous tissue, one saturable
and one non-saturable. Many authors reported two saturable systems
- a low-affinity and a high-affinity transport - in rat brain
slices 28 , rat cortex synaptosomes 49 , mouse brain synaptosomes 30
and rat retinae 34 • The high-affinity transport obviously corre-
sponds to the S system described in Ehrlich ascites cells by
Kromphardt 26 , i.e. a transport site which is specific for S amino
acids. The low-affinity system, named w transport by us 28 , exhi-
bits features which resemble a relatively undefined saturable
system mentioned by Christensen and Liang 4 in Ehrlich cells.
The concept of high-affinity and low-affinity transport of
taurine in the CNS has not been unanimously approved. Honegger ~
240 S.S. OJA, P. KONTRO, AND P. LAHDESMAKI

"0
Q. A B
~ •
c:
!300
"0
E
c:
..............

".. 4
.>t.
a.
:l .........
------
---....................
"c: 2
'§
t!!!
2 4 6 8 10 12 20 40 60 80 100
Taurine concentration mmol/l Taurine concentration I'molll

Fig. 1. The influx of taurine into rat whole brain synaptosomes.

The synaptosomes were isolated according to Gray and Whittaker 16
They were incubated in Krebs-Ringer phosphate-glucose medium at
pH 7.4 under 02 for 30 min with varying concentrations of 135 sl-
taurine. The total influx was divided into its three components,
diffusion and high-affinity and low-affinity saturable uptake sys-
ms, and the transport parameters were estimated as indicated by
Lahdesmaki et al. 30 . A typical experiment: 14 different taurine
concentrations:-each of them in triplicate (Kontro and Oja, unpub-
lished).

al. 19 found only one saturable component for taurine transport in

rat cortex slices. They think that this would correspond to the
unspecific low-affinity component of the other investigators. Also
in retinae and glial cells only a single saturable system has been
reported 40 ,55. This obvious discrepancy may result from the low
transport capacity of the high-affinity transport. The whole high-
affinity component may have been totally overshadowed by the more
capacitive low-affinity transport. For instance, we have not
always been able in all experiments to extract successfully the
small high-affinity component from the total transmembrane taur-
ine transport. Fig. 1 depicts, however, an experiment in which
all three components (one non-saturable and two saturable) of
taurine transport were discernible.
In Table 2 we have compiled transport constants for taurine
reported in the literature. The constants for the high-affinity
and low-affinity transport are of the order of 10 to 60 ]lmol/l
and 0.2 to 6 mmol/l, respectively. The maximal velocities of
transport could not be easily shown in a simple table for com-
parison, since they have been calculated by using different tis-
sue components as reference. It can be stated, however, that the
variation among them is very large.
TAURINE TRANSPORT IN THE eNS 241

Table 2. Transport constants (K ) of saturable transport of

m
taurine in CNS preparations

Reference Preparation High-af fini ty Low-affinity

transport transport
llmol/l unnol/l

Starr and Voaden SS Rat retina 1.0

Pasantes-Morales Chicken
et al. 40 1.S3
retina
Kaczmarek and Rat cortex
Davison 22 slices
so
Uihdesmaki Rat brain
60 6.1
and Oja 28 slices
Ehinger 12 Rabbit retina 43
Rat cortex
Honegger et al. 19 0.173
slices
Neal et a1. 34 Rat retina 27 0.S37
Schrier and Rat glia
10-17
Thompson SO tumour cells
Rat cortex
Schmid et al. 49 20 0.4S7
synaptosomes
Uihdesmaki Mouse brain
33 1.1
et a1. 30 synaptosomes

- means that the authors have not detected this component in the
saturable taurine influx

So far, the published investigations do not corroborate the

assumption that the high-affinity transport system would be
confined to the synaptic region, whereas the low-affinity trans-
port would operate in membranes of neuronal perikarya 3 • Both pro-
cesses seem to function simultaneously in synaptosomal prepara-
tions 30 ,49. Sieghart and KarobathS2 have evidenced that taur-
ine is accumulated by a synaptosomal subpopulation which pos-
sesses slightly different sedimentation properties than norepi-
nephrine- or GABA-accumulating subpopulations. It is not known,
however, from which part of the brain a possible taurine-accumu-
lating synaptosomal subpopulation originates, and whether it dif-
fers from other synaptosomal particles with regard to its taurine
content. The distribution pattern of taurine in the brain is in-
homogeneous 23 ,24,but the uptake of taurine by slices from differ-
ent brain parts does not exhibit any distinct correlation with
the endogenous taurine levels S ,23.
In the brain, intraneuronal transfer of at least some free
amino acids occurs between dendrites or axon and perikaryon15,Sl,
but it has not been studied whether this mechanism could transfer
242 S.S. OJA, P. KONTRO, AND P. LAHDESMAKI

Table 3. Uptake of taurine by some preparations from nervous

tissue in the presence of compounds structurally related to
. a
taur1ne

Inhibitor Ciliary Retinae c Brain Brain Cortical

body-iris cortex d slices e synapto-
prepara- slices somes f
tions b

Amidosulphonic acid 88
Glycine 66 62 125 87 72
Hypotaurine 9 58 50 11
S-Alanine 14 21 42 49 17
L-Alanine 94 82 70
L-Cysteine 54 86 68
L-Cysteic acid 86 75
L-Cysteine sulphonic acid 83 71
y-Aminobutyric acid 38 39 93 65 31
S-Aminoisobutyric acid 62
a-Aminoisobutyric acid 87
N-Methyltaurine 47 117 77 55
S-Guanidinopropionic acid 74
6-Aminovaleric acid 37
£-Aminocaproic acid 80
Isethionic acid 85

aThe results denote the relative rates of taurine uptake in the

presence of the structurally-related compounds tested for their
binhibitor capacity. Control incubations without inhibitors = 100.
Rabbits, taurine concentration 2 x 10-5 mol/I, inhibitor concen-
tration 5 x 10- 3 mol/l (Reddy47)
cRats , taurine concentration 2.065 x 10- 5 mol/I, inhibitor concen-
dtration 10- 3 mol/l (Starr and Voaden 55 )
Rats, taurine concentration 1.65 x 10- 6 or 5 x 10- 7 mol/I, in-
hibitor concentration 10- 3 mol/l (Kaczmarek and Davison 22 )
eRats~ taurine concentrati?n 5 x .. l?-5 mOl(12 inhibitor concen-
f tratlOn 5 :: 10- 3 molll (~ahdesmak1 and OJ a 8)
Rats, taur1ne concentrat10n 2 x 10- 5 mol/I, inhibitor concen-
tration 2 x 10- 3 mol/l (Schmid et al.49)

taurine molecules from the soma to presynaptic nerve terminals. In

synaptic vesicles the concentration of taurine is the highest of
all amino acids; twice as high as the concentration of glutamic
acid ll . Taurine is not bound to synaptic vesicles in the same way
as acetylcholine 45 • Our preliminary experiments with isolated
synaptic vesicles from the brain show, however, that taurine can
TAURINE TRANSPORT IN THE eNS 243

be taken up by synaptic vesicles against a concentration gradient

(Lahdesmaki, Kontro and Oja, unpublished).
The uptake mechanisms for taurine appear to be rather speci-
fic. They are generally not influenced by other amino acids. Of
various amino acids and structural analogues of taurine, S-alanine
and hypo taurine are the most potent competitive inhibitors (Table
3). Their steric configuration closely resembles that of taurine.
GABA seems to be a moderately strong inhibitor. We have inferred
from such inhibition studies that hypothetical carriers in cell
membranes recognize the strongly ionized electropositive and
electronegative groups at the two ends of an acceptable substrate
molecule which should have a two or three carbon atoms long
aliphatic backbone 28 • It has not been feasible, however, to dif-
ferentiate the specificities of the high-affinity and low-affin-
ity components of taurine transport. It is likely that both
transport systems are very similarly affected by inhibitors 28 •
We do not know any means of blocking selectively only one of the
saturable transport systems and thus exposing the other for stud-
ies. Such a circumstance considerably hampers attempts to charac-
terize more closely the properties of the two saturable transport
components, if they really are separate entities.
The influx of taurine is energy-dependent. Dinitrophenol
moderately inhibits the uptake in rat brain slices 22 ,28 and
synaptosomes 25 • In isolated retinae an inhibition was observed in
one study40, but not in another 55 . Iodoacetate and cyanide seem
to be more potent inhibitors than dinitrophenol l ,22,40. The
effect of metabolic inhibitors is more pronounced at low than at
high taurine concentrations in the incubation medium. This
suggests that the high-affinity component of transport is par-
ticularly sensitive 49 , but the failure to separate experimentally
the two components of transport prevents quantitative inferences
as to their relative energy-dependence. An inhibition of the
uptake of taurine under anaerobic conditions and at low incu-
bation temperatures further witnesses the active nature of taur-
ine uptake (Table 4). Similar temperature effects have been re-
ported by some other authors in a variety of eNS preparations 22 ,
40,49,50,55. On the other hand, electrical stimulation sig-
nificantlyenhances taurine influx into rat brain slices 27 , mouse
brain synaptosomes 30 and rat brain synaptosomes 25 and causes a
concomitant increase in their oxygen consumption.
The uptake of taurine is sodium-dependent. Lajtha and
Sershen 31 replaced Na+ with Li+, Rb+, choline or mannitol in the
incubation media. In brain slices the absence of sodium elimin-
ated the concentrative uptake at both low and high taurine con-
centrations. Schrier and Thompson50 have shown with rat glial tu-
mour cells that Na+ ions may also affect the non-saturable influx
of taurine in addition to the rapidly saturable component. In our
recent experiments, the uptake of taurine in the absence of so-
dium was more inhibited at low than at high taurine concentrations
244 S.S. OJA, P. KONTRO, AND P. LAHDESMAKI

Table 4. Effects of incubation temperature and atmosphere on taur-

ine influx into rat brain synaptosomes

Incubation conditions Taurine influx

nmol/min/g protein

O2 atmosphere 37 0 1.00 ± 0.04 (12)

20 0 0.68 ± 0.04 ( 9)
10 0.12 ± 0.02 ( 8)
Air 37 0 0.84 ± 0.03 ( 9)
N2 atmosphere 37 0 0.54 ± 0.06 ( 6)

Synaptosomes were incubated with 10 ~mol/l 1 35 SItaurine under the

incubation conditions indicated. Other experimental details as in
Fig. 1. Means (±S.D.) are given. Number of experiments in parenth-
eses (Kontro and Oja, unpublished).

Table 5. Influx of taurine into rat brain synaptosomes at varying

Na+ ion concentrations
+
Na concentration Taurine influx % of the control
Taurine concentration in the medium
mmol/l 10 ~mol/l 15 mmol/l

130 (control) 100.0 ± 8.8 ( 8) 100.0 ± 6.7 (12)

90 71.5 ( 3) 74.5 ( 2)
60 56.9 ± 3.7 ( 6) 71. 0 ± 4.4 ( 6)
40 51.3 ± 14.0 ( 5) 73.9 ± 6.4 ( 4)
15 28.2 ± 5.1 ( 5) 59.4 ± 4.5 ( 4)
0 30.8 ± 3.9 (10) 54.3 ± 6.1 (10)

Synaptosomes were incubated as described in Fig. 1 with Na+ and

1 35 SItaurine concentrations as indicated. Na+ ions in the incu-
bation medium were replaced by equimolar concentrations of chol-
ine. Means (±S.D.) are given. Number of experiments in parenth-
eses (Kontro and Oja, unpublished).

(Table 5). Schmid et al. 49 reported that taurine uptake at low

concentrations almost-entirely ceased in Na+ free media. Our pre-
liminary kinetic analyses on taurine influx into rat brain syn-
aptosomes in the absence of sodium suggest that the high-af-
finity uptake may have been entirely blocked, and that the re-
sidual uptake at low taurine concentrations results only from
the action of the low-affinity transport and from non-saturable
migration. Such a strict Na+ dependence at low concentrations of
taurine in the medium resembles the strict Na+ dependence of the
TAURINE TRANSPORT IN THE eNS 245

high-affinity uptake systems of other putative neurotransmitter

amino acids 2 • The effects of other ions on taurine influx are less
well known. In the absence of K+ and Ca 2+ ions the influx of taur-
ine into rat brain synaptosomes is, however, moderately inhibited,
whereas the omissions of Mg2+ ions alone or Mg2+ and Ca 2+ ions to-
gether have no significant effects 25 .

TAURINE EFFLUX IN VITRO

Less is known on taurine efflux than on its influx. Efflux

has been studied with brain slices, isolated synaptosomal prep-
arations or retinae, mainly in order to elucidate the postulated
role of taurine as an inhibitory neurotransmitter. The spontaneous
efflux of taurine from brain slices is very slow even in taurine-
free incubation media. Brain slices from 7-day-old rats retain
better their intracellular taurine than slices from adult rats at
different concentrations of taurine in the incubation medium 3S .
The spontaneous efflux of taurine from brain slices is directly
related to the intracellular taurine concentration 27 • The efflux
of taurine from rat retina is also a slow process occurring in two
stages, thus indicating a relatively firm binding of taurine to
retina SS . The efflux from chicken retina is only about one tenth
of the influx under similar incubation conditions 40 •
The molecular mechanisms of spontaneous efflux of taurine
have not yet been specified. The efflux from rat brain slices is
slightly accelerated by a homoexchange with extracellular taurine.
A heteroexchange with GABA, but not with glycine, appreciably en-
hances the efflux 7 • In the retina of domestic fowl a heteroex-
change with GABA, glutamate, aspartate and B-alanine has also been
observed 41 . These results suggest that the efflux of taurine'might
be at least partially mediated by the agency of a transport site
shared by the amino acids exhibiting stimulatory transeffects.
Very little is known on the ion dependence of the spontaneous
taurine efflux, but at least from glioma cells it seems to be
sodium-dependent SO . 10 24
Electrical stimulation of brain slices ' , spinal cord
slices l8 , brain synaptosomes 30 and retinae 41 in vitro has in all
instances enhanced the efflux of taurine. Collins and Topiwala6
found an enhancement with slices of cerebral cortex, but not with
slices of spinal cord. Light flash stimulation of retinae in
vitro causes a manifold increase in the efflux of taurine but does
not affect so much the release of other amino acids 41 ,42. The
concentration of taurine in the retina may vary on ambient il-
lumination. It increases nearly twofold in chicks reared in com-
plete darkness 39 • 2+
The stimulus-induced release of taurine is particularly Ca
dependent. In the absence of calcium the efflux from cortex
slices 6 and retinae 41 is markedly reduced. A replacement of cal-
 246 S.S. OJA, P. KONTRO, AND P. LAHDESMAKI

~t 0.15/
E~
"0
E
c
0.10
c
:8
1!
C
~ "C
C ;,
8 .8 0.05

1 2 3 4 5 1 2 3 4 5
Taurine concentration mM Taurine concentration mM

Fig. 2. Binding of I 35SItaurine to isolated calf brain synapto-

somal membranes. Membranes were isolated according to Whittaker 59 .
They were incubated for 10 min in 1 ml Krebs-Ringer phosphate me-
dium (pH 7.4) at 37 0 C with varying concentrations of 1 35 SItaurine
(0.25 ~Ci). They were then sedimented at 20.000 g, washed twice
with taurine-free incubation medium and dissolved in 0.01 molll
NaOH. Radioactivity of the samples was determined as described by
Lahdesmaki and Oja 27 . Means (±S.E.) from four experiments. A
linear transformation of the curve in the right-hand-side graph
(S/v vs. S) (Lahdesmaki and Raasakka, unpublished).

cium with magnesium somewhat slows down the rate of stimulus-in-

duced taurine release from rat brain slices 22 • In its Ca 2+ depen-
dence, the stimulus-induced efflux of taurine mimics the mechan-
isms of stimulus-induced release of all known neurotransmitters.
A high concentration of potassium ions which depolarizes neuronal
membranes enhances the efflux of taurine less than that of other
putative neurotransmitter amino acids. In fact, the efflux of
taurine has been found to increase about threefold from brain
cortex slices 6 , by only 36 per cent from synaptosome beds ll , and
not at all from spinal cord slices 6 and isolated retinae 4l •

TAURINE BINDING TO SYNAPTOSOMAL MEMBRANE S

It is likely that taurine is temporarily bound to presynap-

tic or postsynaptic receptors if it has a specific synaptic func-
tion. A carrier-mediated transport system at synaptosomal mem-
TAURINE TRANSPORT IN THE eNS 247

iii
£
40
'0 Elution order
111.
30 )
~I)
::J
iii 20

05
I)
10
C
0;c:
::J
~

.
0
ill

z
0
~
it
""Z'"
I»
'"""
Z
I»
0
g
Z
0
g
Z .g
0 0
;;
~
::a
CD
III
c:
C
I» Q.
Q Q J: Z ~ C; !!!.
Q ~ '"VI I»
0 C;
::J
::J
3
I» ..0 J:
>:< It
~ >:< 00 3
g-
is
o I»
::J
CD
III

Fig. 3. Differential elution of 1 35 SItaurine from calf brain s~n­

aptosomal membranes. Membranes were incubated in 3 mmol/l 1 35 sl-
taurine solution (0.05 ~Ci) for 30 min, sedimented and washed as
described in Fig. 2. After washing they were eluted with the sol-
utions indicated, and the radioactivities of these eluents were
measured. The results, mean (±S.E.) of seven experiments, are
given as percentages of the total amount of the label attached in-
itially to the membranes (Lahdesmaki and Raasakka, unpublished).

branes presumably also involves an interaction of taurine with

membrane components. The nature of any attachment of taurine to
presynaptic and postsynaptic membranes must thus be explored if
one seeks to prove or disprove the hypothesis of taurine being an
inhibitory neurotransmitter. The possible receptors of taurine
have been so far completely undefined, even if the receptors of
glycine and GABA, two other inhibitory neurotransmitter candi-
tates, have been characterized to some extent. At the postsynaptic
membrane taurine is able to displace strychnine from its bindin~
sites, which probably constitute postsynaptic glycine receptors 3.
On the other hand, the depressant actions of taurine and GABA are
quite differently blocked by convulsant alkaloids, which circum-
stance would indicate that the possible taurine receptors and the
GABA receptors are separate entities 17 •
248 S.S. OJA. P. KONTRO. AND P. LAHDESMAKI

We have tried to ascertain the existence of some kind of

specific combination of taurine with isolated synaptosomal plasma
membranes. It is hard to separate by gradient centrifugation pre-
synaptic and postsynaptic membranes from each other, and therefore
our fractions are a mixture of both. Preliminary experiments have
disclosed that taurine saturates our membrane fractions. The kin-
etics of this saturation resemble an association-dissociation
equilibrium between free taurine, free membrane binding sites and
taurine-loaded binding sites (Fig. 2). The binding constant was,
however, very large, amounting to about 5 x 10- 4 mol/I. The bound
taurine was extracted from membranes by differential elution (Fig.
3). In spite of the use of hypertonic salt solutions, acid and
alkaline solutions and detergents, about 40 per cent of the radio-
active taurine still sedimented with the membranes, thus indi-
cating a rather firm attachment. Our synaptosomal membrane frag-
ments were electronmicroscopically very pure, but even so, some
contamination with other particles cannot be wholly excluded. Mem-
branes may also curl to form pouches filled with incubation sol-
ution. We have not yet ascertained whether our results reflect
solely binding of taurine to membranes; an influx of taurine into
resealed membrane pockets or into other contaminating particles
would greatly affect our binding constant estimation and alter the
profile of the taurine elution pattern.

CONCLUSIONS

The concentration of taurine in electrically excitable tis-

sues, brain, retina, heart and muscles is high, but the rate of
taurine exchange across cell membranes is low. In the brain there
seem to be three transport mechanisms for taurine, high-affinity
and low-affinity components and non-saturable migration. The in-
flux of taurine is sodium- and energy-dependent and temperature-
sensitive. Only those amino acids which are structurally closely
related to taurine competitively inhibit its influx. The high-af-
finity component of influx exhibits many features common with the
high-affinity transport mechanisms of other putative neurotrans-
mitter amino acids. The efflux of taurine is enhanced by electri-
cal stimulation of brain or retina preparations and by light
stimulation of the retina. The stimulus-induced release of taurine
is calcium-dependent. A characterization of the nature of possible
interaction of taurine with synaptosomal membranes is in progress.
The properties of transmembrane taurine transport are not in con-
trast with the postulated role of taurine being an inhibitory
neurotransmitter, although the low overall rates of metabolism
and membrane transport leave open whether or not taurine could be
eliminated fast enough from synaptic clefts. At least taurine
could act as a modulator of synaptic excitability.
 TAURINE TRANSPORT IN THE eNS 249

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TAURINE TRANSPORT IN THE eNS 251

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TRANSPORT OF ADENINE DERIVATIVES IN

TISSUES OF THE BRAIN

Henry McIlwain

Institute of Psychiatry
The Maudsley Hospital
Denmark Hill, London, England (U.K.)

INTRODUCTION

It will be valuable first to note the ubiquity of adenine

derivatives in the brain and some of the effects which can be
ascribed to the appearance in extracellular fluids of a small
proportion of the tissue's adenine compounds. Adenine derivatives
are prominent components of the small molecular-weight metabolites
of the cells of the brain, and also of important categories of
their macromolecules, especially of the nucleic acids. Because
adenine nucleotides are associated with energy-metabolism, almost
all changes in cerebral activities are to some degree associated
with changes - of place or of chemical nature or both - in
cerebral adenine derivates. The converse is also true; several
adenine derivatives when administered to animals alter their
behavior in fashions which can be ascribed to changes in cerebral
functioning. Examples are quoted in Table 1, which lists also
the effects of applying an adenine derivative directly to the
mammalian brain and to preparations derived from it.

Translocation and central effects of adenosine

Adenosine has been chosen as the compound displayed in Table

1, though several of the actions described have been given by 5'-
AMP and ATP and some by cyclic AMP, often at similar molar
concentrations {some relevant interconversions are described below}.
Actions of the nucleotides, however, have on some occasions
(eg. lO ) been ascribed to chelation, perhaps resulting in changed
concentrations of Ca ions. This cannot be the case with adenosine,
which in addition has many of its actions extracellularly: a

253
254 H. MciLWAIN

region in which phosphorylation to a nucleotide is unlikely to

occur.

Table 1. Actions of adenosine

ADMINISTRATION ACTION REFERENCE

Intracerebral micropipette, At single cerebellar

electrophoresis; rat neurons, depressed cell 9
firing

Solution bathing olfactory l-lOO~M depressed 29

cortex, isolated, mouse negative wave from
stimulating l.o.t.

Intraventricular near lOO-200~M : sleep and 14

hypothalamus, fowls changed EEG (with
aminophylline)

Intraperitoneal, mice with lO-130mg kg sedates and 12

audiogenic convulsions protects from
convulsions

Intraperitoneal, to mice; 50mg kg antagonists 4

hot-plate, stretch tests morphine analgesia

Subcutaneous to mice, in lO-15mg kg favours gain 15

maze-test of learning in learned behavior

It is with this background that we approach questions of

translocation of adenine derivatives. Do such compounds, after
their general administration, arrive at the brain or are their
central actions more indirect? On arrival, are they assimilated,
and if so which compounds and to which histological elements?
If such assimilation is an ongoing process, so also must be the
removal of adenine derivatives from the brain, for cerebral
composition is relatively stable over long periods. Adenine
derivatives, it may be noted, greatly preponderate among the
purines and pyrimidines of the brain: when compared with other
nuc1eotides in the cerebrum and cerebellum of several animal
species; when compared as nucleoside triphosphates in a wide
range of vertebrates at various periods of growth; and when
compared with other free nuc1eotides in nuclei and cytoplasm13 ,18.

Studies on transport of adenine derivatives must note also

the endogenous synthesis of such derivatives which takes place in
the mammalian brain, as in other parts of the animal body. By
intracerebral injection or by incubation with isolated cerebral
TRANSPORT OF ADENINE DERIVATIVES 255

tissues, isotopically-labelled formate, glycine and also inosine

yield similarly-labelled adenine nucleotides and polynucleotides
5, 13, 25. This and other evidence shows that synthesis of
derivatives by routes established in other organisms occurs also
in the mammalian brain 6 , 18.

Again, by modifying the supply of blood and of labelled pre-

cursors between liver and brain it was shown 19 that~4Cladenine
derivatives were acquired by the brain of the rat from t~e liver;
the l4C was transported between the organs preponderantly as
hypoxanthine. Such interconversions of adenine derivatives with
non-adenine precursors and products are however relatively slow
in comparison with reactions which interconvert adenine derivatives
within the brain l6 .

Entry of adenine derivatives into tissue of the brain

Two main types of experiment have shown the occurrence of

this phase of adenine-derivative transport. (i) The first concerns
incubated cerebral tissues in vitro and in simple glucose salines;
these tissues maintain defined levels of simple adenine derivatives,
but at concentrations below those found in vivo. The concentrations
were increased towards normal values by addition e.g. of adenine
and adenosine (ii) Isotopically labelled precursors added to
such incubation fluids have been found to become incorporated into
the adenine compounds of the tissues, without necessarily changing
their molar concentrations. Some of the entering compounds are
now considered individually.

Adenine. Adenine is suspected to be present as such in the

blood, though at quite low level 32 When added to incubating
guinea pig neocortex at markedly greater concentration, approaching
millimolar, adenine was found free in the tissue though at concentra-
tions lower than those of the incubating fluids. Under these
conditions, the adenine nucleotides of the incubating tissues were
increased by the added adenine 32 •

Using ~4CJ adenine with chopped mouse cerebrum33 , maximal

rate of l4C incorporation to adenine nucleotides occurred with
10VM adenine. T~e presence of O.5mM-guanosine stimulated adenine
incorporation; it was suggested that the nucleoside supplied
5-phosphoribosy1-1-pyrophosphate (PRPP) for reaction (1):

(1) PRPP + adenine ....= ......~.. AMP + PP

The circumstances under which the phosphotransferase of

reaction (1) operates in the normal brain in vivo, and whether
256 H. MciLWAIN

it does so with native adenine as substrate, are uncertain. (14 C]

Adenine has been searched for, but with negative results, as a
product from or intermediate in uptake of l:4C] adenosine to rab-
bit heart 11 and cerebral tissues (Pull and McIlwain, unpublished).

Adenosine. Adenosine contrasts with adenine in being well-

established as a free tissue constituent, which under some cir-
cumstances can rise to considerable levels: a normal cerebral
level of 11.4 ± 1.2 nmoles/g in the dog, rising to 130 nmoles/g
with 5 min ischemia l . Normal csf concentrations were 0.22 ± 0.5
nmoles/ml.

Addition of aden.osine to incubating neocortical tissues

markedly increases their content of adenine nucleotides, by some
400/0, and detailed studies have been made of the entry of the
isotopically labelled nucleoside to cerebral tissues of guinea
pigs and rats. The entry proceeded at a rate 7 to 8 times that of
adenine under comparable conditions 33. Uptake experiments with
isotopically-labelled adenosine 30 showed the tissue's free adenosine
to be consistently lower in concentration than that of the incuba-
tion fluid. The greater part of the added adenosine was
converted to nucleotides and found as ATP; the data thus suggest
as probable processes in the assimilation the reactions (2) and
(3) .

(2) A* ---~, A*.

e , 1

(3) A*. +ATP A*MP + ADP

1 ?
Here A* is isotopically-labelled adenosine; e and i refe~ to
extracellular and intracellular adenosine; and the A *MP is
pictured to be in equilibrium; through adenylate kinase, with the
tissue's adenine nucleotides available at the points of uptake.

The uptake of adenosine to the incubating tissue was

concentration-dependent, increasing in the rat between 4 and 20 ~M
external adenosine while in guinea pig tissue the entry was half-
maximal at 20 ~M. Adenosine kinase (process 3) was demonstrated 30
to proceed in cerehral extracts, and as a partly purified enzyme,
at appreciable speed; the maximal rate was, with some assumptions,
about 15 nmoles/mg protein.h. Moreover, after partial purifica-
tion the Km of the kinase was 20~M, coinciding with the apparent
Km of the uptake process. The phosphorylation thus appears to be
the critical step in uptake of adenosine to the tissue. This was
supported by finding two structural analogues of adenosine which
inhibited both adenosine uptake and adenosine kinase. Previous
belief that the transferase (1) might be involved were discounted
by observations with [2, 8- 3H, U-14C] adenosine which showed the
adenine-ribose link to be unbroken during uptake.
TRANSPORT OF ADENINE DERIVATIVES 257

Hypoxanthine. Hypoxanthine was noted above to be the probable

form in which adenine precursors were carried by the bloodstream
to organs including the brain, and conversion of hypoxanthine to
purine nucleotides by chopped mouse cerebrum has been measured 33
Supplied at 100~M, ~ypoxanthine uptake proceeded at 70 nmoles/g
tissue.h: some 600/00f the rate observed with adenine supplied
at similar concentration. Formation of nucleotides from hypoxan-
thine was accelerated by guanosine and this was concluded to be
due to supply of 5'-phosphoribosyl pyrophosphate (PRPP; reaction
4) :

(4) Hypoxanthine + PRPP .,======='~ IMP + PP;

subsequent formation of AMP proceeded through adenylosuccinate.

To sliced guinea pig neocortex and piriform cortex we

supplied [14~ hypoxanthine at 80 nM and found after 5 and 15 min,
l4C distribution much in favor of the tissue (Pull and McIlwain,
unpublished; mean tissue medium ratios 2.1 and 5.1). Some 90%
of the total tissue l4C at 15 min was ~s nucleotides; the amount
identified chromatographically as free hypoxanthine was 6% of the
total, and thus constituted an intracellular concentration much
lower than that existing extracellularly. The processes of
hypoxanthine uptake and conversion to tissue nucleotides are thus
very efficient and operate at low hypoxanthine concentrations.

Adenosine mononucleotides. These are the most abundant

of the simple adenine derivatives of the brain, but. their
transport into cerebral tissues from extracellular fluids is
a sluggish process in comparison with other changes which they
undergo when so presented. Rather, they are dephosphorylated 20 .
The quantity which was assimilated to the tissue was some 109b of
the material converted to adenosine, and thus adenosine may have
been the compound assimilated.

When similarly added to guinea pig neocortex and piriform

cortex, 5 ' -AMP was similarly dephosphorylated (Pull and McIlwain,
unpublished): 32% of the l4C of [14C] AMP was lost from surrounding
fluids during 5 min exposure to the tissues, and this amount incl-
uded 25. SOlo which was found as adenosine in the fluid. The fluid
contained also inosine (2.30/00f the added l4C) , hypoxanthine (0.60/q
and cyclic AMP (0.~1o), while 3% of the l4C supplied was incorpor-
ated to the tissue during the course of the changes noted. The
adenosine deaminase responsible for forming hypoxanthine has been
characterized 22 .

[L4~ Cyclic AMP was added to fluids superfusing cerebral

tissues in experiments parallelling those which involved 5'-AMP
(Pull and McIlwain, unpublished). Some t>/o of the l4C added at
258 H. MciLWAIN

17 nM was assimilated in 5 min. Of that in the fluid most rem-

ained as cyclic AMP but 10%was converted to 5'-nucleotides and
2.4910 to adenosine; a lesser amount was found as hypoxanthine.
Uptake of [8_3~ cyclic AMP added at 10nM to rat cerebral tissues
has. been reported to occur directly, by a high-affinity process 7,
though double-labelling experiments may he needed to establish
this.

Output of adenine derivatives from cerebral tissues.

The processes, several and potent, for assimilating

adenosine and related compounds to cerebral systems lead to
enquiry about processes by which the compounds reach extracellular
regions of cerebral tissues. Arrival from the bloodstream has
been noted above: but the main environment of a cellular component
in the brain is other cellular tissue components. We have indeed
found that an appreciable output of adenine derivatives is a normal
feature of cerebral tissues, and that the magnitude of the output
is affected by a variety of circumstances which modify tissue
activity.

Many details of the processes of release have been summarized

previously 16, 17. Briefly, when the adenine nucleotides of
cerebral tissues are labelted with [14q] adenine, superfusion
gives effluents carrying C as 5'-nucleotides, cyclic AMP, adeno-
sine and hypoxanthine. A variety of circumstances increases such
output. Hypoxia does so, and increases also the release of
adenosine from dog brain in vivo 1 Electrical excitation, and
increased (K+) in superfusion fluids, also gives augmented output
from cerebral tissues.

The molar quantities of adenosine and its metabolites released

electrically have been measured and over a 10 min period release
proceeded at about 1 ~mole/g tissue.h. This corresponded to 7
pmole/g tissue per stimulating pulse 21, a quantity interestingly
similar to the release of ATP reported from a nonadenergic nerve
putatively purinergic 2 •

The adenine derivatives released electrically from cerebral

tissues included adenine nuc1eotides and adenosine, but the pre-
ponderating compounds were inosine and hypoxanthine 20, 31 (Fig.l),
especially in later collection-periods. ATP added to fluids super-
fusing the tissues is however converted to adenosine, inosine and
hypoxanthine and it is thus possible that a larger part of the
adenine derivatives are first released as adenosine or ATP. The
significance of this lies in the much greater biological activities
of adenosine and ATP: in most of the systems listed in Table 1,
inosine and hypoxanthine are inactive.
TRANSPORT OF ADENINE DERIVATIVES 259

140

.!:
.. 120

~Q.

0
rlOO
...
Q
Q.
C

..,
E
~
U 60
£

....
'0

.
> 40
0
u
a:
u 20
!

AMP inosine Cyclic Hypoxanthine Adenine

ADP AMP Adenosine
ATP

Fig.l. Adenine derivatives from hypothalamic tissue released on

electrical excitation; a, b, c, d: successive 6-min periods; stimu-
lation during periods band c. Preincubation: [14C] adenine 3l

A further property of adenosine and its 5'-mononucleotides

is not shared by inosine and hypoxanthine. This is the ability
to augment the cyclic AMP of cerebral tissues 26 , probably
through an adenylate cyclase activated by adenosine and antagonized
by theophylline. A theophylline-opposed augmentation of cyclic
AMP was first noted 8 as resulting from electrical excitation.
A number of situations in which theophylline-opposed augmentation
of cyclic AMP had been reported were examined recently23, 24
(Table 2). Thus among neurohumoral agents, glutamic acid was
outstanding in the large output of adenine derivatives which it
caused from cerebral tissues; this was correlated with a large,
theophylline-sensitive, augmentation of cyclic AMP. Neither out-
put nor cyclic AMP was increased by ~-aminobutyric acid or by
acetylcholine.

Among centrally-acting drugs a similar correlation was shown

in actions of desipramine and 0.1 mM-chlorpromazine. Chlorproma-
zine at 20~M, and pentobarbital, had the reverse action. We
therefore conclude that extracellular adenosine plays a role in
the various actions of Table 2.
260 H. MciLWAIN

Table 2. Adenosine output and cyclic AMP

of cerebral tissues : actions of applied agents

AGENT ADENOSINE CYCLIC AMP

Output formation

Electrical excitation Increased Increase, opposed by T

Glutamate l-SmM Increased Increase, opposed by T

Desipramine 3011M Increased Increase, opposed by T

Chl;rpromazine 100lJM Increased Increase, opposed by T

20lJM Decreased Post-mortem increase

opposed

Pentobarbital
66lJM ; 60lJmoles kg Decreased Post-mortem increase
opposed

T : theophylline at c. 0.1 - lmM. Adenosine data from 23,24

where references to cyclic AMP formation are quoted.

Intracellular movements of adenine derivatives

Intracellular movements of macromolecular derivatives of

adenine have been recognized for some time as involved in the nuclear
synthesis of nucleic acids, and the subsequent migration of some
RNA species to elsewhere in a cell. Histological demonstration
of this in the brain using CI4C) adenine, has been described 3 .

More immediately relevant to present themes is the axonal

transport of simpler adenine derivatives: evidence for its
occurrence in central parts of the visual system has been
obtained 2 7, 28. Tritium of [3EO adenosine injected locally to
the exposed striate cortex of rabbits was initially found mainly
in neuron cell-bodies at the injection site; subsequently it was
in white matter bundles and within one day reached a localized
1 mm3 of the ipsilateral geniculate nucleus. There it was present
in axonal terminals and post-synaptic neurons; none was found
contralaterally. Of the material transported, 939fo was trichloro-
acetic acid-soluble; actinomycin D administered to the striate
cortex diminished the material 'fixed' there but had little effect
on the arrival of (3~ adenosine derivatives at the geniculate.
In separate experiments, (3HJ adenosine iontophoresed intracellularly
TRANSPORT OF ADENINE DERIVATIVES 261

to cat spinal motoneurons and 0.25 to 8h later was again found to

be axonally transported. The arrival post-synaptically in the
geniculate is interpreted as occurring by release from nerve
terminals and accumulation in target neurons 27 • We have analyzed
such a process by making a nerve-terminal preparation and observing
release from it of adenine derivatives lOa ; the material released
was increased by electrical stimulation and included adenosine and
hypoxanthine, compounds susceptible to neuronal uptake.

CONCLUSION

In conclusion it may be suggested that adenine derivatives

in the brain go through the following sequence of movements
(Fig.2) coordinated with chemical change. The movements are
alternately intracellular and extracellular, and at present the
cerebral systems in which they have been observed are diverse
and the sequence is to that extent hypothetical. The sequence
can be stated as comprising the following processes, I to IV.

...
Transport from extracerebral regions:
as PURINES and PURINE PRECURSORS
Extracerebral

.:
Extracellular transport:
as NUCLEOSIDES or PURINES
Brain,
ext [a-
cellular Release and
Receptor sites
metabol ism
Brain"
cellular
Cellular transport
as NUCLEOfIDES

Fig.2. Categories of purine-derivative transport especially

in relation to adenine derivatives in the mammalian brain.
Identified sites of uptake: nerve cell bodies; of release:
nerve terminals.

1. The adenine derivatives arrive at extracellular spaces in the

brain from two potential sources: (1) from the bloodstream, probably
as hypoxanthine; and (2) from the cerebral tissues themselves
which yield to surrounding fluids 5'-adenine mononucleotides to-
gether with adenosine, inosine and hypoxanthine. The latter
compounds may be released as such or may arise extracellularly
from the 5'-nucleotides. An appreciable part of this release
takes place from nerve terminals.

II. Of the compounds named, adenosine and hypoxanthine are

taken up by cellular components of the tissue and promptly appear
as 5'-nucleotides. An appreciable part of the adenine derivatives
262 H. MciLWAIN

liberated from nerve terminals are taken up by the post-synaptic

cell.

III. Adenosine taken up by nerve cell bodies is partly converted

to polynucleotides, and these and the mononucleotides travel by
axonal flow to cell regions including th~ nerve terminals.

IV. (see I.2) Of the compounds released by nerve terminals,

adenine nucleotides and adenosine have a number of pharmacological
actions, associated with the formation of cyclic AMP through an
adenosine-activated adenylate cyclase. Inosine and hypoxanthine
do not have these actions; the processes forming them constitute
a means of terminating the pharmacological or neurohumoral actions
of adenosine while retaining the metabolites, which (see above)
can be reconverted to tissue adenosine nucleotides. Adenosine
uptake (process II) is a further means of terminating its neuro-
humoral action. In addition to actions extracellularly, the
released adenosine can be suggested as having post-synaptic
effects akin to trophic actions, after its uptake by the post-
synaptic cell; for the quantities released are appreciable.

ACKNOWLEDGEMENT

I am indebted to the Medical Research Council for support

for these investigations.

REFERENCES

1. Berne, R.M., Rubio, R. and Curnish, R.R., Release of

adenosine from ischemic brain., Circulation Res., 35 (1974)
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2. Burnstock, G., Pharmacol.Rev., 24 (1972) 509-581.
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4. Gourley, D.R.H., and Beckner, S.K., Antagonism of morphine
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5. Held, I., and Wells, W., Observations on purine metabolism in
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cyclic AMP in rat brain slices., Brain Res. 59 (1973) 451-3.
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electrical stimulation on the accumulation of adenosine 3 ' ,
5 ' -phosphate in isolated cerebral tissue., J.Neurochem.,
16 (1969) 485.
TRANSPORT OF ADENINE DERIVATIVES 263

9. Kostopoules, G.K., Limacher, J.J., and Phillis, J.W., Action of

various adenine derivatives in cerebellar Purkinje cells., Brain
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10. Krnjevic, K., Chemical nature of synaptic transmission in verte-
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lOa.Kuroda, Y., and McIlwain, H., Uptake and release of l4C adenine
derivatives at beds of mammalian cortical synaptosomes in a
superfusion system., J.Neurochem. 22 (1974) 691-699. 14
11. Lin, M.S., and Feinberg, H., Incorporation of adenosine -8- C
and inosine - 8 - l4C into rabbit heart adenine nucleotides.
Amer.J.Physiol., 220 (1971) 1242-48.
12. Maitre, M., Ciesielsky, L., Lehmann, A., Kempf, E., and Mandel,
P., Protective effect of adenosine and nicotinamide against
audiogenic seizure., Biochem. Pharmacol. 23 (1974) 2807-16.
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Neurochemistry, Plenum, Vol.5A, 1971, 249-281.
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adenosine derivatives given into the brain of fowls. Brit.J.
Pharmacol. 46 (1972) 619-636.
15. Mascherpa, P., Psychostimulierende Wirkung von Adenosin bei
der Maus. Artzneimitt-Forsch., 21 (1971) 25-6.
16. McIlwain, H., Regulatory significance of the release and action
of adenine derivatives in cerebral systems., Biochem.Soc.
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17. McIlwain, H., Adenosine 3 ' : 5 ' -cyclic monophosphate and its
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18. McIlwain, H., and Bachelard, H.S., Biochemistry and the central
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derivatives by cerebral tissues, superfused and electrically
stimulated., Biochem.J., 126 (1972a) 965-973.
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agents in the brain. The quantities liberated on excitation
of superfused cerebral tissues. Biochem.J., 130 (1972) 975-981.
22. Pu1l,I., and McIlwain, H., Rat cerebral cortex adenosine deami-
nase activity and its subcellular distribution. Biochem.J.,
144 (1974) 37-41.
23. Pull, I., and McIlwain, H., Actions of neurohumoral agents and
cerebral metabolites on output of adenine derivatives from
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24. Pull, 1., and McIlwain, H., Adenine derivatives from cerebral
tissues. Biochemical Pharmacol. (1975b) accepted for publication.
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Nucleotide metabolism in rat brain., J.Neurochem. , 15 (1968)
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nucleotides on the cyclic AMP of guinea pig cerebral cortex
264 H. MciLWAIN

slices., Mo1.Pharmaco1., 6 (1970) 13-23.

27. Schubert, P., and Kreutzberg, G.W., Axonal transport of adeno-
sine and uridine derivatives and transfer to postsynaptic
neurons., Brain Res., 76 (1974) 526-53a.
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neural connectivity., Brain.Res., 90 (1975) 317-319.
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KINETICAL ANALYSIS OF THE UPTAKE OF GLUCOSE ANALOGS BY RAT

BRAIN CORTEX SLICES FROM NORMAL AND ISCHEMIC BRAIN

Henrik Lund-Andersen and Christel S. Kjeldsen

Institute of Medical Physiology, Dept. A

University of Copenhagen
Juliane Maries Vej 28
DK-2l00 Copenhagen 0, Denmark

INTRODUCTION

The present chapter describes experiments which were carried

out in order to investigate the glucose transport across glial and
neuronal cell membranes and the possible effect of ischemia upon
the membrane transport. In order to clearly separate transport
from metabolism, the two nonmetabolizable glucose analogs a-methyl-
glucoside and 3-0-methyl-glucose were used lO • The membran; transport
was investigated by studying the uptake into the cellular compartment
of brain cortex slices.

In slice preparations the exchange of substances between cells

and incubation medium is delayed by diffusion through the extra-
cellular space. If the diffusion is rapid in comparison with the
membrane transport the delay is not significant. However, if the
diffusion is comparable with or slower than the membrane transport
the diffusion process will be rate limiting for the uptake of
substances by the slice. In that case the intracellular amounts
obtained with the conventional calculation procedures will mainly
reflect the rate of diffusion in the extracellular space instead
of the rate of membrane transport. Changes in membrane transport
will accordingly be overlooked.

The first part of this chapter will deal with a comparison

between diffusion and membrane transport of the glucose analogs in
0.5 mm thick rat brain cortex slices. A more detailed description
of this part is given elsewhere 9 • The second part will deal with
an investigation of possible effects of ischemia upon the membrane
transport.
265
266 H. LUND-ANDERSEN AND C.S. KJELDSEN

UPTAKE OF GLUCOSE ANALOGS BY SLICES FROM NORMAL BRAIN

A model of the brain slice. It has been possible to determine

the rate of diffusion and to compare it with the rate of membrane
transport by fitting a model of the slice to uptake data. The
general assumption of the model is that the slice can be regarded
as a two compartment serial system. The permissibility of this
assumption appears from the following.

The diffusion process in the extracellular space can be

described by a single rate constant (ko ) and a step function, which
ascribes to the extracellular space a concentration which is 19%
of that in the incubation medium8 ,9. If the slice is pre incubated
for 30 min so that the sugar concentration is equal everywhere in
the slice the transport of tracer across the cell membranes can
also be described by a single rate constant (kl). The exchange
between slice and incubation medium can thus be compared with the
exchange of thethreecompartment serial system which is illustrated
in Fig. 1.

INCUBATION EXTRACELLU LAR INTRACELLULAR

MEDIUM SPACE SPACE

k1
kO
k2 = t· k,

H ....
Vt V2

Figure 1. Schematic presentation of the three compartment serial

system which describes the exchange between slice and incubation
medium. V indicates the magnitude of the compartments, k the
rate constants for the transfer between the compartments and
~ the ratio between k2 and k l ; (, = 1 indicates symmetry of
the transport). H represents the stepfunction, which describes
the initial influx into the extracellular space before the
influx is described by a single rate constant.

The extracellular diffusion rate (k ) and the extracellular

space (Vl) can be determined by mannitolOefflux experiments as
described below. The membrane transport (k l ) can be determined by
fitting the model to the uptake of the glucose analogs by slices,
as also described below.
UPTAKE OF GLUCOSE ANALOGS BY NORMAL AND ISCHEMIC SLICES 267

Determination of the model parameters ko and VI. The

extracellular space VI was determined as the space from which
mannitol was washed out with a half-time corresponding to simple
diffusion. Mannitol was chosen as extracellular marker since
mannitol and the glucose analogs have the same diffusion coefficient 4
and since mannitol by its passage through the cell membranes deter-
mines the passive (non carrier) permeability.

The magnitude of the space, its half-time and corresponding

k-value appear from Table I, upper row.

Table I

Magnitude and Exchange Rate of the Extracellular Space

Extracellular space Half-time

(!-L1/100 mg) (min)

Normal 52.3 2.7 0.26

brain ±1.l :to. 4
Ischemic 52.7 2.8
0.25
brain ;t4.6 :to. 3
Magnitude, half-time (t~ ), and corresponding k-value of
the extracellular space of 0.5 mm thick rat brain cortex
slices. The space was determined as the space from which
(14C)-mannitol washed out with a half-time corresponding to
simple diffusion. The k-value was calculated from k = ln2/t~
The upper row gives results obtained on slices from normal
brain. The lower row gives results obtained on slices from
ischemic brain. Results are means ± S.E.M. of 3-4 experiments.

The half-time is 2.7 min. This is in accordance with a theo-

retically calculated half-time of about 2 min. The space is 52.3
~1/100 mg (52.3%). This is a large space in comparison with the
extracellular space in viv0 6 • It is also large in comparison with
the extracellular sp~e~25-30% which was delineated in a combined
morphological and biochemical study where inulin was used as extra-
cellular marker. ll The anatomical substrate for this large space
is not known. It is reasonable to suggest that it represents both
~he extracellular space, as delineated by inulin; and cellular
compartments which during the ~ ~ procedure were damaged to
such a degree that influx and efflux of mannitol could not be
distinguished from simple diffusion.

Since mannitol and glucose analogs have the same diffusion

coefficients 4 this large space has to be regarded as the extra-
cellular space for the sugars. V2 was 32.7 ~1/100 mg (32.7%) when
calculated from the term V2 =
100-Vl-d, where d is dry matter
content (15%).
268 H. LUND·ANDERSEN AND C.S. KJELDSEN

Uptake of glucose analogs and mannitol. Fig. 2 shows the

uptake of mannitol, ~-methyl-glucoside, and 3-0-methyl-glucose
by 0.5 mm thick brain cortex slices. It appears that each of the
compounds has its characteristic uptake pattern. The sugars
equilibrate with the whole water phase (space of 85 ~l) within the
60 min period, whereas mannitol does not. The uptake of 3-0-methyl-
glucose is faster than that of ~-methyl-glucoside.

~MANNITOL

5 10 20 30 40 50 60
TIME OF EXPOSURE TO ISOTOPES,MIN.

Figure 2. Uptake of l4C-labelled mannitol (e-e) a-methyl-

glucoside (0-0) and 3-0-methyl-glucose (0- D) into 0.5 mm
thick rat brain cortex slices. The slices were preincubated
30-60 min before the addition of 0.5 ~Ci of labelled material.
The uptake was followed as a function of time of exposure to
the isotopes, and it was calculated as a space (~1/100 mg
final wet weight) according to the term (c.ptm. per 100 mg
tissue) x (c.p.m. per ~l incubation medium)-. The media
contained 115 mM NaCl, 2.8 mM KC1, 1.4 mM CaC1 2 , 1.0 mM
MgS04, 20 roM NaHC0 3 , 6 mM D-glucose plus either 3 mM D-
mannitol, 3 mM ~-methyl-D-gluco-pyranoside or 3 mM 3-0-methyl-
D-glucose. The results are means of 5-6 experiments with
S.E.M. shown by vertical bars, when exceeding the symbols.

Determination of the Cellular Transport (kl) by Model Fitting.

The determination of the membrane transport (kl) was performed by
fitting the model to the uptake data. VI was 52.3 ~l; V2 , 32.7 ~l;
and k o was held as close as possible to 0.26, cf. the mannitol efflux
UPTAKE OF GLUCOSE ANALOGS BY NORMAL AND ISCHEMIC SLICES 269

data (Table I). k1 and +

were varied to give the best fits. ,
was 1 for both mannitol and sugars indicating symmetry of the
transport process. The ko and k1 values of the best fits are given
in Table II.

Table I I

Best Fitting Values of ko and k1

Mannitol a.-methy1- 3-0-methy1-

glucoside glucose
ko 0.26 0.26 0.35
Normal
(min- 1 )
k1
Brain 0.015 0.18 3.0
(min- 1 )

Ischemic k1
0.015 0.18 3.0
brain (min- 1 )

The model (Fig. 1) was fitted to the uptake (Fig. 2). The
table shows the values of ko and k1 , which gave the best
fits. The k1-va1ues given in the lower row were obtained
on slices from ischemic brain.

It appears from the table that k1 for mannitol was at least

ten times smaller than it was for sugars. It is therefore permissible
to neglect the passive permeability as was done in the model.

Extracellular diffusion versus membrane transport. A comparison

.Q;-methy1-g1ucoside ko~ k t and for 3-0-methy1-g1ucose ko <

between ko (diffusion) and k1 (membrane transport) shows that for
k1 •
This indicates that the dLffusion is partly limiting for the uptake
of .Q;-methy1-g1ucoside and totally limiting for the uptake of 3-0-
methyl-glucose. Determination of the maximal membrane transport
(k 1 ) in terms of unidirectional fluxes across the cell membranes
is accordingly difficult for .Q;-methy1-g1ucoside and impossible for
3-0-methy1-g1ucose 9 • The uptake rate will reflect the extracellular
diffusion and not the membrane transport. An attempt to determine
Km and Vmax values will give diffusion values in the form of falsely
large K and Vrnax values. Our own results (unpublished experiments)
and dat~ from the 1iterature 3 ,5 confirm this interpretation.

The glucose analog 2-deoxy-g1ucose, which is phosphorylated, 1

has also been used to determine membrane transport in brain slices •
A similar model analysis of 2-deoxy-glucose uptake indicates, however,
that the phosphorylation and not tge transport is determined by
application of this glucose analog •
270 H. LUND·ANDERSEN AND C.S. KJELDSEN

UPTAKE OF SUGARS BY SLICES FROM ISCHEMIC BRAIN

Ischemia and anoxia are well known causes of irreversible brain

injury. The pathophysiological mechanisms involved are complex
and the factors responsible for irreversibility are only partly
understood; for review see SiesjH and Plum12 • An irreversible
impairment of the glucose transport mechanism during ischemia might
be one of the mechanisms involved.

Originally, the purpose was to study whether 30 min of ischemia

caused irreversible damage of the glucose transport mechanism, as
evaluated by changes in Km and Vmax for the membrane transport in
slices from ischemic brain. As shown above ~ and V could not
be determined due to the extracellular diffusion del~~ It appeared,
however, from Fig. 2 and from the kl-values (Table II) that the
membranes clearly distinguished between mannitol, a-methyl-glucoside,
and 3-0-methyl-glucose. The distinction was qualitatively the same
as observed ~ viv0 2 • This property was taken as an indication of
a well functioning sugar transport mechanism. It was of interest
to see whether this mechanism was affected by 30 min of ischemia.

Rat brains were made ischemic by decapitation. After a 30 min

period the hemispheres were removed from the skull and slices were
cut. These were incubated in oxygenated media under optimal
conditions for 45 min, before addition of isotopes.

~D~e~t~e~rm~i~n~a~t~i~o~n~o~f~t~h~e~m~o~d~e~l~p~a~r~a~m~e~t~e~r~s~ko and Vl~ Determination

of the extracellular space (VI) and the rate of diffusion (kp ) was
carried out as described above. The results are shown in Table I,
lower row. It appears that the values of the two groups of slices
are identical.

Uptake of sugars and mannitol. The time-dependent uptake of

sugars and mannitol was followed in the same way as described for
the normal slices. The values are shown in Fig. 3.

It appears by comparison between Figs. 2 and 3 that the uptake

into the ischemic slices and the normal slices was identical.

Comparison between membrane transport in slices from normal

and ischemic brain. Using the values of Table I, lower row, the
model was fitted to the uptake. The kl values of the best fits are
shown in the lower row of Table II. It appears that the membrane
transport (k,) was identical in slices from normal and ischemic
brain. The fransport mechanism had accordingly not undergone
irreversible damage during the 30 min period of ischemia. Destruct-
ion of the sugar transport mechanism therefore does not seem to be
involved in the irreversibility of ischemic brain damage.
UPTAKE OF GLUCOSE ANALOGS BY NORMAL AND ISCHEMIC SLICES 271

...J
0I- ~
-I-
ZJ:
Z(!)
«-
::::EILI
~
60
0
ZI-
«ILl
If)~
a:...J

~~
:::J-
40
If)IL
IL DI
o E
ILl 8
... -
:0::-
:e- 20
Q.:::L
:::J~

5 10 20 30 40 50 60
TIME OF EXPOSURE TO ISOTOPES, MIN.

Figure 3. Uptake of 14C-1abe11ed mannitol (I-I), ~-methy1-

glucoside (0-0), and 3-0-methy1-g1cuose ([J-c:P into 0.5 mm
thick rat brain cortex slices from brains exposed to 30 min
of ischemia. The experimental conditions were as described
for normal slices in Fig. 2.

SUMMARY AND CONCLUSION

The uptake of the glucose analogs a-methy1-g1ucoside and 3-0-
methyl-glucose by brain cortex slices w;s studied. Analysis of the
uptake course by the aidofathree-compartment serial model showed
that the extracellular diffusion was rate limiting for the uptake.
Determination of Km and Vmax values for the membrane transport in
terms of unidirectional fluxes across the membranes was accordingly
impossible. Falsely large ~ and Vmax -va1ues would appear.

The transport mechanism clearly distinguished between mannitol,

~-methy1-g1ucoside, and 3-0-methy1-g1ucose. This property was
preserved in brains exposed to 30 min of ischemia.

This study was supported by grant no. 512-5210 from the Danish
medical research council.
272 H. LUND·ANDERSEN AND C.S. KJELDSEN

REFERENCES

1. Bachelard, H.S., Specificity and kinetic properties of mono-

saccharide uptake into guinea pig cerebral cortex ia vitro,
J. Neurochem., 18 (1971) 213-222.
2. Bradbury, M.W.B., and BrCJ!ndsted, H.E., Sodium-dependent transport
of sugars and iodide from the cerebral ventricles of the rabbit,
J. Physiol., 234 (1973) 127-143.
3. Cooke, W.J., and Robinson, J.D., On the uptake of hexoses by
rat cerebral cortical slices, J. Neurochem., 18 (1971) 1351-1356.
4. International Critical Tables, Vol. 5, McGraw-Hill Book Company,
New York, 1929, p. 71.
5. Joanny, P., Corriol, J., and Hillman, H., Uptake of monosacchari-
des by guinea-pig cerebral-cortex slices, Biochem. J., 112 (1969)
367-371.
6. Katzman, R., and Pappius, H.M., Brain electrolytes and fluid
metabolism, Williams and Wilkins Company, Baltimore (1973).
7. Kjeldsen, C.S., and Lund-Andersen, H., Uptake of 2-deoxy-glucose
by rat brain cortex slices. Transport versus phosphorylation.
In preparation.
8. Lund-Andersen, H., Extracellular and intracellular distribution
of inulin in rat brain cortex slices, Brain Research, 65 (1974)
239-254.
9. Lund-Andersen, H., and Kjeldsen, C.S., Uptake of glucose
analogues by rat brain cortex slices, I. A kinetical analysis
based upon a model, J. Neurochem., submitted.
10. Lund-Andersen, H., Kjeldsen, C.S., Hertz, L., and BrCJ!ndsted, H.E.,
Uptake of glucose analogues by rat brain cortex slices, II.
Na+-independent membrane transport, J. Neurochem., submitted.
11. M~ller, M., M~11g9rd, K., Lund-Andersen, H., and Hertz, L.,
Concordance between morphological and biochemical estimates of
fluid spaces in rat brain cortex slices, Exp. Brain Res., 22
(1974) 299-314.
12. Siesj8, B.K., and Plum, F., Pathophysiology of anoxic brain
damage. In G. Gaull (Ed.) Biology of Brain Dysfunction, Vol. 1,
Plenum Press, New York 1973, pp. 319-372.
UPTAKE AND EXCHANGE OF GABA AND GLUTAMATE IN

ISOLATED NERVE ENDINGS

Giulio Levio, Ugo Poceo, and Maurizio Raiterioo

°Laboratory of Cell Biology
Via Romagnosi 18/A, Rome, Italy
oOInstitute of Pharmacology
Catholic University, Rome, Italy

INTRODUCTION

In the last years an increasing number of reports have favour-

ed the concept that GABA and glutamate act as inhibitory and excita-
tory neurotransmitters respectively in the mammalian CNS l l,12,25.
One of the essential biochemical steps of neurotransmission is the
rapid inactivation of the active substance liberated in the synap-
tic cleft. With the only known exception of acetylcholine, which
is rapidly hydrolyzed by a specific enzyme, reuptake into the pre-
synaptic terminal is considered as the major means for terminating
the action of most other putative neurotransmitters ll ,1?,21,24,37.
It has been known for many years that brain slices are able to
take up GABA and glutamate from the surrounding incubation medium lO
More recently, kinetic studies performed with minute brain slices
(microprisms) and with crude or purified preparations of nerve end-
ings have revealed the existence of two components in the uptake of
GABA and glutamate, one with low and one with high affinity for the
substrate 7 ,22,25,37.
The discovery of "high affinity" transport systems and, in parti-
cular, the fact that these systems could initially be detected only
fur putative neurotransmitter amino acids, has led to the widely ac-
cepted concept that the supposedly low concentrations of neurotrans-
mitter amino acids present in the synaptic cleft after stimulation,
could be rapidly removed through specific "high affinity" reuptake
processes 21 ,37. Further studies disclosed the presence in synapto-
somes of "high affinity" uptake systems also for amino acids with-
out any effect on neuronal excitability 2,4,5. However, a discrimi-
nation between "high affinity" uptake of neurotransmitter and non-
neurotransmitter amino acids was made on the basis of sodium depen-

273
274 G. LEVI, U. POCE, AND M. RAITERI

dence S • In fact, the "high affinity" uptake of putative neurotrans-

mitters showed an absolute requirement for sodium, whereas that of
neurally inactive amino acids was sodium independent. Interesting-
ly, the low affinlty uptake of neurotransmitter amino acids showed
only a limited sensitivity to the absence of sodium. It was there-
fore postulated that the specific process utilized by nerve endings
to recapture the released neurotransmitter amino acids is a sodium
dependent "high affinity" uptake, and also that a.sodium dependent
"high affinity" uptake into nerve terminals may be a valuable cri-
terion favouring the candidacy of a given compound as a neurotrans-
mitter S ,37. The validity of this criterion, however, is still open
to discussion since "high affinity", sodium dependent uptake systems
for GABA and glutamate have been recently described in isolated glia
from the CNS 1 7,18,and glial cells of spina1 3l ,32,33and sympathetic
ganglia 8 . I t should be also noted that a "high affinity" sodium
dependent uptake of GABA has been described in neuroblastoma 20 cells
which are devoid of nerve endings.
Recent reports on the synaptosomal homo-exchange 26,30,36 show-
ed that the problem of presynaptic reuptake processes is far more
complex than it was originally formulated.
In the present chapter we shall summarize briefly our data on
GABA and glutamate exchange in synaptosomes, and, in the last sec-
tion we shall describe some recent experiments which led us to the
formulation of a hypothesis on the possible functional significance
of synaptosomal exchange processes.

SYNAPTOSOMAL EXCHANGE OF GABA AND GLUTAMATE

Concentration Dependence of Exchange

When synaptosomes, which contain millimolar concentrations of

amino acids such as GABA and glutamate, were prelabeled with a very
low concentration of 3H-GABA (0.5 pM) and then superfused by a tech-
nique which prevents the reuptake of the spontaneously released
neurotransmitters 29 a release of 3H-GABA of about 1% per min was
observed (Fig.l). If, during superfusion, unlabeled GABA was added
to the superfusion fluid, the release of radioactive GABA.was in-
creased in a concentration dependent and saturable way (Fig. 1).
The stimulation of release was evident also at micromolar GABA con-
centrations, that is in the concentration range of the "high affi-
nity" uptake system. The release rates calculated in this concentra-
tion range, on the assumption of a homogenems distribution of the
3H-GABA within the endogenous GABA pool, were very similar to the
initial rates of GABA "high affinity" uptake. In a recent paper in
which the same phenomenon was studied by a somewhat different expe-
rimental technique, Simon Martin and Krol1 36 have shown that the
apparent Km and Vmax of GABA "high affinity" uptake and exchange
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 275

are practically superimposable. These data suggest that radioactive

GABA can enter synaptosomes through a 1 : 1 homoexchange process.
Similar data to those obtained with 3H-GABA were obtained with
l4C-glutamate (Fig.2). However, the glutamate exchange rates, cal-
culated on the assumption of a homoge~s glutamate pool, were al-
most twice as great as the initial rates of glutamate "high affini-
ty" uptake. This finding might suggest that the amino acid taken
up by synaptosomes during the prelabel i ng phase is not homogeneous-

8
111M GIU
100 ,M

-....
c 7 50 ,M

-....
10 ~M

5 ,M
6
1 pM
co.
Cantlll

.
.~

co
5
W
~

.
c:
3

..
.... 2

o~~~~ __~__~__~__~
2 4 6 8 10 12
Fraeli .. number (1 fracllon : lmin · O.47ml J

Fig. 1 - Stimulation of 3H-GABA release from synaptosomes b~ unlabel-

ed GABA. Purified synaptosomes were prelabeled with 0.5 pM H-GABA
in a Krebs-Ringer medium at pH 7.35. Aliquots (1 ml) of the suspen-
sion were then collected on Millipore filters lying at the bottom
of parallel superfusion chambers thermostated at 37°29, and super-
fused with standard, glucose containing, oxygenated medium. After
5.5 min (see arrow) the medium of the chambers was replaced with me-
dia containing varying concentrations of unlabeled GABA. The radio-
activity of each superfusate fraction was expressed as percentage of
the total radioactivity recovered (total fractions plus radioactivi-
ty remaining in the filters at the end of superfusion). Aminooxyace-
tic acid (10 pM) was present in all the media used. Each cU3~e is
the average of 3 separate experiments. (From Raiteri et al. ).
276 G. LEVI, U. POCE, AND M. RAITERI

10

_ Contro l
9
..- ..... 10~M Glu
c:
0 . 'I, " l l - i > 20 ~M
..., 8 .. / \ o - 0 5 0~ M
'" ,/ \ '
t . ·~ '\
• .200 ~M
;;; 7
Co 1/ \ \
> : \ b
.~ 6 ,6 '\ \
~ 1/ \ \
'" f, \ '\ .
\ \'
0
5
"C

~
I \ '\ .
I .................. \,. ~
'"
;:!
4 I / I -.-.. , ' . , 6

I •, .
~

"'0 .... , 4, '6

c: 3 I ,' ....
...,
'" ,
<;; {,,
a.. 2
~-
A'..

2 4 6 8 10 12
fraction number Cl fract io n= lmin = O.6mf)

Fig. 2 - Stimulation of l4C-L-glutamate release from synaptosomes

by unlabeled L-glutamate. Experimental conditions as described in
the legend for Fig.l, except that the amino acid concentration used
for prelabeling the synaptosomes was 10 ]lI1 instead of 0.5 pM in or-
der to minimize the metabolic breakdown of the l4C- glutamate. Each
curve is the average of 3 separate experiments.

ly distributed within the endogenous pool and is released preferen-

tially. In order to test such possibility, the release of preaccu-
mulated exogenous tritiated GABA or glutamate was compared to that
of l4C-GABA or l4C-glutamate metabolically ~erived from a l4C- pre-
cursor (Table 1). Exogenous 3H-GABA, and 1 C-GABA derived from
l4C-glutamate, exhibited the same rates of spontaneous and stimula-
ted release. On the other hand, both the spontaneous and the sti-
mulated release of exogenous 3H-glutamate were about twice as large
as those of l4C-glutamate metabolically derived from l4C-glucose.
These results indicate that the exogenous glutamate does not distri-
bute homogeneously within the endogenous glutamate pools and may ex-
plain why the glutamate exchange rates calculated on the basis of a
single homogeneous pool appear higher than the initial rates of up-
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 277

Table 1 Comparison between the release of exogenous 3H-glutamate or

3H-GABA and endogenous l4C-glutamate or l4C-GABA metabolically de-
rived from a l4C-precursor.

per cent of total radioactivity

3H-glutamate l4C-glutamate 3H-GABA l4C-GABA

unlabeled glu
or GABA added 100 pH 10 flM 100 flM 10 fll1 100 pM 100 flM
(10th min)
released in 18.1 11.3 7.4 5.0 6.4 6.7
min 1-6 +0.4 +1.5 +1.6 +0.6 +0.9 +1.3
released in 5.8 4.9 2.9 2.5 2.7 2.4
min 7-10 +1.7 +0.7 +0.5 +0.1 +0.3 +0.4
released in 20.1 12.0 11.1 6.8 20.5 16.2
min 11-14 +2.4 +0.4 +2.3 +0.4 +3.3 +2.9
left in tissue 55.8 71.5 78.2 85.2 70.0 74.4
+3.9 +1.0 +2.0 +0.5 +4.0 +3.5

Number of expts. 4 4 4 4 7 7

Purified synaptosomes were incubated for 30 min in a Krebs-Ringer

bicarbonate medium containing 10 roM glucose and either 10 ~Ci/ml of
U_1 4C-D-glucose (in the experiments in which the release of gluta-
mate was studied) or 3.3 pCi/ml of U14 C-L-glutamate (290 mCi/mmole)
(in the experiments in which the release of GABA was studied). The
synaptosomal protein concentration was 4 mg/ml in incubations with
l4C-glucose, and 0.4 mg/ml in those with l4C-glutamate. During the
last 5 min of incubation, 1 pM 3H-glutamate (in the glutamate re-
lease experiments) or 1 jlM 3H-GABA (in the GABA release experiments)
was added to the incubation mixture. Aliquots of the suspension
were superfused as described in the legend for Fig. 1, with amino
acid-free medium, which was replaced after 10 min with new medium
containing unlabeled amino acid as indicated in the table. From each
superfusion chamber 3 fractions of effluent were collected in glass
vials maintained at 0°: a first 6 min fraction, and two subsequent
4 min fractions. The superfusates and the superfused synaptosomes
on the filters were treated with 3% perchloric acid (final concentra
tion), and the extracts were applied to small AG50 columns. The ami~
no acids bound to the resin were eluted with 2N NH40H; the eluates
were dried, washed 3 times with water, resuspended in 80% ethanol,
and applied on 'Vhatman 2 paper. The paper was subjected to high vol-
tage electrophQresis, and the radioactivity present in the position
of GABA and glutamate was measured. The results are expressed as per
centages of total 3H or l4C radioactivity (superfusates plus filter)
+ S.D.
278 G. LEVI, U. POCE, AND M. RAITERI

take. The experiments reported above also showed that the unlabeled
glutamate added to the superfusion fluid stimulated the release of
the l4C-aspartate derived from l4C-glucose (data not presented). In
conclusion, it appears that exogenous glutamate can enter the synap-
tosomesby exchanging with a pool of endogenous glutamate and with a
pool of endogenous aspartate. A comparison between total exchange
rates and initial rates of uptake would be possible only if the size
of the endogenous glutamate and aspartate pools exchanging with ex-
ogenous glutamate were known.

Sodium-Dependence of Synaptosomal Exchange of GABA and Glutamate

Another property of the GABA and glutamate "high affinity" up-

take shared also by the exchange process is the absolute sodium-de-
pendence. Fig. 3 shows that the homoexchange of GABA was virtually
abolished when the synaptosomes wer~ superfused in a sodium-free me-

.-. Control

-. .......•..
0··-0 Sucrose
'"
.!!
5 Sucrose +

-.-....
1>-1>.
500 pM GABA
::!
•...•. ConI 101 +
50 pM GABA

--..
"~

4 ..•.
:E
ow
D
:;;
".
3

---
::!
;;
D

-..
'"
2

.-
a.

o~~~ __~__~__~~~~~__
2 4 & 8 10 12
Fraction number ( 1 fraction =1min U 7ml )

Fig. 3 - Effect of sodium deprivation on the release of 3H-GABA

from synaptosomes. Experimental conditions as described in the le-
gend for Fig. 1. In the sodium-free medium used for superfusion,
256 mM sucrose renlaced 128 ~1 NaCl. Averages of 3 experiments are
presented. (From Rai teri et al. 30) .
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 279

dium, a condition which does not alter significantly the spontaneous

release of 3H-GABA. Simon, Martin and Krol1 36 , showed that the so-
dium-dependence curves of GABA "high affinity" uptake and stimulated
release are almost identical. Also in the case of glutamate, the
sodium-dependence curve of exchange was superimposable to that of
"high affinity" uptake (Fig. 4).

100
........
90

80

c 70
:!>'
c;
0
u
60 ...- exchange
-;; w····" uptake
-
~
u
50
~
c...
40

30

20

10

10 25 50 75 100 138
Sodium concentration ImMI

Fig. 4 - Sodium-dependence of l4C-glutamate uptake and exchange in

synaptosomes. The initial rate of 5 pM l4C-L-glutamate accumulation
was measured over a 2 min period as previously described 26 . Gluta-
mate exchange was measured as described in the legends for Figs. 1
and 2, and was calculated after subtracting the rate of spontaneous
release from the total release rate. The concentration of unlabeled
glutamate used for stimulating the release of 14C-glutamate was 20
pM. NaCl was replaced by sucrose in the sodium-deficient media.
The results are expressed as percentages of control uptake and ex-
change respectively. Each curve is the average of 2 separate exper-
iments, differing less than 10% one from the other.

Substrate Specificity of GABA Exchange

One of the features that characterizes the GABA uptake system

is its high substrate specifici ty3,6,22. ~fuen we studied the sub-
strate specificity of GABA exchange in synaptosomes, we found that
all the ~-amino acids tested, biogenic amines and acetylcholine were
ineffective even at high concentrations (10- 3 M). One of the strong-
est inhibitors of GABA "high affinity" uptake, ~-amino-~hydroxybu-
280 G. LEVI, U. POCE, AND M. RAITERI

tyric acid, was also a strong stimulator of 3H-GABA release from sy-
naptosomes 30 , probably through a heteroexchange process. Another
strong, and, according to Schon and Kelly33,34, specific inhibitor
of neuronal GABA uptake, 2,4-L-diaminobutyric acid (DAB), was also
able to stimulate 3H-GABA release (Fig. Sa).

spontaneous release ot 14C-GABA

.'-A release ot 14C-GABA stimulated by 50 ~M GABA
0- - 0 50~M L-DAB

Control P,,,ncubated with 100 ~M l·OAB

u
co 6 6
f
i
t
.::
:; 4 4
o
"'0
co
. / •. - .. -.l ......
f .4_.4 _ ..

'"0 //0--0--0 .... _°_

"'"'"
u
~
0..
®t
4 6 10 4 8 10
traction number (1 fractlon= 1 min= 0.6 mt)

Fig. S - Homoexchange of GABA and heteroexchange of DAB and effect

of preloading the synaptosomes with DAB on these processes. Ex-
perimental procedure as described in the legend for Fig. 1, except
for the following details: the standard superfusion medium was re-
placed with medium containing unlabeled GABA or DAB after 7.5 min
(see arrow); in the experiments of panel (a) the synaptosomes were
prelabeled with 0.2 pM l4C-GABA for 10 min; in the experiments of
panel (b) 100 rH
DAB was present in the medium during a 10 min pre-
labeling phase with 0.8 pM l4C-GABA. Averages of 2 experiments are
presented.

DAB has been shown by Simon and ~~artin to behave as a competi-

tive inhibitor of synaptosomal GABA influx when it is added together
with GABA to the incubation medium, and as a non-competitive inhibi-
tor when synaptosomes are allowed to accumulate it in sufficient a-
mount. In agreement with these findings, synaptosomes preloaded
with 100 pM DAB and tracer amounts of radioactive GABA showed a
large decrease of the homoexchanee of GABA, even if they were wash-
ed for several minutes before adding the unlabeled GABA to the super-
fusion fluid. Interestingly, also the heteroexchange of DAB for
3H-GABA was similarly reduced in these conditions (Fig. 5b). The
reduced homoexchange of GABA or heteroexchange of DAB could not be
attributed to a large heteroexchange of GABA or homo exchange of DAB
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 281

with the preaccumulated DAB. In fact, in experiments with 3H-DAB,

we could show that this process was very limited (unpublished obser-
vations).

Parallel Decrease of GABA Uptake and Exchange

Beside the cases that have been already mentioned, the uptake
and exchange of GABA behaved in a parallel way also in other circum-
stances. In two series of experiments V we obtained a partial de-
pletion of the endogenous synaptosomal GABA content by two different
methods: 1) by washing the prelabeled synaptosomes on Millipore fil-
ters with incubation medium at 0°. This procedure led to a 60-70%
loss of intracellular GABA; 2) by superfusing the prelabeled synap-
tosomes with a depolarizing concentration of KCl, a condition cau-
sing an increased release of GABA and a depletion of synaptosomal
GABA of about 30%. After an equilibration period of 5 or 10 min with

Table 2 Concentration, exchange and uptake of GABA In synaptosomes

depleted by cold shock or 56 mH KCl.

Type of pre-treatment cold shock 56 m 11 KCl

control treated control treated

% of control % of control

Concentration at the
prestimulation time 16.4 28.4 12.4 70.0
(nmoles/mg protein)
Exchange rate (nmoles/ 0.96 33.3 0.56 67.8
mg protein/min)
Uptake of 20 pM 14C-GABA 5.6 31. 9 4.3 81.8
(nmoles/mg.protein)

Synaptosomes were prelabeled with 0.5 pH 3H-GABA and aliquots were

collected on Millipore filters placed at the bottom of the superfu-
sion chambers. In the cold shock experiments the filters were wash-
ed at 0° (treated) or at 25° (controls), then superfused with stan-
dard medium at 37°. After 5 min the medium was replaced with new me-
dium containing 20 pM l4C-GABA and the superfusion was continued for
7 min. In the experiments with high KCl, all the filters were washed
at 25° and superfused for 20 min either with 56 mM KCl (treated) or
with standard medium. Then the superfusion was continued for 5 min
with standard medium in all the chambers, and finally with a medium
containing 20 pt1 l4C-GABA for 8 min. For other experimental details
see ref. 27.
282 G. LEVI, U. POCE, AND M. RAITERI

standard medium at 37°, the depleted synaptosomes were superfused

with a medium containing 20 pM l4C-GABA, in order to study the re-
lease of 3H-GABA and accumulation of l4-GABA in the same experiment.
Table 2 shows that in treated synaptosomes, both conditions cause a
parallel decrease of uptake and exchange, a finding that could hard-
ly be expected if uptake and exchange were two different and inde-
pendent processes. Interestingly, the decrease in uptake and ex-
change was of the same magnitude as the decrease in the synaptoso-
mal GABA pool, which is suggestive of a correlation between pool
size and transport rates.

Effects of Ouabain and of Calcium Ionophore A23l87

In the preceeding paragraphs we have described a number of ca-

ses in which "high affinity" uptake of radioactive amino acid and
exchange (measured as amino acid-stimulated release) behaved in a
parallel way. The experiments that will be described in the present
paragraph will show that ouabain and the divalent cation ionophore
Lilly A23l87 14 have apparently opposite effects on the uptake and
on the amino acid-stimulated release of radioactive GABA and gluta-
mate. Figs. 6a and 6b show that ouabain and the ionophore A23l87
caused only a modest and relatively slow increase in the spontaneous
release of 3H-GABA from synaptosomes. However, when also a small
concentration of unlabeled GABA (10 rt1) was present in the superfu-
sion fluid, the release of 3H-GABA increased without any lag period,
much more than what would be expected just by the addition of GABA.
Since both ouabain and A23l87 strongly inhibited the accumulation of
radioactive GABA into synaptosomes (50-70%, depending on the experi-
mental conditions), the increased stimulated release could not be
attributed to an increased rate of a 1:1 homoexchange of the amino
acid which, in fact, might even be diminished. Therefore, the pre-
sence of either ouabain or A23187 plus GABA in the superfusion me-
dium caused a much greater net release of intracellular GABA from sy
naptosomes than that induced by ouabain or A23l87 alone. The effect-
of extracellular GABA was substrate specific: GABA, either alone or
combined with ouabain or A23l87, did not affect the release of glu-
tamate from synaptosomes.
From these observations one could infer that a large part of
the inhibitory effect of ouabain and A23l87 on the uptake of radio-
active GABA may be due to an enhancement of net release, determined
by the presence of the extracellular amino acid.
Similar results were obtained also with l4C-glutamate (Figs.
7a and 7b), although the potentiating effect of this amino acid was
less pronounced than that of GABA, at least in the experimental con-
ditions tested. It is noteworthy that also the inhibition of l4C-
glutamate accumulation (about 30-40%) caused by ouabain and A23l87
was less than that observed with 3H-GABA.
In previous studies the releasing effecmof ouabain and of
A23l87 were interpreted as due to an increased concentration of in-
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 283

:; 8
u
10 pM GABA •
'" 7- 1001lM ouabain

'"
0. 1O,IMGABA'
:= 6- 19pM A 23187

'"
o
.
! '.
-c
'" 4- /_ ..........., 4· ../ '. .........10p\1GARA
{
/ " 10 pM GABA
I
f I
/ ,0' _0 100 pM ouballl
0: I
'"u 191'M A23187
__ 0 •• 0--0"0-'0 o·
'"
0..
Coni rol
Ii;l;lt-O>-'-._~~_ Con lllli

a I iJ I
4 6 8 4
fraCtIOn numher 11 frac1101l=11l1l1l=O.611l1 ,

Fig. 6 - Effect of the ionophore A23l87 and of ouabain on the spon-

taneous and stimulated release of 3H-GABA from synaptosomes. Synap-
tosomes were prelabeled with 3H-GABA and super fused as described in
the legend for Fig. 1. After 7.5 min of superfusion with standard
medium, the medium of some of the chambers was replaced with new
medium containing one of the following: 19.2 pM A23l87 (panel a) or
0.1 mM ouabain (panel b); 10 pH GABA (panels a and b); 19.2 pM
A23l87 plus 10 }IM GABA (panel a) or 0.1 mlf ouabain plus 10 pH GABA
(panel b). A23l87 was dissolved in absolute ethanol and added to
the superfusion medium immediately before use, 10 pI/mI. An equiva-
lent amount of ethanol was added to control media. Panel (a): avera
ges of 6 experiments (4 run in duplicate). Panel (b): averages of-
2 duplicate experiments.

tracellular sodium 16 or to an increased influx of calcium respect i-

vely14,19. Judging from data obtained in studies on GABA uptake, both
these cations could affect the carrier mediated efflux of GABA. In
fact, it has been shown that the sodium-dependence curve of GABA
influx into synaptosomes has a si~moid shape, and that calcium, which
stimulates GABA uptake at low, but not at high sodium concentrations,
abolishes the sigmoid character of the curve 28 . If GABA efflux had
similar ionic requirements, the outward transport of the amino acid
would be normally limited by the unfavourable ionic concentrations
(low sodium and negligible "free" calcium). Our data indicate that
an increase of intracellular sodium (produced by ouabain) or calcium
(produced by A23l87) strongly stimulate the carrier mediated release
of 3H-GABA activated by a low concentration of extracellular GABA
284 G. LEVI, U. POCE, AND M. RAITERI

c:
0

'" 6 6
~ lO~MGlu.
0-
19 ~M A23187 10 ~M Glu •
~ 5 5
100~M ouabain
.~
u
4 4 \
'"
!:!
"0
• IO~M Glu
~
3 3
'"
0

-:; 2 o •• o··o··OI9~M A23187 2 7' •• 0 •• 0 -- 0 --0 100~M ouabain

• ... ... 0
......
;: ~o

Cont rol Cont rol

~ 1
1
'"
® t t
c...
(0)
2 4 6 8 2 4 6 8
fraction number Ilfraction=lmin=O.6mll

Fig. 7 - Effect of the ionophore A23l87 and of ouabain on the spon-

taneous and stimulated release of l4C-glutamate from synaptosomes.
Experimental details as in the legend for Fig. 6, except that synap-
tosomes were prelabeled with 10 pM l4C-glutamate, and 10 pM gluta-
mate was added to the superfusion fluids, instead of GABA. The curves
presented in panel (a) are averages of 6 experiments, 2 of which run
in duplicate. The curves of panel (b) are avera~es of 5 experiments,
2 of which run in duplicate.

(Fig. 6) and suggest that, in the new ionic conditions determined by

the two drugs, the stoichiometry of the homoexchanse process is chang-·
ed in a direction leading to net outward transport.
Ouabain has been reported to stimulate the influx of 45 Ca2+ into
synaptosomes1 5 ,38,39 (according to other authors the drug does not
affect calcium uptake 23 ). At any rate, the intracellular level of
"free" calcium might increase in ouabain treated synaptosomes as a
consequence of the altered intracellular concentrations of sodium
and p~tassiuml. Experiments from our laboratories (Levi and Raiteri,
unpublished) indicate that the efflux of 45Ca 2+ from synaptosomes
into a calcium-free medium is sreater in the presence than in the
absence of ouabain (+22%, average of 10 determinations), suggesting
a greater intracellular availability of "free" calcium. Thus, the ef-
fects of ouabain on amino acid release (Figs. 6 and 7) mieht be due
to the increased intracellular concentration of calcium, rather than
to that of sodium. However, this possibility seems unlikely, since
the effects of ouabain on GABA and glutamate uptake and release re-
EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 285

mained unchanged when a calcium-free medium, containing 0.1 mM EGTA,

was used. In contrast, the effects of the ionophore A23l87 on GABA
uptake and release were abolished when calcium (standard concentra-
tion in control experiments: 2.7 mM) was omitted from the incubation
media. Moreover, calcium is not expected to influence GABA transport
when the concentration of sodium is relatively high 28 , as it should
be in the intrasynaptosomal space after ouabain treatment.
Interestingly, the effect of ouabain on the GABA-stimulated
3H-GABA release was larger than that of A23l87 (Fig. 6), a finding
in keeping with the concept that sodium is more effective than cal-
ciur.l in stimulating GAB A carrier mediated transport 28 .

CONCLUDING RE~ARKS

The following conclusions can be drawn, at present, on the de-

bated problem of exchange versus "high affinity" net uptake.
1) The synaptosomal homo exchange of GABA (and glutamate) resem-
bles very much the "high affinity" uptake system in its properties.
Since net uptake has never been demonstrated to occur when using
exogenous amino acid concentrations in the range of the "high affi-
nity" uptake system, whereas exchange has been demonstrated to occur,
we must conclude that accumulation of radioactivity by exchange can
simulate the presence of a "high affinity" uptake system.
2) Isolated nerve endings are capable of a net accumulation of
GABA and glutamate, which, however, can be demonstrated at relative-
ly high amino acid concentrations, falling in the range of a trans-
port system of lower affinity and less sodium dependent than the
"high affinity" uptake system.
3) The problem of the affinity of the transport system perform-
ing the net accumulation is probably not as crucial as it has been
proposed. The limits set between high and low affinity uptake sys-
tems are most arbitrary. For example, GABA uptake systems with KID
values ranging from 0.15 to 40 f.}~ are all accepted to be of "high
affinity", but most people would be hesitant to define as "high af-
finity" uptake a system with a KID over 100 or 200 fM. Moreover,
nobody estimated the concentration of GABA and glutamate in the syn-
aptic cleft after stimulation. This concentration might not be far
from the KID of a "low affinity" uptake system, if one agrees with
Fonnum's calculations 13 , according to which the average concentra-
tion of GABA in "gabergic" nerve terminals is around 100 mM.
4) Hhat seems more important for the reuptake theory of neuro-
transmitter amino acid inactivation is that the system capable of
performing a net uptake is localized in the nerve terminals utili-
zing that particular amino acid as a neurotransmitter. Hilkin et
al. 40 , showed that in isolated cerebellar glomeruli the radioactive
GABA taken up is localized in the axon terminals of Golgi cells,
which are believed to utilize GABA as an inhibitory neurotransmitter.
These considerations do not solve the problem of the possible
functional significance of a process which performs an apparently
286 G. LEVI, U. POCE, AND M. RAITERI

1:1 exchange. Simon et al. 36 suggested that synaptosomes may be in

a functionally impaired condition, which does not allow them to ope-
rate a highly energy-requiring net uptake. However, it has been de-
monstrated that synaptosomes are metabolically active and stable for
relatively long periods after preparation 9 .
If exhange existed also in the living brain, we should wonder
why presynaptic nerve terminals are provided with a very specific
homeostatic mechanism capable of maintaining, with little or no ex-
penditure of energy, cartain concentrations of putative neurotrans-
mitter amino acids such as GABA and glutamate in the synaptic cleft.
Could the presence of small intrasynaptic concentrations of GABA or
glutamate modulate some essential biochemical step of neurotransmis-
sion?
It is generally accepted that under physiological conditions
neurotransmitter release is triggered by the increased availability
of free Ca 2+ ions in the presynaptic terminal consequent to the de-
polarizing stimulus. If the calcium movements induced by the cal-
cium ionophore A23l87 were representative of those taking place du-
ring physiological stimulation, one might speculate that the GABA
and glutamate maintained in the synaptic cleft by the homoexchange
process could play a role in enhancing their own release during the
stimulation process. Furthermore, the decrease of sodium concentra-
tion in the vicinity of the presynaptic membrane occurring in the
depolarization phase might be large enough to slow down the highly
sodium dependent homoexchange, while the "low affinity" net uptake
process, which is less senS1t1ve to a decrease in sodium concentra-
tion, could remove the excess of amino acid from the synaptic cleft.

Acknowledgments: 1-Ie thank Mr. Alberto Coletti for the excellent tech-
nical assistance, and Dr. Peter J. Roberts for collaborating in some
of the more recent experiments. The ionophore A23l87 was a generous
gift of Eli Lilly and Company (Indianapolis). This investigation
was partly supported by Research Grant No. 922 of North Atlantic
Treaty Organization.

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288 G. LEVI, U. POCE, AND M. RAITERI

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EXCHANGE OF GABA AND GLUTAMATE IN NERVE ENDINGS 289

38. Stahl, \-1.L. and Swanson, P.D'., Uptake of calcium by subcellular

fractions isolated from ouabain treated cerebral tissue, J. Neu-
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39. Swanson, P.D., Anderson, L. and Stahl, H.L., Uptake of calcium
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MECHANISMS OF TRANSPORT FOR THE UPTAKE AND RELEASE OF

BIOGENIC AMINES IN NERVE ENDINGS

Donald F. Bogdanski

Section on Biochemical Pharmacology, Hypertension-

Endocrine Branch, National Heart and Lung Institute
National Institutes of Health, Bethesda, Maryland 20014

INTRODUCTION

Reports on the amine transport mechanism located at the plasma

membrane of nerve endings have been extensively reviewed for general
characteristics 30,31,49, and for the effects of electrolytes 46.
The nerve ending is uncommon, or unique in the sense that the product
of secretion is recaptured almost immediately by the secretory organ.
Transport, therefore, may be a bidirectional phenomenon in nerve
endings. As a result of investigations, a synaptic sub-unit is en-
visaged in which electrolytes play an important role in the mobili-
zation of amine as well as for transport in both directions.
Generally, transport phenomena involving norepinephrine (NE) are
similar in the central and peripheral nervous systems. Reference to
reports on both systems will be given, although each will not be given
separate consideration. Moreover, NE, 5-hydroxytryptamine (5-HT) and
dopamine (DA) are all transported by a Na+ dependent mechanism, appar-
ently by cotransport.

General Characteristics of Transport

The term transport is used for the mediated translocation of

amines across a membrane barrier. Uptake refers to inward transport.
According to Iversen 29 the initial rates of uptake of 3H-NE are de-
scribed by Michaelis-Menten kinetics. This finding implies that trans-
port is mediated by a saturable mechanism which is the hypothetical
carrier.
Additional evidence for a carrier mechanism for transport is the
finding that amines structurally related to NE can be transported and
are also competitive inhibitors of NE transport. Tryptamine is a com-
petitive inhibitor of 5-hydroxytryptamine transport. Metabolic pro-

291
292 D.F. BOGDANSKI

cesses are involved in uptake, as indicated by the value of about 2

for the QlO of uptake and by the inhibition of uptake by metabolic
inhibitors 24,31.
The saturable, metabolically-dependent transport mechanism is
stereo-specific; its affinity for (-) 3H- NE and (-) metaraminol is
greater than that for (+) 3H-NE 27 or (+) metaraminol 44, respec-
tively. Steric specificity is also indicated by the greater uptake
of the erythro- than the threo- isomer of metaraminol.

Functions of Transport. The nerve ending recaptures transmitter that

has escaped spontaneously 8 or has been released during synaptic
transmission. In this sense, transport participates in storage and is
a continuous process. More importantly, transport helps terminate
synaptic transmission by removing free transmitter from the synaptic
cleft 11 During times of rapid and continuous nerve activity trans-
port helps to conserve an adequate supply of transmitter. Inhibitors
of transport of NE potentiate and prolong synaptic transmission.
It is becoming increasingly apparent to us that transport can
occur in the reverse (outward) direction. Transport might be an al-
ternative to exocytosis as a mechanism for secretion, or it may be a
factor in the release of transmitter 4 (see also below section on
outward transport).

MECHANISMS OF TRANSPORT

Ion Gradient Hypothesis, General

Insight into possible mechanisms of amine transport depends upon

current theories of transport of monosaccharides and amino acids. For
many of these organic solutes the ion gradient hypothesis, to be de-
scribed, is considered to define a significant, if not the major source
of energy for active transport. Critical data on the concentrations
of free intracellular electrolytes and transported organic solute are
required for assessing the validity of this theory. However, such data
have not yet become available to investigators of amine transport. The
proposed mechanisms of amine transport, therefore, are the result of
parallel observations on transport phenomena relating to amines and
other organic solutes. Thus, discussion of at least the electrolyte
gradient hypothesis for the transport of organic solute?, first pos-
tulated by Christensen and co-workers 40, must be a part of the dis-
cussion of amine transport.
A proposed mechanism of transport for biogenic amines is based on
the sodium gradient hypothesis of Crane 16. The basic supports of this
hypothesis are: 1) Monosaccharide transport is strongly dependent upon
the presence of extracellular Na+. 2) Sodium and substrate transport
are directly related. Both species of solute are transported together
(cotransport). 3) Active transport is dependent upon a close but in-
direct relationship to Na+, ~-ATPase activity. Active transport is
directly energized by the electrolyte gradients dependent upon the
activity of the enzyme. The Na+ gradient is stressed although the K+
SYNAPTOSOMAL AMINE TRANSPORT 293

gradient also participates in transport. 4) To these two asymmetries

Crane added a third, an affinity gradient, which was demonstrated by
studies of the kinetics of transport. These studies indicate that Na+
decreases the apparent ~ for substrate, suggesting that Na+ increases
the affinity between carrier and substrate.
The affinity gradients confer an active characteristic to transport,
which is otherwise a passive equilibrating system if all other factors
affecting the distribution of substrate are the same on both
sides of the plasma membrane. Thus, Na+ decreases the ~, that is,
the concentration of substrate at which the velocity of transport is
half the Vmax . The normally lower concentration of intracellular
Na+ reduces the affinity between substrate and carrier, thus increa-
sing the Km. Active transport can be energized as follows: For any
concentration of extracellular substrate, free intracellular substrate
will accumulate until the velocity of transport is the same in both
directions. If the intracellular affinity is lower than the extra
cellular affinity, the rates of bidirectional transport will not be
equal until the concentration of free intracellular substrate is
greater than the concentration of extracellular substrate. Hence,
active transport (or accumulation of substrate against an electro-
chemical or osmotic gradient),will occur. The energy required for the
uphill, or active transport, is expended to maintain normal electrolyte
gradients. Another important characteristic of cotransport is that
each transported solute can energize the transport of the other. Thus,
an organic solute can energize the transport of Na+, a circumstance
that may be important for the outward transport of amine (see below).
In actual operation, the transport and accumulation of solute
are influenced by numerous factors including metabolism of substrate and
by protein binqing or other intracellular sequestering of organic
and inorganic solute in nuclei or other organelles. The Na+ ~radient
hypothesis is also applicable to amino acid transport l8,40,4L,50

Ion Gradient Hypothesis for Transport of Biogenic Amines

Evidence supporting the hypothesis has been reported. Bogdanski

and co-workers have proposed a single model, based upon the model of
Crane, for the transport of NE and 5-HT in peripheral 8 and central
nerve endings 6,9,10. A summary of the data upon which this hypothe-
sis is based follows.
The discovery that Na+ is essential for the transport of NE by
peripheral adrenergic nerve endings was first reported by Iversen and
Kravitz 32, Bogdanski and Brodie 7, and Gillis and Paton 22. Sodium
is also required for the transport of NE 9,14 and 5-HT 2 by synapto-
somes. Acetylcholine 41 and DA 15 are also transported by Na+ de-
pendent mechanisms in brain. The participation of Na+, ~-ATPase
activity in amine transport was indicated by the response of amine
transport to various conditions known to affect the enzyme. For ex-
ample, both transport and the enzyme require not only Na+ but also ~
in low concentrations ranging between 1 and 25 mM. Potassium-deficient
media or high [K+] inhibit the enzyme activity 8,9,14,38. Ouabain is
294 D.F. BOGDANSKI

also inhibitory 1,9 and the effect of ouabain is decreased by 75 roM

~ 45. A variety of evidence suggests that the relationship between
amine transport and Na+, ~-ATPase is indirect 9,47. The essential
evidence is the rapid inhibition of the enzyme activity in the presence
of ouabain whereas the inhibitory effect of ouabain upon amine trans-
port develops more slowly. Presumably, the time required for the
development of transport block is the time required for the elimination
of electrolyte gradients at the outer and inner membrane surfaces in
the vicinity of the carrier 19. Moreover, the apparent outward trans-
port of amine is not blocked by ouabain, which does not eliminate re-
versed electrolyte gradients (see below, section on outward transport).
Sodium decreases the apparent Km for transport, suggesting that
Na+ increases the affinity between carrier and NE or 5-HT, whereas
the Vmax is less affected. According to the kinetic analysis of
Vidaver 51, the same data suggest that amine and Na+ are transported
stoichiometrically at a 1 to 1 ratio. Thus, NE increases the affinity
of the carrier for Na+ in synaptosomes 10, and in peripheral adrener-
gic nerves 8. In any event, the occurrence of cotransport is sugges-
ted by this evidence.
As an equilibrating system, the amine pump will translocate amine
until bidirectional rates of transport are equal. However, asymmetries
in the disposition of intracellular and extracellular amine would tend
to favor accumulation even if carrier mediated transport did not occur.
These asymmetries include the binding of amine to cytoplasmic sites
and in vesicles. Active transport of amines is generally assumed to
have occurred on the basis of inhibition of transport by inhibitors
of ATP manufacture. The fact that the inhibition of metabolism also
decreases ion gradients must also be considered. An incontrovertible
demonstration of amine transport against an electro-chemical, or osmotic:
gradient is probably still lacking. A model transport mechanism·based
upon the principle of cotransport of amine and Na+ is shown in Fig. 1.

Criticism of the Ion Gradient Hypothesis in General

The ion gradient hypothesis for transport of organic solutes has

dominated the field in recent years. However, opposing pOints of
view are also reported (see ref. 26) and the hypothesis may require
modification. A frequent criticism has been that the Na+ gradient
alone cannot provide the necessary energy for the observed transport.
Recent models of the transport mechanism include the ~ gradient as
part of the driving force 18.
A critical problem has been the estimation of the concentrations
of free Na+ and of the transported organic solute in the cytoplasm.
Thus, neither the energy requirements nor the available energy are
known with certainty. However, reports of binding and sequestering
of inorganic ions to intracellular and extracellular sites in stri-
ated muscle 36 and intestinal epithelial cells 34, and of the seques-
tering of Na+ in nuclei of Ehrlich ascites cells 39 indicate that the
gradient of electro-chemically active Na+ may be greater than that
SYNAPTOSOMAL AMINE TRANSPORT 295

OUTSIDE INSIDE

i:
.
x
Co n
:II
o
m
" Z
""
::;
:s
:II
., o
Z
c: i:
m
'" ~
......
OJ

~ ..
M
low
INEI

II:
(0 ..

Fig. 1. The Na+-amine cotransport model for biogenic monoamines in

nerve endings. The model for uptake was originally presented in 8
after Crane 16. The above model is oriented to represent efflux as
mediated by the same carrier which mediates uptake. This model repre-
sents an equilibrating system for the bidirectional cotransport of
amine and Na+ across the diffusion barrier imposed by the plasma mem-
brane. Carrier mediated exchange of NE has been shown in support of
this hypothesis (Table 1). The hypothetical carrier (C) has access
to the outer ahd inner membrane surfaces. At the outer surface, where
the [Na+] is high and the [~] low, the affinity between carrier and
amine is high, and amine, Na+ and carrier form a ternary complex.
Potassium competes with Na+ for a site on the carrier 8,28. Amine
and Na+ are cotransported to the inner surface of the membrane where
the concentrations of electrolytes are reversed in a microenvironment
for the carrier. Amine is released and bound ~ in the cytoplasm
or vesicles bound to the membrane. Amine can be deaminated by mono-
amine oxidase (MAO) located in the outer membrane of mitochondria.
The carrier is maintained at a generally Na+-unloaded state by the
electrolyte pump dependent upon Na+, K+-ATPase activity (not shown).
When the electrolyte pump is inactivated by ouabain or K+-free media,
the amine carrier becomes generally Na+-loaded and uptake becomes
correspondingly inhibited. When bound amine is mobilized by Ca++ then
the amine and Na+ are cotransported out of the nerve ending by a process
similar to that which mediates uptake.
The Na+ and K+ gradients render the system capable of uptake of
amines against an osmotically active concentration gradient (active
transport) whether Na+ affects primarily the apparent Km or the Vmax '
Translocation in either direction can be inhibited by cocaine or other
inhibitors of uptake (COC.XXXX). Additional details in text under
"MECHANISMS OF TRANSPORT" and under "EVIDENCE FOR OUTWARD TRANSPORT."
296 D.F. BOGDANSKI

calculated on the basis of an even distribution of Na+ throughout the

cytosol. These tissues are often used for studies of transport. Cor-
rections for sequestering in the Ehrlich ascites cells indicate that
the Na+ gradient may be sufficient to account for the energy require-
ments of the observed transport.

Criticism of the Ion Graqient Hypothesis for Amine Transport

For the transport of biogenic amines by nerve endings, the situ-

ation is even more problematical than described above. The overall
[Na+] in synaptosomes must be considered an open question at this time.
The concentration of unbound or unsequestered amine as well as electro-
lyte is unknown.
Since the ion gradient hypothesis for the transport of biogenic
amines is an adaptation of the hypothesis for the transport of other
organic solutes, mutual criticism may be relevant. The necessary data
have not yet been reported, however. But first of all, there is over-
whelming agreement on most aspects of amine transport, including
the requirement for Na+ and K+ and the saturability of the process as
indicated by Michaelis-Menten kinetics. Also, it is agreed that trans-
location is mediated by cotransport of NE and Na+, whether Na+ affects
primarily the ~ or the Vmax . Differences of opinion relate to the
ion-gradient hypothesis as a source of energy for amine transport.
One report has been vigorously employed as evidence against the
ion gradient hypothesis and deserves careful consideration. This
report describes the apparent failure of an imposed Na+ gradient
(raising the [Na+] from 156 to 286 mM) to re-establish NE transport
in synaptosomes whose oxidative phosphorylation was blocked by meta-
bolic inhibitors 52. Under control conditions, the imposed gradient
increased NE accumulation by about 20% (estimated from the published
charts). In the presence of powerful inhibitors of general metabolism
and in the absence of data on initial rates of uptake, the elimination
of this small increment does not appear to be a convincing argument,
in our op~n~on. In fact, the initial uptake appeared to be overwhelmed
by outward movement of NE caused by the osmotic effect of hypertonicity
in the control and in the experimental situation. Apparently, the
membrane was porous to amine as well as to water.
In the same paper, at least two other consequences of metabolic
inhibition, which could seriously affect accumulation, were not taken
into consideration. Metabolic inhibitors block electrolyte transport
with a rapid onset of action in synaptosomes 35 thus decreasing the
imposed Na+ gradient. Moreover, metabolic inhibitors mobilize
bound NE 5,13. According to the concepts of cotransport, free intra-
cellular amine, including some 3H-NE, could energize the outward
transport of Na+, counteracting the accumulation of 3H- NE energized
by extracellular Na+. Thus, a variety of forward and reverse flows
make these experiments extremely difficult to interpret. Their
relationship to transport is questionable for quantitative purposes.
Based partly upon the foregoing paper 52, the Crane model was
SYNAPTOSOMAL AMINE TRANSPORT 297

rejected and two others were adopted for amine transport 54. In
their original form, the two models provided for an effect of the Na+
gradient.
Another report rejected the ion-gradient hypothesis on the basis
that Na+, ~-ATPase was not involved 53. Reports on the effects of
ouabain generally suggest that amine transport persists after enzyme
activity or ion gradients have been inhibited or diminished but not
blocked or abolished. Amine transport and enzyme activity are both
abolished in the presence of high 'concentrations of inhibitor, how-
ever. Also, the indirect linkage between electrolyte and amine trans-
port renders proportionate inhibition unlikely. In any event, the
magnitude of the electrolyte gradient may be greater than indicated
by the incorrect assumption that the electrolyte is evenly distributed
in the cytosol. Moreover, the data on inhibition of transport by low
concentrations of ouabain 53 are quantitatively at variance
with data reported elsewhere 1,25,28,45.
It has been reported that the transport of metaraminol in rat
uteri was energized by Na+, ~-ATPase activity and not by the Na+
gradient 38. Thus, in the presence of ouabain, an imposed Na+ gra-
dient did not energize the accumulation of metaraminol. These data
are consistent with data for NE reported eqrlier by us 47. However,
an imposed Na+ gradient energizes initial NE as well as 5-HT trans-
port in synaptosomes in which Na+, ~-ATPase activity was inhibited
by a ~-free medium 45, and by the ~-free medium or ouabain, re-
spectively 9,47. Moreover, the effectiveness of either inhibitor
was decreased by a low concentration of extracellular Na+ in accord
with the hypothesis. Sustained transport, unlike initial transport,
required Na+, ~-ATPase activity 9,45,47 in order to maintain ion
gradients.

Electrolyte concentrations in synaptosomes. The difficulty of this

areaof study is perhaps exemplified by reports of electrolyte concen-
trations expressed as ~mol/mg protein after earlier attempts to express
actual concentrations 19. Reported ~ concentrations ([K+]) range be-
tween 30-103 mM whereas [Na+] are generally ignored or expressed in
terms of protein. One report suggests approximate equality of [Na+]
across the synaptosomal membrane although a strong gradient for [K+]
was established 6. References to synaptosomal [Na+, ~] are obtained
or may be pursued in 6,19,35,52.

Recent Developments in Transport Mechanism Research

The uptake processes for amines were classified as "allosteric"

or "stoichiometric", depending upon whether Na+ affected the apparent
Km or Vmax,respectively. The processes representing the two classes
may not be mutually exclusive. The graph showing stoichiometric trans-
port of Na+ and metaraminol 43 (not DA 28) shows a cut-off rectilinear
relationship between uptake and [Na+]. It might be expected that a
process which is saturable and stoichiometric for two cotransported
substrates would show similar plots. The plot of uptake vs concen-
298 D.F. BOGDANSKI

tration of amine in the presence of a given [Na+] is a rectangular

hyperbola as expected. Neither the Km nor the Vmax for Na+ can be
calculated from these experiments. By contrast, another report shows
that the transport of Na+ and NE are interrelated so that either can
affect the Km of the other 10.
A different approach has been taken for studies on DA transport
and the effects of ouabain 28. Veratridine and batrachotoxin, which
increase the permeability of the membrane to Na+, and ouabain, all
block the transport of DA. The effects of veratridine and batracho-
toxin are blocked by tetrodotoxin, which decreases the permeability
of the Na+ channel. Like ouabain, veratridine and batrachotoxin in-
crease the intracellular [Na+] and depolarize the membrane but by a
different mechanism. All three compounds block transport. The infer-
ence that ouabain blocks transport secondarily to changes in intra-
cellular salt concentrations, and/or membrane potentialtis not incon-
sistent with, or necessarily different from, the ion gradient hypothesis.
Studies on the transport of 5-HT in fetal synaptosomes 48 indicate
that the accumulation (internal:medium ratio) and uptake (accumulation
plus metabolism) are Na+-dependent, and that accumulation but not metab·-
olism of transported 5-HT is prevented by reserpine. Accumulation is
partly independent of Na+, ~-ATPase activity and a Na+ gradient. Uptake
increases after birth when Na+, ~-ATPase activity, electrolyte gradi-
ents, nerve conduction and synaptic transmission of impulses develop.
We can use these findings to illustrate various principles of
uptake and accumulation of amine in nerve endings. The mobile carrier
translocates amine and the neuron can accumulate amine without active
transport in the presence of amine binding mechanisms. The binding
but not translocation is abolished by reserpine. During the develop-
ment of Na+, ~-ATPase and ion gradients, active transport of amine
against a chemical gradient is mediated by the transport mechanism
described above.
An apparent second transport mechanism in nerve endings is satur-
able, activated by low [Na+], stereo-specific, and inhibited by reser-
pine 43,44. This pump may be related to the so-called readily-avail-
able pool of NE.
Efforts to show the involvement of membrane glycoproteins in Na+
transport linked to NE but not to 5-HT, DA, choline or GABA transport
in brain, have been reported. The membrane was cleared of glycoprotein,
and NE transport was inhibited by trypsin 27.

Effect of Na+ on the kinetic constants of transport of monoamines

There is general agreement that the apparent Km for the uptake

of NE, DA and metaraminol by tissues incubated in physiological [Na+]
is about 0.1 ~M to 1 ~M 10,15,28,30,31. However, the Km for 5-HT
approaches 0.01 ~M 10 Sodium has mixed effects on the carrier,
primarily decreasing the apparent Km but also increasing the Vmax
for 5-HT and NE uptake 10. However~ Na+ increases the Vmax for the
transport of DA 28 or metaraminol 4~. The effect on the Vmax is
small. Raising the [Na+] from 0 to 120 roM, or more, increased the
SYNAPTOSOMAL AMINE TRANSPORT 299

Vmax by a factor of about 2.5. Since the two effects represent a

basic difference in the effect of Na+ on the carrier, more work will
be required to determine whether the mechanisms for transport of the
various amines actually differ in that regard. It is important to
realize, however, that cotransport is assumed in both cases. Thus,
active transport and the equilibrium distributions will be influenced
by concentration gradients of electrolytes.
Unresolved technical factors may determine which constant is
affected by Na+. These factors include the specific substrate employed,
the concentration range, the incubation medium, the preincubation
time to re-establish the internal milieu, and the species of animal
employed. For example, Na+ decreases the apparent Km 16 or increases
the Vmax 23 of monosaccharide transport by intestinal epithelium,
depending upon the animal species. The Vmax is generally reported
to be in the range of m~mol/mg protein per hour.

ELECTROLYTES AND THE STORAGE AND MOBILIZATION OF AMINES

Douglas and Rubin17 reported that retention of catecholamines

in perfused adrenal glands was abolished ip the absence of Na+ and
~ simultaneously, but the absence of either electrolyte alone had
little effect. Bogdanski 7,9 reported that the retention of NE in
rat heart slices was dependent upon Na+ whether or not ~ was also
present. Sodium is also required for the retention of catecholamines
in other organs 21,38. Potassium in low concentrations, helps to re-
tain stored NE 6,8
Calcium is required for the release of NE from heart slices in-
cubated in media containing K+ or choline+ as osmotic replacements
for Na+ 33. The Ca++ dependency is decreased in media containing Li+
or sucrose as replacements for Na+ 3,21.
The Ca++-dependent release of neurotransmitter from slices ap-
peared to us important to study in the light of the Ca++-dependent
release of transmitter during synaptic transmission. The Ca++-depen-
dent release of NE 3 from slices, or of acetylcholine during synaptic
transmission 20 can be expressed by Michaelis-Menten kinetics. More-
over, Na+ and Ca++ are mutual antagonists, non-competitive in the for-
mer, and competitive in the latter instance. This type of relation-
ship suggests that Ca++ takes part in some reaction other than the
binding of storage vesicles to the plasma membrane which would be all
or none in character. Calcium may mobilize amine, which is then se-
creted from the nerve ending, perhaps mediated by outward transport.

EVIDENCE FOR OUTWARD TRANSPORT

The efflux of NE in slices or synaptosomes is increased by re-

placing the extracellular Na+ (reversed Na+ gradient) with isosmotic
concentrations of sucrose, Li+, choline+, K+ or Cs+. The effect of
electrolytes on the side of the substrate was tested by loading the
slices with Na+, Li+, choline+ or K+, by a process of autodialysis 4
300 D.F. BOGDANSKI

As expected, the efflux of NE was most rapid in slices autodialyzed

with Na+ and incubated in the Na+-deficient (choline+) medium con-
taining Ca++. These data suggest that efflux is dependent upon co-
transport with intracellular Na+. However, a Michaelis-Menten analy-
sis of the kinetics of efflux is not possible in the absence of accu-
rate analysis of free intraneuronal amine and electrolyte concentration.s.
One of the above supporting facts for the ion gradient hypothesis
is necessarily inconsistent with outward transport. Indeed, the in-
ability of ouabain to block efflux, unlike inward transport, is taken
as supportive evidence for the idea that transport is independent of
direct metabolic participation by Na+, ~-ATPase and the manufacture
of ATP. Thus, ouabain does not inhibit the carrier directly, and can-
not inhibit outflow indirectly by eliminating energy requiring electro-
lyte gradients in the experiments described (Table 1).
Other evidence for the similar nature of efflux and uptake is
the inhibition of both translocations by cocaine and desipramine
(Table 1). The concentration of desipramine that inhibits efflux
by 50%, the ID 50 , is about 0.03~. This concentration is
almost identical to the ID50 reported for uptake 30. Thus, the
efflux of NE from heart slices incubated in Na+-deficient (choline+)
media containing Ca++ appears to be mediated by a Na+-dependent mecha-
nism similar or identical to that which mediates uptake. Moreover,
desipramine selectively decreased the Na+-dependent efflux in the ex-
periments on tissue loading of electrolytes 5

Various Effects of Transport Inhibitors

Why do uptake inhibitors fail to block synaptic transmission if

outward transport is a factor in transmission? Experiments have shown
that inhibitors of transport can increase, decrease or have no effect
upon efflux depending upon various conditions of incubation. For
example, added K+ increases efflux in slices incubated in Krebs-
HC0 3 medium. The increment is increased by desipramine, thus mimick-
ing the increased overflow from stimulated nerves that occurs by in-
hibition of uptake in the presence of these drugs. By contrast,
trans~ort inhibitors decrease the efflux of NE from tissues incubated
in Na -deficient media (choline +) containing Ca++, an effect which
depends upon inhibition of outward transport.
The remaining alternative response, that desipramine would have
no effect upon efflux, was demonstrated for Ca++-dependent efflux in
the Na+-deficient (choline+) medium containing high [K+]. The lack
of inhibition of efflux by desipramine in these conditions is more
difficult to explain, but a plausible explanation is available. As a
competitive inhibitor of the transport of NE 37 desipramine has a high
affinity for the site on the carrier that also attaches NE. Potassium
inhibits transport by competing with Na+ for the electrolyte site on
the carrier. Possibly, ~ produces a conformational change at the car-
rier which lowers the affinity between the carrier and NE 16. This
effect of K+ might produce a greater inhibition of attachment of
SYNAPTOSOMAL AMINE TRANSPORT 301

desipramine to the carrier as the inhibitor is a larger and more complex

molecule (Table 1). In essence, the ability of desipramine to inhibit
transport is affected by the state of membrane polarization and/or [K+]
in the vicinity of the membrane.

Role of Outward Transport in Synaptic Transmission

The facts are consistent with a possible role for outward transport
in transmitting nerve impulses. Transport is rapid and complete 4,33.
Also, the proposed shuttle of transmitter out of and into vesicles
attached to the presynaptic membrane has some merit for nerve endings
in which the theory of exocytosis raises some problem of transmitter
supply, large molecules are not secreted in the proportion in which
they exist within the cell, and in which a single impulse may not
produce a response by the effector organ. In any event, there is now

TABLE 1

Effect of Inhibitors of Uptake on the Efflux of 3H-Norepinephrine

in Rat Heart Slices

Half-lives of Retention (min)

Incubation
Medium No Inhibitor Desipramine Cocaine

Krebs Bicarbonate 523

Krebs Bicarbonate + K2 S0 4 239 175 170
Krebs Bicarbonate + 3H- NE 152 391

Sodium deficient 64 342 385

Sodium deficient + ouabain 68 338 363
Sodium deficient + K2 S0 4 134 173

Rats were injected with 12.5 ~Ci of dl- 3H-Norepinephrine (3H- NE ) and
killed 18 hrs later. Ventricle slices were incubated in Krebs HC03
medium at 37°, then desipramine or cocaine was added to a concentration
of 3 ~M. Ouabain was added to a concentration of 0.5 roM after 20 min.
Preincubation was continued to 60 min for all. Slices were then trans-
ferred to modified Krebs HC03 containing 12.5 roM Na+; choline chloride
replaced deficient Na+. Other slices were transferred to fresh Krebs
HC03 medium. As indicated on the table, 3H- NE , and potassium sulfate
(K2S04) were added to concentrations of 0.1 ~M or 50 roM, respectively.
The medium was sampled every 20 min. to 140 min. The figures represent
the half time of tissue 3H- NE .
302 D.F. BOGDANSKI

a plausible explanation for the failure of transport inhibitors to

inhibit transmission in most, but not all 12 instances. However,
there has been a failure to recognize outward transport as a possible
mechanism for release until recently 4.
The possible explanation for the failure of transport inhibitors
to block synaptic transmission follows from the observation that de-
polarization and/or high [~] inhibit the effect of desipramine.
Desipramine might still be able to increase overflow in the presence
of high [K+] and high [Na+] between impulses because the Na+ decreases
the inhibitory effect of ~ on the attachment of NE 8 and desipramine
to the carrier. Thus, the NE that might have been released after
membrane depolarization during transmission can be recaptured by the
transport mechanism, and the recapture process can be blocked by
desipramine. +
The inwardly directed Na gradient is probably not a problem
for outward transport. The concentration of transmitter in the
vesicles, if mobilized, approaches 1 M, whereas the ~ for uptake is
about 0.1 ~M. Thus, NE could energize the outward transport of Na+
according to the theory of cotransport.

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47. Tissari, A.H., Schonhoffer, P.S., Bogdanski, D.F., and Brodie, B.B.
Mechanism of biogenic amine transport. II. Relationship between
sodium and the mechanism of ouabain blockade of the accumulation
of serotonin and norepinephrine by synaptosomes. Mol. Pharm.
5 (1969) 593-604.
48. Tissari, A.H., and Suurhasko, R.U.A., Transport of 5-HT in
synaptosomes of developing rat brain. Acta Pharmacol.et Toxicol.
29, Suppl. 4 (1971) 59.
49. Titus, E.O., and Dengler, H.J., The mechanism of uptake of nore-
pinephrine. Pharmacol. Rev. 18, Part I (1966) 525-535.
50. Vidaver, G.A., Glycine transport by hemolyzed and restored pigeon
red cells. Biochemistry 3 (1964) 795-799.
51. Vidaver, G.A., Transport of glycine by pigeon red cells.
Biochemistry 3 (1964) 662-667.
52. White, T.D., and Keen, P., The role of internal and external
Na+ and ~ on the uptake of [3H] noradrenaline by synaptosomes
prepared from rat brain. Biochim. Biophys. Acta 196 (1968)285-295.
53. White, T.D., and Keen, P., Effect of inhibitors of (Na+ + K+)-
dependent adenosine triphosphatase on the uptake of norepinephrine
by synaptosomes. Mol. Pharmacol. 7 (1971) 40-45.
54. White, T.D., and Paton, D.M., Effects of external Na+ and ~ on
the initial rates of noradrenaline uptake by synaptosomes prepared
from rat brain. Biochim. Biophys. Acta 266 (1972) 116-127
CHARACTERISTICS OF THE UPTAKE AND RELEASE OF GLUTAMIC ACID IN

SYNAPTOSOMES FROM RAT CEREBRAL CORTEX. EFFECTS OF OUABAIN

G. Takagaki

Department of Neurochemistry
Tokyo Metropolitan Institute for Neurosciences
Fuchu-shi, Tokyo, Japan

It is often described that glutamic acid seems probably to be

a major excitatory transmitter in the mammalian CNS. Glutamic acid
is very rapidly taken up from the medium into mammalian cerebral
slices and synaptosomes. And it is released from either of them on
depolarization effected by electrical pulses and elevated medium
potassium. These properties of glutamic acid have been studied ex-
tensively and discussed in relation to its possible physiological
transmitter role in CNS, although the in vitro techniques certain-
ly involve highly unphysiological conditions 4 ,8,9.

In the present studies, it was intended to analyze the more

precise mechanisms of the uptake and release of glutamic acid in
nervous tissues, and the comparison was made between the natural-
ly occurring L- and non-natural D-isomers using guinea-pig cere-
bral cortex slices as well as rat cerebral cortical synaptosomes.
Although on the whole some similarity was observed between two isom-
ers on their uptake and release by the cerebral slices, the proper-
ties of the uptake of D-glutamic acid into rat cortical synapto-
somes were found to be different from those of L-isomer, especial-
ly in respect of the sensitivity to ouabain: On double reciprocal
analysis, the uptake of D-glutamic acid into the slices as well as
synaptosomes revealed an excellent fit for a single affinity com-
ponent system, the affinity of which was lower than that of low
affinity component for L-isomer. The mechanisms of these observa-
tions are discussed, although further investigations should be
needed before their functional significance can be clarified. A
preliminary report of these results has been given ll

307
308 G. TAKAGAKI

MATERIALS AND METHODS

The standard medium which was used in these studies in incu-

bating (or superfusing) slices (guinea-pig) and synaptosomes (rat)
from cerebral cortex consisted of (final concn): 128.1 mM-NaCl; 5.0
mM-KCl; 2.7 mM-CaC1 2 ; 1.2 mM-MgS0 4 ; 1.2 mM-Na2HP04; 30 mM-glycyl-
glycine buffer, pH 7.4; 5 mM-glucose. All the incubations were
carried out at 37°C in air, except when otherwise stated.

To study the uptake and release of glutamic acid by slices and

synaptosomes in vitro, the conventional radioisotopic technique
were utilized. D- Pm Glutamate was prepared from DL- D- 3 l!1 glutamic
acid using L-glutamic acid decarboxylase (from E. coli) as previous-
ly described lO •

Slices were cut free hand from guinea-pig cerebral cortex lO .

Only the first slices from the surface of the cortices were utilized.
Slices were pre incubated (approximately 50 mg fresh weight in 2 ml
medium) in the standard medium for 20 min, then labelled compound
was added and incubation continued for various durations. After
incubation, the slices were quickly picked up and separated from
the medium with suction, then homogenized and extracted with 5%
trichloroacetic acid. Amino acids were fractionated by ion exchange
resin chromatographyl. The radioactivity in each amino acid
fraction was counted in 10 ml Bray's solution in a liquid scintillat-
ion counter. The uptake was usually expressed as ~ol of glutamic
acid accumulated per g initial fresh weight of slices.

To study the release from slices, the pre loaded slices with
labelled glutamic acid (10 ~) for 15 min were transferred to a
superfusion chamber and the slices were super fused with oxygenated
medium for 10 min. Thereafter 2-min fractions (4 ml) were collected"
The high-potassium stimulation was effected by switching the super-
fusion solution to that containing 50 mM K+, over a 10-min period.
Amino acids in each fraction were isolated and counted as described
above.

Synaptosomes (usually P 2 f~~tion) were prepared from rat

(Wistar strain) cerebral cortex. After centrifugation, the pellet
of P 2 fraction was gently resuspended in 0.32 M sucrose solution to
give a final concentration of approximately 20 mg of protein per mI.
Portions of this suspension of P 2 fraction sufficient to give a final
concentration of approximately 1.0 mg protein per ml of the medium WE~re
incubated. To study the uptake and release processes of glutamic
acid by synaptosomes, the methods described by Nicklas et a1 7 were
utilized. The synaptosomes were preincubated for 10 min in the
standard medium before labelled glutamic acid (10 ~) was added.
At various time intervals after the addition of labelled glutamic
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 309

acid as well as after the further addition of sufficient 1 M KCl to

increase the concentration of K+ up to 50 mM, 0.5 ml portions of
the suspension were removed and passed through a single 0.65 pm
Millipore filter(25 mm diameter). The filters were washed thrice
with 0.5 ml of 0.15 M NaCl. The whole Millipore filter was counted
in 10 ml Bray's solution in a liquid scintillation counter. The
uptake of radioactivity was expressed as nmol of glutamic acid
accumulated per mg protein.

Protein determination was carried out by the method of Lowry.

RESULTS

Metabolism of Glutamic Acid in Cerebral Slices. After the

slices were incubated with L-[14c]- and D-[3H]glutamic acids(lO pM)
in oxygen for 15 min, the distribution of radioactivity among amino-
acids in the slices as well as in the medium was determined(Table 1).

Table 1
Metabolism of Glutamic Acid in Guinea-pig Cerebral Cortex Slices

Glutamate Aspartate Glutamine GABA Recovery*

(% of activity in TCA extract)

L-Glutamate(lO ~)
48.75 4.17 43.95 0.79
Slices 97.66
± 2.66 ± 0.46 ± 3.64 ± 0.13

Medium 33.37 0.97 52.52 0.63

87.49
± 1.85 ± 0.66 ± 0.82 ± 0.51

D-Glutamate(lO ~M)
96.32 0.16 0.99 0.06
Slices 97.53
± 1.92 ± 0.05 ± 0.31 ± 0.01

99.37 0.38 0.23 0.15 100.13

Medium
± 1.77 ± 0.08 ± 0.20 ± 0.19

The radioactivity in each fraction is expressed as per cent of

the activity in TCA extract. Means of four independent exper-
iments ± SD are given. For details see the Materials and Methods
section.
* The activities in percentage in four amino acid fractions
were added and the sum is shown as 'Recovery'.
310 G. TAKAGAKI

Glutamine was formed in large amounts from L-glutamic acid, and

more than 50 %of the total radioactivity in the medium was found
in the glutamine fraction. When labelled D-glutamic acid was added
to the slices, however, negligible metabolism was observed and al-
most all the radioactivity remained in the glutamic acid fraction •

A B •
OA OA

D.2

JDI
......
40
-a
5. 12 C 12 0 •
8 8
2 2
4 • • 4
•
1~ __---JD.5

~~-40~-6~0 0 0 10 20 40
Time(min)
Fig. 1
Uptake of L- and D-Glutamic Acids into Guinea-pig Cerebral Cortex
Slices. Effects of Ouabain(0.05 mM) and of omitting Sodium Ions
from Medium.
The radioactivity in glutamic acid fractl.on which was isolated
from the TCA extract of incubated slices was expressed as pmol of
glutamic acid per g initial fresh weight of slices. To examine the
effects of ouabain and of omitting sodium ions from medium, the
former was included in and the latter was left ~ut from the medium,

.:
from the beginning of the preincubation.
A: L-Glutami c Acid, 10 pM Control(left-side scale)
B: D-Glutamic Acid, 10 pM A: Ouabain, 0.05 mM(right-
C: L-Glutamic
D: D-Glutami c
Acid,
Acid,
0.5 mM
0.5 mM .: side scale)
Sodium-free(right-side
scale)
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 311

2000
A

~ 50
"'E'
~1000 40
30ii
...'"
1:!!
e
.!!. 20;;
10
0 0
0 10 20 30 40 50
Time (min)

B
0.75

Fig. 2. Release of L- and D-Glutamic Acids from Guinea-pig Cerebral

Cortex Slices. Superfusion Experiments.
Pre loaded cerebral slices with labelled glutamic acid (10 ~) for 15
min were superfused. After an initial 10 min superfusion with oxygen-
ated control medium, 2-min fractions (4 ml) were collected. Depolari-
zation o·f the slices was effected by switching the superfusion solut-
ion to that containing 50 mM K+, over a 10-min period indicated by
solid bar on the time scale. For potassium concentrations (0 ) in
the superfusate refer to the right-side scale.
A: The slices were pre loaded with L_[14C) glutamic acid (10 ~).
The radioactivity in the superfusate (A), and that in the glutamine
fraction (.) from it are given.
Band C: The release of radioactive glutamic acid into the super-
fusate is shown as the fractional rate of release per min 3 . In B,
the slices were pre loaded with L- l4C glutamic acid (10 ~), and in
C, with D-[3H]glutamic acid (10 ~).
312 G. TAKAGAKI

D-Glutamic acid was thus considered not to be actively metabolized

in cerebral slices. It was therefore used in the present studies
as a possible tool to analyze some aspects of the uptake and re-
lease of L-glutamic acid by nervous tissues in vitro.

Uptake and Release of Glutamic Acid by Cerebral Slices. As

shown in Fig. 1, the accumulation of glutamic acid into guinea-pig
cerebral cortex slices was found to be almost the same between two
isomers, either when they were added to the medium at 10 ~ or at
0.5 roM concentration. In the case of the former (10 ~), the
apparent accumulation of L-glutamic acid thus measured leveled off
after about 20 min of incubation, although that of D-isomer con-
tinued to increase even after 60 min. This is thought to be due
to an active metabolism of L-glutamic acid in cerebral slices.

In agreement with an earlier observation 6 , the radioactivity

in superfusate of the pre loaded cerebral slices with labelled
L-glutamic acid was mainly accounted for by glutamine fraction,
the radioactivity of which was not significantly increased in
response to high potassium stimulation (Fig. 2, A). When L-
or D-glutamic acid was used, the radioactivity in the glutamic acid
fraction from the superfusate was very significantly increased
by elevating the medium potassium concentration (Fig. 2, B and C).
The amount of glutamic acid released by high potassium depolarizat-
ion relative to that released spontaneously was greater when the
slices were loaded with L-glutamic acid than with D-isomer.

Kinetics of Glutamic Acid Uptake into Cerebral Slices as

well as into Synaptosomes. In agreement with previous workers 5 ,9,
L-glutamic acid accumulation into guinea-pig cerebral slices as
well as into rat cortical synaptosomes consisted of high and low
affinity components, the high affinity component having a lower
Michaelis constant (Km) and lower maximal velocity (Vmax) than
the low affinity component. Double reciprocal plots of D-glutamic
acid uptake into the slices and synaptosomes, however, yielded
respectively one straight line indicating a one component system,
Km value of which was lower than that of the low affinity component
for L-glutamic acid uptake. Maximal velocity (Vmax) of D-glutamic
acid uptake seemed equal to that of the low affinity component
for L-glutamic acid in either the slices or the synaptosomes
(Fig. 3, A and B).
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 313

Effects of Ouabain and of Omitting Sodium Ions from Medium on

Glutamic Acid Uptake. Nicklas et all reported that the rate
of uptake of L-glutamic acid into synaptosomes was less effective-
ly inhibited by ouabain(O.l mM) than that of GABA, dopamine and nor-
epinephrine. Fig. 2 shows that ouabain(0.05 mM) inhibited almost
completely the uptake of D-glutamic acid into the cerebral slices,
either when it is added to the medium at 10 pM(Fig. 2, B) or 0.5 mM
(Fig. 2, D) concentration. L-Glutamic acid uptake was less inhibit-
ed by ouabain than D-glutamic acid uptake(Fig. 2, A & C). The
inhibition by ouabain(0.05 mM) was greater when L-glutamic acid was
added to the medium at 0.5 mM than at 10 pM concentration. Either

00 10 25 50~ 66.7 100

1,mM

Fig. 3
Double Reciprocal Plots of Uptake of Glutamic Acid.
Each plotted point represents the mean of 4-5 independent determi-
nations. . , L-Glutamic Acid; 0, D-Glutamic Acid
A: Uptake into guinea-pig cerebral cortex slices. Velocity(V) is
expressed as ;mol of glutamic acid accumulated in 5 min per ml
of non-inulin space of the slices.
B: Uptake into P2 fraction from rat cerebral cortex. Velocity(V)
is expressed as nmol of glutamic acid accumulated in 2 min per
mg protein.
314 G. TAKAGAKI

L- or D-glutamic acid uptake into the slices was completely inhibit-

ed in Na+-free medium, either when glutamic acid was added at 10 pM
or 0.5 mM concentration(Fig. 2).

L-Glutamic acid uptake into rat cerebral cortical synaptosomes

was found to be resistant to ouabain(0.05 mM), although it inhibit-
ed greatly D-glutamic acid uptake. By the addition of 50 mM K+ to
the medium, L-glutamic acid was very much released even in the
presence of ouabain, similarly to that observed in its absence(Fig.
4). Again, omission of Na+ from the medium inhibited completely

B
:;

\\ 0.5
i····....... I
~
.
'. ' .•........... D-
••••<;>0..
r
.......'0
"
10.25

o~ ___~___~___~___~
o 5 10 15 20 5 10
Time(min)
15 20
Time(min)

Fig. 4
Uptake and Release of Glutamic Acid by Synaptosomes(P2 fraction)
from Rat Cerebral Cortex. Effects of Ouabain and of omitting
Sodium Ions from Medium.
0, Control; e, Ouabain(0.05 mM); ~,Na+-free.
A: L-Glutamic Acid(lO pM); B: D-Glutamic Acid(lO pM)
Each plotted point represents the mean with SD of 4-5 independent
determinations. At arrows, 50 mM KCl was added.
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 315

the uptake of L- and D-glutamic acids into the synaptosomes(P2

fraction) (Fig. 4, A & B). In confirmation of previous workers
(Snyder et aI, 1973), strong inhibition by ouabain of GABA uptake
into the synaptosomes was observed using the methods which were
the same to those for glutamic acid.

Subfractions from P2 Fraction and Glutamic Acid Uptake.

Subfractions, A, B & C, were obtained from P2 fraction from cat cere-
bral cortex exactly as described by Whittaker & Barker 12 The
uptake of L-glutamic acid was measured with each subfraction, and
the results are shown in Fig. 5. The uptake into subfraction B
amounted to 82.8 %of the total uptake into three subfractions.

...
~a.
r4
.....
"'0
e...

C
2

0~__~__~~-7.~~~
o 5 10 15 20
Time(min)

Fig. 5
Uptake and Release of L-Glutamic Acid(lO pM) by Subfractions from
Crude Synaptosomal Fraction(P2) from Rat Cerebral Cortex.
See the legend for Fig. 4. Uptake of L-glutamic acid(lO pM) into
the P2 fraction subjected to hypo-osmotic shock is also shown( • ).
A: O. Subfraction A(myelin)
B: 0, Subfraction B( synaptosomes)
C: ~, Subfraction C(mitochondria)
316 G. TAKAGAKI

The specific uptake activity of L-glutamic acid into subfraction B

per mg protein was almost twice that into P2 fraction.

After P2 fraction from rat cerebral cortex was subjected to

hypo-osmotic shock, the uptake of L-glutamic acid into it was
measured in the medium fortified with 2 roM ATP. And it was found
that the uptake activity was almost completely lost by the hypo-
osmotic treatment(Fig. 5).

DISCUSSION

The results may indicate that the high affinity uptake system
for glutamic acid is specific to L-isomer and localized in nerve
terminals. When L-glutamic acid is added to the medium at 10 pM,
the concentration per mg protein reached in the synaptosomal
fraction(subfraction B) is more than threefold higher than in the
slices. D-Glutamic acid may be taken up only by the low affinity
system. In the cerebral cortical slices the high affinity component
is probably masked by the predominant uptake due to the low affinity
system. It is highly likely that the bulk of the low affinity up-
take is due to non-terminal structures which are included in the
slice preparations? It is known that L-glutamic acid is accumu-
lated at a markedly higher rate and reaches a higher level in the
glial fraction than in the neuronal fraction, and the release
by high potassium stimulation was also observed from the glial
fraction by Hamberger 2 • The high affinity uptake system for
glutamic acid seems to be peculiar in view of its high resistance
to ouabain. Or, it may easily be substituted by a transport system
which is resistant to ouabain. The uptake by the low affinity
system is greatly inhibited by ouabain. Either high or low affinity
system is dependent on the presence of sodium ions in the medium.

The uptake systems for transmitter candidates into various

nervous tissue structures are generally believed to be involved in
terminating the actions after their synaptical release. However,
the functional significance of the present results is difficult
to discuss unless further details of the mechanisms are clarified.

Acknowledgements. The author is indebted to Mrs. H. Konagaya

for assistance during these experiments. Financial support from
the Ministry of Education of Japan is also acknowledged.
GLUTAMATE TRANSPORT IN SYNAPTOSOMES 317

REFERENCES

1 Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H., Metabolic
compartments in vivo. Ammonia and glutamic acid metabolism in
prain and liver. J.biol.Chem., 237(1962) 2562-2569.
2 Hamberger, A., Amino acid uptake in neuronal and glial cell frac-
tions from rabbit cerebral cortex. Brain Research, 31(1971) 169-
178.
3 Hopkin, J., and Neal, M.J., Effect of electrical stimulation and
high potassium concentrations on the efflux of [14c]glycine from
slices of spinal cord. Br.J.Pharmacol., 42(1971) 215-223.
4 Lajtha, A., Amino acid transport in the brain in vivo and in vitro.
In G.E.W. Wolstenholme, and D.W. Fitzsimons(Eds.), Aromatic Amino
Acids in the Brain, Elsevier, Amsterdam, 1974, pp. 25-49.
5 Levi, G., and Rai teri, M., Detectabili ty of high and low affinity
uptake systems for GABA and glutamate in rat brain slices and
synaptosomes. Life Sci., 12(1973) 81-88.
6 Matsui, S., and Yamamoto, C., Release of radioactive glutamic
acid from thin sections of guinea-pig olfactory cortex in vitro.
J.Neurochem., 24(1975) 245-250.
7 Nicklas, W.J., Puszkin, S., and Berl, S., Effect of vinblastine
and colchicine on uptake and release of putative transmitters by
synaptosomes and on brain actomyosin-like protein. J.Neurochem.,
20(1973) 109-121.
8 Quastel, J.H., Amino acids and the brain. Biochem.Soc.Trans.,
2(1974) 765-780.
9 Snyder, S.H., Young, A.B., Bennett, J.P."and Mulder, A.H.,
Synaptic biochemistry of amino acids. Federation Proc., 32(1973)
2039-2047.
10 Takagaki, G., Developmental changes in glycolysis in rat cerebral
cortex. J.Neurochem., 23(l974) 479-487.
11 Takagaki, G., Uptake and release of glutamic acid in vitro in the
mammalian CNS. Proc.Vth International Meeting of the Inter-
national Society for Neurochemistry, Barcelona, 1975, p. 178.
12 Whittaker, V. P., and Barker, L.A., The subcellular fractionation
of brain tissue with special reference to the preparation of
synaptosomes and their component organelles. In R. Fried(ed.),
Methods of Neurochemistry, Vol. 2, Marcel Dekker, New York, 1972,
pp. 1-52.
RELEASE OF BIOGENIC AMINES FROM ISOLATED NERVE ENDINGS

Maurizio RaiteriO, Alberto Bertollinio,

Renata del Carmineo, and Giulio Levioo

°Institute of Pharmacology, Catholic University

Via Pineta Sacchetti 644, Rome, Italy
o°Laboratory of Cell Biology, CNR, Rome, Italy

INTRODUCTION

Two essential biochemical steps of the aminergic neurotrans-

mission are the release of the neurotransmitter and its reuptake by
presynaptic nerve terminals. Uany neuroactive drugs are believed to
modify the availability of the neurotransmitter amines in the synap-
tic cleft by interacting with these processes 8 ,lO,l4,20,23.
Due to the complexity of the phenomena occurring in the living
brain, many of the studies on uptake and release of biogenic amines
and on the effects of drugs on these processes have been performed
in isolated nervous tissue preparations. In most cases, drug ef-
fects on release were analyzed by prelabeling the tissue with a gi-
ven radioactive neurotransmitter (or one of its precursors) and mea-
suring the radioactivity accumulating in the incubation medium in
the presence of the suspect releasing agent. Hhat is generally
found in the medium after incubation in the absence of the drug is
the net result of two opposite processes taking place almost con-
comitantly: spontaneous release and reuptake of the neurotransmitter
released. For this reason, the effect of a drug which directly sti-
mulates the release of a given neurotransmitter can not be easily
distinguished from the apparent releasing effect of a drug which
inhibits reuptake, unless the experiment is run in conditions in
which reuptake can not occur. This problem has been at times ap-
proached by incubating different brain tissues with the suspect re-
leasing agent, in the presence of a compound believed to be a pure
reuptake inhibitor 2 ,6. This approach has some limitations, although
it is probably the only possibility of minimizing the reuptake com-

319
320 M. RAITERI ET AL.

ponent in in vivo perfusion experiments and when relatively intact

nervous tissues, such as brain slices, are utilized.

SUPERFUSION OF SYNAPTOSOHES

Hith synaptosomes, the reuptake process can be completely pre-

vented by the superfusion apparatus 26 schematically shown in Fig. 1.

,
, I

,
I
THERMOSTATIC
BAT H

' ••7""
:::J PUMP

'1-_ _- - - MllliPORE Fil TER

Fig. 1 - Schematic representation of one unit of the superfusion ap-

paratus used in release studies.

The apparatus consists of several superfusion chambers (18 in our

set-up), provided with a thermostatic jacket. Aliquots of synapto-
somes suspensions, prelabeled with the radioactive substrate under
study, are collected on Millipore filters layin8 on sintered glass
plates constituting the bottom of the superfusion chambers. The
filters are rapidly washed with medium under moderate vacuum. The
chambers are then connected with a multi-channel peristaltic pump
and simultaneously filled with the oxygenated, glucose-containing
medium, already thermostated in the upper reservoirs. Superfusion
is started at a rate of 0.5 ~l/min and 1 min fractions of the super-
fusate can be directly collected in liquid scintillation vials.
This technique has several advantages over the incubation pro-
cedures described in the literature: for example, synaptosomes can
be utilized immediately after prelabeling with the radioactive sub-
strates; one can obtain a complete pattern of the release process;
the composition of the medium can be quickly changed during the su-
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 321

perfusion, utilizing the upper reservoirs for adding the new medium.
The main advantage of this superfusion technique, however, is that
the transmitter is removed by the stream of the superfusion fluid
as soon as it is released, so that its reuptake is prevented. This
is demonstrated by the fact that drugs or experimental conditions
known to inhibit reuptake of a given neurotransmitter do not cause
any increase of its spontaneous release (see ref. 27 and 28, and
following paragraphs).

SOME ASPECTS OF NOREPINEPHRINE RELEASE FROH SUPERFUSED SYNAPTOSOHES

The release rate of 3H-norepinephrine (3H- NE ) from superfused

synaptosomes was much greater than that observed using the traditio

12 -

A--d ToUI J H. no ulclUm

.~ 11
- - . TO"I ~1-i , 2 , JI'\"IM CICI,
.... 0-'"<0 ' H- (l!a mlAlled ml!! t.l»hln, no calCium
~ 10 ·
_ lH_dumlnaltd mllabahlU, 2.1mM CaCI,
~ 9-
>-

~€
'"o

.
:;;;
~ 6-

~
'0
.. . '

..
co
....
a..
2-

-- --~

6_

Fraction number (1 fr action ~ 2 min . 0.9 mil

Fig. 2 - Effect of calcium on the release of 3H-norepinephrine from

synaptosomes superfused with 56 w1 KC1. Purified synaptosomes from
rat cerebrum were prelabeled with 0.2 uM 3H-NE for 10 min and then
superfused with standard medium, in the' absence of calcium. After
7 min the superfusion medium was substituted with new medium con-
taining 56 mM KCl (with or withour 2.7 ~M CaC12)' The total radio-
activity of the superfusate fractions and that present in the form
of deaminated metabolites were measured. The results are expressed
as percentages of total radioactivity recovered (that is, total frac-
tions plus radioactivity remaining in the filters) . The curves
were taken from experiments run in duplicate, using four parall e l
superfusion chambers. The pattern shown was reproduced in four ex-
periments run in different days. (From Raiteri et al. 30 ).
322 M. RAITERI ET AL.

nal incubation technique 26 . This is due to the fact that the spon-
taneously released 3H-NE can not be recaptured by the nerve endings
in the superfusion conditions~
When synaptosomes prelabeled with 3H-NE, in the absence of cal-
cium ions, were superfused with a medium containing 56 mM potassium
chloride, a calcium-dependent stimulation of the radioactivity re-
leased was observed. Fig. 2 shows a sharp increase in the total
tritium released when calcium ions were added in conditions of mem-
brane depolarization. A similar finding has been reported by seve-
ral authors with various neurotransmitters and in different brain
tissue preparations 3 ,6,15. In our experiment the increased release
of radioactivity following stimulation was totally accounted for by
unmetabolized 3H-NE; the release of deaminated metabolites was al-
most unchanged. This indicates that the calcium-dependent release
involves a pool of norepinephrine which is protected from monoamino-
oxidases both during the storage and the "release step and may sug-
gest the involvement of an exocytotic mechanism I6 ,35.
Most of the NE in nerve terminals seems to be localized in the
vesicles and very little in the cytoplasm I9 ,25. Any increase in the

13

12

..,
~ JD

A.\\
~"~,~
" 4..
""l
.......... --6~---A

._- -0- - --(l----o

Fraction number (1 fract ion . 2 min . 0.9 mil

Fig. 3 - Effect of reserpine on the release of 3H-norepinephrine

and 3H-deaminated metabolites from synaptosomes. Experimental de-
tails as in the legend for Fig. 2, except that calcium (2.7 ~1) was
present in incubation and superfusion media. Reserpine was added at
the beginning of superfusion. This pattern of release was typical of
four experiments run in duplicate in different days. (From Raiteri et
a1. 30 ) •
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 323

level of the free amine in the cytosol is expected to result in an

increase in deaminated metabolites. Fig. 3 shows that reserpine,
which is known to release NE from the vesicles into the cytoplasm,
essentially stimulated release of deaminated metabolites.
On the other hand, p-tyramine stimulated the release of large-
ly unmetabolized- 3H-NE from synaptosomes (unpublished observations)
which confirms previous data of Colburn and Kopin l3 . This result
was interpreted by these authors as due to release of radioactive
NE from a pool protected from monoaminooxidases. However, other ex-
planations are possible. For example, p-tyramine might displace
3H-NE from its normal storage sites and protect it from deamination
through a competition for monoaminooxidases, particularly in view
of, the high concentrations of p-tyramine used in these experiments
(between 10-Sand 10-4M).
The data presented in this paragraph, together with observa-
tions made with other putative neurotransmitters 28 , indicate that
synaptosomes trapped on Hillipore filters retain most of their cha-
racteristic properties and could be advantageously utilized for the
study of the effects of neuroactive drugs on neurotransmitter trans-
port processes at the presynaptic level.

EFFECTS OF d-NfPHETAHINE ON THE RELEASE OF BIOGENIC NUNES

It is widely accepted that amphetamine acts by increasing the

availability of catecholamines in the synaptic cleft. This may be
obtained by a direct releasing effect and/or through an inhibition
of the reuptake of the released amine. There is no agreement among
the various authors on which of these two mechanisms is predomi-
nant 4 ,S,7,II,12,17 t 8,24,32,34,36.
In the superfused synaptosomes from whole rat cerebrum, the
release of 3H- NE was only slightly stimulated by 10-SM d-ampheta-
mine 27 . No effect was observed with synaptosomes from hypothalamus,
an area in which noradrenargic terminals are particularly abundant
(Fig. 4a). The drug was ~ijeffective also in synaptosomes from ce-
rebellum and pons-medulla . A modest but significant stimulation
of 3H-NE release was observed in cortical synaptosomes (Fig. 4b)
and a substantially higher effect was detected in synaptosomes from
corpus ~riatum, prelabeled with 3H- NE (Fig. 4c). The effect present
in the latter two areas probably accounts for the slight effect ob-
served in synaptosomes from whole cerebrum27 .
The observation that the largest stimulation of 3H- NE release
was present in synaptosomes from corpus striatum (the area richest
in dopaminergic nerve terminals) prompted us to analyze the effect
of d-amphetamine on the release of 3H-dopamine (3H- DA). The drug
strongly enhanced 3H-DA release in synaptosomes from corpus stria-
tum (Fig. Sa), substantially increased 3H-DA release in the cerebral
cortex (Fig. Sb) and affected only modestly the release of the a-
mine in hypothalamic synaptosomes (Fig. Sc). The effect in striat-
al synaptosomes was still detectable at a concentration of 10- 8 M
324 M. RAITERI ET AL.

c:
COIPU~ Slft.tum
.~ HYPOll lil-l!II l,IS
u

-= 1
Conllol
ConUCl I
d - Amptil!:lfll'l lflt IO-$, M 6
(OI1IUII d · A mp~!llm l ne lO -5 M
CI · Al'IIllheUmlflf: IO-" M

.. 3
o
c:
~
u
~
0.. I 2 3 4 5 7 8 I II
1 1 3 4 I 6 7 8 9 1 11
1 1 1 4 ~ 6 J J 11

fraction number

Fig. 4 - Effect of d-amphetamine on the synaptotomal release of

3H- NE from hypothalamus, cortex and corpus striatum. Crude synap-
tosomal fractions were washed once with 0.32 M sucrose, resuspend-
ed in 0.32 M glucose at a protein concentration of about 6-8 mg/ml,
~iluted 1:10 in a Krebs-Ringer medium and prelabeled with 0.2 rM
H-NE for 10 min at 37°. Then 1 ml aliquots of the suspension were
placed on Millipore filters lying at the bottom of parallel super-
fusion chambers, washed with 10 ml of medium at 37° and superfused
with glucose-containing (10 roM) oxygenated medium at 37° at a rate
of 0.5 ml/min. Fractions were collected every minute. After 7 min
(see arrow), the superfusion medium was replaced eit~5r with iden-
tical medium (controls) or with medium containing 10 M d-amphe-
tamine. The incubation and superfusion media contained 12.5 pH
nialamide and 1 rl1 ascorbic acid. The radioactivity in each frac-
tion was measured and is expressed as percentage of total radio-
activity recovered (that is, total fractions plus filter). The first
2-3 fractions gave at times more erratic results and are not pre-
sented in this and in the following figures. Hhen the number of ex-
periments allowed statistical evaluation, bars representing stan-
dard deviations are shown in this and in the following graphs (data
from Raiteri et al. 29 ). a) Each curve is the average of 4 experi-
ments run on ~different days. Some experiments (not presented) per-
formed using purified synaptosomes gave identical results. b) Each
curve is the average of 2 duplicate experiments on crude synaptoso-
mal fractions, performed in 2 different days. c) Each curve is the
average of 3 experiments on purified synaptosomes run on 1 day.
Several other experiments performed on crude as well as on purified
synaptosomes gave essentially identical results, and could not be
averaged for slight modifications in the experimental procedure.

(unpublished observation) and fairly large at 10- 7 t! (Fig. Sa).

In order to investigate the mechanism of the amphetamine-
induced release of dopamine, experiments were carried out in which
specific inhibitors of biogenic amine uptake were present together
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 325

"o tt'fPolr." ,," ul

.
u

..
m
COAIfIllI • • CelII"ClI . . - COIloI'",'
' -Amphtl. 'O~ l M o d - Amphllurllne 10 - 5 M
c. -- 10- " M
-0
0.-0 d- AmphEII'IUlllll0- )M
~ 10-~M S
:~
u
m
o

~ "
§
'0
"..
~ I
C>.. ®
~'~1~]~'~S7.'1·8·9~IO"1I-- 1 2 10 11 11l"56J8~1[I11
fracl illn nllmher

Fig. 5 - Effect of d-amphetamine on the release of 3H-DA from stri-

atal, cortical and hypothalamic synaptosomes. a) Each curve is the
average of 3 experiments on crude synaptosomal fractions run on 1
day. The concentration of 3H-DA used for prelabeling the synapto-
somes, in the experiments presented in this figure, was 0.1 pli. 15
parallel superfusion chambers were utilized (unpublished data).
b) Each curve is the average of 4 experiments on purified synapto-
somes run on 2 different days. Comparable results were obtained
with crude synaptosomal preparations. 3H-DA concentration used for
prelabeling was 0.4 pH. Some experiments were also repeated using
0.08 pH 3H-DA but no difference in the release pattern was observed.
c) Each curve is the average of 6 experiments on crude synaptosomal
fractions run on 2 different days. Other details as in the legend
for Fig. 4.( b) and c) are taken from Raiteri ~ al. 29 ).

with d-amphetamine in the superfusion fluid. Fig. 6a shows that the

amphetamine-stimulated release of 3H- DA in striatal synaptosomes was
almost unaffected by the presence, in the superfusion fluid, of a
concentration of desmethylimipramine equimolar to that of d-ampheta-
mine. On the other hand, when benztropine was present, together
with d-amphetamine, in the superfusion fluid, the effect of d-amphe-
tamine was largely decreased (Fig. 6b).
3 d-Amphetamine was a strong stimulator of 3H-5-hydroxytryptamine
( H-5-HT) release, both in striatal and in cortical synaptosomes.
The effect of d-amphetamine on 3H-5-HT release was practically abo-
lished, in both striatal and cortical synaptosomes, when desmethyl-
limipramine was added to the superfusion medium (Fig. 7).
Since the striatal uptake of DA is scarcely affected b tricy-
clic antidepressants and strongly inhibited by benztropine 2 ,22,
6
whereas the uptake of 5-HT is severely inhibited by desmethylimipra-
mine 23 , the data reported in Figs. 6 and 7 suggest that d-ampheta-
mine can utilize both the DA and the 5-HT uptake systems and stimu-
late the release of the two amines from their specific nerve termi-
nals. The specificity of the effects observed is particularly im-
portant since DA, NE and 5-HT may enter each other's nerve terminals
 326 M, RAITERI ET AL.

9 9
C!lonl rOI
" CDIll 1101

." ~
Ir - it.
0/,11 10"/,1
d -Am"heUtnmt 10· S M
d ·Alf'lptltllmln,.IlM I
d-J\mphtt.mln. 10- 5 M
"- Aflllphll,mlftt·
81"ltfO,'ft, ( ltIoth IO-5 MI
...
..
I bo" 10' 5/,1 1 0--0 811UlloplnllQa 5 M

6
~

...."
;; 4 4
;;
;;

~
.
"-

@
2 3 4 5 6 7 8 9 10 11 12 13 1 2 3 4 5 6 7 a 9 10 II 12 13
fraction number

Fig. 6 - Effect of desmethylimipramine and benztropine on the amphe-

tamine-stimulated release of 3H-dopamine from striatal synaptosomes.
After prelabeling with 0.1 fIH JH-DA, synaptosomes were superfused
with standard medium; the medium was then substituted with a new
medium (see arrow) containing drugs as indicated. Each 'curve is the
average of 2 experiments run in duplicate in 2 different days, using
8 parallel superfusion chambers. Other details as in the legend for
Fig. 4.

,
durlng the 'lncu b '
atlon 'h t h e ra d'loactlve
Wlt , ,21,32 .
amlnes
It is also important to exclude that the releasing effects ob-
served are due to inhibition of reuptake of the spontaneously re-
leased amines. That an effect on reuptake is not involved is demon-
strated by the following observations: desmethylimipramine, a strong
inhibitor of NE and 5-HT uptake, did not stimulate the release of
the two amines (ref. 10 and Fig. 7); benz tropine , an inhibitor of
DA uptake stronger than d-amphetamine 22 , was only a weak stimulator
of 3H-DA release from striatal synaptosomes (Fig. 6b).
In summary, d-amphetamine was unable to stimulate 3H- NE release
from the hypothalamus 3 an area rich in noradrenergic nerve endings,
but it could release H-NE from striatal and cortical synaptosomes.
As dopaminergic terminals are particularly abundant in the striatum
and are also present in good amount in the cortex 33 , and since do-
paminergic terminals are lik~ly to accumulate 3H- NE in the labeling
step32, it is possible that H-NE is released by d-amphetamine only
after being artificially stored in dopaminergic nerve endings. This
hypothesis is supported by the fact that the amphetamine-stimulated
release of 3H-NE from striatal synaptosomes was only slightly dimi-
nished by the presence of desmethylimipramine (Fig 8), thus showing
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 327

Corpus striatum COl t ex

9

Control
~
0 ~
8 Cont I 01
~ OMI 10- 5 M OMI 10- 5 M
u
'" ........ d - Amphetamine 10- sM "0 d· Amphetamine IO-5M
~ d· Amphetamine + OMI 6--6 d- Amphelamlne+DMI

0
~
c. I bOl h 1O- 5 M I I both 1O- 5 M I
> 6 6
>
u
'"0
"0

'"
-;;; 4 4
0 "' ...8.- _.6,

o ",
C 3
0 '6
o ......~
~
-'6--.
t
~

t
u
~
c..

® (6)
1 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 1011 12
fr act ion number

Fig. 7 - Effect of desmethylimipramine on the amphetamine-stimulated

release of 3H- 5-hydroxytryptamine from striatal and cortical synap-
tosomes. After prelabeling with 0.1 pM 3H- 5- HT , superfusion was
started with standard medium which was then substituted with a new
medium (see arrow) containing d-amphetamine, desmethylimipramine or
both. Each curve is the average of 3 experiments run in triplicate,
using 12 parallel superfusion chambers. Other details as in the le-
gend for Fig. 4.

the same characteristics of the amphetamine-induced release of DA

in the striatum (Fig. 6a).
The possibility exists that d-amphetamine is able to stimulate
release of NE only during depolarization. In fact, Von Voigtlander
and Moore 34 found in in vivo perfusion experiments that d-ampheta-
mine, at concentrations without effect on the spontaneous release of
tritiated dopamine from the caudate nucleus, potentiated the efflux
of the amine elicited by electrical stimulation of the nigro-stri-
atal pathway. Fig. 9 shows that d-amphetamine, in our superfusion
conditions, had no effect on the calcium-dependent release of 3H-NE
induced by 56 mM KCl in synaptosomes from the hypothalamus.
A final possibility that can not be excluded by the experiments
presented is that d-amphetamine releases NE from an endogenous pool
of hypothalamic synaptosomes which does not equilibrate with the
3H-NE used for labeling the synaptosomes. This possibility seems,
however, unlikely in view of the fact that several compounds struc-
turally related to d-amphetamine do release 3H- NE from hypothalamic
synaptosomes (see next section).
In conclusion, d-amphetamine, very often considered as a direct
releaser of NE, could not affect the release of 3H-NE from noradre-
328 M. RAITERI ET AL.

10

ConfJol
DMllO 'M
"o Jf-----i( d Amphelamlne 10 5 M
d Amphetamine +DMI
I both 10 5 MI

6·

1 2 3 4 5 6 1 8 9 10 11 12 13
fraction number

Fig. 8 - Effect of desmethylimipramine on the amphetamine-stimulated

release of 3H-norepinephrine from striatal synaptosomes. After pre-
labeling with 0.1 pM 3H-NE, superfusion was started with standard
medium which was then substituted with a new medium (see arrow) con-
taining d-amphetamine, desmethylimipramine or both. Each curve is
the average of 3 experiments run in triplicate, using 12 parallel
superfusion chambers. Other details as in the legend for Fig. 4.

nergic nerve terminals; from these in vitro results one could de-
duce that the reported increased availability of NE in the synaptic
cleft caused by the drug is essentially due to inhibition of reup-
take of the released amine. On the other hand, d-amphetamine seems
to influence the dopaminergic transp~rt system both by inhibiting
DA uptake and by a direct releasing effect.

RELATIONSHIPS BETWEEN STRUCTURE AND RELEASING ACTIVITY OF

S-PHENYLETHYLAMINE DERIVATIVES

In order to characterize the structural basis of the behavior

of amphetamine described above, a study was undertaken on the re-
leasing effect of compounds structurally related to ~-phenylethyl­
amine. The structure of the compounds studied is shown in Fig. 10.
Some of these compounds are either normal brain constituents or
major metabolites of amphetamine.
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 329

Control

""'
. KCI 56 mM
KCI 56 mM + d -Amphetamine 10. 5 M

.
.
"
~

1 2 3 4 5 6 7 8 9 10 11 12 13 14
fraction number

Fig. 9 - Effect of d-amphetamine on the calcium-dependent release

of 3H- NE induced by 56 ~~ KCl in hypothalamic synaptosomes. After
prelabeling with 0.1 rt1 3H-NE, synaptosomes were superfused with
standard medium. The medium was then substituted (s~5 arrow) with
new medium containing 56 mIl KCl, with or without 10 M d-amphetamine.
Each curve is the average of 2 experiments run in triplicate on two
different days.For other experimental details, see legend to Fig. 4.

BENZYL AMINE
- CH2"NH 2
@

JI'PHE NY LETHY LAMINE

-CHiCH2"NH2
@
H3 y A MPHET AMINE
-CHiCH-NH2
@

p - TYRAMINE
H O - @ -CH2" CH i NH 2

y H3 p - O~-AMPHETAMINE
H O - @ -CHiCH-NH2

Fig. 10 - Structure of benzylamine and of the phenylethylamine de-

rivatives utilized in the present study.

Effects on norepinephrine. Fig. 11 shows that ~-phenylethyl­

amine, which differs from amphetamine for lacking the methyl group
in the ~ position, had a substantial releasing effect on 3H-NE from
330 M. RAITERI ET AL.

Cont rol
<=
0
Benzylamine 10- 5 M
0--0 p-OH-d-Amphetamlne
u
.....----..
'"
.;:
~-Phenylethylamine
p- Tyramine
'"
Co
>
,.
u 4
'"
0
-c
'"
'"
;:
~
<=
'"u
t
'"
Cl.

1 2 3 4 5 6 7 8 9 10 11 12 13
fractIOn number

Fig. 11 - Effects of benzylamine and of some phenylethylamine deri-

vatives on the release of 3H-norepinephrine from hypothalamic synap-
tosomes. For experimental details see legend to Fig. 4. Each curve
is the average of 3 experiments run in triplicate. The drugs were
tested simultaneously on the same synaptosomal preparation, using
15 parallel superfusion chambers.

hypothalamic synaptosomes. A side chain with two carbon atoms is,

however, necessary for a releasing activity since the release of
3H-NE was unaffected by benzylamine. The releasing effect was po-
tentiated by the addition of a para-hydroxyl group to the phenyl-
ethylamine structure, as demonstrated by the stimulation caused by
p-tyramine. On the other hand, the addition of a ~ methyl group
in the molecule of p-tyramine leads to a compound (p-hydroxy-am-
phetamine) with very modest releasing activity.
The structural requirements for uptake inhibition differ from
those just described for release. In fact, the order of potency
with regard to 3H- NE uptake inhibition in hypothalamic s;maptosomes
was: p-hydroxy-d-amphetamin~d-amphetamine>p-tyramine> p-phenyl-
ethylamine.

Effects on dopamine. The effects of phenylethylamine deriva-

tives on 3H-DA release from striatal synaptosomes were strikingly
different from those on 3H-NE release in the hypothalamus. Fig. 12
shows that d-amphetamine was stronger than~-phenylethylamine as a
stimulator of 3H-DA release. p-Tyramine was as potent as d-ampheta-
mine. Interestingly, p-hydroxy-d-amphetamine, which has both the
~-methyl group and the p-hydroxyl group, was a somewhat weaker re-
leaser than either p-tyramine or d-amphetamine. The order of po-
tency of the four drugs in inhibiting 3H-DA uptake in striatal syn-
aptosomes was the following: d-amphetamine> p-hydroxy-d-amphetamine
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 331

............ Control
<=
0
............ ~·Phenvlethvlamine 5 ·10-7 M
0--0 p.OH·d·Amphetamine
" p-Tyramine
.::'" ~ d·Amphetamine
Q;
c.
>-

.:!
"'"0 4
~
'"
e
(;

t
<=

"'"Q;
0-

1 2 3 4 5 6 7 8 9 10 11 12 13
fraction number

Fig. 12 - Effects of phenylethylamine derivatives on the release of

3H-dopamine from striatal synaptosomes. The particles were prelabel-
ed with 0.1 pM 3H-dopamine. Each curve is the average of 2 experi~
ments run in triplicate on 2 different days, using 15 parallel su-
perfusion chambers. Other details in the legend for Fig. 4.

>p-tyramine> f'-phenylethylamine.

Effects on 5-hydroxytryptamine. Fig. 13 (a and b) shows that

d-amphetamine (10- 5 ~1)stimulated the release of 3H-5-HT from stri-
atal and cortical synaptosomes more efficiently than ~-phenylethyl­
amine. Hhen various phenylethylamine derivatives were compared at
a concentration of 5.10- 7 M (Fig. 14) the following order of poten-
cy in stimulating 3H-5-HT release was observed in striatal synapto-
somes: p-tyramine> p-hydroxy-d-amphetamine> d-amphetamine> -phenyl- f3
ethylamine. The latter was inactive at this concentration.·
The structural requirements for uptake inhibition of 3H-5-HT
in striatal synaptosomes were completely different from those for
release stimulation: in fact, if one excludes ~-phenylethylamine,
which was also in this case the least active compound, the order of
potency on 3H-5-HT uptake inhibition was: p-hydroxy-d-amphetamine>
)d-amphetamine) p-tyramine. Benzylamine was devoid of any effect
on 5-HT transport in striatal synaptosomes.

Concluding remarks. Although these results on structure-acti-

vity relationships of phenylethylamine derivatives are still incom-
plete and do not allow an overall picture, certain conclusions can
be drawn from the data presently available:
1. A side chain with two carbon atoms is necessary, both for uptake
inhibition or release stimulation of the three biogenic amines examin-
ed.
332 M. RAITERI ET AL.

Corpus striatum Cortex

Control Cont rol

Jt---I( d- Amphetamine 10- 5 M ......... d ·Amphetamine lO- S M
~- Phenylethylamine 10- 5 M ~. Phenyhlhylamme IO-5 M

®
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
fraction number

Fig. 13 - Effect of d-amphetamine andp-phenylethylamine on the re-

lease of 3H-5-hydroxytryptamine from striatal and cortical synapto-
somes. The particles were prelabeled with 0.1 pM 3H-5-HT. The concen-
tration of the drugs was 10- 5 11. Each curve is the average of 2 ex-
periments run in triplicate on 2 days, using 9 parallel superfusion
chambers. Other details as in the legend for Fig. 4.

Cont TO\
"
0
o-<:J ~·Phenylethyl'm,"e 5.10- 7 M
u ~ d-Amphetamine
ro
0---0 P-OH-d-Amphetamme
~ p-Tyramme
=
~
,.
ro 4
0
"0
ro

ro
0

-;
"
~

"-
t
1 2 3 4 5 6 7 8 9 10 11 12 13
fractIOn number

Fig. 14 - Effect of phenylethylamine derivatives on the release of

3H-5-hydroxytryptamine from striatal synaptosomes. The particles were
prelabeled with 0.1 p11 3H-5-HT. The concentration of the drugs was
5.10- 7 M. Each curve is the average of 2 experiments run in tripli-
cate on 2 days using 15 parallel superfusion chambers. Other details
as in the legend for Fig. 4.
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 333

2. The presence of an ct-methyl group strikingly decreases the re-

leasing activity in the case of 3H-NE, but strongly potentiates it
in the case of 3H-DA and 3H-5-HT. This group enhances the inhibi-
tory activity on uptake of 3H-NE and 3H-DA. but has very little ef-
fect on that of 3H- 5- HT •
3. The para-hydroxyl group enhances the effect of phenylethylamine
on the release of the three amines examined. In the case of 3H-DA,
this enhancement is quantitatively similar to that caused by the
~-methyl group; however. in the case of 3H-5-HT release. the p-
hydroxyl group is more effective than the ~ methyl group. The para-
hydroxyl group also enhances the inhibitory activity on the uptake
of all three biogenic amines.
In conclusion. a given substituent in the ~henylethylamine
structure may have rather selective effects on the activity of the
molecule towards uptake and release of biogenic amines in nerve end-
ings isolated from different areas of the brain. This may be par-
ticularly relevant in view of the possibility that some of the phe-
nylethylamine derivatives. such as p-tyramine, octopamine. phenyl-
ethanolamine and ~-phenylethylamine itself, act as modulators of
the aminergic neurotransmission l • 9 • 3l •

Acknowledgment. Partly supported by NATO Research Grant n.922.

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vol. 3, Plenum Press, New York, 1970, pp. 91-114.
20. Horn, A.S., Coyle, J.T. and Snyder, S.H., Catecholamine uptake
by synaptosomes from rat brain. Structure-activity relation-
ships of drugs with differential effects on dopamine and nore-
pinephrine neurons, ~10l. Pharmacol., 7 (1971) 66-80.
21. Iversen, L.L., Neuronal uptake processes for amines and amino
acids. In E. Costa and E. Giacobini (Eds.) Biochemistry of Sim-
ple Neuronal Models. Advances in Biochemical Psychopharmacology,
vol. 2, Raven Press, New York, 1970, pp. 109-132.
22. Iversen, L.L., Catecholamine uptake processes, Brit. !fed. Bull.,
29 (1973) 130-135.
RELEASE OF SYNAPTOSOMAL BIOGENIC AMINES 335

23. Kannengiesser, }!.H., Hunt, P. and Raynaud, J.P., An in vitro

model for the study of psychotropic drugs and as a criterion of
antidepressant activity, Biochem. Pharmacol., 22, (1973) 73-84.
24. Mg, K.Y., Chase, T.N. and Korin, I.J., Drug-induced release of
3H-norepinephrine and 3H-serotonin from brain slices, Nature,
228 (1970) 468-469.
25. Raiteri, M. and Levi, G., Antisynaptosome antibodies affect syn-
aptosomal permeability to neurotransmitters, Nature New Biol.,
245 (1973) 89-91.
26. Raiteri, H., Angelini, F. and Levi, G., A simple apparatus for
studying the release of neurotransmitters from synaptosomes,
Eur. J. Pharmacol., 25 (1974) 411-414.
27. Raiteri, M. Levi, G. and Federico, R., d-Amphetamine and the
release of 3H-norepinephrine from synaptosomes, Eur. J. Pharma-
col., 28 (1974) 237-240.
28. Raiteri, ~1., Federico, R., Coletti, A. and Levi G., Release and
exchange studies relating to the synaptosomal uptake of GABA,
J. Neurochem., 24 (1975) 1243-1250.
29. Raiteri, M., Bertollini, A., Angelini, F. and Levi, G., d-Am-
phetamine as a releaser or reuptake inhibitor of biogenic amines
in synaptosomes, Eur. J. Pharmacol., 34 (1975) (in press).
30. Raiteri, M., Levi G. and Federico R., Stimulus-coupled release
of unmetabolized 3H-norepinephrine from rat brain synaptosomes,
Pharmacol. Res. Comm., 7 (1975) 181-187.
31. Sabelli, H.C. and Hosnaim, A.D., Phenylethylamine hypothesis of
affective behaviour, Am. J. Psychiatry, 131 (1974) 695-699.
32. Snyder, S.H., Kuhar, M.J., Green, A.I., Coyle, J.T. and Shaskan,
E.G., Uptake and subcellular localization of neurotransmitters
in the brain, Int. Rev. Neurobiol., 13 (1970) 127-157.
33. Thierry, A.M., Blanc, G., Sobel, A., Stinus, L. and Glowinski,
J., Dopaminergic terminals in the rat cortex, Science, 182
(1973) 499-501.
34. Von Voigtlander, P.F.and Moore, K.E., Involvement of nigro-stri-
atal neurons in the in vivo release of dopamine by amphetamine,
amantadine and tyramine~ Pharmacol. expo Ther., 184 (1973)
542-552.
35. Heinshilbaum, R.H., Thoa, N.B., Johnson, D.G., Kopin, 1.J. and
Axelrod, J., Proportional release of norepinephrine and dopa-
r;
mine- -hydroxylase from sympathetic nerves, Science, 174 (1971)
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36. Ziance, R.J., Azzaro, A.J. and Rutledge, C.O., Characteristics
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294.
TRANSPORT OF DOPAMINE IN DISCRETE AREAS OF THE STRIATUM

AND OF CEREBRAL CORTEX IN THE RAT

J.P. Tassin, G. Blanc, L. Stinus, B. Berger,

J. Glowinski, and A.M. Thierry

Groupe NB - College de France

11, place Marcelin Berthelot, Paris 50, France

I - INTRODUCTION

For many years, the catecho1aminergic innervation of the

cerebral cortex in the rat was assumed to be only represented by
the arborisations of the dorsal noradrenergic pathway. However,
a quantity of dopamine (DA) slightly higher than that expected if
DA was only the precursor of noradrenaline (NA) was found in this
structure. It was thus postulated that the relatively high content
of DA was related to the presence of dopaminergic terminals 26 •
Two complementary approaches were available to test this hypothesis.
The first one consisted of detecting some properties of dopaminergic
terminals following the selective destruction of the noradrenergic
ascending pathway. The second was to Simultaneously identify
specific properties of each type of catecho1aminergic terminal in
the cerebral cortex of normal rats. Since DA and NA are taken up
in dopaminergic and noradrenergic terminals respectively by specific
uptake processes, the estimation of the uptake of these amines
could be used in these studies as an index of the specific properties
of each type of catecho1aminergic terminal.

II - Demonstration of the presence of dopaminergic

terminals in the rat cerebral cortex

Thierry et a1.,25 in their earlier studies (1973) specifically

eliminated the noradrenergic innervation of the rat cerebral cortex.
For this purpose, they injected 6-hydroxydopamine (6-0HDA) stereo-
taxically in the pedoncu1us cerebe11aris superior (PCS). Fifteen
days later, although the NA had completely disappeared in the cortex,
the level of DA was barely affected and synthesis of 3H-DA from

337
338 J.P. TASSIN ET AL.

3H-tyrosine still occurled in slices as well as in synaptosomes.

Later on, Berger et al. , using similarly lesioned animals, still
observed a catecholaminergic fluorescent innervation in cerebral
cortices completely devoid of NA.

III - Reuptake process as a tool to estimate and differentiate

catecholaminergic innervation in brain structures

a General characteristics
As already mentioned, dopaminergic and noradrenergic terminals
can be differentiated by their reuptake processes. In earlier
studies of peripheral noradrenergic neurons, it was found that the
estimation of 3H-NA uptake provided a good index of the noradrener-
gic innervation12 • A direct relationship was observed between the
NA content and the intensity of 3H-NA uptake in various structures
innervated by the sympathetic system. The elimination of this
innervation by 6-0HDA resulted in a decline of both NA levels and
3H-NA uptake in most organs 4 ,13,14. The uptake process of catechol-
amines in central catecholaminergic neurons was also used to
specifically label these neurons IO • In addition to these studies
Snyder and COIle examined more precisely the regio~rl differences
in 3H-NA and H-DA uptake in rat brain homogenates • They describ-
ed differences between the uptake affinities of 3H-NA and 3H-DA and
found that in most structures 3H- DA could be taken up by a high
(Km = 8.l0-8M) and a low (1.4 lO-6M) affinity uptake process. If
both 3H-DA and 3H-NA can be taken up by noradrenergic or dopaminergic
neurons, there exists nevertheless, a specific uptake process in
each type of terminal. Indeed, specific competitive inhibitors
can block one carrier or the ~ther: desipramine (DMI) acts specifical-
lyon noradrenergic terminals whereas benz tropine interferes more
markedly with the high affinity transport of 3H-DA in dopaminergic
terminals 5 • In the striatum, the 3H-dopamine uptake decreased in
direct relation to endogenous DA levels after the selective
destruction of nigrostriatal dopaminergic neurons induced by the
injection of 6-hydroxydopamine into the substantia nigra 14 • During
ontogenesis, the development of 3H-catecholamine upta~e in various
structures parallels that of endogenous amine content. Jonsson
et al. 15 estimated the uptake of 3H-NA to confirm the sprouting
of noradrenergic neurons in the brain stem following a 6-0HDA induced
lesion of this region: no change could be seen in the affinity of
the transport process, but a two fold increase of the Vmax was
obtained. These few examples illustrate the use of catecholamine
uptake estimations as a tool for the determination of the respective
importance of dopaminergic and noradrenergic innervation in the CNS.

b Evidence for a specific 3H-DA uptake in the cerebral

cortex of the rat
In control studies, the effect of desipramine and benztropine
on 3H-DA (lO-7M) high affinity uptake was estimated in sucrose
DOPAMINE TRANSPORT IN CORTEX AND STRIATUM 339

homogenates of the cerebellar cortex and of the striatum. In the

cerebellar cortex, a structure containing noradrenergic terminals
and devoid of DA nerve endings A desipramine (5 10- 7M) completely
inhibited the uptake of 3H-DA £4. In contrast, in the striatum,
which is rich in dopaminergic but poor in noradrenergic terminals,
desipramine had no effect. Nevertheless, benztropine (10-~) which
appeared to be less specific, inhibited 3H-DA uptake by 85% in the
striatum and by 30% in the cerebellar cortex.

The cerebral cortex represented an intermediate structure

containing both populations of catecho1aminergic terminals: Although
desipramine diminished the uptake of 3H-DA in cerebral tissues,
the remaining uptake still represented 24% of the total uptake
estimated in control sucrose homogenates. Benztropine was able
to further decrease the remaining uptake. A confirmation of these
results was obtained by repeating these experiments on P.C.S.
6-0HDA 1esioned rats (without noradrenaline in the cortex). An
uptake insensitive to desipramine was still present and it could
be inhibited by benztropine (_60%)24.

IV - 3H-dopamine uptake on microdiscs of cerebral tissues

After the demonstration of the presence of dopaminergic termi-

nals in the cerebral cortex of the rat, two questions remained
still unanswered: the anatomical distribution of these terminals
was not precisely known nor was the exact location of the cell
bodies. In their histofluorescent study, H8kfe1tet a1. 11 indicated
that dopaminergic terminals were particularly concentrated in
the frontal, cingu1ar and entorhina1 cortex in the rat. In order
to obtain quantitative information about the extent of dopaminergic
innervation in those areas, biochemical techniques were used. A
method was available by which microdiscs (0.5 mm 3 ) are punched out
of finely sliced tissues from well-defined structures of the brain 19
Unfortunately, the quantity of dopamine in the cortex was too low
to measure precisely the amine levels in a single micro disc despite
the use of a very sensitive radioenzymatic assay8. On the contrary,
if an uptake of 3H-DA (in presence of desipramine) could be measured
on these micro discs, the sensitivity of the method would be
sufficient to allow a quantitative topography of these terminals in
the cortex. However, in the Pa1kovits's method, the tissue had to
be frozen at -20 0 C. Under these conditions no more uptake of 3H-DA
could be measured. This was not improved by a mild freezing at
-7 0 C. Therefore, we tried to homogenize the frozen (-7 0 C) micro
discs in a small volume of isoosmotic sucrose. This procedure
allowed the reappearance of the uptake activity (Table 1).

a Distribution of dopaminergic terminals in the rat striatum

Before the estimation of 3H-DA uptake activity in sucrose
homogenates of microdiscs from the cerebral cortex, the method was
tested on the striatum which contains numerous dopaminergic terminals.
340 J.P. TASSIN ET AL.

Using the method described by Tassin et al.,23 the uptake was linear
for at least 2 minutes, unaffected by desipramine (Table 1) and
inhibited by benztropine (-75%). Moreover, no loss of uptake
activity was seen when compared to that obtained with unfrozen
tissue when the uptake activity was expressed per mg of tissue.
Differences were observed when the results were expressed in nCi
of 3H-DA per min per mg of protein; this is due to a two fold
purification of synaptosomes occurring during the centrifugation
when the activity of homogenates of fresh tissue is measured (Table 1).

TABLE 1

Comparison of 3H- DA uptake in various preparations of striatal tissue.

tOC D.M. I. 3H-DA uptake 3H-DA uptake

(5.lO- 7M) (nCi/min/mg (nCi/min/mg
2 rot 2 fresh striatum)

Microdiscs _7 0 C + 9.1 + o. 5~'" 1.1 ± 0.06*

Crude microdiscs
homogenates -7 0 C + 150''<* 20.0*''<
+4 o C 273 + 20 20.4 + 1.5
Homogenates of -7 0 C 310 + 10 23.2 + 0.7
whole striatum +4 oC + 289 + 15 21.6 + 1.1
-7°C + 285 + 13 21.3 ±0.9
3H- DA (6 10-8M) uptake was measured on microdiscs punched
out from frozen slices (-7 0 C), on homogenized microdiscs
and on sucrose homogenates from whole striata. D.M.I.
(5 10-7M) was added in most cases (+). Results are the
mean ± SEM N = 4.
* this value is the mean of determinations made on micro-
discs dissected on three slices of the striatum.
** this value represents the mean of 3H-DA uptake obtained
on the rostro-caudal curve of the 3H-DA uptake activity in
the striatum.

The striatum was cut on the frontal plane in 9 slices each 500 ~
thick. Three punches were made in each slice in the dorsal, medio
latera~ and ventral positions except for the 1st, 2nd, and 9th slice
in which only 1, 2 and 1 microdiscs respectively were punched out.
The 3H-DA uptake activity was rather homogene~ in the dorso-ventral
plane but very heterogeneous in the rostro-caudal plane. The uptake
activity decreased progressively from the rostral to the caudal part
of the striatum23 • Similar distributions were obtained when the DA
endogenous content 23 and the DA sensitive adenylate cyclase 3 from
identical micro discs were measured.
DOPAMINE TRANSPORT IN CORTEX AND STRIATUM 341

After the selective degeneration of the nigro striatal dopaminer-

gic pathway, 3H-DA uptake activity could no longer be measured
except in the most rostral slice (40% of the control value). This
could be explained by the presence of dopaminergic terminals in
the rostral end of the striatum which originate from cell bodies
outside the substantia nigra.

b Distribution of 3H- DA uptake in the cerebral cortex of

the rat
The coherence of the results obtained from the striatum led
us to study the distribution of the dopaminergic terminals in the
cerebral cortex 22 • The incubations were done in the presence of
desipramine (5 x 10-7M) to selectively inhibit 3H-DA uptake in
the noradrenergic terminals. The distribution of the 3H-DA uptake
activity in the serial frontal slices is shown in Fig. 1.

The intensity of 3H- DA uptake was schematically represented

as low, medium,or high activities. No significant differences
appeared when the same experiments were done on pes 6-0HDA lesioned
rats (Fig. 2). On these bases, dopaminergic nerve terminals were
mainly found in the medial and in the sulcal prefrontal cortex as
defined by Leonard 17 • Numerous dopaminergic terminals were also
detected in the cingular cortex. These result~ are in agreement
with the fluorescent studies of Berger et al., •

In other rats another determination was done in the rostro-

caudal plane. One micro disc was taken from each slice. This
micro disc was punched out in the medial cortex where the highest
density of dopaminergic terminals was detected in previous studies.
Results are shown in Fig. 2. They are rather homogeneous, however,
a peak seems to occur at about 10,500 ~ using the co-ordinates
described in the Atlas of K8nig and Klippel 16 and regarding the
beginning of the striatum at 9,800 ~ as a reference point.

c Localization of the cell bodies of the cortical dopaminergic

terminals 18 7
Recently Lindvall et al. and then Fuxe et al. observed by
histofluorescence that the cell bodies of dopaminergic neurons
projecting in the cerebral cortex were localized in the A 10 and
A 9 areas. These two areas correspond to the sites of the dopami-
nergic cell bodies of the mesolimbic and nigrostriatal dopaminergic
systems respectively. Therefore, lesions were made at these sites.
The effects of such a lesion on 3H_DA uptake in the cerebral cortex
are illustrated in Fig. 2. After an electrolytical lesion of the
A 10 group the 3H-DA uptake was decreased by 50% in the frontal
cortex but not in the cingular cortex (Fig. 2). This lesion did
not affect the 3H- DA uptake activity in the striatum but decreased
the amine uptake in the nucleus accumbens by 80%. When an
electrolytical lesion was made in A 9, no change in uptake activity
could be seen.
342 J.P. TASSIN ET AL.

_ _ C4;mtrol,
____ '+ ___ PC s. 60HOA l es ion
- - .- - A,O e LectrolytlC.• L IH lon

ROSTRAL CAUDAL

IJOCO

Fig . 1 Fig . 2

Fig. 1: Distribution of 3H-DA uptake activities in frontal

slices of the rat cerebral cortex. Microdiscs were punched
out, homogenized in 15 ~l of 0.25 M sucrose and incubated for
5 min in 400 ~l of a physiological medium containing 6 x
10-8M 3H-DA and 5 x 10-7M desipramine. The drawings are
from the Atlas of K8nig and Klippel 16 •
3H-DA uptake activity is expressed as nCi/5 min/disc.
~ 3H-DA~ 0.5 ~ 0.5"" 3H-DA~ 1 ... 1," 3H_DA, 2
111 2represents the high density region described by Berger et
aI., (1<3H-DA~3).

Fig. 2: Rostro-caudal distribution of 3H- DA uptakes in the

frontal and the cingular cortex. The conditions are identical
to those of Fig. 1. The arrow shows the distance where the
beginning of the striatum takes place. We used this landmark
to figure the separation between the frontal and the cingular
cortex. Results are the mean + SEM of four determinations.
Significantly different (lJ P< :-05 .. P <: .02 It If P <: .01
1t' .. JIC P < .001) from controls.

Simultaneous 6-0HDA lesions of the A 9 and A 10 groups of DA

cell bodies induced a 50% decrease in the uptake activity of 3H-DA
in the cingular cortex as well as in the frontal cortex.
7
These results seem to be in agreement with those of Fuxe et al.
However, in some lesioned rats the DA content could be decreased in
the frontal cortex, the DA levels not being affected in the nucleus
accumbens; it is thus possible that the cell bodies of the mesocortical
DOPAMINE TRANSPORT IN CORTEX AND STRIATUM 343

and of the mesolimbic systems can be found in overlapping regions,

while some of them are localized in distinct areas. Studies
employing fine electrolytical lesions controlled by histological
examination are in progress to make precise the localization
of the cell bodies of the mesocortical dopaminergic system.

V Conclusion and Discussion

Noradrenergic and dopaminergic terminals can be distinguished

by their reuptake process. Specific inhibitors of one reuptake
system or the other can be valuable tools to differentiate their
presence in any brain region. We have shown that there was a
specific uptake of DA in dopaminergic terminals of the rat cerebral
cortex. Furthermore, a method allowing measurement of the uptake
on micro discs (0.5 mm3) of frozen brain tissue was used to
determine the distribution of dopaminergic terminals in the striatum
as well as in the cerebral cortex. It can be assumed that formation
of intact synaptosomes occurs when the homogenization is done in
sucrose. Therefore, the uptake can be measured even after freezing
the tissues. It is of interest to note that this very sensitive
micro uptake method can be applied to the topographical estimation
of the activities of the choline and serotonin high affinity uptake
processes (unpublished results) which appeared to be effective
markers of the cholinergic and serotoninergic innervation in the
central nervous system.

The highest density of dopaminergic terminals in the cerebral

cortex seems to be concentrated in a zone which is conside,ed as
phylogenetically equivalent to the human prefrontal cortex .
Moreover, self-stimulation behaviour can be elicited from this
area 20 • The relation between DA, self-stimulation and emotivity
favors the role of cortical DA in emotional behaviour.

REFERENCES

1. Berger, B., Tassin, J.P., Blanc, G., Moyne, M.A., and Thierry,
A.M., Histochemical confirmation for dopaminergic innervation of
the rat cerebral cortex after destruction of the noradrenergic
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2. Berger, B., Thierry, A.M., TaSSin, J.P., and Moyne, M.A., Dopa-
minergic innervation of the rat prefrontal cortex: fluorescence
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3. Bockaert, J., Premont, J., Glowinski, J., Thierry, A.M., and
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344 J.P. TASSIN ET AL.

4. Breese, G.R., and Traylor, T.D., Effect of 6-hydroxydopamine on

brain norepinephrine and dopamine: Evidence for selective dege-
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tion of dopamine uptake in the corpus striatum as a possible
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6. Coyle, J.T., and Axelrod, J., Development of the uptake and
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7. Fuxe, K., H~kfelt, T., Johansson, 0., Jonsson, G., Lidbrink, P.,
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neurons, Brain Research, 82 (1974) 349-355.
8. Gauchy, C., Tassin, J.P., Glowinski, J., and Cheramy, A., Isola-
tion and radioenzymatic estimation of picogram quantities of
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nephrine in the intact rat brain by imipramine and structurally
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655-669.
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nergic nerve terminals in the rat limbic cortex: aspects of the
dopamine hypothesis of schizophrenia, Science, 184 (1974) 177-179.
12. Iversen, L.L., The uptake of biogenic amines. In J.H. Biel and
L.B. Abood (Eds.) Biogenic amines and physiological membranes in
drug therapy, Marcel Dekker, New York, 1971, pp. 264-266.
13. Iversen, L.L., and Uretsky, N.J., Biochemical effects of 6-hydro-
xydopamine on catecholamine-containing neurons in the rat central
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pamine and catecholamine neurons, North Holland, Amsterdam,
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14. Javoy, F., Sotelo, C., Herbet, A., and Agid, Y., Specificity of
dopaminergic neuronal degeneration induced by intracerebral
injection of 6-hydroxydopamine in the nigrostriatal dopamine
system, Brain Research (1975) in press.
15. Jonsson, G., Pycok, Ch., Fuxe, K., and Sachs, Ch., Changes in
the development of central noradrenaline neurons following
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16. K~nig, J.F.R., and Klippel, R.A., The rat brain: A stereotaxic
atlas of the forebrain and lower parts of the brain stem,
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17. Leonard, C.M., The prefrontal cortex of the rat. I. Cortical
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DOPAMINE TRANSPORT IN CORTEX AND STRIATUM 345

18. Lindvall, 0., Bj8rklund, A., Moore, R.Y., and Stenevi, V.,
Mesencephalic dopamine neurons projecting to neocortex,
Brain Research, 59 (1974) 332-337.
19. Palkovits, M., Isolated removal of hypothalamic or other brain
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cortex of Rattus norvegicus, Behav. BioI., 7 (1972) 567-572.
21. Snyder, S.H., and Coyle, J.T., Regional differences in 3H-norepi-
nephrine and 3H-dopamine uptake into rat brain homogenates,
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Thierry, A.M., Distribution of dopaminergic terminals in the
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Glowinski, J., Topographical distribution of dopaminergic
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Factors Influencing Transport
ENERGETICS OF LOW AFFINITY AMINO ACID TRANSPORT

INTO BRAIN SLICES l

Miriam Banay-Schwartz, David N. Teller,2 and Abel Lajtha

New York State Research Institute for Neurochemistry

and Drug Addiction
Ward's Island, New York, N.Y. 10035, U.S.A.

INTRODUCTION

A.I. Scope. This review concentrates on the following: What

is the energy source for low affinity amino acid transport into brain
slices? Is it glycolysis, or ion flux, or both? In this paper,
glycolysis refers to "breakdown of carbohydrate to pyruvate by the
Embden-Myerhoff-Parnas pathway, irrespective of the subsequent fate
of pyruvic aCid,,40. Neither the Pasteur or the Crabtree effect were
examined in our studies 25 ,40.

A.2. Rationale for Studying Slices. There are several reasons

for studying energetics of active transport in brain slices rather
than in other systems: a. Such studies cannot be done ~ ~ at
present. b. Subcellular particles - i, are partially damaged during
preparation; ii, contain only one or a few portions of an integrated
transport system; iii, are obtained impure, or from mixed cell
populations; iv, are not obtained rapidly enough to avoid break-
down; v, are obtainable in (relatively) too small quantities for
study of bulk (ca. I roM) transport; ~,are not sufficiently
mechanically stable for study of efflux or sequential transfer through
various media.

lpreliminary accounts of some of the work presented here were

reported at New Orleans, La. (Am. Soc. Neurochem., Trans., 5 (1):
93, 1974) and Mexico City, (Am. Soc. Neurochem." Trans., ~ (1): 94,
95, 1975).
2present address: Dept. of Psychiatry and Behavioral Sciences;
Univ. Louisville Medical School, Health Sci. Ctr.; P.O. Box 1055,
Louisville, Ky. 40201 (U.S.A.).

349
350 M. BANAY-SCHWARTZ, D.N. TELLER, AND A. LAJTHA

Brain slices, in contrast, do not restrict experimental

protocols. Slices are somewhat more flexible in their requirements
for transport: ii, iv, v, and vi do not cause problems in experi-
ments with slices, whil; i and-rii can be minimized by using thick
slices (0.4 mm) from specific b~n regions, e.g., olfactory bulb.
Enviably, there are great experimental and theoretical advantages
in using preparations of a composition "simpler" than slices, but
interpreting the results from vesicles and relating these findings
to erain function are more difficult than after experiments with
integrated, relatively intact pieces of brain.

B.I. Background: a. Other Reviews. Recent research concern-

ing mechanisms of active transport has been reviewed by Wilbrandt 42 ;
hypotheses concerning coupling of energy producing and utilizing
systems have been reviewed by Green l9 , by Bennun 7 , and by Meijer
and Van Dam32 , while specific defects in cerebral intracellular
oxidative processes have been linked with rare storage diseases in
man l7 Reviews have appeared of ion-dependency of transport in
other tissues 36 and of high-affinity amino acid uptake 6 ,14 that are
beyond the scope of this article.

B.I. Background: b. Brain Slices. Many of the amino acids

in mammalian brain are accumulated across the blood-brain barrier
by active trans~ort systems that are structurally specific and
require energy27. The properties of cerebral amino acid transport
systems have been studied primarily by incubating brain slices for
the reasons noted above. The levels to which different amino acids
are concentrated ".i£~" by brain slices under uniform experi-
mental conditions depend on the amino acid and its concentration in
the medium 29 • Concentrative uptake (C.U.), the increase of the
amino acid in the brain slice to a level above that of the medium,
requires energy because inhibition of production or utilization of
energy blocks such accumulation l •

Neither the driving forces for such accumulation ~ a concentra-

tion gradient nor the identity of the specific metabolic reactions
producing the energy source for amino acid transport in brain are
known. ATP levels were shown to be parallel to amino acid uptake
in a number of experiments. However, we indicated that the level
of ATP in incubated cortex slices is not a limiting factor for
metabolite transport under certain conditions. By using several
metabolic inhibitors that interfere with the energy metabolism as
well as with the ion distribution in the brain slices, it is possible
to obtain high amino acid uptake in the presence of low ATP levels
in the brain slices, or low uptake in the presence of high ATP
levels 3 . Amino acid transport studies in a variety of systems have
shown that ATP levels do not control active amino acid uptake 3 ,4,8,33
Here, we review some experimental results that indicate a mitochondrial
origin for the energy that supports active transport, a finding that
ENERGETICS OF AMINO ACID TRANSPORT 351

moots the question of ATP involvement.

Another indirect source of energy for active transport is a

presumed "ion-gradient" which could function by exchanging kinetic
energy with a potential carrier molecule in the membrane 28 . In one
view, the carrier may undergo a conformational or translocational
movement that is energized by the ion flow. Here, we review some
recent evidence that suggests the ion flow coupling to active
transport may be coincidental but not necessarily causal.

Finall¥, there is considerable speculation on the involvement

of Na+ or K in active transport systems l4 , particularly with
respect to "high-affinity" uptake. We have begun investigating the
ion-dependency of low-affinity systems and can show that the K+
dependency is not specific; that Na+ dependency may be primarily an
indicator of the treatment of the tissue; that powerful inhibitors
of ion-dependent ATPase are not inhibitors of uptake under certain
conditions; and that the rate of uptake is maximal for several amino
acids when ion-flux is lowest or non-existent, or after tissue levels
of exchangeable amino acids have been reduced by sequential washing
steps.

B.2. Does Active Transport of Amino Acids Require Glycolysis,

Phosphorylation and ATP? a. Evidence from Bacterial Studies:
Energy for transport from glucose can be obtained independently from
two major sources: ATP from oxidative phosphorylation (requiring e-
transport and a functional Ca++, Mg++ATPase) or from substrate
phosphorylation of glycolysis. Energy-rich membrane systems for
pumping processes may be fueled by ATP hydrolysis (in the presence
or absence of respiration) or by respiratory chain oxidations that
can bypass the requirement of functional ATPases. Berger 8 has
reviewed the data from bacterial transport studies which indicate
that the role of ATP is probably indirect (although there may be
certain exceptions, see 24 ); either ATP hydrolysis or electron
transport can drive proline transport in E. coli, while glutamine
uptake is directly fueled by phosphate bond energy from oxidative
or substrate level phosphorylations. Bacterial membrane vesicles,
from E. coli ML 308-225, exhibit at least nine amino acid transport
systems, most of which receive energy by the coupling of respiratio~
between a primary NADH or D-Iactate dehydrogenase and cytochrome b l 0
These transport system models at first appear to be much simpler
than slice preparations, but are actually quite complicated and
sophisticated 31 . Nonetheless, the uptake of amino acids into isolat-
ed bacterial vesicles is inhibited by the same poisons and drugs,
and at very similar concentrations, as those we use to inhibit
transport in brain slices 30 • No oxidative phosphorylation occurs in
the bacterial vesicle systems 21 ,31,35, and a priori there does not
seem to be a requirement for ATP or high-energy phosphorylation to
fuel either active amino acid transport, or glucose transport. For
352 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

example, in membrane vesicles of Azotobacter vinelandii, transport

of amino acids or glucose is coupled to oxidation at a site proximal
to ubiquinone S • Inasmuch as uptake by the vesicle and erythrocyte 39
preparations shows (a) competitive inhibition by analogs, (b)
"metabolic inhibition" by electron transfer inhibitors, (c) saturable
kinetics, (d) temperature dependency, (e) exchange, (f) inhibitor-
stimulated efflux, and (g) sensitivity to SH-inhibitors, all propert-
ies that are shared by active transport in Ehrlich or KB cells as
well as in brain slices, it seemed appropriate to try to determine
whether brain tissue could use energy sources other than glucose
for active uptake of amino acids.

B.2. b. Comparison of Properties of Isolated Vesicles, Cells

and Slices: There ~ major qualitative and quantitative differences
between uptake in vesicles and in slices: in vesicles monovalent
cations are usually inhibitory, while phenazine methosulfate (PMS)
plus ascorbate stimulates uptake 30 . The reverse obtains in brain
slices. (However, we have not yet tried to couple PMS with tetra-
phenylboron or other agents 22 that might permit the use of PMS
despite its poor membrane penetration.) Furthermore, in brain slice
or cell preparations, an entire spectrum of metabolic inhibitors
that block glycolysis (absent, any effects on transport in vesicles)
decisively blocks amino acid uptake in slices, ~ glucose-containing
media.

Melbourne and Charalampous 33 have presented data to show that

a-aminoisobutyric acid (AlB) transport fueled by ATP in KB cells does
not involve participation of high energy intermediates generated
intramitochondrially, nor does phosphoenolpyruvic acid serve as the
energy donor. However, in their experiments, there was no attempt
made to substitute other carbon sources, and the demonstration of
glucose-dependency of KB cells' ATP and transport activity under
anaerobic glycolytic conditions tested with oligomycin and its
analogs is not sufficient to preclude alternate energy sources. For
instance, we observed that ATP levels in slices can be reduced
20-40%, as in KB cells. Under these conditions there is a 60-70%
decrease in AlB entry rate in both systems 3 ,4. However, the AlB
uptake in slices can be restored to control levels while the ATP
is lowered further 4 • Moreover, 2 mM CN- or incubation under N2
severely depresses, irreversibly, AlB uptake by brain tissue slices3,
but only inhibits 20% of the AlB uptake by KB cells 33 . Therefore,
in the absence of additional data concerning the inhibitor sensitivity
of KB cell uptake of AlB to compare with AlB uptake into brain slices,
it appears that energy coupling of one amino acid transport system
in brain slices may be very different from that in KB cells.

C. Experimental Dissociation of Transport from Glycolysis:

Fig. I shows the effects on mouse brain slices of incubation for
various periods in HEPES-2 medium lacking glucose (HEPES-G medium)
ENERGETICS OF AMINO ACID TRANSPORT 353

lL.. 100 r - - - - - , . - - - - - - r - - - - - - - - - r - - - - - - ,
o
/0' UPTAKE AT 37°

40

20
~-GLUTAMIC
O~------L-------L---------------~--~
o 15 30 60
o~
DURATION OF INCUBATION WITHOUT GLUCOSE
BEFORE AMINO ACID ADDITION - MINUTES
Fig. 1. Brain slices were incubated at 37 0 in HEPES-G medium
i.e., HEPES-2 medium 3 ,4, minus glucose, for 15, 30 or 60 min
then l4C-amino acids were added for additional 10 min periods.
Initial concentration of l4C-amino acids in the medium: D-glu,
1 mM; a-AlB and L-1ys, 2 mM. Control levels are those reached
30 min after incubation in HEPES-2, followed by 10 min incubation
with l4C-amino acid. C.U. (concentrative uptake) = ~oles of amino
acid m1- 1 intracellular H2 0 (T), above the medium concentration (M),
at the end of incubation; C.U. = T- [(MxE) / l-E] -M, where E =
(extracellular space), m1 inulin/m1 tissue H2 04. Thirty min
incubation at 37 0 in the absence of glucose is sufficient to block
amino acid uptake. For HEPES-2 medium composition see ref. 44.

on the uptake of three characteristic amino acids. Inhibition of

uptake becomes relatively constant after 30 min. of glucose depletion.
The results of experiments shown in Fig. 1 and 2, and Tables I-III
indicate that when endogenous energy stores in brain slices are
depleted by incubation in media lacking glucose, if lactate, pyruvate,
succinate, or other tricarboxylic acid substrates are provided as
energy sources, then amino acid uptake by the slices can proceed.
Indeed, uptake can continue in the presence of 10 mM NaF, an
inhibitor concentration that blocks glycolysis as well as many
ATPase enzymes, Table II.
354 M. BANAY-SCHWARTZ, D.N. TELLER, AND A. LAJTHA

140~ o-GLU oAIB L- LYS L-VAL

ImM 2mM 2mM ImM
(/00% -/5.6)
.:::.'9~
(/OO% -3.0) (7.0)
''0

100"- \..1 : "S~I rn ~F'n I n

80

60

40
L~
20

Fig.2. Amino acid uptake after incubation in medium lacking glucose

is restored to different levels with various energy sources and the
per cent of the restoration also depends upon the particular amino
acid_ Incubation conditions: brain slices were shaken (2Hz) at 37 0
for 30 min in 4.5 ml oxygenated HEPES-G; then 0.5 ml of test substrate
was added for an additional 30 min. "restoration": OIGP .. a-glycerophosphate;
LAC - L-lactate; MAL = L-malate; PYR = pyruvate; SUC =
succinate; SMP = succinate 20 mH· malate, 5 mH; and pyruvate,
5 mH (all sodi~alts in HEPES-G at ~H 7.35). UNDEPLETED = preincuba-
tion in HEPES-2 (with glucose, 10 mH) followed immediately' by 20 min.
l4C-amino acid uptake. The arrow head and base for each bar is the 20
min. uptake of the l4C-amino acid after 60 min. in medium without glucose
or other energy sources_ All l4C-amino acid uptake measurements were
for 20 min. and were calculated from the "control", (glucose at 10 mH
added for the "restoration" and incubation period = 100%) _ The 100%
C_U. value with glucose restored is shown in parentheses under the
initial l4C-amino acid concentration in the media. The vertical bar,
"r e_s.e." is the 'r~estimated standard error of the mean of the control,
SMP, and undepleted uptake levels.

A comparison (Table l) of tissue constituents of slices incubated

in complete medium, in medium lacking glucose, and in media containing
glucose or (succinate + malate + pyruvate, as the sodium salts) =
(SMP) , indicates that ATP drops and is only partially restored. Na+
increases (caused partly by Na+ added with the Krebs' cycle inter-
mediates, K+ decreases (and is only partially restored), and tissue
water increases. However, the 10 min uptake of D-glu was restored to
86 or 119% of control levels and that of AlB was restored to 77 or
ENERGETICS OF AMINO ACID TRANSPORT 355

TABLE I. Tissue components change during incubation although

amino acid uptake can be restored
ATP )leq/ml (d) Per cent Concentrative Per cent of
111M tissue H20 Swelling (e) Uptake (f) Control Uptake
Na+ K+ D-Glu AlB D-Glu AlB

Samples taken after:

40' incub. in HEPES-2
"Control" (a) 1.18 112 70 49 13.5 9.9 100 100
40' incub. in media
lacking glucose
"Depletion" (a) 0.07 148 20 58 2.0 2.5 15 25

Depletion and
restoration with
glucose-(b) 0.45 134 43 11.6 7.6 86 77

Depletion and
restoration with
SNP (c) 0.36 177 44 66 16.1 8.8 119 89

Table I. (a) Includes 30 min. temperature equilibration at 37 0 before amino acid addition.

(b) Includes 30 min incubation in medium lacking glucose (HEPES-G), 30 min in control
medium, + 10 min in presence of 14C- amino acid. (c) Includes 30 min in medium lacking
glucose, 30 min in presence of SNP (HEPES-G, + 20 mM succinate, 5 mM malate, and 5 mM
RYruvate) + 10 min in presence of l4C-amino acid. id) Na+ and ~ are expressed as
)lequiv. ml- l tissue H20. (e) Per cent swelling. [H20 in slices after incubation
(ml gm-l frozen weight)/H20 in unincubated slices~x 100 (-) 100. Swelling measurements
were performed only with D-glu. Thel~swe1ling of slices 30 min after restoration in the
SNP medium, but before addition of C-amino acid for uptake, was 53. (f) Concentrative
uptake, C.U. (see legend Fig. 1). Initial amino acid concentrations in the medium:
D-glu, lmM; a-AlB, 2 mM.

88%, with glucose or SMP respectively, after 30 min depletion

plus 30 min 'restoration' of energy sources.

The sensitivity of amino acid uptake into brain slices to

inhibitors varies not only for individual amino acids 4 but is also
strongly dependent upon the substrate utilized for energy production.
Table II shows eleven selected examples of differential inhibition:
200 ~ arsenite inhibits uptake very strongly with pyruvate
or SMP, but not with glucose; 10 mM NaF has just the opposite effect.
Fluoroacetate and iodoacetate (lAc) are not as inhibitory when
pyruvate is the substrate, but amy tal inhibits uptake much more when
SMP is used instead of glucose. Rotenone is not as inhibitory at
40-50 nM when glucose or a-glycerophosphate are substrates. As
could be expected, antimycin A is more inhibitory when pyruvate
(or SMP) is used, although a-glycerophosphate also sensitizes the
uptake to this antibiotic.

a-Glycerophosphate (a-GP) was relatively ineffective as an

energy source for brain slice uptake of glu, AlB, lys, val, his,
and gly, suggesting that the shunt of NADH+H+ around the triose
 356 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

TABLE II. Effects of inhibitors depend upon the substrate used

to replace glucose.
HEPES-2(a) GLUCOSE (b) LACTATE PYRUVATE SMP a-GLY-P
GLU AlB GLU AlB GLU AlB GLU AlB GLU AlB GLU AlB

INHIBITOR M PER CENT OF CONTROL (c) UPTAKE

None 100 100 86 86 71 71 59 24 106 99 21 32

Arsenite 2x10- 4 101 76 88 62 20 8 4 10 8 8 9 9
NaF 1xlO-2 11 18 25 12 2 16 43 36 103 75 1 4
FAA 2x10- 2 86 69 99 55 27 15 81 18 85 49 1 4

lAc 3x10- 4 23 52 22 56 57 43 56 32 99 46 1 4

Amy tal 4x10- 4 75 60 69 48 34 17 35 9 11 22 10 18

2,4 DNP 2x10- 5 102 94 77 89 25 32 51 19 79 88 8 28

Rotenone 5xlO_-87 75 64 58 54 19 20 8 13 30 41 8* 18*

Antimycin 5x10 56 46 40 40 13 22 7 8 15 28 6* 10*
Va1inom. 9x10- 7 65 66 65 41 19 16 4 12 14 15 3 10

Ouabain 2x10- 6 73 42 47 35 50 26 40 18 58 20 2 4
01igom. 9.2xlO- 7 86 97 71 87 33 25 14 17 33 45 8* 7*

Table II. a) Inhibitor added at t(incubation) = 0 for 30 min before amino acid was added.
b) All other columns of data were obtained after incubations that depleted the endogenous energy
stores of the slices for 30 min in HEPES-G, followed by addition of inhibitor and energy source
for 30 min (restoration period) prior to addition of l~C-amino acid for 10 or 20 min uptake.
FAA = f1uoroacetate; lAc = iodoacetate; 2,4-DNP = 2,4-dinitropheno1; SMP = succinate, 20 mM;
malate, 5 mM and pyruvate, 5 mM. a-G1y-P = a-glycerophosphate, 20 mM; glucose or lactate, or
pyruvate, 10 mM. *Rotenone, 0.04 ~M; antimycin; 0.2 ~M and oligomycin, 0.46 ~M.
c) 100~values = uptake by undep1eted slices in HEPES-2, without inhibitor; for 1 mM D-g1u
control, 100~ of 10 min C.U. = 13.5; 100~ of 20 min C.U. = 22.9 ; for 2 mM a-AlB, 100~
of 10 min C.U. = 9.9; 100~ of 20 min C.U . • 18.1. The following procedure will give the
concentrative uptake values with any of the substances used for "restoration", or after treatment
with the inhibitor: [(~ of control x control C.U.)/100J.

leve1 3 is not sufficient for transport. Indeed, except with AlB

in DNP and amy tal, uptake was usually pretty well inhibited with
a-GP as substrate. The relative insensitivity of D-glu uptake
fueled by pyruvate to inhibition by lAc, and the lack of uptake when
a-GP was substrate, tends to rule out the transhydrogenase shunt
as an energy source. Pyruvate also decreases the sensitivity of
AlB uptake to 2 ~ ouabain and increases the sensitivity of D-glu
uptake to valinomycin. The uncoupling agent, 2,4-DNP, is much less
inhibitory to uptake when SMP is present than when lactate or
a-glycerophosphate are the energy sources, while amy tal, a different
type of uncoupler, inhibits most with SMP.

What was unexpected was the relative specificity of the

inhibitor/substrate effects. Glutamate uptake was increased more
than 40% over basal levels obtained with pyruvate when fluoroacetic
acid was added, while AlB uptake also increased if IAc was present
when pyruvate was used as an energy source. One possible explanation
of the apparent stimulation of glutamate uptake might be due to
increased conversion to l4C-glutamine, although glutamine does not
accumulate in the mouse brain slices. Indeed, amino acid analyses
ENERGETICS OF AMINO ACID TRANSPORT 357

TABLE III. Concentrative uptake can be restored by substitutions

for glucose ££ K+, these may alter the sensitivity to inhibitors.

Per cent of 30 min. uptake of control (undep1eted) slices

(a) in media containing:

Control
C.D. +gluc (b) -gluc +gluc -gluc -gluc +gluc -gluc
+K -K -K +K -K -K -K
+Rb +Rb +Rb
+SHP
D-g1u 28.6 86 4 11 16 24 100 99
a-AlB 22.1 82 14 20 33 52 73 93
GABA 28.3 103 18 36 50 55 101 98
L-his 18.0 94 32 22 20 38 91 95
gly 20.7 98 12 27 30 44 87 100
L-leu 3.57 73 24 39 41 81 81 89

L-val 7.63 88 28 22 24 46 61 93
L-orn 5.50 110 57 65 70 91 91 91
L-1ys 5.44 97 54 76 97 85 104 89

D-glu 22.9 79 (in 20 roM fluoroacetate for 20 min. uptake) 81 61

a-AlB 18.1 59 " " "
D-glu 22.9 36 (in 10 mH sodium fluoride for 20 min. uptake) 27 85
a-AlB 18.1 19 " 27 75

Table III. a) Brain slices were incubated for three successive 10 min intervals in 5 ml
of oxygenated media lacking both glucose and K+. This was followed by a 30 min incubation
in media containing the substances listed <It the column heading (b) for "restoration" of
energy, and thereafter 30 min uptake of l4C-amino acids was measured after incubation
under the same conditions as the restoration. Inhibitors were added for both the 30 min
restoration period as well as for the 20 min uptake period. The initial concentration of
the amino acids in the uptake media waS-l mH, except for a-AlB and L-lys, which were 2 mH.
When present, glucose = 10 roM; ~ or Rb+ = 5 mH; SMP = succinate, malate, pyruvate
(20, 5, 5 mH).

of the supernatants from the tissue pellets and of the HEPES-2 or

SMP media after 30 min. incubation showed that 30% (+ 1% S.D.) of the
~ radioactivity of added D~glu was in glutamine,-but 97-99% of
the total glutamine radioactivity was in the medium.

Clearly, the utilization of energy sources and the coupling of

each source to the particular transport system (acidic, D-glu; or
neutral, a-AlB) are quite distinctive. Therefore it is not feasible
to generalize or specify the energy linkage to transport beyond
suggesting that ATP hydrolysis (inhibited by F- and lAc) is probably
not obligatorily linked; that glycolysis is not required, although
a shunt through a-glycerophosphate that is amy tal and rotenone-
insensitive may be minimally used; that mitochondrial respiratory
chain inhibitors are most effective at the level of ubiquinone
(rotenone and antimycin A).
358 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

Therefore, in answer to the question above (B.2.), at least two

low affinity transport systems in brain slices appear to lack
obligatory coupling to glycolysis and ATPase activity, because AlB
and D-glu uptake can be demonstrated to proceed at rapid rates under
conditions that are extremely inhibitory to these potential energy
sources.

D. How Does Active Transport of Amino Acids Require K+ or Na+?

1. The KT Requirement is not Specific: Our findings then led us
to speculate upon the essentiality of other possible energy sources,
e.g., ions. We became aware that various experimental treatments
of the slices could inactivate or frustrate subsequent restorative
processes for uptake after first trying to deplete the slices of K+
at low temperatures, or after using ouabain, etc. The exit of K+
was facilitated by 3H-ouabain, but the inhibitor caused some irrevers-
ible effects and could not be completely removed by successive
transfers into ouabain-free media. However, at 370 in the"absence
of glucose, successive ~ransfers of brain slices through media lacking
potassium reduced the K level by 90-95% (Fig. 3, a, c). In the
absenc f of K+, i f glucose or SMP is "added, no uptake ';;ccurs.
When K is supplied, transport is restored. Table III shows the
amino acid uptake capabilities of brain slices that were depleted
of glucose and potassium by three successive incubations in media
devoid of these substances. After 30 min of incubation in various
combinations of glucose and K+, or Rb+, we were able to restore low
affinity amino acid uptake capability (for at least 30 min. additional
incubation periods) with media containing glucose and K+ or Rb+, but
generally, equally good results were obtained with SMP plus either
K+ or Rb T • To test whether SMP-energized uptake was actually sensitive
to "metabolic" poisoning, we examined the effects of fluoroacetate
and fluoride. As expected with tissues whose energy sources bypass
the fluoride-inhibited triose pathway, in SMP media, plus either K+
or Rb+, 10 roM NaF had no effect (Table III). Further, in SMP media
the inhibition by fluoroacetate was increased, probably due to its
increased conversion to the active fluorocitrate. Moreover, the
lack of inhibition of F- in the SMP + Rb+ medium suggests that
Mg~a~+ATPases may not be obligatorily involved in amino acid
uptake, because 2-4 roM NaF is considered sufficient to block most
NaK-ATPase 16 • More importantly, Rb+ can be substituted for K+, and
thus, there is no obligatory coupling of transport with potassium.

D.2. The Role of Na+ is more Difficult to Determine: a. Changing

Na+ and K+ Simultaneously: Finally, we are still examining the
effects of Na+ depletion; Na+ exits into Na+-free media whether or
not glucose is present in the successive washes Wig. 3 ~). In accord
with current concepts of energy-dependent ion balance, the depletion
of K+ occurs most rapidly if glucose is absent, whereas sodium actually
drops faster if glucose is present. If sodium salts are used to
adjust the pH of K+-free media, the Na+ rises at 2/3 of the rate that
ENERGETICS OF AMINO ACID TRANSPORT 359

e,e_
e-e-e
K~--

o 10

Fig. 3. Slice ion contents can be changed very rapidly. a) HEPES-G

(-) K+ (pH adjusted to 7.35 with Na+ salts). b) HEPES-G (-) Na+
(pH adjusted to 7.35 with Tris, K+ = 5 mM). Initial ion levels are
those of freshly sliced unincubated mouse cerebral cortex. c) ATP
and K+ level in tissue incubated as in (a), showing 2 mM l4C-AIB
uptake (C.U.) capability in comparison with that of undepleted,
incubated tissue (control, = 100%).

K+ decreases during the first 5 minutes (Fig. 3 a). If tris-base

is used to adjust the pH of the medium and if 5 ~ K+ is present,
the Na+ decreases, again at 2/3 the rate of the K+ for the first
5 min (Fig. 3 Q). The rates of change in K+ and Na+ are related
even when the initial ion levels are quite different.

To compare the results of K+ depletion with those obtained

when Na+ is depleted, we summarize: 1. If brain slices are
transferred at 10 min. intervals in media lacking glucose and K+,
the ATP level and uptake capability decrease faster than the K+
level (Fig. 3 c). 2. However, the AlB uptake capability of the
tissue is easily restored by K+ or Rb+ with an appropriate energy
source, and then the C.U. may even exceed untreated control levels
(Table III). 3. It is possible to show nearly normal uptake of
AlB or D-glu when K+ is exiting or entering the tissue, or even when
it is driven out by Rb+ (Table IV). 4. Therefore, it seems that
the K+ level or direction of net flux does not affect transport,
although it is very important to keep the tissue functional during
the ion-change protocol. 5. If the uptake systems are destroyed
by the experimental handling procedure, then the results may be
misleading.
360 M. BANAY-SCHWARTZ, D.N. TELLER, AND A. LAJTHA

TABLE IV. Amino acid uptake may be independent of the tissue

K+ level and direction of K+ flux.
Key to
tissue Tissue K+ level lleq/ ml at Net K+ Concentrative uptake
treatment start end change per cent of control
and medium of uptake lleq/ ml D-glu a-AlB
ion content

a 70 27 -43 85 73

b 70 40 -30 97 76

c 5 57 +52 105

d 5 44 +39 80 92

e 5 2 -3 97

f 5 25 +20 90

g 70 16 -54 103 84

Table IV. Most of the uptake of D-Glu or a-AlB was maintained whether K+ went into
the tissue, or came out, from high or low initial ~ levels in the tissue. In all
cases, the brain slices were depleted of their endogenous ~ levels by 3 transfers
at 10 min intervals, in HEPES-G (-~), followed by 30 min preloading, either in
HEPES media containing glucose and: 50 mH K+ (a,b,g); 50 mH Rb+ (e,f); or no K+
(c,d). Then the slices were washed and transferred to media containing the amino
acid, 10 mH glucose, and the following combinations of ions: (a) 0 roM ~;
(b,f) 5 mH KT; (c) 15 mH t+; (d) 10 mH~; or (e,g) 5 mH Rb+. The duration
of uptake was 10 min. in: (a), (b), (e), (f), (g); and 30 min. in (c), and (d).
Control values of D-glu uptake from initial 1 mH medium concentration for 10 min •
13.5; from 2 mH AlB = 9.90; for 30 min uptake, the control C.U. values were 26.6
and 22.1, respectively.

D.2 b. Na+ Flux Relationship to Amino Acid Uptake: Sodium

levels and direction of ion-flux are considered the sine qua ~
of active transpor~. This is probably incorrect. We show (in Fig.
4 a), 'typical' Na -dependency of D-glu uptake. However, note that
theK+ levels of the slices in this medium are elevated because KOH
was used to neutralize the medium*. If we used only 5 mM K+ + Tris
for pH adjustment, we observed that the C.U. of D-glu changed in
proportion to the ~ increase in the tissue, and not in proportion
to the rise of tissue Na+ (kept approx. equal to that of the medium),
Fig. 4 b. Further, we could then show that the greatest flux of
amino acid entry occurred when the tissue Na+ flux was nearly zero,
for AlB, D-glu, and GABA. If we compare the change in tissue Na+
at 3 min. intervals with the change in C.U., Fig. 5, from the third

~( Choline, as a substitute basic ion, can also be extremely inhibitory

towards amino acid uptake. We have not investigated the mechanism
of such choline inhibition.
ENERGETICS OF AMINO ACID TRANSPORT 361

24 120~
::i c
u .;::
"20
~
<t
~/6
:::)

lIJ
">12
i=
c;(

~8
~
~4
c
u

Fig. 4.(a). Uptake of D-glu is proportional to the tissue Na+ when

K+ is high. Slices were depleted of Na+ and ~lucose by two suc-
cessive 15 min. incubations in HEPES-G (-) Na (+) 100 mM sucrose,
with pH adjusted to 7.35 using KOH. The slices were transferred
into HEPES-G (-) Na+ (+) 100 mM~crose (+) KOH-neutralized SMP
for 30 min before a 20 min uptake measurement in various levels
of Na+. Left-hand ordinate is the C.U. of 1 mM D-glu; the
right hand ordinate is the ~quiv of Na+ and K+ in the tissue
after uptake. Correlation coefficient for Na+/C.U. = 0.96, for
K+/C.U. = 0.98; r2 = 0.93 and 0.97, respectively.
(b) Uptake of D-glu is not proportional to the tissue Na+ when
K+ is kept low. Depleti~ two 15 min incubations in HEPES-G (-)
Na+, (+) 5 mM K+ (+) 100 mM sucrose (medium neutralized with Tris);
30 min restoration in presence of Tris-SMP; slices were trans-
ferred for uptake measurement into this medium with additional
Na+ as marked on the abscissa. Note that the uptake capability
rises independently of the tissue Na+. Correlation coefficient
for Na+/C.U. = 0.89, forK+/C.U. = 0.98, r 2 = 0.79 and 0.97,
respectively.

to the sixth minute, the rates of uptake are maximal while the rate
of Na+ change is nearly zero. Only with valine does the uptake rate
fall continuously in this case. From these results it is also
obvious that the sodium level in the tissue does not have to be
set at some arbitrarily high (i.e., 100 meq/l) level for maximum
rates of transport, 20-40 meq/l appear to keep the tissue uptake
system quite 'happy'.
362 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

2·0 • 25

1.5
20 IJ
Na+
f1
c.u. 0
15 MIN.
MIN. 1.0
GABA
/0
/
I.~£-·-£
0.5
iK. e,<;AIB 5
6. [J ....
"'[J=- VAL
o - ~- Ih I~ 21 0
TRANSFER
Fig. 5. The maximal rate of GABA, AlB and glu uptake occurs after the
flux of Na+. Slices were depleted by two 15 min incubations in
HEPES-G (-) Na+ (+) 5 roM K+ (+) 100 roM sucrose, Tris-adjusted to
pH 7.35. They were transferred into this medium (+) Tris-SMP for
30 min, and were then transferred into this medium (+) 80 roM Na+,
containing l4C-amino acid. The ordinate is the change (delta) per
min of tissue Na+ or C.U., for 3 min intervals after the last transfer.
Na+ flux (in) is essentially complete in 2 min (cf. Fig. 3). Uptake
rate is maximal for three "sodium-dependent" amino acids after the
Na+ flux, but decreases steadily for L-valine.

D.2. c. Summary of Na+ Relationship to Uptake: We have not

found a comparable substitute for Na+ (as Rb+ is for K+); neither
Li+ or Cs+ can stimulate transport when Na+ is absent. Indeed, they
are inhibitory when there is only a low Na+ level in the tissue. So
we have not yet obtained a clear answer to the Na+-dependency question.

Active transport of Na+ is not linked to a particular step in

the tricarboxylic acid cycle 32 ,38, and the tightness of the linkage
of Na+ flow and amino acid transport is not yet well-defined 2 ,20
We tested neutral amino acid (AlB) accumulation by the A system,
energized by a potential gradient of Na+ (in/Jut) and K+ (out/in).
ENERGETICS OF AMINO ACID TRANSPORT 363

The linkage is loosely coupled and the energization basis may be

somewhat different from that examined by Heinz and Geck 20 because
the maximum rate of uptake of AlB occurs after Na+ entry stops,
while the tissue level of Na+ does not rise, i.e., we did not
observe the expected symport with AlB, nor did low initial internal
K+ rising to 50 mM appear to inhibit the AlB uptake.

Therefore, we could demonstrate that Na+ levels and net flux

directions may be irrelevant to transport: 1. Na + flux in or out
(i.e., from high Na+ medium inward, or outward from high Na+ tissue
levels) does not block transport. Under the most unfavorably low
Na+ conditions, 25-60/0 of the ''best'' uptake can be maintained.
2. The Na+ level in the medium needs to be only 40 mM (not 100-
120 mM) for nearly maximal uptake of D-glu (Fig. 4~. 3. If the
Na+ flux inwards is measured at brief intervals, it has ceased
when the D-glu or AlB flux is maximal (Fig. 5). 4. It is possible
to demonstrate an absolute correlation of Na+ levels in the tissue
with each degree of uptake that is restored. However, by decreasing
the K+ level in the medium, one can show that the correlation of
Na+ with uptake is coincidental or artefactual (Fig. 4 ~ , ~).

DISCUSSION

Glycolysis, ATP and Mitochondrial Electron Transfer as Energy

Sources for Uptake.

Two opposite views of the coupling of ATP hydrolysis to AlB

transport into mammalian (Ehrlich or KB) cells have been reported
recently15,33. However, we examined the effects of oligomycin and
other uncouplers on the concentrative uptake of AlB in slices with-
out observing evidence for mediation by unknown phosphorylative
processes or Na+,K+-ATPase. Our results indicate that 10 mM NaF
stimulates AlB transport in the presence of pyruvate above the level
reached with glucose, while 0.4 mM amy tal is extremely inhibitory.
This suggests that ATPase enzymes (inhibited by 10 mM NaF and
stimulated by 0.4 mM amy tal) may compete with AIB transport for
energy. Such enzymes may be inhibited or unutilized in the presence
of pyruvate, while with SMP, some activity of the enzymes and
sensitivity to inhibition by NaF is restored, although not to the
level observed with glucose. Similarly, AlB uptake was most sensitive
to 2,4-DNP when lactate or pyruvate were the energy sources, and no
inhibition was observed with glucose or succinate, malate and pyruvate
(SMP). Amy tal, which inhibited AlB uptake approx. 40% when glucose
was the energy source, became much more inhibitory when lactate,
pyruvate or SMP was used. Probably, the energetics of AlB transport
may be derived from a number of enzymic steps in glycolysis or mito-
chondrial electron transfer. Moreover, it is possible that certain
of the processes that normally support cellular activity may also
364 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

compete with those energizing AlB transport. If conditions in the

medium and tissue are favorable to supply energy for the AlB uptake
process, but are simultaneously relatively inhibitory towards the
other competitive processes, e.g., ATPase activity, AlB uptake may
appear to be stimulated above control levels. Similar effects could
be expected with other amino acids.

Inhibition of D-glu or AlB uptake (fueled by all sources) when

oligomycin was present at 0.92 ~ or less indicates that one key
step in energy transfer to amino acid transport may be the Pi-H 2 0
exchange because this low concentration of oligomycin (0.4 ~/mg
protein) stimulates oxidative phosphorylation, energy linked trans-
hydrogenation, and ATP-Pi exchange, while inhibiting this "catalytic"
energy exchange 43 of mitochondrial ATP synthetase.

Replacement of K+ or Na+ in Incubated Brain Slices

Inhibition by valinomycin increased when Rb+ was substituted

for K+ with SMP as the energy source (Table V). This is in agreement
with the order of magnitude diffe+ence in the_5elat~ye m~~ility and_ 4
dis~£ciat~on constants of 10 mM K /Rb~ 8 x 10 ohm cm I 8 x 10
ohm cm- in the presence of 10- 7 valinomycin, even though their ion
radii differ by only 6_8%28 Furthermore, Cs+ in the presence of
valinomycin has the same effect on conductance of a membrane as K+
and valinomycin, even though Cs+ has a much larger ionic radius.
However, o¥r results with Cs+ indicate that it can not substitute
well for K in the medium. Thus, it appears that rather than ion
mobility, it is the size of the ion as well as that of the caged
complex that may be involved in the inhibition of transport. More-
over, this interpretation is further supported by our results with
Rb+ or Cs+ and valinomycin; Cs+ plus valinomycin was more inhibitory
than either Rb+ plus valinomycin, or K+ plus valinomycin, and despite
the greater potential gradient that was represented by the lower
mobility of the Cs+ alone in comparison with those of the other two
ions, yet the equally mobile Cs+-complex produced much greater inhibit-
ion. Such interpretation, if substantiated by other methods, would
suggest that K+ (or Rb+) function in transport is coincidental because
previous investigators have focussed on mobilities of ion complexes,
and we would have expected identical inhibition regardless of the
ion. For instance, our results are not at variance with what has
been reported for valinomycin activity in mouse or Ehrlich ascites
cells ll ,13. In these cells, as well as in slices, valinomycin
inhibits transport.

One source of artifact in Na+-dependency studies is the utilizat-

ion of K+ as a substitute for Na+ to neutralize media. As shown in
Fig. 4(a), at high K+ levels, uptake appears to parallel Na+ levels
very closely. However, such uptake is really inhibited by the high
K+, for when the elevated K+ level is reduced to 5 mM and osmolarity
ENERGETICS OF AMINO ACID TRANSPORT 365

TABLE V. Ionophore inhibition varies with energy source.

Substrate: Glucose SMP (Na)

Valinomycin (llM) Valinomycin (llM)

0.45 4.5 9.0 0.45 4.5 9.0

Control Per cent of Control Per cent of

C.U. control C.U. control
(100~ (100~

K+ 23.7 78 60 41 23.2 62 16 14
Rb+ 23.7 77 57 34 22.6 43 9 10
Cs+ 15.9 35 16 15.6 8 10

Table V. Inhibition of D-Glu uptake by valinomycin was increased when

Rb+ was substituted for K+,in medium fueled by SMP. Brain slices were
depleted of K+ by 2 x 15 min incubations in medium lacking glucose
and K+, then regenerated for 30 min in presence of 10 roM glucose or
SMP (20, 5, 5 roM, Na+ salts) + 5 roM K (or Rb+, or Cs+) + the respective
valinomycin concentrations. D-G1u-1- 14 C (1 roM) uptake was measured
for 20 min in the same medium used for regeneration.

is maintained with sucrose and Tris, the uptake is much better yet
Na+ levels are not so closely related (Fig. 4 b). As reported by
Weiss and Hertz 4l , K+ levels of 50 mM also exert a maximal effect
on slices, causing swelling and increased 02 uptake, and independent-
ly increase the efflux of glutamate (thereby decreasing the apparent
uptake at equilibrium).

There is also the possibility that a small recycling pool of

K+ can maintain oxidative phosphorylation1 8 • However, replacement
of K+ by Rb+ upon reenergization should have produced only a partial
restoration of transport under those conditions, as Rb+ competes
with K+, is less mobile than K+, inhibits ATP symport, and inhibits
ATPase (see below) - all of which should have decreased uptake if
transport were tightly coupled with either ATP generation or K+
transport. Instead, uptake with Rb+ was quite normal.

The increased dependence of slices in high K+ media on glycolysis

(see review by Bull and Lutkenhoff lO ), which does not occur simultan-
eously with a K+-induced respiratory increase, suggests that K+ flux
and the carbon source can interact to alter the observations.
Therefore, most of our observations of K+ and Na+ effects on transport
were made after data was obtained with both glucose and SMP restorat-
ion of uptake. Bull and Cummins 9 , who also used brain slices 0.41 mm
thick, observed that 1-3 min. after addition of K+ (6-60 mM) there
was a sharp oxidation and then a recovery or overshoot of NADPH
reduction from the 4th minute onwards. This corresponds (temporally)
366 M. BANAY-SCHWARTZ, D.N. TELLER, AND A. LAJTHA

very closely with our finding of the initiation of the most rapid
D-glu and AlB transport during the second 3-minute period (after
K+ levels remained relatively constant as shown in Fig. 3 a and
~). If the rate of transport is indeed closely coupled with
NADPH reduction, our finding of pronounced inhibition of transport
by ouabain in SMP medium is in agreement with their observation that
the glycoside also blocks the NADPH reduction.

Dissociation of Ion Pump Activity from Uptake

Another reason for suggesting that Na~+ATPase may not be

obligatorily involved with transport of D-glu or a-AlB is the lack
of effect of Rb+, which at 2-5 mM inhibits cardiac Na~+-ATPase26,
while supporting brain slice amino acid transport. The response
of the slice transport to Rb+ concentrations at high and low Na+
or K+ is not identical to that of ATPase: high Na+ did not increase
the Rb+ inhibition of transport as it is reported to increase the
inhibition of ATPase. There is precedent for this: Terry and
Vidaver 39 have shown that glycine accumulation by the pigeon
erythrocyte depends upon cation gradients, rather than ATP synthesis
or Na~+-pump activity (See also ref. 34). We are in agreement with
their notation that "ATP requirement for the maintenance of normal
membrane structure suggests that maintenance of a functional pump
might also require energy". However, depletion of ATP levels in
our experiments followed by a partial restoration did not inhibit
subsequent uptake in brain slices. Therefore, the question of the
relative importance of ATP for maintenance, as well as energization,
of amino acid transport systems in brain is moot.

There are many questions and possibilities for future studies.

For instance, transport might be coupled to energy producing systems
in competition with ATP synthesis (see Bennun,7 for review). Perhaps
this can be tested experimentally: Southard and Green 37 found that
as the concentration of heavy metals, e.g. mercurials, increased
from two to ten nmoles/mg protein in beef heart mitochondria,
oxidative phosphorylation disappeared, while active transport of
K+ and Mg+ appeared. If active transport in mitochondria and brain
slices is an ionophore mediated function, then fluorescein mercuric
acetate ought to first stimulate, and then inhibit uptake, while
electron transport is maintained by artificial means (ascorbate +
tetramethyl-£-phenylenediamine). Few recent data exist: Colombini
and Johnstone 12 reported inhibitory results with only one, large
concentration (2 mM) of HgC1 2 in a model system of Ehrlich membrane
vesicles. Similar vesicular preparations from brain tissue might
be a useful control for the nonspecific membranal effects of
mercurials, as they preclude the involvement of mitochondrial
energetics.
ENERGETICS OF AMINO ACID TRANSPORT 367

Summary. It appears possible to dissect and study some of

the potential energy sources for amino acid transport in brain
slices despite the apparent complexity of the tissue in comparison
to that of isolated bacterial vesicles 23 • The uptake capability
of the tissue may be inadvertently damaged in some experimental
protocols so that very special controls must be used to ensure that
the treatment did not somehow inactivate the very mechanism that
thereafter will be tested. We have presented some evidence that
brain slice amino acid transport may not be obligatorily linked
to glycolysis, ATP levels, Na+, K+-ATPase activity, K+ levels or
direction of flux, or to Na+ flux. However, the energy source
linkage for different amino acids appears to be rather specific,
so that further generalizations are difficult to sustain. For
instance, the incubation media and conditions we describe here
were experimentally adjusted to maximize uptake of D-glu or a-AlB
in the absence of glucose, or in lowered K+ or Na+. Therefore,
these procedures, the results of which directly challenge some
common assumptions regarding the energy basis for active transport
in brain slices, probably will not be universally extensible to all
other actively transported amino acids.

ACKNOWLEDGEMENTS

The authors thank Mr. Henry Sershen for the suggestion to try
Rb+ as a K+ substitute; Ms. Teresita De Guzman, Ms. Denise McHale,
and Mr. Stanley Reid for their skilled technical assistance. We
are also indebted to Dr. Amos Neidle for help with amino acid
analyses and to Dr. M.A. Verity, whose publications on synaptosomal
energy requirements for ion accumulation encouraged us to attempt
this work. The experiments, summarized here, were supported in part
by NIH grant No. NS-03226, and in the main by the New York State
Department of Mental Hygiene, Research Division, G.C.Salmoiraghi, ,M.D.,
Ph.D., Associate Commissioner.

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368 M. BANAY·SCHWARTZ, D.N. TELLER, AND A. LAJTHA

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ENERGETICS OF AMINO ACID TRANSPORT 369

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Biophys., 154 (1973) 575-582.

36. Schultz, S.G., and Curran, P.F., Coupled transport of sodium
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44. Note added in proof:
HEPES-2 medium contains 119 mM NaC1, 5 mM KC1, 0.75 mM CaC1 2 ,
1.2 mM MgS04' 1 mM NaH 2 P0 4 , 1 mM NaHC0 3 10 mM glucose, 25 mM
HEPES (N-2-hydroxyethy1piperazine N'-2-ethane sulfonic acid),
and the pH is adjusted to 7.35 with 1N NaOH at 25 0 • The final
Na+ concentration is 132 mEq/l.
POTASSIUM EFFECTS ON TRANSPORT OF AMINO ACIDS, INORGANIC IONS,

AND WATER: ONTOGENETIC AND QUANTITATIVE DIFFERENCES

Leif Hertz

Department of Anatomy
University of Saskatchewan
Saskatoon, S7N own, Canada

INTRODUCTION

The potassium ion plays a unique role in brain function since

it is released from excited neurons to an extracellular space
which, at least at several locations, is limited to clefts of 150 R
thickness 23 ,53. Neuronal function can therefore be expected to
evoke an increase in the extracellular potassium concentration,
and such an increase has in recent years been convincingly
demonstrated. The observed increase after 'normal' neuronal activity
is relatively small, but during spreading depression the extra-
cellular potassium concentration may reach such high levels as
60-80 mM 29 ,54. These concentrations are similar to the potassium
concentrations of 30-100 mM which for years have been used for in
vitro studies of potassium effects on energy metabolism and trans-
port of amino acids, inorganic ions and water. The potassium
effects are to a large extent mimicked by application of electrical
stimulation, and they have often been assumed to be secondary to a
potassium-induced depolarization. Another possibility is, however,
that excess potassium might act by a direct stimulation of an ion-
activated system, and there is no reason to take for granted that
different effects by potassium are all evoked in the same way. A
distinction between the various effects exerted by high concentrations
of potassium may possibly be obtained by studying cellular and sub-
cellular localization of the different phenomena as well as their
ontogenetic development. Effects due to a depolarization of excitable
cells (neurons) may all be expected to reach a maximum with the
potassium concentration that is sufficient to evoke a general excitat-
ion. A comparison between the potassium concentrations required to
obtain the different potassium-induced phenomena may therefore also
be helpful in determining their interrelationship and possible
mechanisms of action.

371
372 L.HERTZ

POTASSIUM EFFECTS ON AMINO ACID TRANSPORT

Release

Oualitative differences. Measurements of the release under

different conditions may be obtained by following the efflux of
the radioactively labelled compound. This procedure requires
nreloading with radioactive amino acids, and conceivably exogenous
amino acids may be treated differently from endogenous amino acids*.
In other experiments, the net release of amino acids to the medium
has therefore been measured. Unless inhibitors of the uptake are
used, this method does, however, not distinguish between effects
on release and on uptake.

Good concordance is found in the literature that the release of

exogenous and endogenous glutamate, GABA, and aspartate from brain
slices and synaptosomes is increased by application of high concen-
tration of notassium 2,10, 18, 26, 46, 47 though the effect on
endogenous glutamate may be relatively small 30. Proline is probably
treated in the same way34. In spinal cord slices and synaptosomes
from the spinal medullary region, the release of glycine is also
increased by excess potassium24 , 34, 39. Amino acids other than
glutamate, GABA, aspartate, nroline and glycine show normally
little or no potassium-induced release. If, however, glutamate is
present in the incubation medium, the release of also these amino
acids is increased by excess potassium 2.

Ouantitative aspects. Van Harreveld and Fifkova 52 showed that

very high potassium concentrations are required to evoke a substant-
ial increase in the release of glutamate from chick retina.

FromFig.l it can be seen that also the potassium-induced release

of glutamate from brain slices increases with a rise of the K+ con-
centration over the whole range studied (up to 125 mM) and that
this 'concentration/response curve'differs from those showing pot-
assium effects on swelling and oxygen consumption. An almost
similar correlation has been observed between potassium concentra-
tion in the medium and release of GABA (G.B. Weiss and L.Hertz, un-
nublished exneriments). The further increase in release when the
potassium concentration is raised above 50 mM suggests that the re-
lease of the two amino acids from brain slices is not simnly due to
a notassium-induced denolarization of excitable cells since full
de~olarization is obtained with 50 roM potassium in the medium 2l .
These findings with brain slices are in contrast to results obtained
using spinal-medullary synaptosomes, where maximum release of

* This source of error can probably he neglected if isotope

equilibration is obtained.
K+ EFFECTS ON AMINO ACID AND ION TRANSPORT 373

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"0
0.0 - 55 E
00 20 40 60 80 100 120 ::1-

Potassium concentration in medium (mM)

Fig.l. Swelling (~) expressed in percent, rate of oxygen

uptake (0 ) expressed in ]Jmoles/g, and decrease in halftime
(T~) for washout of (14 CJ-glutamate (0) expressed in min, as a
function of the potassium concentration of the incubation medium.
The changes in T~ were obtained from washout curves like that
shown in Fig.3. Results originate from ZO (OZ uptake),Z8 (swelling)
and 55 (glutamate washout).

glutamate, aspartate, GABA and glycine is evoked by 10 roM pot-

assium39 •

Ontogenesis. The potassium-induced release of glutamate is

absent in brain slices from newborn rats Z in which exposure to
excess potassium even causes a reduced release of glutamate 45 .
From Fig.Z it can be seen that the response to 35 roM K+ changes
gradually during the first postnatal weeks and that the potassium-
induced release barely has reached the adult level at the age of
30 days. The increased release evoked by 80 mM K+ becomes evident
somewhat earlier, and a maximum response is evoked already at 10
days, when addition of 30 mM K+ has no effect. The ontogenetic
development of potassium effects on GABA release is different.
Addition of potassium to a final concentration of 35-45 mM has
some effect on GABA release even in tissue from newborn animals 9 ,45.
The response becomes drastically altered during ontogenesis as
shown by a very pronounced release at 6 days of age, absence of
any effect at Zl days and another release in animals older than Zl
days (Fig.Z). Addition of potassium to a final concentration of
80 roM exerts qualitatively the same effect; at the early devel-
opmental stages, there are no major quantitative differences, but
the release after Zl days is larger and some effect can be ob-
served even at that age 45
374 L. HERTZ

-25

-20

-15
f

I
c
"e -10

~
.= -5
c

•
CI
C
0
"
.s=
U
5

5 10 15 20 25 30 A
Age (days)

Fig.2. Correlation between age of animals and potassium-

induced changes in release of GABA (0) and glutamate (4, "" )
from rat brain cortex. slices preincubated for 1 hr. in physio!~gical
media containing respectively 1 mM 1- [}4C] -GABA and 10 mM U-Ll4C]-
glutamate. Open symbols indicate the effect of 35 mM K+ and closed
symbols the effect of 80 mM K+. 'A' indicates results obtained
with adult animals. The changes are expressed as decreases or
increases in halftimes (T~) for washout of the amino acids and
were obtained from washout curves like that shown in Fig.3.
Results are means of 4 individual experiments with S.E.M. shown
as vertical bars if they extend beyond the symbols. From Schousboe
et al. 45

Cellular and subcellular localization. The quantitative

difference between potassium effects on the release of glutamate
and GABA from brain slices and from synaptosomes may indicate that
K+ EFFECTS ON AMINO ACID AND ION TRANSPORT 375

a major part of the release in brain slices occurs from sources other
than synaptosomes. Evidence for a potassium-induced release of GABA
from satellite cells has been obtained in dorsal root ganglia of
the rat 32 , 33 and it has been suggested that the potassium-induced
release of GABA which develops after the age of 21 days, at least
to some extent, may occur in glia ce11s45. A potassium-induced
release of GABA has been observed both in neuronal-enriched and
glia cell-enriched fractions obtained by gradient centrifugation
(A.Hamberger and A.Sel1strom, personal communication). Attempts
to demonstrate a potassium-induced release of glutamate and GABA
in either all-glial or mixed neuronal-glial primary cultures from

100 I

0
•~. 50
CD 0

-
::J
II)
.!! 0
25
0
c
0
C)
c 0
·c 10 0

.-
.iii 00
E 00
...
CD
5
00
0
>. 0 ....
~
>
;; 0000
u 2.5 000
.~
"C
ca
0:::
10 15 30 45 60 75 90
Time from start of washout, min.
Fig. 3. An example of washout curves showing, as a function of
time, release of radioactivity from a mixed neuronal-glial culture
(dissociated brain cells from a 7-day-old chick embryo cultured for
7 days in a modified Eagle's minimum essential medium as originally
described by M.Sensenbrenner and coworkers I9 ,48). The culture had
been loaded with U-(14cJ- labelled glutamate and the washout was into
a 'physiological' (0) medium (0-45 min+ and 69-90 min.) and (45-69
min.) into a medium containing 80 mM K (e). From such curves, the
halftimes (~) can, after an initial deviation (0-20 min.), be direct-
ly read, and the changes in ~ shown in Figs. 1-2 are the differences
between the ~ before and during, the exposure to excess potassium.
376 L. HERTZ

rat, mouse or chick brains have failed, but the potassium-induced

reduction of the glutamate release found at early postnatal stages
has been observed in mixed neuronal-glial cultures (Fig.3).

Uptake

In view of the vast amount of information about kinetics of

amino acid uptake, remarkably little work has been done on potassium
effects on the uptake. In general, relatively high concentrations
of amino acids have been present in the medium and the effects ob-
served may thus have nothing to do with the high-affinity uptake.
High concentrations of potassium cause a lowered accumulation of se-
veral amino acids, including glutamate and GABA. The decreased con-
tent of glutamate after incubation in potassium-rich media can be
explained by the increased release, and the rate of the glutamate
uptake is not decreased by excess potassium 2 , 3, 55. GABA is,
in contrast, at all ages taken up at a reduced rate when the pot-
assium concentration is increased 30 , 45

POTASSIUM EFFECTS ON TRANSPORT OF INORGANIC IONS AND WATER

Potassium. Addition of 50 roM K+ during the washout of radio-

active potassium causes a considerable increase in the efflux from
a rapidly exchangeable potassium compartment suggested to represent
glia cells ll , 18. Also the potassium concentration in the tissue
increases 18 , 28 and the magnitude of both influx and efflux increase
from 1-2 ~moles~g final wet weight per minute before K+ was added to
about 10 ~moles;'g per minute after the addition. This value is
remarkably close to the net uptake after termination of electrical
excitation 8 . The potassium-induced increase of potassium release
may be observed in slices from newborn rats 43

Chloride. Addition of excess potassium during the washout of

radioactive chloride from brain cortex slices has no effect on the
rate constant 18 , 44. The chloride content increases, however, indic-
ating that the rates of both the release and the uptake are raised.
This is the case in adult as well as in neonatal tissue 44 . In accord-
ance with this, the velocity of the 36 Cl uptake into brain cortex
slices is significantly increased by excess potassium and has been
found to follow Michaelis-Menten kinetics with a Km for potassium
of about 30 roM4 . Similar observations in the NN glia ~ell line
suggest that the chloride uptake occurs into glia cells l .

Water. High concentrations of potassium have long been known

to lead to an increased water uptake in brain slices. This 'swelling'
may conceivably be secondary to an active, potassium-induced uptake
of K+ and Cl-. It is in keeping with this concept that it is pre-
vented by replacement of chloride with the indiffusable isethionate
ion 6 or by lowering of the temperatureS. The potassium-induced
swelling is absent neonatallyll, 44, 51 and appears gradually at
K+ EFFECTS ON AMINO ACID AND ION TRANSPORT 377

about 14 days of age 4l . The threshold concentration of K+ for

swelling of rat brain cortex slices is about 20 roM K+28 and a max-
imum effect is obtained with 50 roM K+ (Fig.l). The potassium- ind-
uced swelling is almost selectively localized to glia cells35~ 56.

The swelling represents a slow net uptake of water. The very

fast exchange of water (half-time less than one minute) is, if
anything, reduced by an increase of the potassium concentration 38

POTASSIUM EFFECTS ON METABOLISM

Energy Metabolism

A potassium concentration of at leasb 20 roM is required to

cause an increase of oxygen uptake (Fig.l) and lactate production
as well as a decrease of the ATP concentration 20 , 49, 50. The pot-
assium-induced stimulation of oxygen uptake is absent in brain
cortex slices from rats younger than 1-2 weeks 22 . Neonatal thyr-
oidectomy abolishes the development of the response 13 .

Almost all available information indicates that glia cells

react to a rise of the potassium concentration with a considerable
increase in rate of oxygen uptake and decrease of ATP content l ,14
17, 19, 42, but it should be noted that excess potassium has no
stimulatory effect on the respiration of the NN astroglia line 37
The question of a potassium-induced increase of neuronal respira-
tion is more ambiguous. Using a micro-spectrophotometric technique,
Hultborn and Hyden 25 observed a stimulation of oxygen uptake in
microdissected neurons, whereas other authors have found the res-
piration of microdissected or bulk-prepared neurons to be unaffected
or only slightly increased by an increase of the potassium concen-
tration in the medium l , 14, 17, 19.

The Na+-K+-ATPase seems of special relevance for potassium

effects on energy metabolism and on transport since this ion-
activated enzyme exerts a major control on energy metabolism and
probably is directly involved in the transport of sodium and potas-
sium. It is therefore peculiar that only 10-20 roM potassium are
required to exert maximum stimulation of the brain enzyme 15 , 40

As shown in Table 1, the activity of the Na+-K+-ATPase is

higher in glia cells than in neuronal perikarya 7 , 15, 31, 36. The
variability between different preparations is, however, consider-
able. The Na+-K+-ATPase activity is thus relatively low in the
established glia cell lines C6 and NN, but considerably higher in
glia cells obtained from primary cultures 48 and in bulk-prepared
glia cells 15 , 27, 31. The Na+-K+-ATPase activity is much lower in
378 l. HERTZ

neonatal than in adult brain. In the rat, the increase in activ-

ity seems to occur especially fast during the first 2-3 weeks
after birth 3l , 36, 40

Table 1

Specific activities of the Na+-K+-ATPase (~moles Pi liberated/mg

protein per hr.) in different neuronal and glial preparations.

Specific K+
PreEaration SEecies activity conc.

Bulk prepared neurons rat 1 20 31

" " " rabbit* 1.5 20 15

" " " rabbit* 2 1015

" " " rat 1.62 ± 0.69 10 27

Neuroblastoma mouse 0.44 + 0.09 10 27

" mouse 0.44 0.62 20 48

Bulk prepared .glia cells rat 7 20 31

" " " " rabbit* 5 20 15

" " " " rabbit* 8 1015

" " " " rat 5.67 +

- 0.77 10 27

Primary glial culture rat 2.33 + 0.06 20 48

C6 glia cell line rat 0.67 + 0.05 20 48

" " " " rat 1.05 + 0.08 10 27

-
NN glia cell line hamster 0.89 + 0.03 20 48

The Na+-K+-ATPase activity was measured as the difference

between the total ATPase activity and that in the absence of
(Na+-K+) or in the presence of ouabain. Only data obtained with
either 10 or 20 mM K+ present during the assay are shown. Authors
are indicated by supercript numbers at the end of each line.

* Results obtained using membrane fractions.

K+ EFFECTS ON AMINO ACID AND ION TRANSPORT 379

CONCLUSION

The comparison of the various effects of excess potassium on

metabolism and on transport of amino acids and inorganic ions has
revealed a certain pattern. In rat brain slices, the potassium-
induced increase of oxygen consumption and the concomitant decrease
in ATP content have a threshold of about 20 mM K+, and maximum
effects are obtained with 50 to 100 mM potassium (Fig.l). The
effects appear at a late stage of brain development and seem
mainly to occur in glia cells. The accumulation of chloride and
the swelling have approximately the same quantitative and histol-
ogical characteristics, and the hypothesis has been forwarded that
the increase in energy metabolism reflects an active transport of
KCl into glia cells which for osmotical reasons leads to an uptake
of water, and that this mechanism serves the function of maintain-
ing potassium homeostasis during neuronal activi ty5, 15, 16. This
hypothesis fits several experimental data, but it is peculiar that
the potassium effects on the transport of both K+ and CI- in con-
trast to the other potassium-induced phenomena are present neo-
natally and that a potassium-induced increase in uptake of chlor-
ide but no metabolic reaction to excess potassium is found in the
NN glia cell line. It might also be expected that the Na+-K+-ATPase
should have the same quantitative and developmental characteristics
as the increase in energy metabolism. This was found not to be the
case although the Na+-K+-ATPase had a predominantly glial localiz-
ation.

The necessity of using very high potassium concentrations to

induce a substantial release of GABA and glutamate from brain
slices indicates a dissociation between potassium effects on amino
acid transport and on energy metabolism and transport of inorganic
ions. The concept of a difference between these phenomena is fur-
ther supported by the widely different ontogenetic development.
The differences between the ontogenetic development of potassium
effects on each of the two amino acids studied (Fig.2) suggests an
even greater complexity, and further studies will be required to
obtain more conclusive evidence of the physiological role played
by the potassium-induced release of glutamate and of GABA and
even of the histological localization of these phenomena. The
great variability between different neuronal and glial prepa-
rations with respect to a relatively stable parameter such as
the activity of the Na+-K+ATPase (Table 1) indicates that the
answers to these questions may not be unequivocally and easily
obtained.

ACKNOWLEDGEMENTS

This work was supported by grant DG No.120 from the Medical

Research Council of Canada.
380 L. HERTZ

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EVIDENCE FOR A SYNTHESIS-DEPENDENT RELEASE OF GABA

Ricardo Tapia

Departamento de Biologia Experimental

Instituto de Biologia
Universidad Nacional Autonoma de Mexico
Apdo. Postal 70-600, Mexico 20, D.F., Mexico

INTRODUCTION

The control of neuronal activity through synaptic inhibitory

mechanisms in the central nervous system (CNS) is presently well
established 3 ,7,29,41. This kind of regulatory mechanism implies
that the activity of neurons is controlled by the degree of the
inhibition exerted upon them by other neurons. This means that a
given neuron will fire when the inhibitory action of the controlling
neuron decreases, that is, through disinhibition.

One of the most important implications of such a control system

is that in the resting condition a given neuron will not fire, not
because of lack of stimulation, but rather because of the inhibitory
action of other neuro~s. This type of control can be defined as
continuous or tonic inhibition.

Neurochemically, the concept of tonic inhibition implies:

a) that the inhibitory synaptic transmitter is being continuously
released from the presynaptic terminal, and b) that any alteration
of the synaptic function, leading to a block of the postsynaptic
action of the inhibitory transmitter, will immediately result in an
uncontrolled firing of the postsynaptic neuron 32 . In the present
paper I will give evidence that these considerations apply to the
GABA-dependent inhibitory mechanisms in the mammalian CNS, and,
further, that the continuous release of GABA is dependent upon its
synthesis, accomplished by the activity of glutamate decarboxylase
(GAD).

385
386 R. TAPIA

GABA-DEPENDENT INHIBITION

GABA is the most widely distributed inhibitory neurotransmitter

in the mammalian CNS6,14,18. Its role has been related to the
control of motor excitability3l,32,42, and recently also to some
neurological abnormalities in man, such as Huntington's chorea l ,40
The synthesis of GABA ~s catalyzed by GAD, an enzyme concentrated
in nerve endings 8 ,25,2 , particularly in those cerebral regions
where GABA has been demonstrated to act as an inhibitory transmitter
10,19

There are two obvious ways of blocking the function of a GABA

synapse (or of any other synapse, with its corresponding transmit-
ter): a) blocking the interaction of GABA with its receptor; or
b) decreasing GABA concentration in the synaptic cleft. According
to the points mentioned in the Introduction, either of these
phenomena should result immediately in an uncontrolled activity of
the GABA-sensitive neurons. Since, as mentioned above, GABA is
involved in the regulation of motor excitability, such uncontrolled
activity should manifest itself as the occurrence of seizures. In
fact, when one of the two antagonists of GABA at the receptor site,
bicuculline 4 ,5, or picrotoxin 12 , is administered to experimental
animals, convulsions occur very rapidly4,20,26,38.

As to mechanism b), a decrease of GABA concentration in the

synaptic cleft should be obtained by decreasing the rate of
spontaneous release. In studies in vitro with brain slices, two
convulsant drugs which do not modify GABA metabolism, pent ilene-
tetrazol and picrotoxin, have been shown to decrease the spontaneous
release of GABA17; whether this effect is involved in the convulsant
effect of these compounds ~ vivo is not known.

A decrease of the concentration of GABA in the synaptic cleft

should also be obtained through an inhibition of its synthesis.
However, this mechanism would be effective only if the pool of
GABA available for release were significantly diminished as a
consequence of the inhibition. In this respect, the evidence that
seizures occur when the activity of GAD is decreased to a certain
extent, even though the total level of GABA in brain may be largely
increased 13 ,33-37,43,44, seems of particular importance. In fact,
correlative studies of GAD inhibition, appearance of seizures, and
level of GABA in brain, indicate that the activity of the enzyme,
rather than the GABA level, is the crucial event regulating cerebral
excitability (for extensive reviews on this subject see Ref. 3l ,32
and 42 ). Furthermore, the inhibition of GAD by some convulsant
substances has been shown to occur in the nerve endings 25 , and the
inhibition of other amine or amino acid-related enzymes has
apparently no connection with seizures 32 ,34.
SYNTHESIS DEPENDENT RELEASE OF GABA 387

GLUTAMATE

GAD

GABA~STORAGE

---
GABA
-
Figure 1. Synthesis-dependent release of GABA from
inhibitory nerve endings •• It is postulated that a certain
proportion of GAD is bound to the presynaptic membrane in
the presence of Ca 2 +, and that the pool of GABA synthesized
by the membrane-bound enzyme is released into the synaptic
space (®). A second pool of GABA, synthes ized by GAD
soluble in the synaptoplasm, would be stored for its
release stimulated by depolarization <@).

These studies, together with the considerations made in the

Introduction, have led us to postulate that GABA is continuously
released from central inhibitory nerve endings by a synthesis-
dependent process 3l ,37. This hypothesis, schematized in Fig. 1,
implies that at least part of the GAD is bound to the presynaptic
membrane, where the enzyme would be acting in such a way that the
product (GABA) would appear outside the cell, in the synaptic
space. That GAD may in fact be bound to membranes has been shown
by Fonnum8 , who found that in the presence of physiological
concentrations of Ca 2 + a certain proportion of the enzyme sediments
with brain membrane fractions, from where it is not eaSily released.
Thus, part of the GABA synthesized would be released in a process
coupled to its synthesis <~ in Fig. 1); another part would be
possibly stored (~ in Fig. 1). The GABA taken up into the
terminal by the Na -dependent uptake process described by other
388 R. TAPIA

authors 14 - l6 would also contribute to this storag~ pool. It seems

possible that the latter pool is used for the Ca 2 -dependent
release of GABA stimulated by depolarization 14 ,23,30.

STUDIES ON THE SYNTHESIS-DEPENDENT RELEASE OF

GABA IN VITRO

In order to obtain more direct evidence for the hypothesis

described above, we have studied the release of labeled GABA
formed from labeled glutamate in superfused brain slices, in the
absence and in the presence of aminooxyacetic acid (AOA) , an
inhibitor of GAD activity. Fractions of the superfusing medium
were collected at lO-min intervals. The experimentaldetails, and
the results for the cumulative 40 min superfusion period, are
shown in Table 1. The release rates of both GABA and glutamate,
expressed either as absolute radioactivity or as percentages, were
linear in all the experimental conditions.

As can be observed in Table 1, about half of the labeled GABA

synthesized from labeled glutamate was released from the tissue in
40 min. In the presence of AOA, which under the experimental
conditions employed has been shown to inhibit GAD activity in brain
slices 28 , both the synthesis and the release of labeled GABA were
decreased. Since the decrease of these parameters was very similar,
the percentage of labeled GABA formed by the tissue which was
released did not change. This is more readily seen in Table 2,
which shows the synthesis and release of labeled GABA, as related
to the amount of the labeled precursor, glutamate, and also the
effect of AOA on these parameters and on GAD activity in brain
slices. Since the release of labeled glutamate was not affected
by AOA (Table 1), this form of expression is an indication of the
percentage of glutamate converted into GABA and of the endogenously
synthesized GABA that was released. Clearly, the release of the
latter pool of GABA was decreased proportionally to its synthesis.

In contrast to the above results, when the slices were given

labeled GABA instead of glutamate before the superfusion, AOA
produced an apparent increase both in the total radioactive GABA,
due probably to the inhibition of GABA aminotransferase, and in
the release of labeled GABA. That this possible stimulatory effect
on release is not just the result of a greater accumulation of
labeled GABA is shown by the difference in the release expressed
as percentage value (Table 1).

Other experiments were carried out to investigate whether,

under the experimental conditions used, AOA had some effect on the
uptake of glutamic acid or GABA. The results of these experiments
confirmed that AOA did not affect the uptake of glutamic acid 28 and
showed that GABA uptake was also unaffected by AOA, under the
SYNTHESIS DEPENDENT RELEASE OF GABA 389

Table 1. Spontaneous release of labeled GABA synthesized from la-

beled glutamic acid, and of labeled exogenous GABA, in superfused
mouse brain cortex slices, and the effect of aminooxyacetic acid (AOA)
Total dptn':' x 10 - 3 GABA released Glu released
Glu GABA dptnt x 10 - 3 %t:
Slices loaded with [14CJglu:
Control (5) 91.1:!" 5.90 5.25t6.77 2.70 to. 44 52.9. t 6.7 34.0 t 8.8 36.5 t 9.0
WithAOA (3) 74.5 t16.4 1.63 to. 56\ 0.82 ± 0.2d 53.0 t 4.4 33.4 ±" 4. 5 46.8 t 6.8
0/0 of control 82 31 30 100 98 128
Slices loaded with [14CJ GABA:
Control (4) 129 1" 29 75.8 -t 22 58.6i6.7
With AOA (3) 250 t 35 207 t 29t 83.2 t 7.7
0/0 of control 194 273 142
Approxitnately 100 tng of tnouse brain cortex slices (0.75 x 0.75 tntn) were incubated twice for 25
min in 2.5 tn1 of oxygenated Ringer-Krebs-phosphate-glucose mediutn, pH 7.0, at 250 C, under
950/0 oxygen attnosphere. as described previously28. The first incubation was in the presence or
absence of AOA (5tnM). After washing the slices by vaccutn filtration, they were incubated again
in the satne tnrdiutn with 0.3 )lCi of j).4- 14 C] DL-glutatnic acid (sp. act. 14 tnCi/tntnole). or
0.3 )lCi of [1- 4C] GABA (sp. act. 2-5 tnCi/tntnole). At the end of this incubation the slices
were washed again. transferred to a chatnber of 1 tn1 capacity. and superfused for 40 tnin at 37 0

activity present as [14c r

at 0.5 tnl/tnin. Fractions of the superfusing tnediutn were collected every 10 tnin. The radio-
GABA and 4 C] glutatnate in the superfusate fractions and in the
superfused tissue was isolated by paper chrotnatography and tneasured by liquid scintillation
spectrotnetry. The results are means of the number of experitnents shown in parentheses t S.E.M.
)~ Released+tissue radioactivity in the amino acid, at the end of the 40 min superfusion period.

*
t Cutnulative radioactivity released in the 40 tnin superfusion period.
Labeled atnino acid released in 40 tnin superfusion as percentage of the total radioactivity in
the atnino acid.
Values significantly different frotn the control values (p":0.02 or p<0.05.l test). All other
differences were not significant (p"0.05).

incubation conditions described in Table 1. Thus, the accumulation

of radioactive GABA produced by AOA has to be ascribed to the
inhibition of its metabolism.

The results shown in Tables 1 and 2 support the hypothesis

that in ~ the newly synthesized GABA is continuously released
in a process dependent upon the activity of GAD. However, they
require further experimental confirmation with a larger number of
experiments and the use of other inhibitors of GAD and other brain
preparations, such as synaptosomal fractions.

FUNCTIONAL SIGNIFICANCE OF THE RELEASE OF NEWLY SYNTHESIZED GABA

AND OTHER NEUROTRANSMITTERS.

Besides newly synthesized GABA, it has been reported that

recently synthesized catecholamines and acetylcholine are preferential-
ly released from central nervous tissue.

The release of endogenously synthesized dopamine and norepine-

phrine is decreased when experimental animals are treated with
inhibitors of their synthesis, especially when the catecholaminergic
neurons are stimulated ll ,39. After a short time, the release of
390 R. TAPIA

Table 2. Effect of aminooxyacetic acid (AOA) on GAD activity, on

the synthesis of GABA from glutamic acid and on the release of
endogenously synthesized and of exogenous GABA, in mouse brain
cortex slices.
GAD Release of Released of
activity in GABA newly synthe- labeled GABA
brain slices* synthesis+ sized GABA+ taken up by
(%) the slices

Control 100 5.8 3.0 59

AOA 55 2.2 1.1 83
"I of control 55 38 37 142
The values of the third and fourth columns were calculated from
Table 1 as described below. Values of the fifth column are the
last ones listed in Table 1.
,,< From ref. 28
+ total dpm in GABA x 100 after 40 min superfusion
+ total dpm in glutamate
dpm in released GABA x 100
total dpm in glutamate
after 40 mill superfusion

catecholamines increases, and this increase has been attributed to

a mobilization from the storage compartment to the pool readily
available for release. Since these results have been correlated
with physiological parameters, such as the ovulation process or the
self-stimulation behavior ll , it seems probable that the release of
newly synthesized catecholamines is effectively related to synaptic
transmission.

Newly synthesized acetylcholine has been shown also to be

preferentially released from the central nervous system 9 • In
studies in vitr0 2l ,22 and in viv0 2 , recently synthesized acetyl-
choline was preferentially released on stimulation. These findings
also suggest the existence of different pools of acetylcholine,
one of them, the newly synthesized, more easily available for release
than the others. No physiological correlation is available to assess
the functional postsynaptic action of the newly synthesized acetyl-
choline released, but the fact that its stimulated release is a
calcium-dependent process 2l makes very probable that under normal
conditions acetylcholine released from the recently synthesized pool
has a neurotransmitter role.

The probable functional role of the release of newly synthesized

GABA has been already indicated above. In the case of this particular
neurotransmitter, a decrease of its rate of synthesis leads to an
uncontrolled firing of neurons, which is dramatically manifested as
SYNTHESIS DEPENDENT RELEASE OF GABA 391

convulsive activity; this can be observed, both clinically and

electroencephalographicallYi in the absence of any type of stimulat-
ion, in several species 13 ,3 -33,42. Since the appearance of
convulsions seems to be specifically related to GAD inhibition34,
and is observed even when the level of GABA, and therefore presumably
the storage GABA pool, has been greatly increased by previous
inhibition of GABA aminotransferase 33 ,35,37, it can be concluded
that the continuous, synthesis-dependent release of GABA, is
responsible for a tonic synaptic inhibition under normal conditions.
The results shown in Tables 1 and 2 are the in ~ counterpart of
these ~ ~ experiments, since in the presence of AOA, an inhibitor
of both GABA aminotransferase and GAD, the release of endogenously
synthesized GABA decreased whereas that of exogenous GABA (which
apparently mixes with the storage poo124), tended to increase.

Our data also suggest that the equilibrium between the newly
synthesized pool and the storage pool of GABA is very slow, or,
alternatively, that they do not mix. In contrast, the pool of
recently synthesized catecholamines or acetylcholine, although also
preferentially released upon stimulation, seems to be in a relatively
rapid equilibrium with the storage pool, which apparently can supply
neurotransmitter for release and thus maintain the synaptic function.

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SYNTHESIS DEPENDENT RELEASE OF GABA 393

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36. Tapia, R., P~rez De La Mora, M., and Massieu, G.H., Correlative
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38. Tews, J.K., Carter, S.H., Roa, P.D., and Stone, W.E., Free
amino acids and related compounds in dog brain: Post-mortem
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TRANSPORT OF AMINO ACIDS AND CATECROLAMINES IN RELATION

TO METABOLISM AND TRANSMISSION

J.S. de Belleroche and R.F. Bradford

Department of Biochemistry
Imperial College of Science and Technology
Prince Consort Road
London SW7 2AZ, Great Britain

I INTRODUCTION

Synaptosomal preparations from many regions of the CNS provide

an excellent experimental opportunity for studying the mechanisms of
release and of reuptake of neurotransmitters which are occurring in
the intact nervous system. The metabolic performance of synaptosomes
and their response to depolarizing stimuli indicates that they are
sealed, cell-like, structures, carrying a membrane ~otential and
performing as isolated, working, nerve-terminals 3 ,1 ,13.

Neurotransmitter-Uptake: The net uptake of externally supplied

neurotransmitters by synaptosomes has been demonstrated for a wi~a
range of compoun~~, including noradrenaline, 5-hydroxytryptamine ,
dopamine 18 , GABA ,21, glutamate 7 , glycine 6 , and acetylcholine 20 •
A common property of these processes is their requirement for
maximal activity, of high sodium concentration (130-150 mM) and low
potassium concentrations (3.9 mM), characteristic of the ionic
composition of the CSF which would normally bathe the nerve-ending
in situ. The likely involvement of Na+-K+ ATPase in their inward
transport is indicated by their common sensitivity to ouabain (see
also section 3), an inhibitor of this enzyme.

The occurrence of a continuous cycle of transmitter release

and reuptake is strongly indicated by the accumulation of transmitters
in the incubation medium when ouabain or amino acid analogues are
included to block the reuptake phase of this cycle (Table 1). This
shows the release of amino acids from an endogenous source rather
than any externally supplied source. The fact that physiologically

395
396 J.S. de BELLEROCHE AND H.F. BRADFORD

active amino acids are the most affected indicates the importance
of this cycle for neurotransmitters.

TABLE 1

Amino acid release from rat cerebral cortex synaptosomes and the
effect of ouabain,~-aminobutyric acid (BABA),2,4-diaminobutyric
(DABA) and N-acetylglutamic acid (NAG).

Amino acid Amino acid released to the incubation medium

Control
Incubat ion,
No 70 of Control value in the presence of added
Additions, agent.
nmol/lOOmg
protein O.lmM Ouabain lmM BABA lmM DABA lmM NAG
Glutamate 90 (±10) +282 +111 +189 +191
Aspartate 46(+ 6) +200 +109 +217 + 39
GABA 28(± 3) +275 +286 +329 0

Glutamine 110 (± 2) + 41 + 16 + 33 ··6

Serine 348 (±11) + 30 + 12 + 24 -16
Glycine 240 (±12) + 15 7 + 58 -7
Alanine 244 (±14) +104 + 57 + 36 +57
No. of
Experiments 6 5 4 4 8

Synaptosome beds were incubated in Krebs-phosphate medium contain-

ing 10 mM glucose for 40 min at 37 0 C. Additions were made after 30
min incubation. Control values are means + S.E. Ms. Values in the
presence of added agents are means with S.E.Ms less than 10%. Data
is derived from ref 1.

Neurotransmitter Release: Depolarizing stimuli also increase

the extent of accumulation of transmitters in the incubation fluids,
and this has been demonstrated for acetylcholine 2 , dopamine 4 ,
noradrenaline 9 , glycine 24 , GABA, glutamate and aspartate l and found
to be calcium-dependant. This occurs in addition to the background
release of transmitters which is detectable in unstimulated control
samples and may be due either to "handling" stimulation, loss by
partial degeneration, or occur by a separate mechanism, possibly
identifiable with the transport cycle referred to in the last section.

Experimental Approach to Study of Transmitter Flux: Since

release and reuptake are so intimately linked, studying "release"
as the net change in level in the incubation medium following
application of a stimulus leaves a degree of uncertainty as to the
true size of the response, and whether it is restricted to enhancement
TRANSPORT, METABOLISM, AND TRANSMISSION 397

of efflux or includes inhibition of uptake. In an attempt to

separate "resting" efflux from "resting" influx a double-label
scheme was devised employing two forms of radioactive dopamine and
synaptosomes isolated from sheep corpus striatum as the experimental
system. This amine was selected in preference to an amino acid
transmitter,. as its involvement in mainstream metabolism is limited,
and a substantial amount of information has already been obtained
on its metabolism and compartmentation in striatal synaptosomes 4 ,25.

2. THE SHUTTLING OF DOPAMINE ACROSS THE MEMBRANE OF CORPUS

STRIATUM SYNAPTOSOMES: THE DIFFERENTIAL EFFECT OF
d-AMPHETAMINE

Experimental Procedure: The system used to separately label the

uptake and release processes involved a prelabelling stage in which
''beds'' of synaptosomes l were incubated with a dotamine precursor,
DL-DOPA (DL-3 (3,4,-dihydroxyphenyl) alanine r2-
4c:l), in order
to label endogenous dopamine (Fig. 1). The [."i4C) -dopamine produced
was used as marker of "resting" release from the synaptosome pre-
paration. The bed was then rinsed in saline and placed in fresh
medium containing dopamine at various concentrations labelled with
tritium. Incubation was continued for 3 minutes. During this
period, breakdown of dopamine was found to be minimal employing a
sensitive autoanalyser which separates all the monoamines and their
metabolic products 5 • The movement of (3H:l-dopamine into synapto-
somes was used to monitor dopamine uptake. Although the extracellular
space of beds is probably large (about 80%)17, this is unlikely to
change during these experiments and gross movements of counts from
saline to bed and vice versa are taken as indices of movements from
or into the intracellular compartment.

Incubate Wash
40' trans'er

~ synaptosomes

Figure 1. Schematic plan for labelling uptake and release of

dopamine by synaptosomes using two radioisotopes.
398 J.S. de BELLEROCHE AND H.F. BRADFORD

Results and Conclusions: The results show that [3H)-dopamine

uptake in the 3 minute incubation period increased as external
dopamine levels were raised (Fig. 2c). Other workers have shown
this to be a saturable process l5 • Inclusion of d-amphetamine
reduced the amount of (3H:J -dopamine recovered in the synaptosome
beds. On the other hand the movement of t:.14C )-dopami ne showed
that although about 30% could be recovered in the salines in
control incubations, this was not significantly increased or
decreased by either raising external dopamine levels, or by the
presence of d-amphetamine (Fig. 2a, 2b). The following assumptions
are made in interpreting these results: (i) Measurement of the
outward movement of Cl4 CJ would not be affected by reuptake of
U 4 c)-dopamine since that effluxed (0.5 nmol) would be diluted by
the excess of external dopamine present (5-80 ~ol), (ii) Following
efflux of (14C) -dopamine, counts rather than total dopamine is
fully acceptable when studying the effect of amphetamine. This
follows from our previous work 4 which demonstrated that even after
60 min incubation in the presence of amphetamine the specific radio-
activity of dopamine generated from~14C:l-DL-DOPA and released to
the medium was the same as that remaining in the bed, (iii) (3HJ-
dopamine enters the same pool as thd:14C]-dopamine already present,
and the absence 0£[l4CJ -dopamine efflux with rising external
dopamine indicates the absence of [3H]-dopamine efflux. A 30%
correction (addition) should be made to C3HJ-dopamine uptake to
allow for the release indicated by [14C)-dopamine efflux in the
control. Thus, although uptake and release are occurring concomitant-
ly, studying them separately in this system shows that in a 3 minute
period {?H)-dopamine influx occurs with riSing external dopamine
with little or no (14CJ-efflux occurring in parallel. d-Amphetamine
appears to inhibit influx and not efflux, and therefore shows the
properties of a transport blocker (Fig. 3) rather than those of a
releasing agent, its commonly assumed action.

These effects of amphetamine may be compared with its action

in increasing the amount of dopamine in the incubation media in our
previous experiments 4 • (Fig. 4.) Here, amphetamine is seen to
cause the release of a labelled pool of dopamine from synaptosomes
(Fig. 4c, 4d), an effect which is in contrast to those of depolariz-
ing stimuli, such as electrical pulses or potassium, (Fig. 4a, 4b)
which release only unlabelled dopamine, though in much smaller
amounts. This differential effect indicates that depolarization
and amphetamine mobilize different pools of synaptosomal dopamine.
Taken together with the results reported here, it suggests that
amphetamine "releases" by blocking the reuptake phase of a continuous
and balanced cycle of dopamine release and reuptake which appears
to be occurring at the synaptosome membrane and whose function is
obscure.
 TRANSPORT, METABOLISM, AND TRANSMISSION 399

14C Dopamine

,..
.. . 3H Dopamine uptake
.. . ., ..
"
a) Synaptosome total n mo n mol "
per 100 mg protein per
100 mg
b) Per cent released protein

,. L:.!-,
II !
••
--'-____ . . ,'.. . .- - ----1 DA external !JM
,. . '0 •

• .1 n It

DA external !JM
Fig. 2. - Synaptosome beds were incubated in Krebs-bicarbonate med-
ium (10 mM glucose) containing 3.9 !JM DL-3(3,4-dihydroxyphenyl)ala-
nine-[2- l4 C]at 37 0 for 40 min. The beds were then rinsed twice in
medium containing no radioisotope and placed in fresh-b carbonate
medium (10 mM glucose) containing C3Badopamine (10 ~Ci) present at
the concentrations of dopamine indicated. After 3 min the incubation
was terminated and the (3H1and[14CJcontents of the final incubation
medium and synaptosomes were determined by double isotope scintillat-
ion spectrometry, Individual values are indicated by filled circles
for control incubations, those carried out in the presence of d-
amphetamine (0.12 mM) are indicated by open triangles. The total
synaptosome content of~14CJdopamine is shown in (a) expressed as nmol/
100 mg protein, with the proportion (%) released to the medium being
shown in (b). The synaptosome content of(3H)is shown in (c).

Figure 3
400 J.S. de BELLEROCHE AND H.F. BRADFORD

.
DOPAMINE RELEASE
DOPAMINE RELEASE

a' CONTROL )

n mol

"

40' I

40 'I

b)

n Ci
I
100me

100

20' I

Figure 4

(a) and (c) Synaptosome beds were incubated in Krebs-bicarbonate

medium at 37°C for 40 min. The dopamine released to the medium
was determined fluorometrically4 and the mean values are represented
as histograms (nmol/lOO mg protein), the bars indicating the S.E.Ms
with the number of experiments beside. KCl was added after the
first 30 min to give a final concentration in the medium of 56 mM.
d-amphetamine (d-amph) was present at 0.12 mM as indicated throughout
the incubation. (b) and (d). Synaptosome beds were incubated for
20 min at 37 0 in Krebs-bicarbonate medium (10 mM glucose), following
a 40 min period of incubation in similar medium containing 3.9 ~
3 ,4-dihydroxyphenyl-alanine- ()_14CJ (51 nCi/nmol). KCl was added
to give a final concentration 56 mM after the first 10 min of the
final 20 min incubation and d-amphetamine (0.12 mM) was present as
indicated for the whole 20 min period. Values are means (nCi/lOO
mg protein) with the bars indicating the S.E.Ms with the number
of experiments beside. Dopamine was determined by scintillation
spectrometry following ion-exchange chromatographic separation4 •
*denotes that value is significantly greater (p<O.Ol) than control
open histogram (no additions).
TRANSPORT, METABOLISM, AND TRANSMISSION 401

Over the 3 minute incubation period employed, about 30% of

l:14G]-dopamine was effluxed whether external dopamine was present
or not. During longer incubation periods in the presence of
d-amphetamine,e.g. 20 min, 40 min, 65% or 77% were released to the
medium respectively~ indicating the presence of a continuous efflux
process cycling the total radioactively labelled pool across the
membrane with a half-time of about 12 minutes.

It is tempting to conclude that a similar continuous shuttling

of dopamine is occurring at the striatal nerve-terminal in ~,
though due account must be taken of the in vitro experimental
conditions which include the presence of~elatively rapid fluid
movements and the possible occurrence of effects due to preparation
and "handling". Certainly, it is attractive to conclude that
d-amphetamine changes dopamine tissue compartmentation in favour of
the extracellular space through an uptake-blocking action. This
would then be the basis of its effects in inducing the complex
locomotory patterns described as stereotypy28 which are blocked by
neuroleptic drugs that specifically affect dopamine turnover 14 and
are presumed to act as blockers of the dopamine receptor.

3. THE EXISTENCE OF A CONTINUOUS MEMBRANE FLUX OF

NEUROTRANSMITTERS IN GENERAL

The main questions to be answered about a continuous cycle of

dopamine efflux and influx at the nerve-terminal membrane are whether
it may be generalized to all transmitters, and possibly to other
membranes, and whether depolarization-induced transmitter release
occurs by a separate mechanism or not. Since depolarizing stimuli
applied to synaptosomes are thought to activate the natural trans-
mitter-secretion system normally operating at the nerve terminal
!£ vivo 3 , its relationship to a continuous cycle of release and
recapture becomes of central interest.

Considering first the uptake, Na+-K+ ATPase is strongly

implicated as being responsible for the transport of a wide range
of compounds including transmitters and their precursors. The
evidence shows good correlation between the activity of this enzyme
and the uptake of transmitter. The degree of inhibition of the
ATPase by transition element ions correlates well with the inhibition
of noradrenaline and choline uptake by rat brain synaptosomes 26 •
Similarly, inhibition of the enzyme by ouabain or incubation with
metabolic inhibitors e.g. cyanide or in the absence of glucose
inhibits the uptake of these and other transmitters20,lO,~~,2U,1.

The uptake process is likely to serve the function of providing

a source of transmitter for utilization and retention within the
terminal. The process of retention or maintaining a steady
concentration of transmitter shows a close link with the process
of uptake.
402 J.S. de BELLEROCHE AND H.F. BRADFORD

The evidence for the existence of a simultaneous efflux comes

from those experiments employing low Na+ and agents that block
transmitter transport. In the absence of an exogenous source,
some endogenous transmitter accumulates in incubation salines contain-
ing low levels of sodium23 or when transport inhibitors are present
10,18,1. The actions of these agents and conditions described above
which affect transmitter transport could be through indirect effects
such as depolarization of a synaptosomal membrane potential ll ,9 or
decreased respiratory activity16 with a consequent efflux of trans-
mitter down its concentration gradient. Equally, they could result
from inhibition of the influx phase of a balanced efflux-influx
cycle which normally produces no net efflux. Such outward transport
must be clearly distinguished from that induced by depolarization
agents. The latter is distinctive in that it is calcium-dependant
1,2,9 and in the case of dopamine, at least, clearly occurs from a
different transmitter poo14.

The ''homeoexchange'' scheme recently proposed by Levi and

Raiteri 19 ,27 to explain the cold GABA-induced efflux of radioactive
GABA from pre loaded synaptosomes is similar to that proposed here
on the basis of our own results. However, we postulate that the
process is occurring continuously and is not dependant on an
external source of transmitter for its activation. Also, in our
scheme, the inward and outward components are not necessarily
linked, uptake blockers or the presence of low sodium concentrations
producing a measurable 23 ,1 net efflux, though sodium-free superfusates
did not increase the rate of efflux of radioactive GABA from
synaptosomes in the Levi and Raiteri system.

In summary, we believe that a case can be made for generalizing

to other transmitters this shuttling of dopamine which appears to
occur across the membrane of the striatal nerve-ending. The function
of such a process/particularly the outward transport, remains
uncertain, especially as it would be expensive in terms of energy
utilization. It is possible that, in a modified form involving
the participation of calcium ion, it forms the basis of a mechanism
for quantitized transmitter release from a non-vesicular source.

ACKNOWLEDGEMENTS

This work was supported by a Programme Grant from the Medical

Research Council, U.K.

REFERENCES

1. de Belleroche, J.S., and Bradford, H.F., Metabolism of beds of

mammalian cortical synaptosomes: response to depolarizing
influences. J. Neurochem., 19 (1972) 585-602.
TRANSPORT, METABOLISM, AND TRANSMISSION 403

2. de Belleroche, J.S., and Bradford, HoF., The stimulus-induced

release of acetylcholine from synaptosome beds and its calcium
dependence, J. Neurochem., 19 (1972) 1817-1819.
3. de Belleroche, J.S.,.and Bradford, H.F., The synaptosome: An
isolated working neuronal compartment, In G.A. Kerkut and J.W.
Phillis (Eds.) Progress in Neurobiology, Vol. 1, Pergamon Press,
Oxford (1973) pp. 277-298.
4. de Belleroche, J.S., Bradford, H.S., and Jones, D.A., A study
of the metabolism and release of dopamine and amino acids from
nerve endings isolated from sheep corpus striatum, J. Neurochem.,
(1975) in press.
5. de Belleroche, J.S., Dykes, C.R., and Thomas, A.J., The automated
separation and analysis of dopamine, its amino acid precursors
and metabolites and the application of the method to the measure-
ment of specific radioactivities of dopamine in striated synapto-
somes, Analytical Biochem., (1975) in press.
6. Bennett, J.P. Jr., Logan, W.J., and Snyder, S.H., Amino acid
neurotransmitter candidates: sodium-dependent high affinity up-
take by unique synaptosome fractions, Science, 178 (1972) 997-999.
7. Bennett, J.P., Logan, W.J., and Snyder, S.H., Amino acids as
central nervous transmitters: The influence of ions, amino acid
analogues, and ontogeny on transport systems for L-g1utamic and
L-aspartic acids and glycine into central nervous synaptosomes
of the rat, J. Neurochem., 21 (1973) 1533-1550.
8. Blaustein, M.P., and Goldring, J.M., Membrane potentials in
pinched-off presynaptic nerve terminals monitored with a fluores-
cent probe: evidence'that synaptosomes have potassium diffusion
potentials, J. Physiol., 247 (1975) 589-615.
9. Blaustein, M.P., Johnson, E.M., and Needleman, P., Calcium-
dependent norepinephrine release from presynaptic nerve-endings
in vitro, Proc. Nat. Acad. Sci. U.S.A., 69 (1972) 2237-2240.
10. Bog~i, D.F., Tissari, A., and Brodie, B.B., The role of
sodium, potassium, ouabain and reserpine in uptake, storage
and metabolism of biogenic amines in synaptosomes, Life Sci.,
1 (1968) 419-428.
11. Bradford, H.F., Membrane potentials and metabolic performance
in mammalian synaptosomes. In P.F. Benson (Ed.), Cellular
Organelles and Membranes in Mental Retardation, Churchill
Livingstone, Edinburgh (1971) pp. 1-11.
12. Bradford, H.F., Synaptic preparations for studying neurotrans-
mission at the biochemical level, Biochem. Soc. Trans. 2 (1974)
13. Bradford, H.F., Isolated nerve terminals as an in vitro prepara-
tion for the study of dynamic aspects of transmitt~tabolism
and release. In L.L. Iversen, S.D. Iversen and S.H. Snyder
(Eds.), Handbook of Psychopharmacology vol. 1. Plenum Publishing
Corporation, New York (1975) pp. 191-252.
14. Carlsson, A., and Linqvist, M., Effect of chlorpromazine or
haloperidol on formation of 3-methoxytyramine and normetanephine
in mouse brain, Acta Pharmacol. and Toxico1. 20 (1963) 140-144.
404 J.S. de BELLEROCHE AND H.F. BRADFORD

15. Coyle, J.T., and Snyder, S.H., Catecholamine uptake by synapto-

somes in homogenates of rat brain: stereospecificity in different
areas, J. Pharmaco1. Exp. Ther., 170 (1969) 221-231.
16. Diamond, I., and Fishman, R.A., Development of glucose oxidation
in isolated nerve endings, Natur~ 243 (1973) 519-520.
17. Heaton, G.M., and Bache1ard, H.S., Fluid spaces of synaptosome
beds, J. Neurochem. 22 (1974) 561-564.
18. Holtz, R.A., and Coyle, J.T., The effects of various salts,
temperature and the alkaloids veratridine and batrachotoxin on
the uptake of r 3HJ dopamine into synaptosomes from rat striatum,
Mol. Pharmaco1., 10 (1974) 746-758.
19. Levi, G., and Raiteri, M., Exchange of neurotransmitter amino
acid at nerve endings can stimulate high affinity uptake,
Nature, 250 (1974) 735-737.
20. Marchbanks, R.M., Exchangeability of radioactive acetylcholine
with the bound acetylcholine of synaptosomes and synaptic vesicles,
Biochem. J., 106 (1968) 87-95.
21. Martin, D.L., and Smith, A.A., Ions and the transport of y-amino-
butyric acid by synaptosomes, J. Neurochem., 19 (1972) 841-855.
22. Neal, M.J., and Iversen, L.L., Subcellular distribution of
endogenous and /3H/-aminobutyric acid in rat cerebral cortex,
J. Neurochem. 16 (1969) 1245-1252.
23. Osborne, R.H., and Bradford, H.F., The influence of sodium,
potassium and lanthanum on amino acid release from spine1-
medullary synaptosomes, J. Neurochem., 25 (1975) 35-41.
24. Osborne, R.H., Bradford, H.F., and Jones, D.G., Patterns of
amino acid release from nerve-endings isolated from spinal cord
and medulla, J. Neurochem., 21 (1975) 407-419.
25. Patrick, R.L., and Barchas, J.D., Regulation of catecholamine
synthesis in rat brain synaptosomes, J. Neurochem., 23 (1974)
7-15.
26. Prakash, N.J., Fontana, J., and Henkin, R.I., Effect of
transitional metal ions on (Na+-K+)ATPase activity and the
uptake of norepinephrine and choline by rat brain synaptosomes,
Life Sciences, 12 (1973) 249-259.
27. Raiteri, M., Federico, R., Coletti, A., and Levi, G., Release
and exchange studies relating to the synaptosomal uptake
of GABA, J. Neurochem., 24 (1975) 1243-1250.
28. Randrup, A., and Munkvad, I., Special antagonism of amphetamine-
induced abnormal behaviour, Psychopharmaco1ogia, 7 (1965)
416-422.
29. Simon, J.R., Martin, D.L., and Kroll, M.J., Sodium-dependent
efflux and exchange of GABA in synaptosomes, J. Neurochem.,
23 (1974) 981-991.
CHANGES IN CEREBRAL AMINO ACID TRANSPORT DURING DEVELOPMENT

Federico Piccoli

Department of Neurology
University of Palermo
Palermo, Italy

INTRODUCTION

The transport of metabolites to and from the central nervous

system is of considerable interest. To a greater extent than most
other tissues, central nervous system tissue in vitro takes UP amino
acids to well above their concentrations in the incubation medium.
Presumably the transport systems responsible for this uptake and
for efflux in vitro are also those responsible for transport between
brain cells-in living animals 2 .

Studies of changes in amino acid transport during development

are somewhat scarce. With reference to the ontogenetical changes
in amino acid levels, most investigators have found a complex
pattern that is not surprising if we consider that the function of
amino acids itself presents a complex picture 7 . Correlations between
in vitro developmental changes in amino acid transport and the
ontcgenetical change of the level of these compounds should take
into account a number of parameters, most of which are still under
investigation. We will try to give a partial picture of this complex
pattern, taking into consideration the fate of some membrane and
energy related enzymes during development as well as the onto genetical
changes in the composition of the tissue (i.e. ions; degree of
swelling and spaces volume during incubation). Finally some of lIe
available data on changes of transport of amino acids during onto-
genesis will be briefly reviewed.

DEVELOPMENTAL CHANGES OF ENZYME PATTERNS

. 1
Bonavlta has shown that lactate dehydrogenase (LDH) of the

405
406 F. PICCOLI

muscle type (isoenzyme 5) and related hybrids prevail in proliferat-

ing nervous tissues. The data suggest that during the postnatal
neurogenesis an increase in the dependence of normal function on
aerobiosis occurs. This is reported in physiological studies in
the newborn rat 3 .

Jilek et a1. 5 have made observations on anoxia and hypoxia

in newborn and mature rats that indicate the possibility of direct
metabolic adaptation of the immature nervous tissue to hypoxia,
and have suggested that the increase in intensity of anaerobic
glycolysis is probably the basis of this metabolic adaptation. In
fact, studies on LDH offer a molecular and kinetic basis to such
a specu1ation 1 .

Studies on aminotransferase provide data in agreement with a

relatively lower efficiency of the citric acid cycle in the newborn
brain. The enzyme may exist in two functional forms: the pyridoxal
and the pyridoxamine forms, whose interconversion in vitro has been
demonstrated 1 . It has been found that the ratio between these two
forms is dependent on the keto acid concentration, but the prevail-
ing type in various tissues of the adult rat is the pyridoxamine
form. Recent studies on the immature rat brain have shown that
this ratio changes gradually during deve1opment 1 .

Studies on malic dehydrogenase (MDH) do not seem to contribute

to the knowledge of the metabolic organization of the developing
nervous tissue to the same extent as studies on LDH. However,
without molecular changes, both aspartate aminotransferase and
MDH increase during the postnatal neurogenesis by a factor of 4 to
51. This may indicate an increase of the capacity (not efficiency)
of the citric acid cycle and of the sub cycle by which small amounts
of malate triggers the conversion of glutamate to aspartate plus
C02 9 .

The level of ATP and the specific activities of Mg++ and Na+-
K+ ATPase have been investigated in the developing rat brain 11 . The
level of ATP decreases during post-natal development, while Mg++
and Na+ - K+ ATPase increase. The exposure to nitrogen of the fetus
at the 21st day of intrauterine life slightly depresses Mg++ ATPase
activity, while the Na+ - K+ ATPase activity remains unchanged, and
a significant fall in ATP level occurs. Conversely, the exposure
of the fetus to 169'ooxygen increases the Na+ - K+ activity, and
mainly the level of ATP.

The possibility of a dissociated variation of ATP level and

ATPase activity could be explained by assuming that at least during
development the activation of ATPase systems and the pattern of ATP
synthesis are not connected. Moreover, one could exclude a role
of ATP in the activation of ATPase 11
 CHANGES DURING DEVELOPMENT 407

EXTRACELLULAR SPACE AND ION CONTENT

The study of the distribution of tissue fluids, among various

compartments, has been considered important for studies in vitro of
cell membrane function, since it is desirable to differentiate the
intracellular and the extracellular spaces. The changes of water
content of the rat brain during development and the effect of
incubation upon the distribution in two principal compartments are
summarized in table I.

TABLE I
WATER CONTENT AND SPACES IN INCUBATED SLICES OF RAT BRAIN
Age Total Water (ml/g dry wt.) Swelling (percentage Inulin space
(days) before after increase of water (percent of
incubation incubation after incubation) total water)

P 7.73 7.77 0.5 30

Nb 7.06 7.86 11.3 45.6
3 7.56 8.45 13 .0 47.8
15 5.06 6.20 22.5 46.9
30 4.22 5.60 32.7 50.4
Ad 3.78 5.20 37.6 53.3
P = Fetus on 21st day of intrauterine life.
Nb = Newborn rat, less than 10 hold.
Ad = Adult rat, more than 120 days old
see ref. 10

The water content per unit dry wt of the intact brain decreases
by more than 50 per cent from fetus to adult. There is no swelling
in incubated slices from fetal brain, and the extent of swelling
increases with age; consequently the changes in fluid content at
the end of incubation are less than those in unincubated slices as
a function of development. The non-inulin space of incubated brain
slices decreases from fetus to adult by about 50 per cent; this
decrease probably does not imply that as development proceeds the
cellular space shrinks.

A valuable indicator of cell membrane function is the content

of Na+ and K+ in the tissue. The levels of these two ions in neural
tissue, together with the activity of ATPase (as previously mentioned),
have been reported in relation to the appearance of electrical
activitylO Our observation on developmental pattern of Na+ and K+
levels in incubated brain slices deserves a brief comment.

The total amount of Na+ in unincubated brain is higher in the

fetus, and in the newborn is only 8 to 10 percent higher than in the
adult. During incubation of brain slices, only a slight increase
of intracellular Na+ occurs in the fetal samples, but a large
408 F. PICCOLI

increase is measurable in incubated slices of older animals.

By contrast, during development of the brain the change of K+

seems to be in the opposite direction from that of Na+ lO The
fetus exhibits the lower level, and with a final stabilization at
a higher level soon after birth. After incubation the highest
increment of K+ can be measured in slices from fetus.

Concluding this section we should underline how, in slices of

fetal brain, incubation does not affect the total amount of tissue
water, or the distribution of water between the intra- and extra-
cellular compartment. The distribution of Na+ and K+ in cerebral
tissue may provide a partial explanation for these findings. A
high level of Na+ and a relatively low level of K+ characterizes
fetal brain. The values for fetal brain tissue are close, at least
in the case of Na+, to those reported for plasma of adult rat and
for CSF of fetal rat 4 . Moreover, the CSF - to - plasma ratios for
Na+ and K+ in the fetus are close to the Donnan distribution values,
suggesting that active transport of Na+ or K+ has not yet developed.
The sudden lowering of Na+ and increase of K+ in the brain soon after
birth presumably reflects activation of the Na+-K+ pump. The possible
role of oxygen in activating ATPase system, described above, seems
in keeping with the hypothesisll.

AMINO ACID TRANSPORT

Although it is generally accepted that in living animals most

substances penetrate into the immature brain to a greater extent
than into the mature brain, this is not too well documented.

When uptake of amino acids by brain slices from newborn and adult
is compared, most compounds appear to be taken up by slices from
adult brain to a greater extent, although some compounds such as
ethanolamine or tyrosine are taken up more by newborn slices 7 .

The comparison between changes in brain levels in the living

animals with those in transport capacity during development shows
that most compounds that are lower in newborn brain are taken up
by newborn brain slices to a lower degree, but compounds that are
higher in newborn brain shows no parallel decrease of transport in
slices during development 7 . Therefore transport changes as measured
with brain slices do not explain the developmental changes in the
level of amino acids in the living brain.

When uptake in brain slices is studied in greater detail, a

somewhat more complex pattern is shown lO

Influx of some amino acids (e.g. glutamate, glycine, and taurine)

is greater in adult than in newborn, but in each case maximal influx
CHANGES DURING DEVELOPMENT 409

z
o
8
•
I-
oct
Ill:
I-
Z
w 6
U
Z
o
()

Ill: 4
oct
...J
~
...J
...J
W
U
oct 2
Ill:
I- ____~________~~/rAU
Z

12 18 24 30 36 42

AGE (days)

Fig. 1. Uptake (influx) of glutamate (GLU), glycine (GLY)

and taurine (TAU). The dotted line represents an average
value for the concentrations of the amino acids in the medium
at the end of the incubation. Short term incubation lO •

is reached at a relatively early age and then declines to adult

levels (Fig. 1).

The maximal flux also does not occur at the time the levels
reach a peak (glycine levels reach peaks at birth and after 32 and
90 days, for example).

In short-term experiments with brain slices, amino acid influx

is the process that determines levels; in longer term experiments,
steady-state levels are reached, which are influenced not only by
influx but also by efflux and exchange. The changes in the steady-
state level of amino acids during development (at least with
reference to glutamate, glycine, and taurine) are less complex than
those of influx (Fig. 2).

Steady state uptake of glutamate and taurine increases gradually

till adult levels are reached; with glycine a maximum is reached
about the same time that the maximum of influx is reached lO . By
contrast aminoisobutyric acid uptake in mouse brain slices shows
a minimum at birth followed by a rapid increase to adult about 10
days after birth 6 .
410 F. PICCOLI

60

w GLY
:..:
<I: f-+-IltLU
~J-o
~
D..
::> 40
w
>
I- f---I ;"'AU
<I:
a:
I-
Z
w 20
()
Z
0
()

12 18 24 30 36 42
LLJuJ
60 Ad.

AGE (days)

Fig. 2. Steady-state uptake of glutamate (GLU), glycine

(GLY), and taurine (TAU). Long term incubation lO •

Recently a more detailed kinetic analysis of aming acid transport

during development in chicken brain slices by G. Levi showed that
there is little change in the apparent Km of transport, while rates
(Vmax) increase with age. Since this system Krn probably measures
the affinity of amino acid to the carrier, there seems to be little
change in the association of amino acid carrier complexes. The
increase in maximal rates of transport without a concomitant increase
in affinity to carriers can be best explained, not by a change in
the properties of the carriers, but by an increase in the relative
concentration of carriers during development in a way that parallels
the developing pattern of most enzymes.

With reference to Levi's data it is noteworthy to recall how

GABA seems to be transported by more than one carrier. One of the
GABA carriers shows behavior similar to that of the other carriers,
during development, while the second carrier on transport system
either decreases during development or, if present at all in adult
contributes little to the overall measured rates of GABA transportS.

CONCLUSIONS

The changes during development in the level, fate, and movement

of amino acids presents a prevailing complex picture now. They are
CHANGES DURING DEVELOPMENT 411

undoubtedly related to functional and structural changes, but the

detailed connections still escape us.

Transport mechanisms are present in immature brain with proper-

ties very similar to adult transport, although a number of carriers
increase in level during development. Transport processes, obviously
at very high level in the brain, are clearly connected with changes
in levels, regional metabolism, compartments, and metabolism, with
influx and efflux and maximal transport showing complex patterns
similar to those of the levels and metabolism.

REFERENCES

1. Bonavita, V., Developmental changes of enzyme patterns in the

nervous tissue. In: H. Peeters (ed) Protides of the Biological
Fluids, Elsevier Publ. Co., Amsterdam, (1966) p. 163.
2. Cohen, R.S., and Lajtha, A., Amino acid transport, In: A. Lajtha
(ed.) Handbook of Neurochemistry, Plenum Press, N.Y. (1972) p.
545-574.
3. Fazekas, J.F., Alexander, F.A.D., and Himwich, H.E., Tolerance
of the newborn to anoxia, Amer. J. Physiol., 134 (1941) 281.
4. Ferguson, R.K., and Woodbury, D.M., Penetration of l4C-inulin
and l4C-sucrose into brain, cerebrospinal fluid, and skeletal
muscle of developing rats, Exp. Brain Res., 7 (1969) 181.
5. Jilek, L., Fischer, J., Krulick, L., and Trojan, S., The
reaction of the brain to stagnant hypoxia and anoxia during
ontogeny. In: W.A. Himwich and H.E. Himwich (Eds.) The Develop-
ing Brain, Elsevier, Amsterdam, (1964) p. 113.
6. Lahiri, S., and Lajtha, A., Cerebral amino acid transport in
vitro. I. Some requirements and properties of uptake, J. Neurochem.,
11 (1964) 77.
7. Lajtha, A., and Piccoli, F., Alterations related to the cerebral
free amino acid pool during development, In: D.C. Pease (ed.)
Cellular Aspects of Neuronal Growth and Differentiation, V.C.L.A.
Forum Med. Sci. Vol. 14 (1971) p. 419.
8. Levi, G., Development of amino acid transport systems in incubated
tissue. In: W.A. Himwich (ed.) Biochemistry of the Developing
Brain, Dekker, N.Y. (1973) p. 187-218.
9. Lowry, O.H., Roberts, N.R., and Lewis, C., The quantitative
histochemistry of the retina, J. Biol. Chern., 220 (1956) 879.
10. Piccoli, F., Grynbaum, A., and Lajtha, A., Developmental changes
in Na+, K+ and ATP and in the levels and transport of amino acids
in incubated slices of rat brain, J. Neurochem. 18 (1971) 1135.
11. Piccoli, F., Guarneri, R., Savettieri, G., and Bonavita, V.,
ATP e ATPasi nell'encefalo di ratto durante 10 sviluppo, Acta
neurol., 27 (1972) 501.
 Relationship of
In Vivo and In Vitro
Studies
THE USEFULNESS OF STUDIES IN VITRO FOR UNDERSTANDING

CEREBRAL METABOLITE TRANSPORT IN VIVO

Abel Lajtha and Miriam Banay-Schwartz

New York State Research Institute for Neurochemistry

and Drug Addiction
Ward's Island, New York, N.Y. 10035, U.S.A.

Arguments between proponents of experimentation in vivo and

those who favor studying systems in vitro are quite old. The form-
er say that comnlex living systems cannot be studied in vitro
because the changes in these preparations cause observations of
artifacts, while the latter say that complex systems have first
to be broken into simpler ones before we can hope for meaningful
interoretations of our observations. We belong to what we perceive
to be the majority, who feel that both aporoaches are very useful
and necessary for studies of nervous system function, and are aware
of the need to know the advantages and liulitations of both. Similar
discussions can still be heard among researchers studying neural
barriers: "the blood-brain barrier (and other brain barriers) can
be studied only in living animals; oerhaps the biggest change in
any system in vitro is the absence of the barrier!' is the statement
often heard. Clearly, if we want to study the initial rate of
transport from the circulating plasma into brain, studies in vivo
are the most suitable; but if we want to study the factors that
determine the equilibrium and influence flux, cellular transport
processes (requiring observations in vitro) should not be neglected.
Hence this chapter.

BARRIERS IN VIVO AND IN VITRO

It has been shown in a number of studies, with short term

infusions, and with longer term administration, that the oenetra-
tion into brain of non essential amino acids, e.g., aspartate, glu-
tamate, GABA, glycine, and taurine, is small and slow as comoared
to that of the large, neutral class of (essential) amino acids, e.g.,
leucine, valine, and phenylalanine 6 ,55. In brain slices the
situation is the opposite. The uptake of the former group (Asp,Glu,

415
416 A. LAJTHA AND M. BANAY·SCHWARTZ

GABA, Gly, Tau) is faster, and accumulations (tissue medium con-

centration gradient) are greater, than those of the latter 12 , 30
It is also well known that the concentrations in the brain of the
nonessential amino acids are much higher than are those of most of
the essential amino acids 2 7, 41. If, for the sake of the oresent
discussion, we assume that cerebral uptake of the amino acids ad-
ministered in vivo is mainly limited by caoillary transport, while
uptake in slices is determined by cellular and subcellular transport,
then we must conclude that the activity of capillary transport is
not the final determinant of the level of cerebral metabolites, but
that cellular transport and metabolism also play an important role,
those compounds that penetrate slowly through the capillary having
higher levels in the brain in vivo than those taken up rapidly.
Capillary transport is clearly important, and if exit from the tissue
via the capillaries is also considered, it may have decisive infl-
uence on cerebral metabolite levels. However, it is likely that
the role of cellular transport is equally or even more decisive.
Capillary transport can be studied well in vivo, while cellular
transport can be observed better in vitro. Thus it would be re-
grettable if any approach ill vivo or in vitro were to be abandoned,
or if their advantages or limitations were ignored.

Numerous changes occur in preparations in vitro. Membranes are

partially damaged and some metabolites leak out; metabolic activity
is altered (autolysis of proteins could rapidly double the level of
some amino acids in the free pool); compartmentation may be altered;
ion composition and ion gradients are changed. All these and many
other changes justify the necessity to test the findings from isola-
ted systems in the intact brain as much as possible. In turn, the
discovery of high-affinity transport of putative neurotransmitters
in synaptosomal preparations 8 ,9, with no easy opportunity for stud-
ying high-affinity transport in vivo, should further delineate the
necessity for studies in vitro.

RATES OF CEREBRAL PROTEIN SYNTHESIS IN VIVO AND IN VITRO

Studies of protein metabolism can illustrate advantages alld

pitfalls of studies in vitro. The major advances in molecular
biology that we witnessed during the past years, some of which are
still in progress, were made possible only by the use of isolated
and purified systems. Without such exacting studies the mechanism
of protein synthesis would not have been clarified. Any present
study of factors influencing protein metabolism - growth, stimula-
tion, drugs, or regeneration - would not be complete if it did not
include studies of the detailed mechanism of the process, problems
that are only possible to study by the use of experimentation in
vitro. An area of great interest, which is subject to a number of
studies in vivo and in vitro is alterations of protein metabolism
during development.
USE OF IN VITRO TRANSPORT STUDIES 417

Technical problems of measuring protein synthesis are beyond

the scope of this article, but of the three methods commonly used,
measurement of the incorporation of administered labeled amino acid
is likely to give values closest to true turnover. Of the other
two methods: determination of the release of radioactivity from
prelabeled proteins is likely to be less precise; analysis of
changes in protein levels (enzyme recovery after inhibition, enzyme
induction) probably involves changes in the physiological equilibr-
ium. Measurement vf rates of protein synthesis that utilize the
incorporation of amino acids requires both knowledge of the specific
activity of the precursor amino acid used for measurement and also
maintenance of this specific activity to be constant over a period
of time. Possible compartmentation of the precursor has to be
considered.

TABLE I. Rates of protein synthesis in brain in vivo and in slices

Age of animals Incorporation of valine Incorporation in slice

(dogs) nmoles mg protein h per cent of in vivo
in vivo in slices

2 9.9 7.2 73

7 9.8 5.6 57

9 9.9 5.3 54

14 7.0 3.4 49

Adult 2.8 0.4 14

Initial rates of incorporation (30 min - 2 h period) of

a large dose of valine were measured in rats 32 . incorporation
in young in vivo is high and is fairly constant between 2 and
9 days of age; initial rates of incorporation in adults are
about 1 3 of that in young. Incorporation in slices from
young brain are close to that in vivo; in slices from adult
brain incorporation is a small fraction of in vivo rates.

Recently, we attempted to define the experimental conditions

that would provide optimal conditions for measurement of amino acid
incorporation. These consisted of administration of a large dose
of the amino acid (flooding), or the continuous administration of an
amino acid over a long period of time (infusion, subcutaneous im-
plantation of a pellet)34, 63 for experiments in vivo; in brain
slices we sought the optimal composition of incubation medIa, and
the amino acid levels that gave the highest rate of incorporation 20
Table I shows the measured rates of incorporation in young and adult
41B A. LAJTHA AND M. BANAY·SCHWARTZ

brain in vivo and in vitro. The rates shown in vivo in Table 1 are
comparable to those reported recently from other laboratories 19 ,24,52.
Although the incorporation rates il, slices shown are the highest we
can obtain, still they are below the levels in vivo. The difference
between slices and in vivo is not constant; it is smaller in the
young brain and much larger in the adult. The greater stability of
the system for protein synthesis in young brain has been observed
previously59. Table 1 illustrates how studies of changes in rates
of protein synthesis during decelopment must take into account
changes that are due to such factors as this stability before meas-
urements in vitro can be related to those in vivo. It also is of
interest that most studies of mechanisms of protein synthesis used
systems in vitro in which activity is only a very small fraction
of that in vivo ..

o Lysine uptake
• AI B uptake

Sliced bulb

.•
20

,--.-
Intact bul b
•
.•"'-
E
"
" 12
'E"

4 ..1---..A_--'
. - ___-:.:!e=--=---e---e---..a
'" _.-::r
~-
30 60 90 120 I~O

Time of incubation, min

Fig.l. Uptake (Tissue-to-medium concentration ratio) of lysine and

r amino isobutyric acid in intact and in sliced olfactory bulb
following incubation in a medium of 2.0 roM. Equilibrium uptake
is reached at approximately 120 min with untake in the slices
several fold higher than in the uncut tissue 50 .

OBSERVATIONS ON IN VITRO ALTERATIONS

There are only a few studies that compare transport in various

preparations under identical conditions. In one study we compared
USE OF IN VITRO TRANSPORT STUDIES 419

the uptake of amino acids in isolated olfactory bulbs of young mice

before and after the tissue was sliced 50 The olfactory bulbs are
small enough that the access of oxygen to the major part of the
tissue is satisfactory. The uptake of amino acids was lower in the
intact bulb than in the slices of olfactory bulb (Fig.l). This was
found with all (Gly, Val, His, Glu, AlB, and Lys) at several con-
centrations, 0.1, 1.0, and 2.0 mM. The relative uptake was similar
in bulbs and slices, i.e., uptake of non essential amino acids (bly,
Glu) was high, that of the essentials (Val, Lys) was low. The
difference in uptake did not seem to be due to changes in the levels
of energy or ions or rates of respiration; the content of substances
that may influence transport (ATP, Na, and K) was similar in the in-
tact and sliced tissue. The major difference seemed to be in the
inulin space, which was 2-to 3-fold higher in slices. The measured
apparent affinities (Km) were similar in the two preparations; the
maximal velocity of uptake (Vmax) was 4-fold higher in slices. This
indicated greater activity, but no qualitative change, in transport
processes. Perhaps part of the brain tissue is latent, or is not
active under many conditions, and becomes active in slices.

The increase in inulin and dextran space indicates one of the

most significant changes in slices: the increase in water content
(swelling). We studied the effect of water uptake on amino acid
transport in slices l . Under most conditions the increase in water
in incubated slices 0f brain is approximately 50 per cent; the
presence of Dextran or polyethylene glycol in the incubation medium
abolished this increase in water but did not affect the uptake of
amino acids by the slices (Table II). Dextran or polyethylene gly-
col reduced, but did not abolish, the increase in extracellular
marker space in vitro; nevertheless, both extracellular and intra-
cellular swelling was reduced, without changes in amino acid uptake.
Similarly, the effect of metabolic inhibitors 011 transport was in-
dependent of tissue water content l . This illustrates that the
effects of changes of the tissue in vitro on transport processes
can be studied. Such studies not only could help us to understand
the changes that occur in vitro, but also could help clarify the
factors involved in transport mechanisms.

Alterations in other preparations were not studied in detail.

Such knowledge would be important for correlating the results obtained
in vitro with function in vivo. For example, both isolated neurons
and glia transport amino acids 24 , 25 These studies in vitro clearly
established the presence of transport mechanisms in both types of
cells and made it possible to gather information about the properties
of such uptake systems. However, evaluation of quantitative compar-
isons are more difficult. Transport activity was found to be higher
in glia than in neurons 25 • Nevertheless, this may not reflect the
situation in vivo, but may be only the result of the greater damage
during preparation to neurons (with their processes shorn off) ,unlike
420 A. LAJTHA AND M. BANAY-SCHWARTZ

glia. On the other hand, the determination (with preparations in

vitro) that high affinity amino acid transport is not restricted to
neurons or synaptosomes, but is present in glia 7 , 26, is of help in
clarifying the role of such transport processes in vivo.

TABLE II. Independence of amino acid uptake of swelling in incubated

slices of brain.

Measurement Unit in tissue Control Control plus

water 4 per cent
Dextran 80

Swelling Water increase 48 2

(per cent)
Extracellular space Inulin space (per cent) 43 39

Tissue Na+ pmol/ml 111 102

Tissue K+ pmol/ml 70 73

Gly uptake (2mM) pmol/ml 26 25

Glu uptake (lmM) pmol/ml 28 28

ATP pmol/ml 2.0 2.0

The inclusion of 4 per cent Dextran in the incubation medium

reduces the increase in tissue water content but does not alter
the uptake of amino acids. Na+, K+, ATP levels, and inulin space
are also unaltered l , 31. Slices were incubated for 60 min.

Damage to membranes may not necessarily cause decreased transport

activity, since latent transport may be activated; but damage is
likely to resulL in a greater leakiness of the membrane. Brain
slices retain fairly closely the composition of the living brainS
even when the slices are incubated in amino acid-free media 5l , 5 ,
although a few compounds such as glutamine may leak out of the
tissue. Mo~t other preparations from brain, such as cells 60 or
particulates 46 , lose a greater portion of their amino acid content.
It is of interest that although the total level of free amino acids
is lower, the relative composition is altered less; that is, the
nonessential amino acids remain higher than the essential ones, a
similarity to whole brain that exists even in synaptosomal vesicle
preparations 17 . The leakiness may explain the lower tissue/medium
concentration gradient in particulates than in slices 49 In slices,
a third compartment was defined by computer simulation; it affects
the metabolism of glutamate i glutamine, GABA, and the Krebs' cycle,
and is due to tissue damage 3. This third compartment equilibrates
USE OF IN VITRO TRANSPORT STUDIES 421

rather slowly and thus strongly influences measurements of metabol-

ism in short-duration experiments.

The method of preparation may influence the transport activity

and metabolite pools of the various cellular and subcellular systems.
In slices, the effects of temperatu:r:e, ions, and duration of anoxia
on swelling have been reported. In our hands, brain slice amino
acid uptake was not sensitive to previous handling, although it was
influenced by slice thickness 6l . Glucose and electrolyte metabolism
was found to be closer to that in vivo in carefully prepared slices
of defined thickness than in minced brain or in slices of varying
thickness 4S . Dependence of amino acid transport activity on the
method of preparation of synaptosomes has been reporteti 43 .

The method of handling preparations during the experiment is

also of influence. Washing of preparations to remove extracellular
components may leach cellular contents; cooling a preparation to
stop transport may cause it to approach an equilibrium that is closer
to the one usually attained at lower temperatures 12 .

SUBSTRATE SPECIFICITY OF TRANSPORT IN VIVO AND IN VITRO

Although transport rates are expected to differ, in many cases

it was found that the basic mechanisms and their properties are sim-
ilar in vivo and in vitro. However, a difference in rate limiting
steps results from an apparently different distribution of the
transport systems, as shown by studies of substrate specificity.

Studies of substrate specificity indicate that fewer transport

systems for amino acids are active in capillary transfer than can be
observed in brain in vitro. In short-duration capillary perfusion
experiments Oldendorf distinguished three transport systems, one
each for the neutral, the basic 6 and the acidic amino acids, with
no overlap of the three classes S . Transport of other amino acids
(Gly, Pro, Tau, and GABA) was absent or below detectable limits 55 .
Transport of the neutral class seems to be only through the "L"
system66 of Christensen - a system primarily active in exchange
rather than uphill net transport, that does not require Na+ 15
In brain slices we found separate uptake systems with high activity
for those amino acids (Gly, Pro, Tau, and GABA) for which no such
capillary transport was detectable 13 , and we found evidence for
additional systems besides the "L" type for neutral amino acid
transport S7 We could not detect a Na+ requirement in capillary
amino acid transport in vivo 62, in contrast to the strong Na+ de-
pendence in brai~ slices 36 . We also found a separate transport
system in slices for diamines (putrescine and cadaverine)35 that
we could not detect in capillary transport 62 (Table III). Appar-
ently a number of transport systems that are present and show
high activity in brain cells are not detectable in capillaries.
These results indicate that capillary transport is not necessarily
422 A. LAJTHA AND M. BANAY·SCHWARTZ

representative of brain tissue transport, and that if equilibrium

between tissue and blood is determined by transport, tissue (cellular)
transport processes cannot be neglected.

Does the rapid capillary penetration of essential amino acids

(and a barrier without transport for the nonessential amino acids)
indicate that the essential amino acids are supplied from blood while
the nonessential ones are synthesized within the brain? There is no
indication that this is the case. Arteriovenous difference measure-
ments 2l , 22 indicate that valine and leucine are among the amino
acids removed from the blood at the highest rate. Metabolism of leu-
cine in vivo is very high, while that of valine is low in the brain,
but the A/V difference of leuciue is only half of that of valine.
High A/V differences have also been found for proline and glycine 2l ,
despite undetectable capillary transport for these amino acias. The
significant A/V differences of other nonessential amino acids, such
as alanine, serine, and threonine, also indicates that there is a
supply of these compounds via the circulation.

TABLE III. Inhibition of cadaverine and putrescine uptake in vivo

and in slices.

Uptake Per cent inhibition

in vivo in slices in vivo in slices

Cadaverine 3.8 5.0

+ putrescine 3.2 2.6 16 48

Putrescine 3.7 4.0

+ cadaverine 3.2 1.9 14 52

Uptake units are given in vivo as per cent taken up from tracer
i~fusion55, 62, uptake in slices as tissue medium concentration
ratio after 30 min incubation. The inhibition in vivo is not
significant, the small uptake possibly being via diffusion;
the concentrative uptake in slices (with tissue levels 4-5 fold
higher than medium) is significantly inhibited by analogs 35 at
low or high concentrations and in short and long time experi-
ments.

It is important to realize that, although the metabolic inter-

conversion of some amino acids in the brain is very rapid, we do not
know the net rate of utilization of any cerebral amino acid. This
can be illustrated with our information about cerebral metabolism
of glutamate, which is generally assumed to be one of the most rap-
idly metabolized amino acids. The isotopic equilibration between
oxoglutarate and glutamate is ve~y rapid because of very high amino-
USE OF IN VITRO TRANSPORT STUDIES 423

transferase activity. Although this may not cause any net utiliza-
tion of glutamate, it will cause the appearance of label from glut-
amate in a variety of compounds. The rate of net glutamate in brain
is unknown, and we still cannot exclude the possibility that the
major replacement for the glutamate that is utilized in the brain
comes from plasma glutamate. In turn, the final distribution and
regional level (perhaps even the regional metabolism) of glutamate
depends on cellular transport processes 30 •

ASPECTS OF TRANSPORT THAT CAN BE STUDIED BETTER IN VITRO

In a discussion of advantages and limitations of various

approaches to transport studies it should be emphasized (although
perhaps it is obvious) that numerous aspects can be studied only in
vitro at present. The best examples are studies of ion and energy
requirements, since ion and enrrgy content cannot be manipulated to
the necessary degree in vivo 3 .
Ion requirements of amino acid transport in brain have been ex-
amined in some detail. In synaptosomes, high and low affinity
transport can be distinguished, as only the high-affinity transport
shows strong Na dependence 64 • Further examples of studies of ion
requirements in brain slices from our laboratory are shown in Table
IV. The following findings are illustrated in this table: a) Uptake
of most amino acids in slices shows an absolute dependence on Na+ 36,
although it is possible that the effect of absence of Na+ is indirect;
b) The lowering of tissue Na+ below optimal decreases transport in
general, but affects the uptake of various compounds to different
degrees 32 , 47; c) Uptake of diamines is independent of Na+ 35, of
basic amino acids only partially dependent 36 ; d) There is less de-
pendence on K+, but depletion of tissue of K+ inhibits uptake (in-
directly), and K+ can be replaced by Rb+ 5; e) K+ also affects var-
ious compounds to different degrees; f) Under most conditions net
Na+ flow into tissue increases, net Na+ exit from the slices inhibits,
amino acid uptake 37 . Although some of these experiments involve
milieu unlikely to occur under physiological conditions, in addition
to yielding information about the properties of transport systems,
such demonstrations show the influence of physiological variations
in ion grddients. Ion changes have wide effects on brain prepara-
tions, as illustrated in this volume 4.

Tissue preparations are also suitable for studying energy requ-

irements; they make possible tests of the effects of metabolic in-
hibitors, and of the effects of depleting or replacing substrates or
high energy compounds, that are not feasible in vivo. Illustrations
with slices from our laboratory are shown in Table V; results are
discussed in more detail elsewhere 2 , 3 and in this volume 4 The
following conclusions are illustrated in Table V: a) the level of
ATP is not necessarily rate limiting for amino acid uptake, since
it is possible to have high ATP with low uptake and low ATP levels
424 A. LAJTHA AND M. BANAY·SCHWARTZ

TABLE IV. Effects of ions on amino acid uptake in brain slices

EX:Qeriment Amino acid Per cent of control

No Na+ Glutamate 0
Lysine 33
Cadaverine 116
Low Na+ Glutamate 42
GABA 3
No K+ Glutamate 10
Ornithine 65
No K+ + Rb+ Glutamate 94
Ornithine 91
Na uptake Glutamate l33
GABA 149
Na exit Glutamate 54
GABA 51
Results are expressed as intracellular uptake per cent of
control (optimal Na+ and K+ in incubation medium). Low Na:20rnM'
Rb:5rnM:Na uptake: transferring slices from 74 to l64rnM Na+ ;
Na exit: transferring slices from 164 to 74rnM Na+.Results are
taken from 3, 31-32, 35-37.

TABLE V. Metabolic energy and amino acid uptake in brain slices

Incubation Amino acid Per cent of control

medium Uptake ATP
NaF, lmM AIB 39 51
Leucine 29 50
Fluoroacetate,lOmM AlB 89 52
Leucine 71 47
NaCN, 0.25mM AlB 95 37
Leucine 102 36
Gramicidin D, 0.2mM Glutamate 37 32
NaF, 3mM Glutamate 81 11
Ouabain, 2)lM Lysine 68 81
Iodoacetate, 0.3mM Lysine 85 9
No glucose + SMP Glutamate 119 38
Glucose + 10mM NaF Glutamate 25 9
No glucose + SMP Glutamate 95 7
(20-S-SmM) + 10mM NaF
Glucose + Rotenone Glutamate 75 30
No Glucose + SMP + Roten. Glutamate 30 4

Slices of mouse brain were incubated for 30 min in the medium,

labeled amino acid was added,and uptake was measured in the 30-90
min period.AIB: aaminoisobutyric acid;SMP:succinate malate pyru-
vate; Rotenone was 10-8M.The effect of lowered ATP levels on
transport depends on the inhibitor,amino acid,and substrate meta-
bolized; there is no direct relationship between ATP level and
degree of uptake.
USE OF IN VITRO TRANSPORT STUDIES 425

with high uptake; b) the effect of lowering ATP on uptake is depend-

ent on the particular inhibitor used; c) lowering ATP affects var-
ious amino acids in different degree; d) glucose as substrate can
be replaced by other compounds that also modify or bypass the effects
of metabolic inhibitors 65 ; e) mitochondrial energy metabolism is of
importance in fueling amino acid uptake in brain slices 6s . In the
interpretation of kinetic measurements of transport the use of iso-
lated and purified systems offers great advantages s3 ; here again
measurements in slices seem to be more precise than those in vivo.

FUNCTION OF THE BARRIERS

Information about the various transport processes in brain is

considerable, but we know very little of the function of the barriers.
It has already been discussed in this chapter that with most meta-
bolites it is not possible to relate capillary transport rates to
cerebral metabolic rates. Another proposal that can be examined is
that the function of capillary transport is to supply amino acids
for protein synthesis. In the adult brain most proteins are in a
dynamic state 38 . If the half-life is 10 days for the bulk of cerebral
proteins 34 , at this rate of turnover the amount of most amino acids
present in the free pool would be incorporated in about 2 hours,
giving a half-life of exchange between free and protein bound amino
acids of one hour. In the adult brain there is no net protein depos-
ition, so incorporation does not cause a net utilization of protein.
In young brain there is net protein synthesis that was estimated as
0.5 per cent/h in the fast growing stage 33 . This alliount of synthesis
utilizes the total brain free amino acid content of many amino acids
within one hour. From this, protein metabolism would not require
additional free amino acids in the adult, but would require a rapid
supply during development. Since initial studies did not indicate a
significantly higher rate of ca~illary transport in young brain,
although diffusion was greater 6 , the activity of capillary amino
acid transport does not seem to parallel developmental changes in
amino acid requirements for protein synthesis.

We also examined the relationship of transport activity in brain

slices to amino acid levels and to alterations in amino acid levels.
The rationale of these studies was to examine whether cellular trans-
port activity or capacity (if this is what can be measured in slices)
is the determinant of amino acid distribution. Some paralellism was
observed between transport activity in vitro and amino acid levels
in vivo, as already mentioned, in that the rate and extent of uptake
in slices of those amino acids that are at high levels in the brain
(Glu, Asp, GABA, Gly) is much higher than that of the other amino
acids ll . (Table VI). This parallelism was not very close; for
example, the differences between the levels of glutamate and aspartate
were not reflected in similar differences in their slice uptake.
Similarly, the developmental (Table VI) or regional (Table VII) diff-
erences in uptake were not parallel with developmental or regional
426 A. LAJTHA AND M. BANAY·SCHWARTZ

changes in amino acid levels 29 • The results thus show parallel

behavior in some cases, but in many cases differences between
transport activity and physiological levels.

TABLE VI. Levels of amino acids in vivo and uptake in brain slices

Concentration Uptake
Adult Newborn Adult Newborn
Glutamate ---g:g- 4.4 ~ 50
Taurine 8.3 19 44 17
Glycine 0.91 2.30 53 34
Lysine 0.19 0.23 8.8 10
Leucine 0.04 0.08 5.1 6.5
Phenylalanine 0.05 0.11 3.3 4.7

Concentration in brain in vivo is expressed as u moles per g

fresh tissue; uptake is expressed as intracellular concentra-
tion (p moles/ml) after incubation for 90 min with2mM amino
acid 4l . Amino acids at high level are taken up in slices to a
greater degree; developmental changes in level do not parallel
developmental changes in uptake.

TABLE VII. Comparison of regional amino acid levels with uptake

by slices

Relative distribution, whole brain = 100

Hemisphere Midbrain
in vivo slice in vivo slice
Glycine 55 90 137 155
Glutamate 112 III 73 92
Taurine 125 83 47 265
Lysine 75 110 l33 107
GABA 101 100 164 148

Slice uptake was measured in rat brain after 90 min incubation

with 2mM amino acid 29 . Regional levels and uptake are parallel
with GABA but not with taurine.

In spite of the negative correlations, it seems likely that tran-

sport activity on the cellular level is an important mechanism in de-
termining the distribution and concentration gradients of amino acids.
The high uptake capacity for nonebsential amino acids indicates that
one of the functions of amino acid transport is to remove from the
extracellular space compounds that have high physiological activity;
obviously the level of compounds with direct effect on neural act-
ivity and the level of other amino acids that serve as precursors
for neurotransmitter amines have to be carefully controlled.
USE OF IN VITRO TRANSPORT STUDIES 427

The developmental pattern of the free amino acid pool is complex.

Quantitatively, the most significant changes are the increase in glut-
amate and the decrease in taurine with age. Some compounds have sev-
eral maximal or minimal levels during development l6 , 57. There are
particularly rapid changes for a few amino acids such as alanine in
the period around birth 39 . Developmental patterns of transport sys-
tems have not been studied in detail to explain perinatal changes.
We found that the cellular transport systems for the nonessential
amino acids develop rapidly around birth - that for the essentials
before birth 62 . The proportion of uptake of GABA by a high affinity
system decreased, perhaps disappeared, during development, either
because there was a great increase in the low affinity system or
because of a decrease in the high affinity system40 .

One possible explanation for the many examples of negative

correlation between uptake and levels may be that uptake rates as
measured in slices do not reflect the rates of uptake in vivo. There
are many examples that changes in enzyme levels as determined in vitro
do not necessarily reflect changes in enzyme activity in vivo. We
found, as already mentioned, that the rates of protein turnover and
therefore of protein breakdown decrease during development l9 , 33. In
spite of this decreasing proteolytic activity, the content of pro-
teinases increases in brain during development 48 • The reason for
this must be that proteinase activity measured in vitro reflects the
total capacity of the tissue, including latent enzymes, rather than
activity in vivo. If transport activity is similarly different,
then slice uptake may show activity that quantitatively is not a
good measure of cellular transport rates in vivo. It remains to be
shown whether it is an indicator of transport capacity.

The high affinity amino uptake systems for putative neurotrans-

mitter amino acids are not as easily demonstrable in slices as in
synaptosomal preparations. The reason for this may not be any dam-
age or changes in slices; it may be due to the presence of the low-
affinity and high-capacity systems responsible for the bulk of trans-
port 42 In assigning function to transport systems their capacity
should also be considered: a high capacity, low affinity system may
be more efficient for removal of neurotransmitters than a low capac-
ity, high affinity system.

Another important aspect to consider in assigning function to

transport is the role of exchange. The role of homoexchange in
synaptosomes that shows the chardcteristics of high affinity uptake 44
and may be mistaken for it (Levi et al.,this volume) is puzzling.
Both homo- and heteroexchange were detected in the brain in vivo
and in vitro 31 ; heteroexchange may gain significantly in amino
acidemias.
428 A. LAJTHA AND M. BANAY-SCHWARTZ

METABOLITE C0MPARThENTATION IN VIVO AND IN VITRO

In studies of transport the cerebral compartmentation of amino

acids cannot be neglected. The question that is pertinent to the
present discussion is whether such compartmentation changes in vitro
and the uptake of amino acids occurs in different compartments in
vitro. It has been suggested, for example, that endogenously formed
glutamate is primarily located in neurons, while uptake in slices
occurs primarily in glia S4 . Indication that glutamate taken up by
synaptosomes is present in a special pool not in complete equilibrium
with the endogenously furmed glutamate is discussed by Dr.Levi in
this volume. The compartmentation of glutamate metabolism in the
brain in vivo is well established lO . Although it was claimed that
such compartments cannot be detected in slices, if brain slices are
prepared at 37 0 instead of 0 0 , or allowed to recover by incubation
at 37 0 , compartmentation of glutamate metabolism can be shown ll

At present there is no suitable method available to measure

the quantitative distribution of the free amino acid pool in various
celiular and subcellular elements, although fixation with gluter-
aldehyde (binding to local proteins?) followed by autoradiography
could localize a significant portion of some amino acids 28 .

We investigated whether the free amino acid pool in the brain

in vivo or in slices is exchangeable with external amino acids. In
continuous infusion of tracer doses of labeled amino acids in vivo
for 6 h the major portion of cerebral amino acids exchanged with
plasma amino acids, although complete isotonic equilibrium was not
reached 63 ; but these experiments did not exclude the presence of
a small pool that equilibrates very slowly or not at all. Part of
the reason for not reaching complete equilibrium is the dilution of
the labeled free amino acid pool in the brain by metabolism, princi-
pally by the rapid incorporation of labeled amino acid into and re-
lease of unlabeled amino acids from proteins through rapid cerebral
protein turnover 63 . In slices we found a similar situation, with
the bulk of most amino acids of the slice measured in rapid exchange
with tracer labeled amino acids in the medium, and a sequestered com-
partment of less than 6-20 per cent SI • Studies of regional distri-
bution of the labeled amino acid within incubated slices 61 did not
show large differences. Uptake is more rapid as expected into the
more damaged surface of the slic~ but within the slice distribution
is close to equilibrium. Metabolism and protein turnover make it
difficult also in slices to precisely estimate this slowly equilibr-
ating pool. A pool that is the possible substrate for high affinity
transport in synaptosomes or in glia may also be present in slices,
although its detection may not be easy if the activity of the low
affinity system is much higher 42 . A detailed discussion of compart-
mentation of amino acids for protein synthesis is beyond the scope
of this article. In some systems evidence was found for preferential
USE OF IN VITRO TRANSPORT STUDIES 429

utilization of external or internal pools for protein synthesis;

our experiments indicated that the average free amino acid (not
compartmented) is the precursor for proteins in brain l8 . Experi-
ments studying amino acid metabolism, amino acid exchange and ex-
changeability, and amino acid incorporation into proteins thus do
not furnish conclusive evidence that amino acid compartments present
in vivo are altered or absent in brain slices.

CONCLUSIONS

The homeostatic control mechanism governing metabolite distri-

bution in the brain has 1l1any aspects that are specific for this organ.
Capillary structure, permeability, and transport are part of this
mechanism, but many other elements contribute. Uptake by neurons and
glia, and by synaptosomes, transport by the choroid plexus, exchange
across numerous membranes, bulk flow of spinal fluid, for example,
all contribute to homeostasis, as does metabolism.

The structural heterogeneity of the nervous system is paralleled

by heterogeneous distribution of metabolite transport systems. Only
a part of such systems can be observed in capillary transport; with-
in the tissue transport also is heterogeneously distributed. Although
it is tempting to assign the presence of a specific transport system
in a structure to a specific function - especially to the removal of
neurotransmitters - the distribution is multiple with multiple func-
tions. Relationship of changes in transport to changes in other
functions during development in pathology are obscure.

The variety of transport classes and compartments tha~ can be

studied and the many successful approaches using many preparations
are well described in this book; they illustrate the success, and
the need for multiple approach, and also our present limitations.
Studies in vitro gave us much information on specificity, ion energy
dependence, and other properties of transport; it is likely that
these properties are qualitatively identical in vivo. Kinetic
relationships (rates, activity) in vivo are more difficult to deduce
from studies in vitro. Perhaps the relationship of enzyme content
determined in vitro to enzyme activity in vivo can serve as an
example. An understanding of transport activity and the changes
of such activity in specific structures in vivo will help to clarify
the role of metabolite transport in the metabolism and function of
the nervous system.

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430 A. LAJTHA AND M. BANAY·SCHWARTZ

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432 A. LAJTHA AND M. BANAY-SCHWARTZ

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1976, in press.
35. Lajtha, A., and Sershen, H., Substrate specificity of uptake
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41. Levi, G., Kandera, J., and Lajtha, A., Control of cerebral
metabolite levels.I. Amino acid uptake and ltvels in various
species, Arch.Biochem.Biophys., 119 (1967) 303-311.
42. Levi, G., and Raiteri, M., Detectability of high and low affi-
nity uptake systems for GABA and glutamate in rat brain slices
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44. Levi, G., and Raiteri, M.,Exchange of neurotransmitter amino
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45. Ludt, H., and Dittman, J., Advantages of defined tissue slices
against minced samples in biochemical in vitro investigations
of brain cortex, Acta Biol.Med.Germ., 34 (1975) 189-195.
46. Mangan, J.L.,and Whittaker, V.P., The distribution of free
amino acids in subcellular fractions of guinea-pig brain, Bio-
chemical Journal, 98 (1966) 128-137. ----
47. Margolis, R., and Lajtha, A., Ion dependence of amino acid
uptake in brain slices, Biochim.Biophys.Acta, 163 (1968) 374-385.
48. Marks, N., Stern, F., and Lajtha, A., Changes in proteolytic
enzymes and proteins during maturation of the brain, Brain
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late fractions from brain,Biochim.Biophys.Acta,173 (1969) 518-531.
USE OF IN VITRO TRANSPORT STUDIES 433

50. Neidle, A., Kandera, J., and Lajtha, A., The uptake of amino
acids by the intact olfactory bulb of the mouse: A comparison
with tissue slice preparations, J.Neurochem. 20 (1973) 1181-
1193.
51. Neidle, A., Kandera, J., and Lajtha, A., Compartmentation and
exchangeability of brain amino acids: Evidence from studies of
transport into tissue slices, Arch.Biochem.Biophys., 169 (1975)
397-405.
52. Oja, S.S., Incorpolation of phenylalanine, tyrosine and trypto-
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53. Oja, S.S., and Vahvelainen, M.L., Transport of amino acids in
brain slices, In N.Marks and R.Rodnight (Eds.), Research Meth-
ods in Neurochemistry, Vol.3, Plenum Press, New York, 1975,
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in cerebral-cortex slices from the rat, Biochem.J., 128 (1972)
1117-1124.
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amines, and hexoses after arterial injection, Amer.J.Physiol.,
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56. Oldendorf, W.H., and Szabo, J., Amino acid assignment to one of
three blood brain barrier amino acid carriers, Amer.J.Physiol.,
1975, in press
57. Piccoli, F., Grynbaum, A., and Lajtha, A., Developmental changes
in Na+, K+ and ATP and in the levels and transport of amino
acids in incubated slices of rat brain, J.Neurochem., 18 (1971)
1135-1148.
58. Pull, I., Jones, D.A., and McIlwain, H., Superfused cerebral
tissues in hypoxia: neurotransmitter and amino acid retention;
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59. Roberts, S., Effects of amino acid imbalance on amino acid
utilization, protein synthesis and polyribosome function in
cerebral cortex.ln Aromatic Amino Acids in the Brain, CIBA
Foundation Symposium 22, American Elsevier, New York, 1974,
pp.299-324.
60. Rose, S.P.R., and Sinha, A.K., Some properties of isolated
neuronal cell fractions, J.Neurochem., 16 (1969) 1319-1329.
61. Sershen, H., and Lajtha, A.,The distribution of amino acids,
Na+, and K+ from surface to centre in incubated slices of
mouse brain, J.Neurochem., 22 (1974) 977-985.
62. Sershen, H., and Lajtha, A., in preparation.
63. Seta, K., Sansur, M., and Lajtha, A., The rate of incorporation
of amino acids into brain proteins during infusion in the rat,
Biochim.Biophys.Acta, 294 (1973) 472-480.
64. Snyder, S.H., Young, A.B., Bennett, J,P., and Mulder, A.H.,
Synaptic biochemistry of amino acids, Fed.Proc., 32 (1973) 2039-
2047.
434 A. LAJTHA AND M. BANAY-SCHWARTZ

65. Teller, D.N., Banay-Schwartz, M., De Guzman, T., and Lajtha, A.,
EnergeLics of amino acid transport into brain slices. Effects
of glucose depletion and substitution of Krebs cycle inter-
mediates, Brain Research, 1976, in press.
66. Wade, L.A., and Katzman, -R., Transport of L-DOPA and related
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presence of the L transport system, Abstracts 5th Meeting
Internatl.Soc.Neurochem., Barcelona, 1975, p.462.
RELEASE OF AMINO ACIDS FROM THE SPINAL CORD

IN VITRO AND IN VIVO

Robert W. P. Cutler

Stanford University Medical Center

Department of Neurology
Stanford, California 94305

INTRODUCTION

In the last decade, considerable evidence has supported the

initial suggestion by Aprison and Werman 2 that glycine is a likely
inhibitory transmitter in the mammalian spinal cord. The concen-
tration of glycine becomes progressively higher in more caudal
areas of the nervous system and it is particularly high, on the
order of 5 ~moles/gram, in regions of spinal cord known to contain
inhibitory interneurons. The concentration falls after partial
spinal cord ischemia in a manner proportional to the loss of inter-
neurons. 12 The action of glycine is antagonized by strychnine,
which is known to block postsynaptic inhibitory mechanisms. 5 There
is reason to believe that other amino acids may have roles as
neurotransmitters in the spinal cord, based on less complete evi-
dence than that for glycine. For example, GABA has been proposed
as a presynaptic inhibitory transmitter at primary afferent termi-
nals. It is found in high concentrations in the dorsal horn, and
local ischemia of this region leads to a marked fall in concentra-
tion. 23 Deafferentation of the spinal cord by dorsal root section
leads to a reduction in glutamic acid decarboxylase activity in
the dorsal lateral region of cord, together with a fall in GABA
content and a reduction of [3H] GABA uptake. 14 Both glutamic
acid and aspartic acid have been cited as potential excitatory
transmitters in the spinal cord. The higher concentration of
glutamic acid in dorsal than in ventral roots and horns suggested
that it might function as a primary afferent transmitter 15 , and
the reduction in aspartic acid following ischemic loss of inter-
neurons suggested that it might be an intrinsic spinal excitatory
transmitter. 12 The fall in spinal content of these amino acids
after dorsal root section is consistent with these views. 22

435
436 RoWoP oCUTLER

Further support for a transmitter role of glycine, GABA,

aspartic acid, and glutamic acid comes from the finding of distinct
high affinity transport systems which mediate the uptake of these
amino acids in the spinal cord. 3 Such uptake systems are generally
held to represent inactivation mechanisms at the synapse, although
recently it has been proposed that high-affinity transport may in
fact represent simple exchange diffusion, not net uptake. 28

While extensive studies have characterized the transport

systems which mediate uptake of amino acids by tissues, relatively
few studies have been directed toward the mechanisms of release
of amino acids. This chapter reviews pertinent published work and
presents some new data on amino acid release from spinal cord.

RELEASE OF MUNO ACIDS FROH SPINAL CORD SLICES

Release of [14C] glycine from electrically stimulated

spinal cord slices was first demonstrated by Hopkin and Neal. 18
Shortly thereafter fuller report~ were published which character-
ized some of the properties of [ 4C] glycine release. 17 ,19 In
these studies, rat spinal cord slices were preloaded with a variety
of isotopically labeled solutes and then superfused with artificial
media under conditions which permitted study of the exit process
in isolation from reuptake. 9 It was found that the application of
electrical pulses resulted in a two- to threefold increase over the
resting release of labeled glycine, GABA, and taurine, a somewhat les-
ser increase in release of labeled glutamic acid, and no sig-
nificant release of labeled urea, alanine, lysine, proline, or
cycloleucine. l7 ,19 The rate of release was not a simple function
of the quantity of labeled amino acids taken up by the tissue. For
example, GABA and taurine were released to the same degree despite
the fact that the uptake of labeled GABA was lS-fold greater than
the uptake of the labeled taurine. 17 Thus, there appeared to be
some degree of specificity of electrically induced release of amino
acids in that those amino acids suspected of having a transmitter
role were preferentially released. When the slices were incubated
and superfused in calcium-free medium containing EDTA, the electri-
cally induced release of [14C] glycine and [3H] GABA was markedly
reduced. 17

The spontaneous release of [14C] glycine from spinal cord

slices was found to be mediated by a process consistent with mem-
brane transport. 9 Efflux was retarded at 4°C and was accelerated
by exchange diffusion w!th unlabeled gl~cine in the superfusing
medium. Exchange of [1 C] glycine or [ H] GABA with amino acids
of other classes was relatively slight when compared with the
de~ree of autoexchange. The release of [14CJ glycine, but not
[1 C] urea, was accelerated by ouabain and cyanide.
AMINO ACID RELEASE FROM SPINAL CORD 437

Mulder and Snyder studied potassium-induced release of

labeled amino acids from spinal cord and cerebral cortex slices. 24
Of interest was the finding that glycine was released by potassium
to a greater extent from the spinal cord than from the cerebral
cortex, while the reverse was found for GABA. As mentioned above,
this difference is not likely to be the result of regional dif-
ferences in uptake of these amino acids. The extent of release of
glutamic acid and aspartic acid was comparable in the two tissues,
while several amino acids not suspected of possessing transmitter
properties were not released by elevated potassium from either
tissue. The potassium-induced release of the transmitter amino
acids was shown to be calcium dependent. In addition, release
appeared to occur from tissue pools of amino acids which were
labeled by sodium dependent, high-affinity transport systems.

The finding that spontaneous release of the transmitter

amino acids from nervous tissue was mediated by membrane transport
prompted the study of the relationship between spontaneous and
stimulus-induced release. It was found that graded acceleration
of the spontaneous release of [14C] glycine from spinal cord slices
by exchange diffusion was accompanied by a graded reduction in
electrically induced release. IO In addition, other factors, such as
ouabain or cyanide exposure, which accelerated the spontaneous
release, reduced or abolished the electrically induced release of
glycine. Similar findings for GABA, aspartic acid, and glutamic
acid in cerebral cortex slices led to a hypothesis that membrane
transport may be rate limiting in the stimulus-induced release of
transmitter amino acids from nervous tissue. 8 ,IO,16

RELEASE OF AMINO ACIDS FROM SPINAL CORD NERVE ENDINGS

One major objection to the use of tissue slice preparations

for study of labeled transmitter release is the uncertainty of the
cellular localization of the labeled solute. It has been estimated
by autoradiography that approximately 70% of the [3H] GABA taken
up by brain slices is localized in nerve terminals 20 , the remainder
presumably being present in neuronal perikarya and glial cells.
Release from these latter elements could influence the interpreta-
tion of results of tissue slice studies.

An approach toward greater homogeneity has been taken by

Bradford and coworkers, who have prepared synaptosomes from the
medulla and spinal cord of rats and have developed methods for
measurement of the endogenous amino acids in these preparations. 26
Medulla/spinal cord synaptosomes were found to have a high rate of
respiration which increased in response to electrical stimulation,
indicating the Viability of the preparation. Both electrical sti-
mulation and elevated potassium concentration induced the differen-
tial release of endogenous glycine, GABA, aspartic acid, and
glutamic aCid, the response being greatly reduced in a calcium-free
438 RoWoPo CUTLER

medium containing EOTA. Of great interest was the finding of a

reduction in the electrically stimulated release of glycine {and
other amino acids} from spinal cord synaptosomes prepared from
animals that had been pretreated with tetanus toxin. These valuable
studies should yield important information on the neurochemical and
pharmacological properties of transmitter amino acid release.

RELEASE OF AMINO ACIDS FROM ISOLATED SPINAL CORD

The hemisected spinal cord of the frog or toad remains

physiologically active for several hours. When dorsal and
ventral roots are preserved, the preparation is potentially useful
for studying amino acid release during synaptic activity. Aprison
reported that [14C] glycine was released from the preloaded, isolated
toad spinal cord when the dorsal roots were electrically stimulated. l
The magnitude of release was proportional to the frequency of stimu-
lation. These results could not be duplicated by Roberts and
Mitchell in their more complete study of amino acid release from
the hemisected toad spinal cord. 27 In their study, the spinal
cord was preloaded with several labeled amino acids and then
superfused. Electrical stimulation of either dorsal roots or
ventral roots failed to induce the release of any of the amino
acids studied. However, when the rostral end of the spinal cord
was stimulated, there was a large increase in the rate of efflux of
labeled glyci~e, GABA, aspartic acid,and glutamic acid. The
release of [14C] glycine was found to increase as the frequency of
stimulation was increased from 5 to 100 Hz. It was substantially
reduced when the spinal cords were incubated and superfused in a
calcium-free medium or when the magnesium concentration in the su-
perfusion medium was increased to 10 mM. As with spinal cord slices,
the specificity of transmitter amino acid release was suggested by
the failure of electrical stimulation to release labeled serine,
threonine, leucine, mannitol, or urea.

RELEASE OF AMINO ACIDS FROM INTACT SPINAL CORD

Jordan and Webster briefly reported results of experiments

on [14CJ glycine release from the intact cat spinal cord. 2l The
central canal of the lumbar cord was perfused in cats with Cl spi-
nal transections. The cord was preloaded with [14C] glycine and
the efflux into artificial CSF was measured. Electrical stimula-
tion of both femoral and sciatic nerves failed to increase the
resting release of [14C] glycine. When p-hydroxymercuribenzoate
was added to the perfusion medium, there was a prominent increase
in the spontaneous release and an additional increase in the sti-
mulus-induced release of [14C] glycine. This agent is known to
block the uptake of glycine by spinal cord slices and to inhibit
the transport of glycine out of the spinal fluid.
AMINO ACID RELEASE FROM SPINAL CORD 439

We have begun to study the release of amino acids from the

intact spinal cord of the rat and have briefly reported our results. 7
A model for perfusion of the spinal subarachnoid space has been
developed and some of the characteristics of bidirectional amino
acid flux between spinal fluid and blood have been examined. II ,13
It is well known that the cerebrospinal fluid contains at its
boundaries (e.g., choroid plexus) biochemical mechanisms for the
removal of a large variety of solutes (organic acids, amino acids,
ions, metabolites, drugs) by specific active transport systems.
The transport site is not confined to the choroid plexus. In
fact, the rate of removal of [14C] glycine was found to be several-
fold greater from the spinal subarachnoid compartment than from
the ventricular compartment of the cat. 25 While the cellular
element involved in transport out of the subarachnoid space has
not been identified, it is of interest that Wright has found uni-
directional active transport of glycine across the isolated arach-
noid membrane of the frog in the direction from CSF to blood. 29 It
is generally accepted that these transport mechanisms serve, at least
in part, to remove waste materials from the brain and to aid in homeo-
stasis of the brain's extracellular chemical environment. It
is possible, therefore, that the high-capacity system for trans-
porting glycine from spinal fluid to blood aids in removing synap-
tically released glycine from the extracellular space. This reason-
ing formed the basis of attempts to measure release of amino acids
from spinal cord to spinal fluid.

Originally, experiments were designed in which the release of

labeled amino acids was examined. It was found during perfusion
of the subarachnoid space with [14C] glycine or [3H] GABA that the
labeled amino acids equilibrated in the total water compartment of
the spinal cord ll , indicating that the method was sufficient for
intracellular loading with these labeled transmitter suspects. The
spontaneous efflux of [14C] glycine from preloaded spinal cord to
glycine-free artificial CSF was analyzed kinetically and found
to consist of three rates of efflux from compartments whose sizes
were consistent with the intracellular (1), extracellular (2), and
spinal fluid (3) compartments (Figure 1). With the use of this
apparently suitable preparation, it was disappointing to find
that electrical stimulation of the sciatic nerves did not modify
the rate of spontaneous efflux of labeled glycine or GABA into the
spinal perfusate (Figure 2). This result was consistent with previ-
ous observations cited above where peripheral nerve stimulation
failed to release labeled glycine from the toad or cat spinal
cords. Because of the possibility that preloading of the spinal
cord from the spinal fluid route failed to label transmitter pools
of glycine, it was desirable to measure the flux of endogenous gly-
cine into sninal fluid. The [3H] dansylation method for amino acid
assay developed by Briel et a1 4 was found to be sufficiently sen-
sitive for this purpose, although only trace amounts of aspartic
and glutamic acids and no GABA could be detected in the perfusate.
440 R.W.P. CUTLER

100
0
0

50

<!>
• <D 56% k ' 002 min·'
~
z 20 ® 2 1 % k ' .040min- '
<i
:;;
w
® 2.3% ~ '. 115 min- '
a:
w
z 10
U
>-
...J
<!>

}'
-=.... 5
~
0

2
,
60
,
90
.
120
.
150
TIME . mi n

Figure 1. Efflux of [14C] glycine from the intact rat spinal cord.
[14C] glycine, 10 ~Ci, was injected into the spinal fluid. After
incubation for 30 min , cisternal-lumbar perfusion with artificial
CSF was carried out (see reference 11). Rate constants and com-
partment sizes were calculated from the desaturation curve.

z 200
0
~
a:
....z
w 15 0
U
z
0
U
w
a.
....
0
0
100
~
w
>
~
...J
50
W
a:

0 ,
0 5 10 15 20
SAMPLE NUMBER

Figure 2. Electrical stimulation of both sciatic nerves (biphasic,

square wave pulses, Smsec, lOrnA) was applied during the washout of
labeled glycine and GABA from the intracellular compartment of the
intact rat spinal cord during subarachnoid perfusion.
AMINO ACID RELEASE FROM SPINAL CORD 441

80 0

o .......... GLYC INE

o 0-----0 ALA I N(
<! Z 600 t:r--l!. SE RI NE
00 c---o liIIle - GL.... cI NE
Z~
- <t
<!:=
W 400
,-"W
ZU
<!Z
1: 0
U u 200 o-CMBA

~ I

o 5 10 15 20
SAMPLE NUMBER

Figure 3. At the point indicated O.lmM p-CMBA was added to the

subarachnoid perfusions medium. The sciatic nerves were electri-
cally stimulated (100 Hz) where indicated by the hatched bar. The
release of endogenous amino acids and the entry of [14C] glycine
from plasma were measured during subarachnoid perf us ions and con-
stant intravenous infusion of [14C] glycine.

The spontaneous entry of glycine and other amino acids was

found to remain relatively constant during perfusion for 2-3 hours. 13
~{hen the proximal end of the transected sciatic nerve was electrically
stimulated at frequencies of 5, 50, or 100 Hz, no increase in the
rate of flux of glycine into the spinal fluid was found. ~en the
perfusate contained p-chloromercuribenzoate (p-CMBA),as in the
experiments of Jordan and Webster 2l , the rate of flux of glycine
and other amino acids into the spinal fluid gradually increased
(Figure 3). The rate of increase was comparable to the increase
in flux of [14C] glycine from blood to CSF, indicating that a gen-
eralized disturbance in blood-CSF permeability had occurred.

However, when the sciatic nerve was stimulated during the time of
action of p-CMBA, an increased rate of release of glycine was in-
duced. Because the glycine was not labeled, it originated in the
spinal cord and was released into the spinal fluid under conditions
which presumably blocked its reuptake. An attempt was then made
to induce the release of glycine into the fluid by blocking its
binding to postsynaptic receptor sites with strychnine. This drug
is not known to affect the uptake of glycine by the tissues. Strych-
nine alone, after intravenous injection, did not alter the resting
flux of glycine, but when strychnine was administered in combina-
tion with simultaneous sciatic nerve stimulation, there was a sig-
nificant release of glycine into the fluid (Figure 4). The enhanced
rate of release continued for at least 40 minutes after nerve stimu-
lation ceased. There was no increase in the rate of release of any
442 R.w.P. CUTLER

180

SAMPLE NUMBER

Figure 4. Release of endogenous glycine from the intact spinal

cord of the rat. Animals undergoing subarachnoid perfusion were
subjected to the following stimuli: electrical stimulation (solid
bar) of the right sciatic nerve at 5Hz (triangle); intravenous
strychnine (arrow), 2.5mg /kg (open circles); combined nerve sti-
mulation and strychnine (closed circles).

I
300
C
"e i
01lio ,5
.. E
"0 ~
E a.
8 u

~ ~- 200
II> ...
<t Z
w w
--' w
~ ~

a
u \
\
~ 'b---'\,
u
~ u
,
100 ....
,
''b''_...D_-"o .......
"'!!
!

II
"'0
!
~ .o IT''----.....,,'''~---....,,
f . .o
5
If
20

o 20 40 60 eo 100 120
PERfUSION Ti ME, min

Figure 5. Increase in rate of release of endogenous glycine with-

out an increase in rate of entry of [14C] glycine from plasma during
spinal subarachnoid perfusion. Strychnine (arrow) and sciatic
nerve stimulation (bar) were applied as indicated.
AMINO ACID RELEASE FROM SPINAL CORD 443

SER IGLU, ALAI

60mM K+
60

o 2 • 10
SAMPLE NUMBER

Figure 6. Release of endogenous amino acids into the spinal fluid

during subarachnoid perfusion with 60mM K+ medium. The results
are the means of six experiments. Values are for lysine (closed
circles), glycine (open circles), and serine (diamonds). The amino
acids given in parentheses followed the general pattern indicated.

of the other amino acids measured -- taurine, serine, glutamine,

lysine, alanine, or arginine -- which represented the principal
amino acids found in the fluid. The released glycine came from
the spinal cord, there being no evidence of enhanced entry of [14C]
glycine from the blood (Figure 5).

When the stimulation frequency was increased to 100Hz, alanine

and taurine were also released into the spinal fluid (44% increase
over resting release), although to a lesser extent than glycine
(117% increase over resting release). It is of interest that strych-
nine antagonizes the depressant action of all three of these amino
acids when they are applied by iontophoresis to the vicinity of
motorneurons. 6 It was not possible to show that glycine release
was dependent upon the presence of calcium. Perfusion with cal-
cium-free medium did not influence the release of glycine, but in
the intact animal, this procedure probably would not deplete the
extracellular space of the spinal cord of its calcium content.
Perfusion with calcium-free medium containing lilim1 magnesium led
to fatal hemorrhage into the spinal fluid.
444 R.W.P. CUTLER

Additional experiments were performed to study the influence

of high concentration of potassium on the release of amino acids
from the spinal cord. The rate of efflux of [3H] glycine from
the preloaded spinal cord was unchanged (K=O.002 min -1) when the
subarachnoid space was perfused with media containing 3~1 or
60mM~. A mixed pattern of release of endogenous amino acids was
found (Figure 6). There was a prominent increase in the perfusate
concentrations of arginine and lysine, a slight, transient increase
in the concentrations of glycine and taurine, and no change in
the concentrations of serine, glutamine, or alanine.

CONCLUSION

Studies of the patterns and properties of release of amino

acids from a variety of spinal cord preparations (synaptosomes,
slices, hemisected toad cord, and intact rat cord) supplement the
biochemical and physiological evidence that glycine is a neurotrans-
mitter. It remains to be shown that glycine is released into a
collecting medium with specific activation of inhibitory neurons.
These experiments seem feasible with the subarachnoid perfusion
experiments discussed here. An important aspect of these experi-
ments was the finding that depolarizing stimuli did not have the
same effect on endogenous spinal cord glycine and on [14C] glycine
previously taken up from the spinal fluid. The difference must
be considered in the interpretation of in vitro experiments with
labeled glycine which are designed to study neurotransmitter release.
It is quite possible that exogenous glycine does not label the
transmitter pool.

REFERENCES

1. Aprison, M.H., Evidence for the release of C14 _glycine from

hemisected toad spinal cord with dorsal root stimulation,
Pharmacologist, 12 (1970) 222.
2. Aprison, M.H., and Werman, R., The distribution of glycine
in cat spinal cord and roots, Life Sci., 4 (1965) 2075-2083.
3. Balcar, V.J., and Johnston, G.A.R., High affinity uptake of
transmitters: studies on the uptake of L-aspartate, GABA,
L-glutamate and glycine in cat spinal cord, J. Neurochem.,
20 (1973) 529-539.
4. Briel, G., Neuhoff, V., and Maier, M., Microanalysis of amino
acids and their determination in biological material using
dansyl-chloride, Hoppe-Seyler's Z. physiol. Chern., 353 (1972)
540-553.
5. Curtis, n.R., Hosli, L., and Johnston, G.A.R., The inhibition
of spinal neurones by glycine, Nature (Lond.), 215 (1967) 1502-
1503.
6. Curtis, n.R., Hasli, L., and Johnston, G.A.R., A pharmacological
study of the depression of spinal neurones by glycine and
related amino acids, Exp. Brain Res., 6 (1968) 1-18.
AMINO ACID RELEASE FROM SPINAL CORD 445

7. Cutler, R.W.P., Glycine release from rat spinal cord in vivo,

Trans. Amer. Soc. Neurochem., 6 (1975) 191. ------
8. Cutler, R.W.P., and Dudzinski, D.S., Release ofl 3H] GABA and
L-14C] glutamic acid from rat cortex slices: the relationship
between the tissue pool size and rates of spontaneous and
electr~ally induced release, Brain Research, 88 (1975) 415-423.
9. Cutler, R.W.P., Hammerstad, J.P., Cornick, L.R., and Murray,
J.E., Efflux of amino acid neurotransmitters from rat spinal
cord slices. I. Factors influencing the spontaneous efflux of
[14CJ glycine and 3H- GABA , Brain Research, 3~ (1971) 337-355.
10. Cutler, R.W.P., Murray, J.E., and Hammerstad, J.P., Role of
mediated transport in the electrically-induced release of[14C]
glycine from rat spinal cord, J. Neurochem., 19 (1972) 539-542.
11. Dudzinski, D.S., and Cutler, R.W.P., Spinal subarachnoid perfus-
ion in the rat: glycine transport from spinal fluid, J. Neurochem.,
22 (1974) 355-361.
12. Davidoff, R.A., Graham, L.T., Shank, R.P., Werman, R., and
Aprison, M.H., Changes in amino acid con~entration associated
with loss of spinal interneurons, J. Neurochem., 14 (1967)
1025-1031.
13. Franklin, G.M., Dudzinski, D.S., and Cutler, R.W.P., Amino acid
transport into the cerebrospinal fluid of the rat, J. Neurochem.,
24 (1975) 367-372.
14. Gottesfeld, Z., Kelly, J.S., and Rayner, C.N., Depletion of
glutamic acid decarboxylase (GAD 1) activity from the spinal
cord following dorsal-root section, J. Physio1., Lond., 231
(1973) 25-26P.
15. Graham, L.T., Shank, R.P., Werman, R., and Aprison, M.H.,
Distribution of some synaptic transmitter suspects in cat spinal
cord: glutamic acid, aspartic acid, y-aminobutyric acid, glycine,
and glutamine, J. Neurochem., 14 (1967) 465-472.
16. Hammerstad, J.P., and Cutler, R.W.P., Efflux of amino acid
neurotransmitters from brain slices: role of membrane transport,
European J. Pharmacol., 20 (1972) 118-121.
17. Hammerstad, J.P., Murray, J.E., and Cutler, R.W.P., Efflux of
amino acid neurotransmitters from rat spinal cord slices.
II. Factors influencing the electrically induced efflux of
:14C: glycine and 3H-GABA, Brain Research, 35 (1971) 357-367.
18. Hopkin, J.M., and Neal, M.J., The release of l4C glycine from
electrically stimulated rat spinal cord slices, Br. J. Pharmac.,
40 (1970) l36-l37P.
19. Hopkin, J., and Neal, M.J., Effect of electrical stimulation
and high potassium concentrations on the efflux of C14 C] glycine
from slices of spinal cord, Br. J. Pharmac., 42 (1971) 215-223.
20. Iversen, L.L. and Bloom, F.E., Studies of the uptake of
3H- GABA and [3 H] glycine in slices and homogenates of rat brain
and spinal cord by electron microscopic autoradiography, Brain
Research, 41 (1972) 131-143. --
446 R.W.P. CUTLER

21. Jordan, C.C., and Webster, R.A., Release of acetylcholine and

l4C-glycine from the cat spinal cord in vivo, Br. J. Pharmac.,
Chemother., 43 (1971) 44lP. - --
22. Jones, I.M., Jordan, C.C., Morton, I.K.M., Stagg, C.J., and
Webester, R.A., The effect of chronic dorsal root section on
the concentration of free amino acids in the rabbit spinal
cord, J. Neurochem., 23 (1974) 1239-1244.
23. Miyata, Y., and Otsuka, M., Distribution of y-aminobutyric acid
in cat spinal cord and the alteration produced by local
ischaemia, J. Neurochem., 19 (1972) 1833-1834.
24. Mulder, A.H., and Snyder, S.H., Potassium-induced release of
amino acids from cerebral cortex and spinal cord slices of
the rat, Brain Research, 76 (1974) 297-308.
25. Murray, J.E., and Cutler, R.W.P., Clearance of glycine from
cat cerebrospinal fluid: faster clearance from spinal subarach-
noid than from ventricular compartment, J. Neurochem., 17
(1970) 703-704.
26. Osborne, R.H., and Bradford, H.F., Patterns of amino acid
release from nerve-endings isolated from spinal cord and
medulla, J. Neurochem., 21 (1973) 407~4l9.
27. Roberts, P.J., and Mithcell, J.F., The release of amino acids
from the hemisected spinal cord during stimulation, J. Neurochem.,
19 (1972) 2473-2481.
28. Raiteri, M., Federico, R., Coletti, A., and Levi, G., Release
and exchange studies' relating to the synaptosomal uptake of
GABA, J. Neurochem., 24 (1975) 1243-1250.
29. Wright, E.M., Active transport of glycine across the frog
arachnoid membrane, Brain Research, 76 (1974) 354-358.
THE DISTRIBUTION OF DRUGS IN THE CENTRAL NERVOUS SYTEM

A.V. Lorenzo and Reynold Spector

Departments of Pharmacology, Medicine, and

Neurology
Harvard Medical School
Boston, Massachusetts

I. INTRODUCTION

The relative exclusion of many drugs from the central nervous

system (CNS) is generally attributed to the blood-brain and blood-
cerebrospinal fluid (CSF) barriers. In the past, this phenomenon
was more or less interpreted as an impedance to the entry of drugs
into the CNS. However, it is now apparent that numerous factors
regulate the entry, as well as the distribution and final concen-
tration of drugs within the CNS 22 For instance, the rate of
penetration from blood into the eNS is not only dependent on the
plasma concentration, the physical-chemical properties 7 ,26, and the
degree of binding of the drug to plasma proteins I5 ,47, but also on
cerebral blood flow and the general permeability of cerebral blood
vessels 8 ,20. Other factors beside the rate of penetration which
affect the distribution of drugs5w~6hin the CNS include regional
vascularity, cerebral blood flow' , the rate of diffusion through
the interstitial spaces 30 and CSF, uptake and metabolism within
neuronal and glial elements, and the rate of efflux or diffusion
from brain to CSF IO or to blood 28 , as well as efflux from CSF to

447
448 A.V. LORENZO AND R. SPECTOR

blood 29 • The role and significance of factors which affect the

penetration of molecules into the CNS will be dealt with in detail
in the chapter by Dr. Oldendorf and will be alluded to by us only
where necessary. The purpose of this chapter is to present the
results of a series of studies conducted in our laboratories, as
well as those of others, which lend support to the concept that the
CSF plays an important role in the distribution and regulation of
drugs within the CNS.

II. DRUG DISTRIBUTION BETWEEN BLOOD AND CSF

In general, the penetration of drugs from blood into the CSF

has been thought to be dependent on the degree of ionization of the
drug and the lipid solubility of the unionized moeity7,26,32.
However, the failure of many drugs to reach the expected concentra-
tion in CSF has suggested the existence of some mechanism which
acts to clear these drugs from the CSF. Evidence that the passage
of some drugs from the CSF to blood is more rapid than in the
opposite direction was first provided by Mayer et a1.27, who
ascribed the loss of drug from the CSF to passive filtration
through the arachnoid villi. However, a second mechanism involving
specialized transport processes was postulated when the loss of
quaternary ammonium compounds from CSF was shown to be greater than
that of inulin35. Iuulin as well as other large molecular weight
compounds and hydrophobic compounds which are not transported are
essentially cleared from the CSF through the arachnoid villi at
rates equivalent to CSF drainage. Since then, numerous drugs have
been shown to be cleared from the CSF presumably by active trans-
port mechanisms comparable to the weak acid transport system first
described by Pappenheimer et al. 29 (Table 1). The significance of

Table 1. Drugs accumulated by the choroid plexus and thought to be

transported from the CSF.
Class Drugs
Primary Amines Norepinephrine, serotonin44
Tertiary Amines Morphine 3 ,43,48; Dihydromorphine, nalorphine,
r~deine, L-methorphan, dextrorphan, levorphan18 ,
; Methadone 17 ; Atropine l ,13,50
Quaternary Amines Hexamethonium, decamethonium, N-methylnicotina-
mide 2 ,45; Methylatropine 13 ,50; Acetylcholine 5l
Organic Acids Iodopyrac~t, phenolsulfonphthalein 29 ; p-amino-
ffPpurate 1l ; Methotrexate34; PenicillinI2,14,40,
; Salicylic aCid 25 ,39; p-Aminosa1icylic acid
38;o-Iodohippurate, iodopamide 14
Lysergic acid diethylamide 52
Aminoglycosides Gentamicin 37
Prostaglandins Prostaglandin E16
DISTRIBUTION OF DRUGS 449

these transport systems in establishing effective concentrations

in the CSF of four therapeutically important drugs has been studied
by us.

1. Salicylic Acid

In man, as in other primates, rabbit and guinea pig. salicylate

is 50 to 90% bound to plasma protein42 . When the concentration of
salicylate in plasma is maintained at about 1 mM or higher, the
concentration in the CSF is approximately 50% that of the unbound
portion in plasma 7 ,26,39. This value is considerably lower than
the theoretically predicted value derived from the following
equation:
CSF 1 + 10PHCSF - pKa

pVHlma (free) 1 + 10pHpiasma - pKa

This equation is based on the concept that the steady state concen-
tration of a drug in the CSF is primarily dependent on the lipid
solubility and the simple diffusion of the undissociated moeity26.
Since the dissociation constant (pKa) for salicylate is 3.0 and the
pH of the CSF is normally a tenth of a pH unit lower than the pH of
plasma (7.31 vs 7.41) the calculated steady state ratio for salicy-
late should be 0.91. The discrepancy between the calculated (91%)
and the experimentally derived value (50%) was first attributed to
the relatively slow entry of salicylate into the CSF coupled with
removal through the arachnoid villi 26 . Evidence that a specific
mechanism which transports salicylate from CSF to blood and con-
tributes to the lower salicylate concentration in CSF has been presented
by us 25 ,39.
The ability of the choroid plexus incubated in vitro to accumu-
late salicylate against a concentration gradient is shown in Figure
1. This process is saturable and dependent on metabolic energy.
Such evidence suggests the presence of a carrier-mediated transport
system and in the past has been generally accepted to indicate that
in vivo the choroid plexus is a locus for the CSF efflux mechanisms
acting to transport drugs from the CSF to blood. The validity of
this assumption can be assessed in part by studying the passage of
drugs from CSF to blood and from blood to CSF during ventriculo-
cisternal perfusions 29 . During ventriculocisternal perfusion
studies with anesthetized cats, the clearance of salicylate from the
CSF was found not to be saturable, nor inhibited by other weak
organic acids such as penicillin or probenecid 39 , even though the
clearance of salicylate from the CSF exceeded that of 125I-human
serum albumin, l4C-inulin and l4C-sucrose (Fig. 2). However, in
rabbits the clearance was saturable, and we concluded that salicy-
late was actively cleared from the CSF by the weak organic acid
transport system but that the affinity (Kt ) for the carrier was
somewhat lower than that exhibited by other drugs (Table 2). The
inability to demonstrate saturation in the cat may have been due to
anesthesia which is known to depress the activity of some choroid
450 A.V. LORENZO AND R. SPECTOR

(6)
12 EXPERIMENTAL 15MIN " OF

It......... ..........
CONDITION UPTAKE CONTROL

10 I
(41 CONTROL
(O.OO/mll)
11.0tO.7 -
I ~, SALICYLATe
{5mMI
3.0!0.3 21.3

I " ......Salicylate
8 -<
95 "No'"
3.1 :!:.0.3 2B.3
I 5" COZ

(6)/ " NO GLUCOSe

d'c
5.7tO.4
2.8:t0.2
52.0
25.0
6
l
I
'" , (6)
DIODRAST
{ImNI
1.5:!:'0.2 13.0

I ' PAH

~
3.3±0.3 30.0
4 I {fOmMl

I MEDIUM
60MIN
C. PLEXUS
HOMOGENATE
SUPERNATE
I ACTIVIlY
(CPll/mll
ACTIVITY ACTIVITY

2 ~ (6)
(CPM/Qm I (CPM/mll

/Inulin 10122 33685 37601

o 10 20 30 40 50 60 TIM£(minlJtes)

Figure 1. Choroid plexus uptake of 14C-sa1icylic acid and 3H-inulin

as a function of time. Choroid plexuses were incubated in artificial
CSF containing 1 mglml glucose, trace concentrations of l4C-sa1icy1-
ic acid and 3H-inulin and at 37 0 C in an atmosphere of 95% 02 and 5%
C02' At the end of the incubation period the plexuses were removed
from the incubation flasks, wiped on a glass slide and homogenized
in distilled water. A1iquots of homogenate and incubating medium
were assayed for 14C activity. Plexus accumulation of salicylate
was corrected for metabolism, binding and extracellular content
using 3H-inulin, and expressed as a tissue to medium ratio (TIM).
Chart on right shows changes in incubation media or conditions that
were imposed to determine competition, energy dependence and extent
of binding of the system. Uptake (Intracellular Cone.lMedium Cone.)

plexus transport systems 1 ,24. This is partly supported by the

observation that the CSF to plasma steady state ratio for unbound
salicylate in unanesthetized animals is approximately 1/5 of that
observed in the anesthetized animal. In Table 3 are indicated the
unidirectional clearances from CSF to blood with tracer concentra-
tions (Ko) and high concentrations (1-5 mM) of drug (Ko s) and from
blood to CSF (Ki ).
DISTRIBUTION OF DRUGS 451

.0

0.8
• • 125I _ HSA
14
C- Sucrose

0.6 • 3H-Salicylate

VjCj- (Vo +Va) Co

0.4 Ko=
Cy

(Vo +Va) Co+KOC y

0.2 Kj =
Cpl

o 20 40 60 80 100 120 140

TIME (minules)

Figure 2. The concentration in the outflow (Co) divided by the

concentration in the inflow (Ci) is plotted vs perfusion time.
Although the recovery of 125I-HSA was quantitative, the steady
state concentration of 125I-HSA in the outflow was 80% of the
inflow concentration due to dilution by newly formed CSF. The
recovery of sucrose and salicylate was less than that of 125I-HSA as
indicated by the lower outflow concentrations at steady state. The
lower recovery of sucrose and salicylate indicates diffusion or
extraction of these substances from the CSF. Efflux (Ko) and influx
(Ki) clearances in milliliters per minute were calculated from the
above equations at steady state from approximately 70 to 140 min-
utes. C represents concentrations of sucrose and salicylate in
counts per minute per milliliter in the inflow (i), outflow (0) or
unbound in the plasma (pI) at 37 0 C.

2. p-Aminosalicylic Acid (PAS)

As shown in Table 2, PAS is accumulated by the isolated chor-

oid plexus incubated in vitro against a concentration gradient (T/M=
6.8 ± 0.6). The uptake process has a saturable and non-saturable
component, is dependent on metabolic energy and is self-saturable.
Indeed, on the basis of the similarity between the transport con-
stants (Kt and Vmax ) for salicylate and PAS, it could be suggested
452 A.V. LORENZO AND R. SPECTOR

Table 2. Kinetics of drug accumulation by the choroid plexus in

vitro.
Kt Vmax
Drug T/M (mM) (pmole/m¥nin) Inhibitors
Salicylate (.OOlmM) 1l.0±0.7 .096 v:ri87 S.S., anoxia, temp
diodrast
p-Aminosalicylate 6.8 ± 0.6 .087 .036 S.S. anoxia, temp
(.005mM) DNP, diodrast,
probenecid,
penicillin

Penicillin (.OOlmM) 10.2 + 1.1 .043 .075 S.S., temp probene-

cid, salicylate,DNP
Gentamicin (2pg/ml) 5.4 + 0.6 51.8+ 1.75+ S.S., DNP, temp
anoxia, kanamycin
Choroid plexuses were obtained from the lateral and 4th ventricles
of cats or rabbits and were incubated at 37 0 C for 15 min (gentamicin,
2 hours) in artificial CSF containing the 14C-Iabelled drugs. The
methods used to obtain the affinity constant (Kt) and transport
maximum (Vmax ) for the saturable transport component are described
in detail in our previous publications 23 ,25,39. (S.S.- self satur-
able; DNP - dinitrophenol).
+The Kt is in ~g/ml and Vmax is in pg/ml/min.

that both drugs are transported by the same system. However, during
ventriculocisternal perfusions in rabbits the non-saturable clearance
of PAS was found to be less than that of salicylate. This was some-
what unexpected since the dissociation constant for PAS (pKa= 4.0)
isgreater than for salicylate. Therefore in CSF, PAS should be less
ionized than salicylate and presumably be able to penetrate from
CSF to blood more readily. This disparity becomes m!)!'e "'pparent
when the clearance from blood to CSF (Ki) (Table 3) is compared for
both drugs. The clearance value for salicylate is some 6 times
greater than for PAS. Since both drugs are bound to plasma protein
to approximately the same extent, the relatively higher influx rate
for salicylate must be attributable to some other factors. Pos-
sibly, the intramolecular hydrogen bonding which converts salicylate
to a chelate (not observed with the para and meta hydroxylated
derivatives of benzoic acid), and/or the interaction with lipids
such as lecithin which markedly enhance the solubility of salicylate
in organic solvents may be the basis for the higher influx rate39.
Also, the classical analysis of Brodie 7 and Mayer et al. 27
which assumes that only undissociated drug can pass through lipid
membranes is known not to be correct l6 ,33, since in the presence of
high concentrations of ionized moeity the relatively small amount of
ionized drug that penetrates across membranes constitutes a signifi-
cant fraction of the total amount (ionized and unionized) that passes
across the membrane. Whatever the explanation, in the same unanes-
DISTRIBUTION OF DRUGS 453

Table 3. Clearance of drugs from and into the CSF during ventricu-
locisternal 2erfusions.
Ko Ko s Ki
K~os Ko/Ki
Drug ~ml/min} ~ml/min}
Salicylic .026 .022 .024 1.2 1.1
.025 .017 1.5
.022 .009 .004 2.4 5.5
.028 .004 .002 7.0 14.0
.011 .0009 12.0
.690 040 17.5
et al. avson and Pollay

thetized preparation (rabbit), the nonsaturable transport of sali-

cylate both into and out of the CSF exceeds the saturable transport
(Table 2). The opposite is true for PAS.
In view of the above data, it would appear reasonable to
suggest that the relatively low levels of PAS in CSF may be attrib-
utable to the slow entry of PAS into CSF coupled to an efflux
mechanism which appears to be at least 5 to 6 times more efficient.
In the case of salicylate, the efflux mechanism 1's relatively less
important and therefore, salicylate achieves higher levels in CSF.

3. Penicillin

The failure of this water soluble cyclic peptide to achieve

therapeutically effective concentrations within the central nervous
system has been recognized for many years. The nearly complete
ionization of its carboxyl group (pKa= 2.6) at pH 7.4 as well as the
drug's extensive binding to plasma proteins (30-65%) act to limit
its rate of entry into brain and CSF. In addition, the level of
penicillin within the CSF which is normally maintained at approxi-
mately 1.0 to 2.0% of the unbound plasma concentration also is
affected by an active transport system which acts to clear penicillin
from the CSF, and possibly brain, to blood. The possible existence
of this efflux transport mechanism within the CSF was first sugges-
ted by Pappenheimer et a1. 29 on the basis that iodopyracet (Diodrast)
and phenolsulfonphthalein (PSP) were transported from CSF to blood
against a concentration gradient. This was later confirmed by
Fishman14 in studies in which the removal of penicillin from the
cisternal fluid was shown to be mediated in part by an efflux
transport process. More extensive studies involving ventriculo-
cisternal perfusions in dogs l2 and in cats and rabbits 40 ,4l demon-
strated the clearance of penicillin from CSF to blood to be against
a concentration gradient and that the process met most of the
criteria ascribed to carrier-mediated transport systems (Tables 2
and 3). The ability of the isolated choroid plexus but not ependyma-
454 A.V. LORENZO AND R. SPECTOR

cortex, incubated in vitro, to concentrate penicillin 12 provides

additional evidence that the choroid plexus is the locus for the
efflux transport mechanism. Whether a similar mechanism is present
at the blood-brain interface remains somewhat unresolved. It may
be argued that if such a mechanism is present at this interface it
would, on the basis of available evidence, be less effective than
that present at the blood-CSF barrier. For instance, it is known
that in the dog, the clearance of penicillin from the eSF to blood
(Ko) is 5 times greater than clearance from blood to CSF (Ki )l2,
while in the rabbit, Ko is some 14 times greater than Ki (Table 3).
Although this type of data is not available for the exchange of
penicillin between blood and brain, the use of probenecid, a known
inhibitor of the weak organic acid transport systems in the CSF, as
well as in other tissues such as the kid ney49, should reveal the
degree of involvement of these systems in regulating the level of
penicillin in the eNS. In the dog, as well as in the rabbit, the
administration of probenecid results in reduction of penicillin
clearance from eSF and consequently to a relative greater increase
in penicillin concentration in the eSF than in plasma l2 ,l4,40. In
contrast, in brain, the increase in penicillin level was not pro-
portional to the increase in plasma so that the brain to plasma
(unbound penicillin) ratio decreased. The decrease in the ratio
was attributed by Fishman 14 to a competition between probenecid and
penicillin for entry into brain. This suggestion impl~es that the
entry of penicillin into the eNS may be carrier-mediated, an assump-
tion supported by the finding that increases in plasma concentration
of penicillin do not lead to proportional increases in brain 21 . An
alternative, more likely explanation for the finding that the
increase in brain penicillin was not proportional to the increase
in the plasma penicillin, is available. Almost certainly, brain,
like plasma, contains non-specific binding sites for penicillin,
and these would become saturated at higher plasma penicillin con-
centrations. This explanation obviates the postulation of carrier-
mediated entry of penicillin into brain. Nevertheless, whatever
the case, the efflux transport mechanism at the brain-blood inter-
face, if present at all, must be less effective than the process at
the eSF-blood interface, since inhibition of the efflux mechanisms
by probenecid at the two interfaces, more than compensates for the
reduced entry of penicillin into the CSF, but not into brain.
The weak acid efflux transport mechanism also plays a role in
regulating the concentration of penicillin within the eSF.during
meningitis. Generally, the increase in CSF penicillin levels which
are observed during meningitis have been attributed to inflammatory
processes which are thought to cause a non-specific increase in the
permeability of the blood-brain and blood-eSF barriers. However,
when l4C-penicillin is injected intracisternally into control and
H. Flu inoculated animals and its clearance from the CSF compared,
it becomes apparent that penicillin clearance from the eSF of H. Flu
inoculated unanesthetized animals is considerably slower even though
the clearance of inulin remains the same (Table 4)41. This would
DISTRIBUTION OF DRUGS 455

suggest that with meningitis the weak acid efflux transport system
is inhibited. The finding that the uptake (T/M) of penicillin by
the choroid plexuses obtained from H. Flu inoculated rabbits was
also inhibited, lends support to this suggestion. The degree of
inhibition does not appear to be related to the severity of the
disease, since the inhibition was similar whether the meningitis
was induced with H. Flu, which produced a mild type of meningitis
that resolved within 5 days, or with S. Aureus, which produced a
severe meningitis that proved fatal within 24-48 hours. Moreover,
the inhibition produced during meningitis was only partial, since
administration of probenecid led to further reduction in the clear-
ance of penicillin from the CSF. As yet we have not resolved the
factor(s) which act to depress the carrier-mediated transport
process of the choroid plexus. The fact that no inhibition was
apparent during ventriculocisternal perfusions (penicillin clearance
(Ke) controls = 0.014 ± .002 ml/min; H. Flu = 0.011 ± .003 ml/min)
could be due to anesthesia as discussed above.

4. Gentamicin

Gentamicin is an organic cation (pKa =7.2) which neither under

normal conditions, nor meningitis, frequently attains therapeutic
levels within the CSF3l. The failure to obtain adequate levels has

Table 4. Penicillin clearance from the CSF and uptake into choroid
plexus in control and H. Flu inoculated rabbits l
CSF Retention! Choroid Plexus 2
Penicillin Inulin 5 min uptake
Group % of in1. dose Day dpm/ml x 104 T/M ratio
Control 4.1 ± 0.2 1 0.85 + 0.19 10.8 + 1.1 (14)
H. Flu 8.9 ± 2.1 1 0.81 ± 0.14 7.5 + 0.7 (16)
3 9.6±1.3 (8)
5 15.8 ± 1.2 (6)
lAnimals were inoculated intracisternally with 5 x 10 7 Hemophilus
influenza type b (H. Flu) in 0.2 ml phosphate buffer. The follow-
ing day, l4c-penicillin and 3H-inulin were injected intraventricu-
larly. The animals were killed 2 hours later and cisternal CSF was
drawn. The amount of penicillin retained in CSF was expressed as
l4C/3H activity in CSF 100
14C/3H activity in injectate x
21n the choroid plexus experiments, choroid plexuses were obtained
from rabbits inoculated with 0.2 ml buffer or H. Flu in buffer
intraCisternally 1, 3 and 5 days after inoculation. The choroid
plexuses were then incubated in artificial CSF containing l4C-peni-
cillin, according to the procedures described in Figure 1.
456 A.V. LORENZO AND R. SPECTOR

20 1 ) - - - - - - - - - - 0 14C ·Penicill in
....
u
c:
0 18
()
E
.2 16
"0
G,)

~
"- 14
u
c:
0
() 12
...RI
::;,
10 14C-p-Amino
G,)
U Salicylic Acid
....c:...
RI
8
.....
UJ
~
«
....el. 14C· Salicylic
:J Acid
~_ _ _ _ _ _ _ 14C-Gentamycin

3H-lnulin
15 30 45 60
TIME (minutes)

Figure 3. Demonstration of the relative ability of the

choroid plexus to concentrate various drugs.

been attributed to poor penetration into the CSF. We decided to

investigate the possibility that gentamicin, much like other cations
such as atropine, methyl atropine, cho1ine 1 , hexamethonin, decame-
thonium and n-methy1nicotineamide Q5 may be cleared from the CSF by
a transport mechanism. As shown in Figure 3, gentamicin is concen-
trated by the isolated choroid plexus incubated in vitro. At a
concentration of 2/Gg m1 in the medium, the TIM ratio is 5.35 0.59 ±
after 2 hours incubation. Since the uptake is dependent on meta-
bolic energy and is reduced approximately 70~by increasing the con-
centration of gentamicin in the medium, it must be assumed that the
uptake involves a carrier-mediated transport process. In this
respect, it is of interest to note that when incubations were
carried out with rabbit CSF, the uptake was only 1.79 0.32, ±
indicating that substances naturally present within the CSF compete
DISTRIBUTION OF DRUGS 457

for the uptake process. The presence of natural substances which

inhibit the cation transport mechanism in eSF has been demonstrated
by Tochino and Schanker 46 • When the uptake of gentamicin by choroid
plexus is compared to that of penicillin it becomes apparent that
the process for gentamicin is considerably less potent (Fig. 3).
Indeed, 2 hours after intraventricular injection of gentamicin, the
amount of l4e-gentamicin present in a cisternal eSF sample was
approximately 71% of the injected dose. When this is compared to
results with penicillin in which the residual amount was 4%, it
becomes evident that gentamicin clearance from eSF by the cation
transport process is not a significant factor in regulating the
levels of this drug within the eSF.

III. DISCUSSION

The failure of many therapeutically important drugs to achieve

adequate levels within the CNS is a problem which still besets
clinicians in the treatment of eNS disease. Limited entry into the
CNS as a result of protein binding, lipid solubility, and extent of
ionization are but a few of the important factors regulating the
level of drugs within the CNS. Active removal of drugs from the
CSF and possibly brain by mechanisms which transport drugs from the
CSF to blood is another factor involved in the regulation of drug
levels within the CNS. For some drugs such as penicillin, the
efflux transport mechanism is of considerable importance, while for
other drugs such as gentamicin, it is less important. However, such
conclusions may be somewhat misleading in that much of the work
performed by us as well as by others was performed with artificial
CSF or other buffers. It is obvious from our studies, in which
endogenous (donor) CSF was used rather than artificial CSF37, that
accumulation of penicillin and gentamicin by the choroid plexus was
significantly reduced. The presence of endogenous substances
within the natural csp46 may very well have important consequences.
Teleologically, the transport system could be said to have consider-
able biological importance, since it provides a means by which the
CNS may be protected from accumulating a variety of potentially
toxic agents of either endogenous or exogenous origin. It is
exactly for this reason that interference with the efflux mechanism
in an att~ toincrease the level of drugs within the CNS must be
approached with caution.
458 A.V. LORENZO AND R. SPECTOR

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 Alterations of
Transport in Pathology
CEREBRAL PERMEABILITY PHENOMENA IN EPILEPSY

Leontino Battistin

C1inica delle Ma1attie Nervose e Menta1i

Universita di Padova, Padova, Italy

In recent years cerebral permeability has become of increasing

interest in neurosciences, and the structural and functional com-
plexity of the brain barriers has been the object of various stud-
ies 15 It is becoming increasingly clear that the movements of
metabolites into and out of brain cells occur through active pro-
cesses of uptake, exit, and exchange; these processes are likely
to play an important role in the control of cerebral metabolite
levels, and therefore in the control of cerebral metabolism and
function 14 , 20, 4, 15.

Brain permeability phenomena have been studied mostly under

physiological conditions, and also during development or under
some pharmacological influences; however, not much investigation
has been done in pathological states, where it is likely that some
kind of alteration may occurl. In convulsive states, where we know
of some membrane phenomena patholo gY l most studies have regarded
metabolite levels or energy supplies 9, and only few have regarded
movements of ions and proteins 11 , 18, finding an increased uptake of
sulfate and of albumin in a number of regions of the cat brain after
convulsions. For amino acids, a facilitated passage of GABA, gluta-
mic acid, glutamine, and glutathione, was found at the site of freez-
ing lesions in the cat brain8 , 23.

The report presented here discusses some aspects of cerebral

permeability to amino acids after Metrazo1-induced convulsions; it
is well known that acute models of epilepsy, like ESK or drug-induced
convulsions, are not the best in studying epilepsy phenomena; never-
theless, such models are currently used in neurochemical research,
since chronic models are more difficult to prepare and to control,

.465
466 L. BATTISTIN

even though they have the great advantage of being much more similar
to human pathology.

In the present chapter, results from our laboratory on uptake

of some amino acids in vivo in the whole brain of the mouse, and in
various regions of the rabbit brain and preliminary results of
uptake in vitro by mouse brain slices will be discussed. Some
reports of these studies have already been presented 6 , 7.

TABLE I. Levels of the free amino acids in the mouse brain

~ moles/g fresh tissue

Control Metrazol

Taurine 6.22 5.41

Aspartic acid 3.31 3.24
Glutamic acid 8.43 8.17
Glycine 1.44 1.52
Alanine 0.60 1.62 **
Isoleucine 0.078 0.112 ~k

Leucine 0.058 0.057

Tyrosine 0.063 0.067
Phenylalanine 0.053 0.072 **
GABA (+NH3) 2.59 4.73
Lysine 0.32 0.39
Histidine 0.11 0.12
Arginine 0.20 0.27

The animals were killed by decapitation; the brain was quickly

removed, frozen, weighed, homogenized in 3 per cent (w/v) of
perchloric acid; the amino acid content of the supernatant
was determined with the Technicon Auto-Analyzer. In the experi-
ment, the mice were injected with 2 mg of Metrazol, killed
immeduately after convulsions, and subsequently treated as the
control group.
Averages of 3-7 experiments are given. * P",0.02 ** P'::::O.OOI
P was calculated by comparing the ~ moles/g brain of experi-
mental and control animals by Student's t-test.

Level and uptake in vivo. The levels of free amino acids in the
mouse brain under physiological conditions and after generalized
convulsions are given in Table I. In the normal brain the levels of
the various compounds are fairly similar to those described elsewhere
5, 16; after convulsions, we found that some amino acids (alanine,
AMINO ACID UPTAKE IN CONVULSIONS 467

isoleucine, and phenylalanine) increased, one (taurine) decreased,

and others did not show any changes in their levels. We were not
able to separate GABA and NH3, so the results for these compounds
should be checked further; similar results were found in previous
investigations, with changes in the levels of alanine, GABA, gluta-
mate, and aspartate in most instances depending on the species and
on the convulsant agent used 19 .

TABLE II. Uptake of L=histidine J L-aspartic acid, and L-methionine

by the mouse bra1n in vivo after convulsions

~ moles/g fresh tissue

Plasma Brain Brain Plasma

L-histidine
o (Control) 0.15 0.17 1.13
o (Metrazol) 0.18
10 (Control) 0.49 0.31 0.63
10 (A) 0.58 0.31 0.53
10 (B) 0.56 0.25 0.45

L-aspartic acid
0 (Control) 0.10 3.27 32.7
0 (Metrazol) 3.43
10 (Control) 4.99 3.77 0.76
10 (A) 9.87 3.33 0.34
10 (B) 8.21 3.29 0.40

L-methionine
o (Control) 0.042 0.061 1.45
o (Metrazol) 0.054 0.073 1.35
10 (Control) 0.41 0.11 0.27
10 (A) 0.55 0.17 0.31
10 (B) 0.52 0.22 0.42

Experimental animals were injected with 2 mg of Metrazol intra-

peritoneally and killed after generalized convulsions; the blood
and the brain were treated as previously described. In A the
amino acid was injected immediately after convulsions, and the
animals were killed at 10 min; in B the amino acid and Metrazol
were injected simultaneously, and again the animals were killed
after 10 min; the animals that did not get convulsions were dis-
carded. In all cases the subsequent treatment was as previously
described. Averages of 3 experiments are given. The amounts of
amino acids injected were histidine 2, aspartic acid 15, and
methionine 2 mmoles.
468 L. BATTISTIN

The changes in alanine are likely to be due to changes in meta-

bolism (pyruvate), while increase in phenylalanine and decrease in
taurine may be due to changes in penetration.

Recently, a marked decrease of taurine at the site of cobalt-

induced epileptogenic lesion and also in human epileptic focus was
found 24 , 25, so that a role of taurine in the pathogenesis of epil-
epsy has been postulated.

Although the endogenous levels of some amino acids may not be

changed following brief convulsions, their uptake through membranes
may be altered. We measured the uptake of L-histidine, L-aspartic
acid, and L-methionine after generalized convulsions, and in control
animals (Table II). Values show that L-histidine and L-methionine
are taken up by the brain fairly rapidly, while L-aspartic acid is 5
not; similar results were found also in our previous study in vivo.
After convulsions, for histidine there was no variation in the uptake
between normal and experimental conditions; similar results were
also found for aspartate, which was not taken up by the brain either
under normal conditions or after convulsions; the uptake of methion-
ine was much higher in the experiment after and during convulsions
than control, and this result was also seen in the brain/plasma
ratio.

TABLE III. Inulin spaces in mouse brain slices after convulsions

Per cent wet weight

medium medium
with glucose without glucose
Control experiment Control experiment

Control 47 46 40 39
L-Aspartic acid 34 35 40 38
L-Glutamic acid 35 36 39 39
L-Lysine 48 45 48 44
Glycine 43 42 43 39
L-Leucine 43 42

Slices were incubated with (C 14] inulin, 40 mg/lOO ml (final

concentration) for 60 min; amino acids were added at the
concentrations used in uptake experiments, that is, 2 ~ moles/
ml; the estimates of inulin space are given as percentage of
wet tissue weight.
Averages of 4-10 experiments.
AMINO ACID UPTAKE IN CONVULSIONS 469

Uptake in vitro. Changes in membranes due to cONvulsions may

be more closely studied by measurement of transport in vitro. Since
we wanted to study the uptake of a number of amino acids by mouse
brain slices taken from animals immediately after they underwent
generalized convulsions, we investigated first the size of extra-
cellular space with inulin in normal and experimental conditions,
and in the presence of various amino acids, representative of the
acidic, basic, and neutral amino acid classes. Table III shows our
results; it can be seen that inulin space decreases significantly
when slices are incubated in the presence of acidic amino acids, as
has been found elsewhere 3 , 10, 17, whereas no differences from
control values were found in the presence of neutral or basic amino
acids; furthermore, a decrease in the inulin space was found when
slices were incubated in a medium without glucose; these results
mean that the absence of glucose in the medium or the presence of
acidic amino acids are the cause of an increased intracellular
swelling, confirming previous studies 9 ; on the other hand, no sig-
nificant differences were found between control and experimental
samples. These results show that convulsions in vivo do not influ-
ence the distribution of water spaces in subsequently incubated
tissue slices.

Changes in methionine uptake in vivo are not paralleled by such

changes in slices as shown by the results of the uptake of a series
of amino acids by mouse brain slices. {Table IV). It can be seen that
at 5 min there is some slight decrease in the rates of uptake of
aspartate and methionine, and a slight increase in the uptake of
lysine; however, in all cases such differences were not statisti-
cally significant; at 60 min no significant variations between
control and experimental samples could be found.

We also studied uptake processes in vitro, incubating slices

in a medium without glucose to see whether the absence of an import-
ant energy supply would affect similarly control and experimental
samples; in fact (Table V), we found an inhibition of the rate of
uptake that was fairly similar in the two conditions, with the except-
ions of lysine and methionine, which showed a minor inhibition of
uptake after convulsions.

Regional uptake in vivo. It is likely that convulsive activity

affects primarily specific areas of the brain. Although very small
parts of tissue cannot be studied with accuracy, some differences in
areas and in amino acids can be studied regionally. Such measure-
ments may reveal differences not detectable by analysis of whole
brain.

The results on physiological distribution of L-histidine in

various regions of the rabbit brain and on the uptake in vivo under
normal conditions, and after convulsions are shown in Table VI. It
can be seen that the physiological level of L-histidine was fairly
homogeneous in the various areas; the uptake of L-histidine after
470 L. BATTISTIN

TABLE IV. Amino acid uptake in vitro after convulsions

Concentrative uptake
5 min 60 min
control experiment control experiment
L-Aspartic acid 10.03 8.47 46.3 44.7
L-Glutamic acid 9.41 10.48 45.7 44.9
Glycine 8.97 8.27 42.5 44.6
L-Alanine 6.68 6.09 32.1 29.8
L-Leucine 2.93 2.65 3.43 3.49
L-Valine 5.09 5.27 7.59 6.89
L-Lysine 2.79 3.54 8.37 9.44
L-Methionine 3.15 2.49 6.28 5.88
L-Histidine 6.16 6.89 38.7 38.9

The slices were incubated at 37 0 C with 2 mM C14 -labeled amino

acid; the concentrative uptake, that is, uptake less medium
(at the end of incubation) was calculated according to the
following formula: (T - MxE/l-E) - M, were T : amino acid
concentrati.on in the tissue (fJ. moles amino acid ml tissue
water), M : amino acid concentratlon in the medium at tl,e end
of incubation (fJ. moles amino acid/ml medium) and E • extra-
cellular (inulin) space of the slice. Averages of 4-5 experi-
ments.

TABLE V. Inhibition of amino acid uptake in vitro after convulsions

in the absence of glucose

Per cent inhibition of concentrati.ve uptake

Control experiment

L-Aspartic acid 75 71
L-Glutamic acid 48 47
Glycine 56 54
L-Valine 73 68
L-Lysine 35 61
L-Methionine 36 45

Amino acid concentration was 2 mM; incubation for 60 min

For experimental details see legend to Table IV.
Averages of 4-5 experiments.
AMINO ACID UPTAKE IN CONVULSIONS 471

30 min was consistent and fairly uniform in the various regions,

being only slightly higher in the occipital areas. After convuls-
ions, the uptake appeared somewhat higher than normal in all the
cortical areas, with a statistically significant difference, where-
as there was no significant difference in the subcortical areas.

TABLE VI. Level and uptake in vivo of L-histidine in various regions

of the rabbit brain before and after convulsions.

~ moles/ g fresh tissue

Regions Control Injected Metrazol

Frontal cortex 0.19 0.55 0.84 ***

***
Temporal cortex 0.19 0.51 0.78
Occipital cortex 0.21 0.73 0.85
Caudate nucleus 0.21 0.61 0.73
Thalamus + Hypothalamus 0.15 0.41 0.54
Mesencephalon 0.14 0.45 0.49
Pons 0.18 0.55 0.62
Medulla oblongata 0.16 0.62 0.69
Cerebellum 0.16 0.46 0.45
Plasma 0.095 0.36 0.29

The rabbits, after anesthesia with chloroform, were killed

by decapitation, the blood was collected in heparinized
dishes, the brain was quickly removed and dissected into
the various regions and the blood, and brain areas were
treated as described elsewhere. The experimental animals
were injected at 0 time with 2mn.oles of L-histidine, and
with the same amount at 15 min; at 30 min the animals were
sacrificed. In the third group, the animals, after being
injected with the amino acid at 0 and 15 min, were injected
i.v. at 20 min with 80 mg of Metrazol, which immediately
induced generalized convulsions; at 30 min they were killed
and treated as already described. The amino acid content was
determined with the microbiological procedure.
Averages of 4-8 experiments. * P~ 0.02. ** P '::'0.005
*** P <0.001. P was calculated by comparing the fl moles/g
brain of injected and Metrazol animals by Student's t-test.

Table VII shows the results on L-aspartic acid. It can be

seen that this amino acid presents a rather homogeneous distribution
in the various regions of the rabbit brain under physiological condi-
tions, differing significantly from what has been found in other
animal species, where aspartic acid levels were very heterogeneous
in the brain 3 , 13, 22 after convulsions, the levels of aspartic
472 L.BATTISTIN

acid appeared slightly increased in all brain regions, particularly

in the subcortical areas, with a statistically significant differ-
ence in the pons.

TABLE VII Level and uptake in vivo of L-aspartic acid in various

regions of the rabbit brain before and after convulsions

~ moles/g fresh tissue

Regions Control Injected Metrazol

Frontal cortex 2.93 2.55 3.13

Temporal cortex 2.79 2.36 3.17
Occipital cortex 2.97 3.02 3.77
Caudate nucleus 1.94 1.84 2.49
Thalamus + Hypothalamus 2.47 2.45 2.95
Mesencephalon 2.79 2.55 3.39
Pons 3.01 2.81 3.77 ~'(

Medulla oblongata 3.19 2.92 3.85

Cerebellum 2.29 2.75 3.12
Plasma 0.094 3.76 4.36

The amount of L-aspartic acid injected was l5mMoles at 0

and 15 min. For further experimental details see Legend
to Table VI. Averages of 4-10 experiments. P< 0.05.

acid appeared slightly increased in all brain 'regions, particularly

in the subcortical areas, with a statistically significant differ-
ence in the pons.

The results on L-methionine are given in Table VIII. It can

be seen that this amino acid is evenly distributed in the various
regions of the rabbit brain, as was found in other species; the net
uptake under physiological conditions was fairly high, particularly
in the cortical areas, where methionine levels were approximately
five times higher than normal after 30 min; after convulsions, the
net uptake in the cortical areas was almost unchanged, whereas in
the subc~rtical regions the uptake was significantly higher than
under normal conditions. The highest degree of uptake occurred in
the cortical areas, while the increase in uptake due to convuls i.ons
occurred in other areas, showing a difference in regional uptake
under normal conditions and following convulsions.

CONCLUSIONS

It is clear that human epilepsy and drug-induced convulsions

are very different conditions; nevertheless, we can take such an
AMINO ACID UPTAKE IN CONVULSIONS 473

acute model of epilepsy as a useful approach for a more extensive

project of investigations on permeability phenomena in epilepsy.
We emphasize this point to avoid any general conclusions from our
results.

Cerebral levels of the free amino acids are somewhat altered

after convulsions; we found a significant increase of alanine,
phenylalanine, and isoleucine; we also found a decrease in taurine
level, as described by other workers 24 , 25, even though our findings
were not as striking as described elsewhere. Nevertheless, we think
that the interest on this amino acid is justified, for both its pec-
uliar distribution in the brain 3 , l2and its regional uptake in vitro
13 Furthermore 2l it seems also that taurine may be an inhibitory
neurotransmitter , and this fact emphasizes the need of a better
understanding of the relationship between taurine and epileptic
phenomena.

TABLE VIII. Level and uptake in vivo of L-methionine in various

regions of the rabbit brain before and after convulsions

~ moles/g fresh tissue

Regions Control Injected Metrazol

Frontal cortex 0.11 0.58 0.66

Temporal cortex 0.13 0.62 0.68
Occipital cortex 0.13 0.72 0.72
Caudate nucleus 0.16 0.49 0.67 ~In'(

Thalamus + Hypothalamus 0.11 0.36 0.53

Mesencephalon 0.11 0.41 O. 59 ,~

Pons 0.12 0.39 0.65 ,'coki'e

Medulla oblongata 0.12 0.44 0.68 *"k

Cerebellum 0.15 0.37 0.62 ";'c*,'C";'(

Plasma 0.068 0.28 0.41

The amount of L-methionine injected was 2 mmoles at 0 and

15 min. For further experimental details see Legend to
Table VI. Averages of 4-12 experiments.
oJ( P < 0.05 *oJ( P < 0.025 *,'d< P -< 0.02 >~oJ<oJ(* P<::0.005

Our results on uptake of amino acids in vivo by the whole brain

after convulsions showed no variations of uptake of L-histidine,
while the uptake of L-methionine was almost doubled. The regional
studies showed that the uptake of L-histidine after convulsions
was higher than normal in cortical areas, while that of L-methionine
474 L. BATTISTIN

was higher in the subcortical areas; in both studies, in the

whole brain and in the various regions, there was no uptake of
L-aspartic acid under physiological circumstances, whereas after
convulsions there seems to be an entry of this amino acid in the
subcortical areas of the rabbit brain. These findings emphasize
the specificity of the effect. Not all amino acids are affected,
and the regional changes of those that are affected varies, per-
haps with each compound influenced in a specific way.

Our findings on the rates of uptake of various amino acids in

~ after convulsions were fairly similar to those found under
normal conditions; there were only some slight differences in the
rates of rapid influx at 5 min, differences that disappeared at 60
min when equilibrium was reached between influx and efflux rates.
The extracellular space was similar in control and experimental
samples; the absence of glucose in the medium affected fairly
similarly the rates of uptake under normal conditions and after
convulsions.

These findings, even though preliminary, seem to indicate

that in vivo there is some alteration in uptake after convulsions,
with an increased entry of the amino acid tested, whereas in vitro
the processes of uptake seem to behave similarly under normal
conditions and after convulsions. This finding suggests the need
for a better studying of the factors that, operating only in living
animals, may have some influence on transport of amino acids; we
are thinking about vascular membranes, blood circulation,energy
supplies, hormonal controls, all factors that have not yet been
investigated in detail under epileptic phenomena.

Uptake by brain slices, especially of the nonessential amino

acids (glutamate, aspartate, glycine, GABA, proline) is much greater
than the penetration of these compounds into brain in vivo. It seems,
at least for these compounds, that the cellular and subcellular trans-
port activity is high while their capillary transport is low or ab-
sent. It is the capillary passage that is rate limiting under most
experimental conditions in vivo, and it is likely that such changes
were observed in our experiments. It is possible that transport
activity is not of maximal capacity in vivo, while it is near max-
imal in slices; thus effects in vivo are more likely to be observed.

It is also interesting to point out that a similar facilitated

passage of substances into the brain after convulsions was always
found in living animals. Increased entry of some amino acids (GABA,
glutamic acid, glutamine, and glutathione) was found at the site of
freezing lesions in the cat brain 8 , 23; increas~entry of sulfate
was foundllin a number of regions of the cat brain during Matrazol-
induced convulsions; an increased uptake of albumin was found 18 in
AMINO ACID UPTAKE IN CONVULSIONS 475

various cat brain areas, particularly in the subcortical areas after

drug-induced convulsions, with the entry of albumin enhanced by in-
creasing the duration of seizure activity.

It seems, at the present time, that cerebral permeability

during convulsions is somewhat altered with a facilitated passage
of substances into the brain, perhaps more in the subcortical
regions; the nature of this alteration remains unknown, even
though it seems that is occurs mainly in living animals rather than
in brain slices; we therefore think that for a better understanding
of the phenomena underlying such alteration in cerebral permeability,
we should also study factors that may influence transport processes
in living animals, such as vascular membranes, hormonal controls,
and energy supplies; clearly, the influence of metabolic needs and
of modified functional activity should also be investigated.

ACKNOWLEDGEMENTS

The investigations reported here were supported in part by

Consiglio Nazionale delle Richerche, Roma, Italy.

We wish to thank Drs.Mary Varotto, Giorgio Zanchin, and Aldo

De Lorenzi for their skillful cooperation.

REFERENCES

1. Bakay, L.,Alterations of the brain barrier system in pathological

states. In A.Lajtha (Ed.), Handbook of Neurochemistry, Vol. VII,
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2. Battistin, L., Grynbaum, A., and Lajtha, A.,Energy dependence of
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3. Battistin, 1., Grynbaum, A., and Lajtha, A., Distribution and
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4. Battistin, L., and Lajtha, A., Regional distribution and movement
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5. Battistin, L., Grynbaum, A., and Lajtha, A.,The uptake of various
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6. Battistin, L., Varotto, M., Zanchin, G.,Trevisan, C., De Lorenzi,
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7. Battistin, L., Varotto, M.,and De Lorenzi, A., Amino acid uptake
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476 L. BATTISTIN

8. Berl, S., Takagaki, G., and Purpura, D.P., Metabolic and pharma-
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10. Cohen, S.R., Blasberg, R., Levi, G., and Lajtha, A., Compart-
mentation of the inulin space in mouse brain slices, J.Neurochem.,
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11. Cutler, R.W.P., Lorenzo, A.V., and Barlow, C.F., Changes in blood-
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in cat brain,J.Neurochem., 19 (1972) pp.43l-435.
13. Kandera, J., Levi, G., and Lajtha, A., Control of cerebral meta-
bolites levels. II. Amino acid uptake and levels in various areas
of the rat brain, Arch.Biochem, 126 (1968)249-260.
14. Lajtha, A., Transport as control mechanism of cerebral metabolite
levels. In A.Lajtha and D.H.Ford (Eds.), Brain Barrier Systems,
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15. Lee, J.C., Evolution in the concept of the blood-brain barrier
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Grune and Stratton, New York, 1971,pp.84-l45.
16. Levi, G., Kandera, J.,Lajtha, A.,Control of cerebral metabolite
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vitro incubation of brain hemispheres. Fluid spaces and amino
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18. Lorenzo, A.V., Shirahige, J., Liang, M., and Barlow, C.F.,
Temporary alteration of cerebrovascular permeability to plasma
protein during drug-induced seizures, Amer.J.Physiol.,223 (1972)
268-277 •
19. Lovell, R.A., Some neurochemical aspects of convulSions, In
A.Lajtha (Ed.), Handbook of Neurochemistry, Vol.6, Plenum Press,
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20. Neame, K.D., Transport, metabolism, and pharmacology of amino
acids in brain. In A.N.Davison and J.Dobbing (Eds.), Applied
Neurochemistry, Blackwell , Oxford, 1968, pp.1l9-l77'.
21. Oja, S.S., and Lahdesmaki, P., Is taurine an inhibitory neuro-
transmitter? Med.Biol., 52 (1974) 138-143.
22. Perry, T.L., Sanders, H.D., Hansen, S., Lesk, D., Kloster, M.,
and Gravlin, L., Free amino acids and related compounds in five
regions of biopsied cat braln,J.Neurochem., 19 (1972) 2651-2656.
23. Purpura, D.P., Girado, M., Smith, T.G., Gomez, J.A., Synaptic
effects of systemic aminobutyric acid in cortical regions of
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(1958) 348-353.
24. Van Gelder, N.M.,and Courtois, A., Close correlation between
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PATHOLOGICAL ASPECTS OF BRAIN TRANSPORT PHENOMENA

Maria Spatz and Igor Klatzo

NINCDS, National Institutes of Health

Bethesda, Maryland 20014

Historically the extravasation of dye compounds such as Evans

blue and fluorescein as dye albumin complexes into the brain have
been t~I ~~d~~t acceptable indicators of blood brain barrier3 (BBB)
injury , , . Subsequently, among radiolabeled substances,
labeled albumin has been most commonly used for the quantitative,
besides the qualitative, e3~l~~tion of BBB permeability to albumin
in pathological conditions ' . More recently, fluorescein
labeled serum protein 30 and horseradish peroxidase 48 (MW 40,000)
have been introduced as tracers for the functional assessment of
BBB status under various circumstances. All of these substances
have a common denominator of enabling the investigation of one of
the many probable parameters of BBB permeability; namely, the pas-
sage of proteins across the membrane barrier. Another dimension
was added in clarifying the selective vulnerability BBB when
altered brain uptake of nutrients was demonstrated in the experi-
mentally damaged BBB55 - 57 • However, for years elaborate quanti-
tative investigations of brain transport phenomenon in disease
have been limited due to lack of suitable methods for such a study.

In this report we will try to review the available information

concerned with some aspects of cerebral transport in pathological
conditions. First, consideration will be given to experimentally
induced BBB injury in order to evaluate one of the barriers to pro-
tein and glucose which physiological properties has been most exten-
sively studied and known 3 ,8,lO,12,15,34,37,39-42,48. These will
include: (a) the effect of mercury on BBB, and (b) the effect of
hyperosmolar perfusate on BBB. Secondly, we will describe and dis-
cuss our recent investigations and studies in progress concerned
with pathological effects on the glucose transport across the BBB
in the synaptosomes and cerebral capillaries. These will comprise:

479
480 M. SPATZ AND I. KLATZO

(1) ischemia, (a) the effect on BBB, (b) the effect on synaptosomes;
(2) the effect of oxygen saturation and pC0 2 tension on the trans-
port from blood to brain, (a) in vivo studies, and (b) in vitro
studies. - -- -- - - -

The Effect of Mercury on BBB

Steinwall reported 20-40% inhibition of l4C glucose brain up-

take with very slight BBB impairment which was demonstrated by the
reduction of the labeled hexose uptake and the presence of slight
extravasation of acid dye in the cerebral hemisphere ipsilateral to
intracarotic perfusion of HgC1 2 in low concentration. On the other
hand, he had shown obvious penetration of both the dye albumin
complex and the l4C hexose into the brain with high concentrations
of HgC12 in the cerebral perfusate 55 ,56. Similar observations were
made using l4C labeled amino acids with the dye albumin complex57 •
In our experiments with the double isotope technique of 01d~ndorf40-42
(which measures the extraction of l4C test substance using H20
internal standard from brain during single microcirculatory passage)
an inhibition of 3H 2-DG (2-deoxy-D-glucose) brain uptake was ob-
served with both low (.01 roM) and high (.08 roM) of HgC12 (brain up-
take index 16.3 + .5 S.E., 29.8 + 3.06 S.E., respectively; control
48.83 + .83 S.E.). The inhibitory effect of the former solution was
equivalent to the maximal self-inhibition of the labeled 2-DG with
the unlabeled (cold) 2-DG in controls (BUr 16.3 + .5 S.E. and 14.35
+ 1.84 S.E., respectively). The Bur of 2-DG was-actually signifi-
cantly higher in rabbits treated with high, as compared to the ones
treated with low concentrations of HgC1 2 . The level of Bur for 3H
2-DG could not be reduced with simultaneous injections of labeled
and cold substrate (29.8 + 3.06 and 26.8 + 3.06, respectively), sug-
gesting an increased passive diffusion of-the glucose analogue into
the brain. We could not demonstrate an increase in BUI abov~ the
control values with this metabolic inhibitor (Passow et al.) 3 except
in grossly hyperemic brain, where BUI was 73%53.

Although no histochemical and ultrastructural correlative studies

are available in this model, but based on other histochemical and
electron microscopic investigations of mercury intoxication, one may
expect that the observed effects of HgC12 in glucose brain uptake
depended on the degree of BBB alteration. The inhibition of the
hexose uptake might occur at the same time as enzymatic reduction
of BBB associated enzymes could have taken place but without greatly
increased endothelial transport of protein 13 ,60. The appearance of
passive diffusion of the labeled hexose correlates well with the
observed extravasation of acid dye and l4C glucose reported by
Steinwall and probably takes place when the capillaries exhibit a
marked mitochondrial degeneration and increased vesicular trans-
port with widening of the endothelial leaflet spaces 60 •
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 481

The Effect of Hyperosmolar Perfusate on BBB

About thirty years ago, Broman and Lindberg-Broman ll suggested

that hyperosmolar solutions caused destruction of cerebrovascular
membranes. Almost three decades later a renewed increased interest
ensued in the investigations of the hyperosmolar effect on BBB.
An augmented uptake of several radiolabeled substances depending on
the molecular size of the components was observed after hyperosmolar
perfusate of rat by Thompson 59 • In another study, Rapoport et al.
described hyperosmolar reversible opening of BBB which was manifested
by extravasation of Evan's blue following topical or intravenous
treatment with hyperosmolar solutions 46 ,47. The authors suggested
that the observed penetration of the various substances above the
normal permeability of these compounds across the BBB is the re-
sult of endothelial cell shrinkage and leakage through open tight
junctions 46 ,59. These suggestions were to some extent substan-
tiated by electronmicroscopic studies with peroxidase reported by
Brightman et al. 9 and Sterrett et al. 58 • Both groups demonstrated
peroxidase spread across the basement membranes, but without evi-
dence of opened tight junctions. Moreover, Sterrett et al. 58 des-
cribed shrinkage of all layers of arterial wall, endothelium,
pericyte, and smooth muscle with an increase in space, while Bright-
man et al. 9 found the peroxidase tracer in the interjunctional pools
between successive tight junctions of some endothelial cells. In
view of these observations and absence of greatly increased number
of pinocytic vesicles, the authors concluded that the transfer of
the peroxidase occurred through reversibly opened endothelial
junctions.

The investigations of the hyperosmolar effect on glucose trans-

port from blood to brain 52 ,53 with the double indicator method of
Oldendorf reveal an increased BUI for 2-DG, except for lactamide
treated animals, as compared to controls (see Table I) determined
10 minutes after intracarotic perfusion. The elevated BUI levels
were inhibited to the same extent or more as compared to the BUI
values of control rabbits perfused with Ringer's solution when the
labeled compound was simultaneously injected with cold substrate.

As may be seen also for Table I, no significant effect on BUI

was seen following intracarotic perfusion of isotonic urea (except
for lactamide) and 40 minutes after hypertonic urea prIfusion.
Similar influence of hypertonic urea was seen on the C 3-0-methyl-
D-glucose (3-MG) BUI (63.75 + 1.85 S.E.) when compared to control
BUI (30.39 + 2.88 S.E.). In-both the experimental and control
rabbits, the l4~ 3-MG BUI could be reduced to the same degree as in
controls when 1 C 3-MG was injected together with various concen-
trations of cold phlorizin (the known inhibitor of glucose carrier
transport) or with 60 roM glucose (cold).
482 M. SPATZ AND I. KLATZO

The obse~~d increase in the saturable BUI of the partially

metabolizable 2-DG and non-metabolizable 8 ,38 3-MG following hyper-
tonic perfusion of rabbit brain (except lactamide) suggests an
augmented rate of unidirectional entry of glucose analogues into
the brain via cerebral capillaries. At this junction, the question
arises whether the BUI changes may be considered equivalent to the
change of the test tracers uptake since the BUI is a ratio of l4C
tracer to the 3H20 uptake "and not an absolute indicator of l4C test
tracer uptake. Any modification of 3H20 uptake produced by the
experimental conditions, whether by changes in blood flow or not,
could alter the BUI without actually changing l4C tracer uptake.
Recently it has been shown that water does not equilibrate freely
with the exchangeable water in the brain and the fractional extrac-
tion of brain water is dependent on cerebral blood flow 20 ,44,45.
As far as cerebral blood flow is concerned, if it was altered by
the experimental conditions, the change was not appreciable and
not evident using radioautography. Although 3H 20 uptake may pos-
sibly have changed, the similar variability of 3 3H 20 uptake control
and experimental brains (1-3% of the injected H20 uptake) suggest
that significant differences did not occur 52 ,53. Although the
mechanism of hyperosmolar stimulation is unknown A similar effects
were reported in adipose tissue and in muscle14,~3. The possible
vasogenic and cytogenic changes which would produce such stimula-
tion were elaborated in a previous paper 52 - to list them again
here would be beyond the scope of this paper.

In conclusion, we presented two examples of an altered BBB

permeability demonstrable by extravasation of Evan's blue dye com-
plex, which apparently have different ultrastructural changes
responsible for abnormal spread of peroxidase across the vascular
channels into the brain. Nevertheless, both are characterized by
increased BBB permeability to protein tracers and a different effect
on glucose transport. As far as the sugar transport is concerned,
HgC1 2 injured BBB leads to decreased glucose brain uptake with the
exception of severe injury in which additional passive diffusion
of the tested labeled hexose took place since the uptake of the
labeled substrate could not be inhibited by unlabeled compound. On
the other hand, the hyperosmolar BBB change, except for lactamide,
results in an increased saturable uptake of labeled glucose ana-
logues from blood to brain. The lactamide effect on glucose trans-
port should be clarified by kinetic studies in order to define its
action as a possible specific inhibitor of the carrier mediated
glucose transport across the BBB.
In the second half of this report, we would like to draw
attention to the experimentally induced pathological conditions
which may produce an altered transport phenomena simultaneously or
in quick succession across different membrane barriers.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 483

Ischemia

A. The Effect on BBB

The BBB permeability has been studied in various ischemic models,

but we will illustrate and discuss the data obtained from cerebral
ischemia produced in Mongolian gerbils subjected to unilateral
common carotic occlusion 25 ,26,29,3l,5l. In this type of ischemia
in the symptom-positive animals 29 , the degree of cerebral injury
depends on the durations of the ischemic insult, which manifesta-
tions are inversely proportional to the time of reestablished
cerebral circulation. Thus, after a short period of brain ischemia,
the extravasation of Evan's blue albumin complex was observed at a
later time than in cerebral ischemia of longer duration. For ex-
ample, all gerbils with left common carotic artery clipping for 3
hours showed blue discoloration of cerebral hemispheres ipsilateral
to occlusion only 3 hours after the clip release, while animals
with 6 hours cerebral occlusion displayed similar changes in 30
minutes after clip release 3l • Ultrastructurally, in symptom-posi-
tive animals, somewhat enhanced transfer of horseradish peroxidase
across the cerebral arterioles, venules and capillaries was seen
with a slightly increased number of endothelial vesicles after 3
hours of brain ischemia and 1 hour of reestablished cerebrovascular
circulation. The gerbils (symptom-positive) subjected to 6 hours
left common carotid artery occlusion and 1 hour release, showed in
the homolateral cerebral hemisphere an increased number of vesicles
containing peroxidase in the endothelium of all three types of ves-
sels with the spread of the tracer from the basement membrane to the
neuropil. In addition, minimal pinocytic uptake of the peroxidase
was seen in the astroglial cells and neurons, as well as swelling
of the astrocytic endfeet and other astrocytes focally. The most
severe changes were found in the neuropil after 18 hours of occlu-
sion and 1 hour release. However, at none of the examined periods
of time were the endothelial cells seen to be damaged and the
peroxidase was never found between the endothelial cells from the
luminal to the first abluminal tight junctions (Westergaard et al. 62 ).
The transport of glucose analogues was studied in the same model
of cerebral ischemia5l , but after 5 minutes of reestablished cerebral
circulation in both the symptom-positive (with histopathologic picture
of ischemic injury) and symptom-negative (without histologic evidence
of cerebral damage) animals. An increased BUI for l4C 3-MG and l4C
2-DG was observed in each group as c~~pared to control. For example,
at 6 hours and 18 hours the BUI for C 2-DG was 80.44 + 1.64 (7)
and 89.32 + 1.23 (5) in symptom-negative, and 81.51 + 7~92 (3) and
91.1 + 2.i7 (3) in symptom-positive g-erbils, respectIvely [control
BUI 51.88 + 1.66 (6)]. Again we would like to mention that the
variability of 3H 0 brain uptake was found in experimental animals
to be similar to the one in control animals (1-3% of 3H20 injectate).
The augmented glucose analogue brain uptake could be
484 M. SPATZ AND I. KLATZO

Table I. The Effect of Various Intracarotic Perfusates on l4C

2-deoxy-D-glucose Brain Uptake
14C Deoxy-D-G1ucose Brain Uptake
Type of Solution No Inhibition Mean Self-Inhibition Mean* % Inhibition
Perfusate ~ S.E.M. ~ S.E.M.

Ringer's 48.83 + 0.83 (10) 14.35 ~ 1.84 (5) 70.6

Isotonic Urea 44.62 :;:: 3.37 (3)
Lactamide 35.10 ! 0.90 (5) 25.0***

Urea 72.92 + 2.32 (14)** 19.95 + 2.02 (4)** 73.7

Acetamide 68.66 :;:: 0.99 (3) 18.67 :;:: 2.26 (4 ) 72.8
Hypertonic NaC1 67.21 :;:: 3.34 (3) 11.48 ! 0.79 (3) 82.9
Lactamide 34.40 ! 0.90 (5) 25.0***
Urea 44.62 ~ 3.37 (3)+

* 60mM unlabeled 2-deoxy-D-glucose

** Number of animals in parentheses
*** Compared with Ringer's perfused animals
+ Determined 40 minutes after hypertonic perfusion

Table II. Brain Uptake of l4C Glucose Analogues and Amino Acids

Percent of Control Brain Uptake Index

14c Test Substance Hypoxia Hypercapnia Hypocapnia

2-Deoxy-D-G1ucose 52 + 1.4 59 + 1.6

3-0-Methyl-D-G1ucose 52 :;:: 1.9 58! 1.3 140 ± 5.1*
L-Leucine 55 + 3.8 137 + 11.9* ll3 + 3.46**
L-Alanine 54 :;:: 5.9 62 :;:: 11.6 III :;:: 10.8**
L-Arginine 48 + 3.3 80:;:: 5.7 130 + 11.6*
L-Histidine 38 ~ 2.8 45 ± 4.3 96:;:: 6.7**

L-Tyrosine 31 + 2.0 51 + 3.5 73 + 5.0

L-Methionine 26 + 1.5 36 + 2.4 77+4.7
D-Leucine 27 ±
3.1 36 ±3.9 101 ±
6.7**
L-Serine 70 + 11 76 + 12 193 + 5.9*
L-G1utamic Acid 104 ±
8.8** 98 ±13.3** 159 ±
19.0*

The injected mixture contained 1.5 ~C of each 3H20 and 14C test
substance in concentrations of .01 - .02 mM except for 3 ~C of
glutamic acid (.04 mM) and serine (.06 mM). Percent of control
Brain Uptake Index values are means ± S.E.M. of 3-9 experiments.
* Increased percent of control BUI. ** No effect.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 485

self and cross inhibited in all the gerbils subjected to cerebral

ischemia with the exception of 18 hours symptom positive animals.
This strongly suggests that the passage of the sugars occurred
by facilitated carrier mediated transport (as defined by Crone 15 ),
except for the 18 hour ischemic symptom positive gerbils. After
18 hours of cerebral ischemia also, the BUI for l4C sucrose doubled
in the symptom positive as compared to controls, and symptom nega-
tive animals (BUI 19.76 + 3.9 (3) and 7.16 + .42 (4), respectively),
and the penetration of E~an's blue complex 3T was seen in the affected
hemispheres. The mechanisms for the apparently increased carrier
mediated transport is unknown, but could have been the result of an
altered blood flow following the release of ischemic occlusion.

These findings and the ultrastructural observations suggest

that severe alteration of BBB permeability may occur without obvi-
ous degeneration of the vascular endothelium and without opening of
the endothelial tight junctions. Nonetheless, the carrier mediated
transport of the BBB remains intact, at least initially after rela-
tively long periods of cerebral ischemia (6 hours).

Before leaving the subject of ischemic effect on BBB, we would

like to add that increased permeability of the BBB was observed
earlier in cerebral ischemia complicated by experimentally induced
hypertension, than by cerebral ischemia or acute hypertension alone.
It is not surprising, since Johansson et al. observed an increased
BBB permeability to Evan's blue complex 27 , while Westergaard et al.
found an increased vesicular endothelial transport 6l in normal brain
due to acute hypertension, which most probably have an additive
effect on the pre-existing stimulated vesicular transport resulting
from cerebral ischemia.

B. The Effect on Synaptosomes

In the last few years, methods became available for obtaining

synaptosomes (pinched off nerve endings) which are enclosed by
membranes arrived from neurons and dendrites exhibiting the bio-
chemical and histological features of in vivo nerve endings and
may mirror the transport properties see; in neurons, axons and
dendrites and therefore, can be useful as a model for studying the
function of these structures under physiologica1 2 ,18,19,24,64,65
and pathological conditions 54 • Moreover, in ischemia characterized
by deprivation of oxygen and glucose, the neurons may be more sensi-
tive than glia cells to each of these substa~ces, since oxidative
metabolism was reported to be greater in the former than the latter
cells. Furthermore, in view of the reported high affinity by
Diamond of 2-DG transport in synaptosomes, which conceivably reflect
a general property of neuronal membrane 19 , it was thought to be of
great importance for the understanding of some cerebral processes
of ischemia to study the effect of ischemic and post-ischemic
period on synaptosomal transport.
.486 M. SPATZ AND I. KLATZO

In symptom positive gerbils, the specific 3H 2-DG synaptosomal

uptake was progressively reduced in ischemia of 30-180 minutes dura-
tion. Although the animals showed a variable percent of control 3H
2-DG uptake (the effect must probably be related to different
vascular abnormalities existing between connective arteries of the
cerebral and vertebral system, but two major groups could be sepa-
rated out showing a decrease from 23-53% and from 45-72% after 30
and 60 minutes of ischemia respectively in the synaptosomes from
the cerebral hemisphere ipsilateral to carotic artery occlusion.
After 3 hours of ischemia, the specific uptake of 3H 2-DG was almost
absent in synaptosomes. Recovery of the synaptosomal transport had
not occurred by addition of metabolites such as Adenosine 5'-Tri-
phosphate (ATP), Adenosine 5'-Diphosphate (ADP), Adenosine 3',5'-
cyclic-monophosphate (cAMP), and phosphoenolpyruvate (PEP) to the
synaptosomes obtained from the brain ischemic for 60 minutes. How-
ever, after the cerebral circulation was reestablished for 5 minutes
following ischemia of 1 and 3 hours duration, the synaptosomes of
four out of two and two out of four animals, respectively, showed
the same uptake values as control sham-operated animals. One hour
after I and 3 hours of ischemia, the recovery of the specific 3H
2-DG uptake was seen in all tested synaptosomes since not only the
uptake values were not different from the controls, but also the
post-ischemic synaptosomal 3H 2-DG uptake could be inhibited to
the same degree as in controls when these synaptosomes were incu-
bated with various concentrations of cold 3-MG. Thus, it appears
that at least initially, the membranes transport f~nction can be
restored following ischemia of 1-3 hours duration54 • This observed
phenomenon could be possibly enhanced by the increased glucose
transport occurring across the BBB51.

The Effect of Oxygen Saturation and pC0 2 Tension on the Transport

From Blood to Brain

The altered transport events in ischemia could be due to mani-

fold of factors. Therefore, we also investigated the effect of
oxygen saturation and C02 tension on the transport of glucose
analogues and amino acids from blood to brain. The model consisted
of two l5-minute controlled ventilation periods: in the first 15
minutes all animals were respirated to stabilize them in the normal
range of blood gas values, and in the second 15 minutes the rabbits
received an appropriate combination of 02, CO 2 and N2 for the desired
stable changes in the blood gases 5 •

In Vivo Studies

The results summarized in Table II were obtained from completed

and unfinished separate studies in rabbits performed by Berson et
al. 5 ,SO and Fujimoto et a1.2l in our laboratory with the 01dendorf
technique. As may be seen from this table, a decreased BUI
(expressed as a percent of the control BUI of each tested substrate)
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 487

14
of C glucose analogues and most of the amino acids was observed
in severe hypoxia SA02 of 15-18% (values derived from rabbit hemo-
globin dissociation curve and corrected for variation in pH4).
The most affected substances appeared to be L-Tyrosine, L-Methionine
and D-Leucine (but the quantitative differences may not be signifi-
cant since precise assessment is not possible by this method) while
little or no effect was seen in the BUI of L-Serine and L-Glutamic
acid, respectively. A similar BUI reduction of these substances
was also found in severe hypercapnia (pCO Z 98-lS0 mmHg) with the
exception of L-Alanine and L-Serine, show~ng only a slight decrease
of BUI, while the BUI of glutamic acid was found normal in this
severe respiratory acidosis (pH 7.1). However, in hypocapnia
(pC02 lS-l8 mmHg) the BUI of L-Tyrosine and L-Methionine appeared
little altered while the other tested compounds revealed the BUI
to be increased or normal. Although a variety of differently
classified amino acids have been screened in this experiment, the
changes observed cannot be attributed to any group39. Only the
glutamic acid, which was unaffected, belongs to the acidic amino
acids. In hypocapnic animals with respiratory alkalosis (pH 7.7S
and pC02 about IS mmHg) the BUI of 3H 3-MG was 3S% higher than the
BUI of controls. Inhibition studies revealed that the hypocapnic
rabbits were more sensitive than the control animals; that is, the
Km or inhibition concentration (cold 3-MG) necessary to decrease
the BUr by SO% was .S mM and S mM, respectively. The control uptake
of all the substances were similar to the BUI reported by Oldendorf 4l
and by different indicator techniques by other investigators 37 ,6S.

Since the uptakes of the listed substances were determined by

the Oldendorf method, which interpretation is much disputed at the
present time, we might consider more carefully the fallacious use
of 3H2 0 as internal standard for determining and interpreting the
uptake of 14C labeled test substances. (1) Both conditions,
hypoxia and hypercapnia, are associated with increased blood flow.
Therefore, according to the results of Raichle's group4S, one
could expect a decreased extraction of 3H20 uptake. However, the
ratio of the injected to the recovered water in most of the experi-
mental brains did not differ significantly from the ratio of the
injected to recovered water in control brains. (2) If an actual
reduction of water uptake had occurred (which was not demonstrable
by our evaluations of this problem) with or without decrease of the
l4C test substance than an increased rather than decreased Bur should
have been seen, since the BUI is calculated as the ratio l4C/3H tissue
uptake divided by the same ratio in the injected mixture:

l4C tissue / 3H tissue

BUI X 100
l4C ~nJectate
. . ..
/ 3H lnJectate
488 M. SPATZ AND I. KLATZO

(3) There is some other evidence to support the contention that the
BUI do not simply reflect blood flow changes: (a) hypercapnia (pC0 2
= 100 mmHg) had been shown in dogs to have a much stronger dilating
effect on cerebral than severe hypoxia 23 , causing possibly a greater
blood flow in hypercapnia. The BUI values in hypercapnia are similar
with one exception, to those observed in hypoxia, Table I. (b)
On the other hand, hypoxia (SA02 less 60%) has been shown in dogs
to abolish autoregulation and results in a passive pressure-flow
relationship23. However, the BUI of 2-DG in severe hypoxia was
found to be independent of a wide range of arterial blood pressure,
which in the absence of autoregulation, would expect to result
in a correspondingly wide range of cerebral blood flow. (c)
Single-step hyperoxia to levels above 300 mmHg has been shown to
decrease cerebral blood flow by at least 20% in man l • In the
rabbit experiments, there were no significant differences in the
BUI found in a similar single-step hyperoxia, and the control
animals 5 • (4) If the BUI of the tested compounds would entirely
reflect the results of uptake caused by increase or decrease of
blood flow, then there should be a more uniform BUI response to
the altered p02 saturation and pC02 tension (Table II).

The mechanisms responsible for the observed decrease in BUI of

glucose analogues remain unclear "(no significant differences in
glucose blood level were found between the experimental and control
animals), since transport of glugose was reported to be independent
of oxygen. However, Betz et al. described a decrease of uni-
directional glucose transport after 10 minutes of anoxia into the
isolated dog bra~~ determined by a different indicator dilution
technique using Na as an intravascular mark~r. These observations,
together with ours, suggest that glucose brain transport may be
indeed energy dependent. On the other hand, most of the tested
amino acids are actively transported and the decreased uptake in
hypoxia is not surprising, but no effect of anoxia (10 min.) on
the uptake of L-Leucine was found in the isolated dog brain by Betz
et al.7.
In severe prolonged hypercapnia, an increased regional (thal-
amus, hypothalamus, midbrain, medulla and ~pinal cord) permeability
to albumin was described by Cutler et al. lb ,17. As far as our
results are concerned, the determinations were performed after
acutely induced pC02 changes (15 minutes) in temporolateral hemi-
sphere only, and therefore, the data cannot be correlated with those
of Cutler. Obviously, more studies are required for the interpreta-
tion of our observations.
Certainly, Raichle et al. 45 studies indicating that butanol is
a flow-limited substance should be used for replacing H20 with
butanol as an internal standard in the double tracer indicator tech-
nique of Oldendorf. Thus, this method will become more reliable
for the assessment of transport phenomena across the BBB in patho-
logical states.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 489

Table III. Effects of Oxygen Saturation and pC0 2 on 2-Deoxy-D-(3H)-

Glucose Uptake by Cerebral Capillaries

Group No. of Range of Range of Mean pC0 2 Mean pH Percent of

Animals paZ SA02 * (mmHg) :+: s. E. Control Uptake
(mmHg) (%) :+: S.E.

Control 3 98-130 100 28.5 + 1.5 7.46 :+: .02 100

Hypoxia 3 15-18 15-18 27.4 + .9 7.45 :+: .02 40

Hypercapnia 3 91-111 100 159 :+: 4.0 7.11 :+: .01 70

Hypocapnia 5 91-111 100 16.5:+:1.2 7.62:+: .04 168

Aliquots of capillary suspension (0.1 ml) containin~ 60-120 u protein

were incubated in duplicate with 2.5 ~C 2-deoxy-D-( H)-glucose in 0.5
ml 1% Albumin-Ringers Solution for 15 minutes at 37 0 C. The final
specific radioactivity of the 2-deoxy-D-(3H)_glucose was 4.8 x 10 6
cpm/~mol.
* Arterial oxygen saturation.

In Vitro Studies

The brain uptake changes could be the result of an altered

transport in capillaries and/or neurons and glia. Since lately,
the isolation of metabolic active cerebral capillaries became
possible, we thought that such a fraction would be of great help
in clarifying certain aspects of transport processes occurring in
physiological and pathological conditions. Hence, pure fraction of
cerebral capillaries free of any other cellular contamination,
histoenzymatically and biochemically active showing saturable 2-DG
uptake, was obtained by Mrsulja et al. 36 using a combin~~ and
modified technique of Joo et al. 28 and Goldstein et al. • In
our first approach we tested the uptake of 3H 2-DG in the capil-
laries from rabbits subjected to the same changes in oxygen satur-
ation and pC0 2 tension as those studied in vivo. Preliminary results
are summarized in Table III showing a greater reduction of 2-DG
uptake in hypoxia than hypercapnia and an i~creased uptake in
hypocapnia when the uptakes were calculated as percent of control
uptake. The effect of an altered p02 saturation and pC02 tension
in this experiment appear to be similar to that obtained by the
in vivo BUI technique of Oldendorf 5 , suggesting that the noted
changes reflected in the BUI of 3H 2-DG have most probably taken
place on the capillary level (Fujimoto et al.). Surely one cannot
equate the uptake of the substances with the permeability properties
of the capillaries in order to clarify the brain uptake of a given
substance from blood to brain.
490 M. SPATZ AND I. KLATZO

It is possible that the capillaries may be permeable to sub-

stances which do not accumulate in the endothelium and these are
probably the ones which cross the BBB by simple diffusion. On the
other hand, the carrier mediated transfer may depend on the capillary
uptake and release into the cerebral tissue. Therefore, one could
have a high uptake in the capillaries with little release into the
brain but back diffusion into the blood. However, the low capillary
uptake would most probably be related directly to the diminished
cellular transport phenomena of a given structure. Based on our
preliminary results, the usefulness of the cerebral capillary
fraction is promising for the study of some parameters of the
transport phenomena occurring in both physiologic and pathologic
states.

In conclusion, the brief review and summary of the transport

phenomena altered by pathological conditions investigated by various
methods illustrates that such studies become and will be more acces-
sible in the future.
SUMMARY

The aim of this paper has been to review and discuss the past
and the recent investigations concerned with the study of cerebral
transport phenomena in pathological conditions which have been
divided into two main parts: (1) the effects of experimentally
induced blood brain barrier (BBB) injury by (a) HgClZ or (b) hyper-
osmolar intracarotic perfusate; and (Z) the effects of ischemia or
of an altered oxygen saturation and pCOZ tension on glucose and/or
amino acids and/or protein transport across the BBB, in the syanpto-
somes and cerebral capillaries. The most important observations
were as follows: (1) HgClZ or hyperosmolar perfusates produced an
increased BBB permeability to protein tracers but the brain up-
take of glucose analogues was found decreased following the former,
and increased (except for lactamide) after the latter treatment.
(Z) (a) In ischemia, the noted increased vesicular transport of
peroxidase, as well as the increased saturable and non-saturable
passage of glucose analogues across the BBB depended on the dura-
tion of cerebral deprivation of blood supply which never resulted
in degeneration of endothelial cells of the brain vessels. (b)
The progressively decreased specific Z-deoxy-D-glucose uptake in
the synaptosomes seen during cerebral ischemia of 30-180 minutes
returned to the level of controls 1 hour after reestablishment
of cerebral circulation. (c) A decrease in brain uptake of glu-
cose analogues and amino acids (with few exceptions) was
observed in severe hypoxia and hypercapnia while an increase
or no change in the brain uptakes was seen in hypocapnia. (d)
Preliminary investigations of the Z-DG uptake by the cerebral
capillaries obtained by fractionation of the brain from animals
subjected to normal or altered oxygen saturation and pC02 tension
suggested that cerebral glucose uptake may be directly related to
its capillary function.
PATHOLOGICAL BLOOD TO BRAIN TRANSPORT 491

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BRAIN DAMAGE AND ORAL INTAKE OF CERTAIN AMINO ACIDS

John W. Olney

Washington University School of Medicine

Department of Psychiatry, St. Louis, Missouri

The primary focus in this chapter will be on glutamate (GLU)

and certain of its structural analogues which I shall refer to as
the excitotoxic amino acids because of specific neuroexcitatory
and neurotoxic properties which characterize these acidic amino
acids as a groupo Brief attention will be given separately to
L-cysteine (CYS) which, when given orally to experimental animals,
induces a different neurotoxic syndrome from that caused by oral
intake of excitotoxic amino acids o Most of the evidence I shall
review pertains to excitotoxic amino acids as a group but derives
from studies on GLU, the prototypic and most studied member of the
groupo

EXCITOTOXIC AMINO ACIDS

GLU neurotoxicity can best be viewed as having species general

characteristics in that GLU-induced lesions have been demonstrated
in the CNS of immature mice, rats, rabbits, guinea pigs, chicks and
rhesus monkeys (reviewed elsewhere 1S ,18)0 The primary form of cellu-
lar pathology induced by GLU following either oral or subcutaneous
administration is acute neuronal necrosis 3 ,9,12,13,16
0 The reaction
is characterized by early pathological changes in dendritic and somal
constituents in the absence of any primary axonal involvement. In
the mouse, the only species studied extensively, the pattern of
lesion distribution following administration of GLU or other excito-
toxic amino acids is as depicted in Fig. 10 Most GLU neurotoxicity
research has been concerned with the arcuate nucleus of the hypo-
thalamus (ARH) both because of its particular vulnerability to GLU-
induced damage and to neuroendocrine disturbances arising therefrom 12 0

Employing microhistochemical techniques to study regional uptake of

GLU in brain, Dr. VoJ o Perez and I have found that the ARH differs
497
498 J.W.OLNEY

Fig. 1. Patterns of GLU and CYS types of brain damage compared.

The regions most vulnerable to GLU, hence the only areas affected
when relatively low doses of GLU are given, are the inner retina
(not shown), the arcuate nucleus of the hypothalamus (ARH) and the
subfornical organ (SFO)o Additional areas affected by high doses of
GLU include the medial preoptic region (PO), rostromedial dendate
hippocampal gyrus (DR), medial habenulum (MH) and superior colliculi
(SC). CYS lesions distribute widely over the cerebral cortex, thala-
mus, hippocampus major and amygdala in a pattern which is curiously
reciprocal to the GLU pattern.

from the brain proper in its tendency to accumulate GLU from blood.
By injecting infant mice soc. with GLU, 2 mg/g, we induced a 4-fold
increase in GLU concentrations in the ARH whereas the immediately ad-
jacent ventromedial nucleus and the more remote lateral nucleus of
the thalamus exhibited no appreciable increase in GLU 2 30 It was
also noted that the time course of GLU uptake and accumulation in
ARH paralleled the time course of the neurodegenerative reaction in
that nucleus. We are currently examining threshold conditions for
inducing necrosis of ARH neurons in infant mice and have obtained
preliminary evidence that (1) acute necrosis of ARH neurons occurs
when ARH levels of GLU become 20% elevated and (2) that conditions
for inducing such ARH levels may be either a transient peaking of
plasma GLU to 20-fold resting levels or a more sustained elevation
at lower, yet to be determined levels (Perez, Olney, Martin and
Cannon, unpublished)u

It is well known that GLU excites central neurons when intro-

duced upon their dendritic or somal surfaces by microelectrophoresis,
and Curtis and colleagues have shown 4 that certain acidic structural
analogues of GLU mimic its excitatory effecto We coined the term
excitotoxic amino acids after finding in recent molecular specificity
studies 17 ,20 that we could reproduce the ARH lesion by SoC o adminis-
tration of GLU excitatory analogues (Table 1) but not by a variety
of control compounds. We also found that several analogues known to
BRAIN DAMAGE BY AMINO ACIDS 499

TABLE I. COMPARATIVE TOXICITY OF GLU AND ITS EXCITATORY ANALOGUES

Compound Potency

L-Glutamic Acid I
D-Glutamic Acid* I
L-Aspartic Acid 1
D-Aspartic Acid 1
L-Cysteine Sulfinic Acid* I
L-Cysteic Acid* 1
DL-Homocysteic Acid* 20
N-Methyl-DL-Aspartic Acid 100
Kainic Acid 200

The severity of ARH damage induced in 10 day-old mice by a 12 mmoles/

kg s.c. dose of L-GLU was assigned a value of 1 and the relative poten-
cy of each other compound was judged according to the s.c. dose re-
quired to induce a lesion of comparable severity. The first 6 corn-
pounds listed were essentially equipotent with L-GLU whereas the lat-
ter three were substantially more powerful (modified from Olney
et al. 17 ,2D). Compounds followed by asterisks were found in sepa-
rate experiments to have markedly reduced or no toxic activity when
administered orally whereas the remainder exhibited 60-90% of their
s.c. activity when given orally. (Olney and Rhee, unpublished).

be more potent than GLU as excitants were similarly more potent as

neurotoxins. These correlations strongly suggest that the toxic ac-
tivity of GLU is mediated by its excitatory recepto~whereas recent
evidence that certain toxic analogues of GLU do not compete with GLU
for its high affinity uptake receptor tends to rule out involvement
of this receptor in GLU neurotoxicity. Several GLU analogues which
have neurotoxic activity when administered subcutaneously have
little or no neurotoxic activity when administered orally (Table 1).
This suggests, and it is not surprising, that the absorption of ex-
citotoxic amino acids from gut into blood is mediated by mechanisms
which do not correspond in their molecular specificities with those
which pertain to uptake of such compounds by ARH.

In recent GLU experiments we compared the oral and s.c. routes

of administration for efficacy in producing the ARH lesion in mice at
several ages (Table 2) and, consistent with prior less systematic ob-
servations from this 12 ,16 and other laboratories 3 ,9,we found that dif-
ferences between the two routes are relatively slight. This is in
keeping with the findings of McLaughlan et al. iD in weanling rats that
plasma levels of GLU rise approximately as high from oral as from s.c.
administration of GLU. That free GLU, unlike GLU contained in food
proteinS, enters blood efficiently after ingestion was shown in early
experiments of Himwich et al. 8 who gave GLU (monosodium salt) orally
to the human adult at 0:2 mg/g body wt and observed a rise in plasma
500 J.W. OLNEY

TABLE 2. ORAL VERSUS SUBCUTANEOUS TOXICITY OF L-GLU AT SEVERAL AGES

Age LEDoral LEDSoc •

(days) (mg/g) (mg/g)

10 0 050 0 35
0

21 1.00 0 080
45 1.50 1.20
60 2000 1.50

The lowest effective dose (LED) at any given age was established by
administering L-GLU either orally or SoCo to mice over a range of
doses and determining histologically the lowest dose which, in > 50%
of the animals treated at that dose (n = 6), produced at least 5 necro-
tic neuronal profiles in a representative section cut through the ARH
at its point of maximal damage (Olney and de Gubareff, unpublished).

GLU to nearly 20-fold resting levels (from 12 to 238 ~moles/100 ml)o

Unneutralized glutamic acid, which is not absorbed as efficiently as
the sodium salt, was nevertheless shown by Bessman et al0 2 to induce
a l2-fold elevation (from 5.4 to 6405 ~moles/100 mi5-inplasma GLU
when given to the human adult at 0 01 mg/g body wto McLaughlan et
al. IO studied plasma GLU curves in weanling rats following ingestion
of free GLU alone and free GLU mixed with food (ground veal) and
found that food accompaniment delayed onset but did not significantly
alter the magnitude of the plasma GLU elevations obtained. Stegink
et al0 26 reported the same finding in neonatal pigs given GLU either
wit~or without infant formula and noted, in addition, that food ac-
companiment caused more sustained plasma GLU elevations o Consistent
with these observations, we have demonstrated in the infant monkey
that oral GLU (1 mg/g) in milk destroys ARH neurons 22 o

In early experiments 16 on infant mice we demonstrated that the

two most commonly ingested excitotoxic amino acids, GLU and ASP, when
administered orally in combination, add to one another's neurotoxi-
city. Elsewhere we demonstrated additive (homergistic) properties
of GLU and ASP in combination with other excitotoxic amino acids 14 0

These observations led us to investigate the toxic potential of As-

partame, a synthetic sweetener recently approved in the United States
for food additive use o Aspartame is a dipeptide which is rapidly
hydrolyzed in the gut and absorbed as its constituent amino acids,
phenylalanine and ASP o A major assumption made in approving Aspar-
tame for food additive use in children's foods was that only the neo-
nate is sensitive to GLU or ASP toxicityo This is contradicted by
such evidence as is available 9 ,12 on adult animals and by evidence
presented here (Table 2) that weanling or older mice do not differ
markedly from infants in their vulnerability to hypothalamic damage
from oral GLUo The appearance of lesions in the 21 and 45 day-old
mouse hypothalamus from oral GLU is depicted in Figs. 2a and b.
BRAIN DAMAGE BY AMINO ACIDS 501

Figs 0 2a-c o A small lesion in the ARH of a 21 day-old mouse (a) and
moderate sized lesion in the ARH of a 45 day-old mouse (b) induced by
oral GLU, 1 and 2 mg/g respectivelyo On the right (c) is a small
lesion in the ARH of a 15 day-old mouse 3 hours following oral Aspar-
tame, 205 mg/g. Neurons rendered necrotic (dark centers surrounded
by clear halos) appear identical to those killed by GLU in Figso 2a
and b (X 200)0 (Olney, Rhee, de Gubareff, unpublished)o

Administering Aspartame by feeding tube to 10-20 day-old mice, we

have found that doses in the range of 2-2 05 mg/g produce the GLU-
type hypothalamic lesion (Fig. 2c), presumably by release of ASP into
blood o In view of the combined toxic potential of GLU and ASP, this
raises questions regarding the use of both GLU and Aspartame as addi-
tives in children.' s foods (see discussion).

Stegink et al. 28 have found in rhesus monkeys that substantial

elevations of~terna1 plasma GLU are required (somewhere between 20
and 70 times resting levels) to cause an increase in plasma GLU
levels of the fetus. This suggests that the primate placenta has
effective barriers or homeostatic mechanisms for preventing fetal
plasma GLU elevations from occurringo Our demonstration 15 that se-
vere GLU-type brain damage can be induced transplacentally in the
fetal monkey does not contradict the Stegink et alo findings, in that
by administering GLU, 2 mg/g intravenously, we-obtained maternal
plasma GLU concentrations nearly 400-fold higher than resting levels.
Stegink et al. 27 also reported that GLU, 001 mg/g, administered orally
to human-adult lactating females does not result in significant
changes in the GLU concentration of breast milko Thus, the human
breast may have a steady state mechanism for regulating the GLU con-
centration of breast milk. It is noteworthy that the concentration
of free GLU in human breast milk is quite low (100-150 ~moles/IOO ml)
so that a suckling human infant weighing 6 kg and ingesting 150 ml
would only receive about 30 ~moles/kg, that is 1/100 the oral dose
(3 mmoles/kg) which damages infant animal brain o
502 J.W.OLNEY

CYSTEINE NEUROTOXICITY

In the course of testing amino acids for neurotoxic poten-

tial,l6,l7 we found one, L-cysteine (CYS) which was unique. Rodents
die a few hours after a single dose of CYS and are more sensitive to
this effect in the 2nd and 3rd weeks of life than at earlier or later
stages. For example, the lethal S.Co dose is approximately 1.5 mg/g
in the 1st postnatal week, 0.5-1.0 mg/g in the 2nd week and 2.5 mg/g
in adulthood. During late gestation and in the 1st postnatal week,
a sublethal dose (1-1.2 mg/g s.c. or 1.5 mg/g orally) given to the
pregnant dam or infant respectively induces a pattern of brain damage
(Fig. 1) different from and more widespread than the GLU-type damage 16 •
The time course of the acutely evolving lesion is slightly delayed
(6-24 hrs) compared to the GLU-type damage (0.5-6 hrs). Whether CYS
is neurotoxic in its own right is uncertain. The CYS type of brain
damage could possibly be explained in terms of CYS distributing widely
across blood brain barriers into areas excitotoxic amino acids cannot
penetrate, then converting to its excitotoxic metabolite, cysteine
sulfinic acid. Cysteine oxidase (cysteine dioxygenase EC 1.13.11.20),
the enzyme which catalyzes the conversion of CYS to cysteine sulfinic
acid, is present in both the adult and infant rodent brainll.

DISCUSSION

The observation that a neuron-necrotizing reaction occurs in ARH

not only when GLU but any of its excitatory analogues is administered
s.c. and that dendritic and somal membranes appear to be the primary
tissue components involved in either the toxic or excitatory response
of neurons to these compounds, suggests that the two phenomena may
have a common mechanism of action and be mediated by a common recep-
tor site. When administered by microelectrophoresis, these compounds
excite neurons in many areas of the CNS and there is preliminary evi-
dence to suggest that neurons throughout the CNS, when directly ex-
posed to these compounds, may be sensitive to their necrotizing ac-
tion 2l ,29. The seeming paradox of GLU being both a specific neuro-
toxin and the most abundant amino acid in brain is best resolved by
viewing GLU as an intracellular anion which only exerts excitatory or
toxic action when present in the extracellular compartment. This is,
of course, consistent with the postulate that GLU is a natural trans-
mitter and that GLU excitotoxic activity is mediated through synap-
ses. Even if GLU excitotoxic activity were mediated at an extra-
synaptic locus on neural membranes, however, the brain would need ef-
ficient homeostatic mechanisms for keeping GLU away from such loci.
Two such mechanisms are known; (1) blood brain barriers which shelter
the extracellular compartment in all but a few brain areas from effec-
tive penetration by exogenous GLU and (2) the high affinity GLU trans-
port system which, by rapid intracellular reuptake of GLU may function
to keep the brain endogenous stores of GLU from accumulating extra-
cellularly. An idiopathic weakness in blood brain barriers would have
BRAIN DAMAGE BY AMINO ACIDS :;03

neurotoxic consequences only when plasma GLU elevations occur. Dr.

M.G. Stemmerman (personal communication) has been following a pedia-
tric patient who may have this type of dysfunction. The child has
a history of petit mal seizures which occur very frequently or not
at all, depending on whether free GLU is used as an additive in her
foods. As discussed in greater detail elsewhere 14 ,15 dysfunction of
the second mechanism, high affinity GLU transport, might result in
a progressive translocation of GLU from the intra to extracellular
compartment of brain with consequent mental or neurological distur-
bances which would be very difficult to trace to GLU. The chances
that cerebral pathology based on a disturbance in excitotoxic amino
acid transport could occur are enhanced, of course, by the fact that
the brain normally contains several excitotoxins (aspartic, cysteine
sulfinic and cysteic acids) in addition to GLU.

Clinical deterioration has been reported in schizophrenic pa-

tients follOWing oral loads of either methionine 24 or CYS25 in com-
bination with MAO inhibitors. The methyl-donor-hypothesis, initial-
ly proposed to explain the methionine effect, does not explain why
CYS, a non-methyl-donor, mimics that effect. The deleterious effects
of either CYS or methionine in schizophrenia could perhaps be ex-
plained better in terms of the demonstrable neurotoxic potential of
CYS itself and several excitotoxic amino acids which are metabolic
products of either CYS or methionine in brain. Whether the reaction
of schizophrenic patients to sulfur amino acid loading implies a
defect state in their mode of transporting or metabolizing such com-
pounds has not been fully explored.

GLU (monosodium glutamate) is widely used as an additive in pro-

cessed foods, including those ingested by immature humans. This prac-
tice has been successfully defended (for regulatory purposes) by the
argument that GLU neurotoxicity occurs as a function of blood brain
barrier maturation and that closure of barriers occurs within weeks
after birth to make GLU a totally safe additive for anyone old enough
to eat processed foods. The problem with this interpretation is that
it ignores potentially important distinctions between barriers per-
taining to special brain regions such as ARH and those pertaining to
the brain proper. Our studies on the ARH of infant mice suggest that
barriers (if any exist) about the ARH either freely admit or perhaps
concentrate GLU from blood23 • In other studies on mice (Table 2) we
have found tha GLU destroys ARH neurons not just in infancy but
throughout life; thus, barriers pertaining to ARH may very well dif-
fer from those pertaining to the brain proper, not just in infancy
but. throughout life. We did find that the dose of GLU required to
damage the ARH increased gradually with increasing age but this could
be explained in non-barrier terms - for example, in terms of a gra-
dual increase in the liver capacity to metabolize GLU. In any event,
the evidence suggests only a gradual increase in dose requirements
with increasing age and, for food safety purposes, this means that
GLU may not be much safer for older infants and children than for
neonates.
504 J.W. OLNEY

It is often represented that GLU "as used" in foods provides a

wide margin of safety. Such claims, being based on adult referents,
are not valid for infants and children who may not only be more sensi-
tive than adults but have a much higher GLU intake/body wt ratio.
For example, when GLU was added to baby foods in 0.6% concentration,
one small jar provided the human infant with 130 mg added GLU/kg body
wt which, when compared to 500 mg/kg, the toxic dose in infant animals,
yields only a 4-fold safety margin. The safety margin for young chil-
dren who ingest foods with similar GLU concentrations and have only a
slightly larger body mass to distribute it over is about 10-fold.
How much that 10-fold margin might be compromised by the introduction
of Aspartame into childrens' foods has yet to be studied. The argu-
ment that GLU is safe for young people because millions of them have
been exposed to high loads without exhibiting signs of acute distress
is totally unacceptable in that experimental animals, including those
which suffer severe enough hypothalamic damage from GLU intake to de-
velop obesity and neuroendocrine disturbances in later life, manifest
no signs of distress at the time arcuate neurons are being destroyed.

It is noteworthy that the primate placenta shields the fetus

from exposure to plasma GLU elevations 2B , that similar protection is
built into the human infant's natural source of nutrition, the mater-
nal breast 27 and that the immature human was further protected prior
to the modern era by the fact that he was weaned late and almost all
of the GLU he ate post-weaning was in the form of food protein which,
unlike free GLU has little tendency to induce significant plasma GLU
elevations S • The potential significance of feeding immature humans
free GLU loads in the range of 100 mg/kg or more can best be appre-
ciated by noting that similar loads induce 12 to 20-fold plasma glu-
tamic acid elevations when ingested by human adults 2 ,8 and that a
transient 20-fold elevation of plasma glutamate is sufficient to in-
duce silent necrosis of ARH neurons in infant mice. In the absence
of oral GLU tolerance data on immature humans, one cannot be confi-
dent that the plasma GLU levels in infants or children would not rise
higher from a given dose of GLU than occurs in the human adult nor
that the marked individual differences noted in oral GLU tolerance
curves among human adults 2 ,8 would not be more extreme during develop-
ment. Whether conditions predisposing the adult to hyperglutamatemia,
such as primary gout 7 or certain viral infections 6 might have a simi-
lar effect on immature humans warrants consideration. These and other
similar unknowns, of course, are the reasons why a 100-fold margin of
safety has traditionally been recommended in the use of identified
toxins in foods.

ACKNOWLEDGMENTS

Supported in part by USPHS Career Development Award MH-38894 to

John W. Olney and USPHS Grants NS-09l56, NS-08909 and DA-00259. I
wish to thank Dr. V.J. Perez for permission to cite recent evidence
arising from collaborative studies conducted in his laboratory.
BRAIN DAMAGE BY AMINO ACIDS 505

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14. Olney, J.W., Occult mechanisms of brain damage. In A. Vernadakis
and N. Weiner (Eds.), Proceedings of Symposium on Drugs and Devel-
oping Brain, Plenum Press, New York, 1974, pp. 489-501.
15. Olney, J.W., Neurotoxic effects of glutamate and related amino
acids. In W.1. Nyhan (Ed.), Heritable Disorders of Amino Acid
Metabolism, John Wiley & Sons, Inc., N.Y o, 1974, pp. 501-512.
160 Olney, J.W. and Ho, 0.1., Brain damage in infant mice from oral
intake of glutamate, aspartate or cysteine. Nature 227 (1970) 609.
17. Olney, J.W., Ho, 0.1. and Rhee, V., Cytotoxic effects of acidic
and sulphur-containing amino acids on the infant mouse central
nervous system. Exp. Br. ~., 14 (1972) 61-76.
18. Olney, J.W., Ho, O.L., Rhee, V. and de Gubareff, T., Neurotoxic
effects of glutamate. New Eng. I. Med. 289 (1973) 1374-1375.
19. Olney, J.W., Ho, 0.1., Rhee, V., Schainker, B., Cysteine brain
damage in infant and fetal rodents. ~~. 45 (1972) 309-313.
506 J.W. OLNEY

20. Olney, J.W., Rhee, V. and Ho, O.L., Kainic acid: A powerful
neurotoxic analogue of glutamate. Brain Res. 77 (1974) 507-512.
21. Olney, J.W., Sharpe, L.G. and de Gubareff, T., Excitotoxic
amino acids (Abstr.) Soc. Neurosciences, 1975.
22. Olney, J.W., Sharpe, r:-and Feigin, R., Glutamate induced brain
damage in primates. I. Neuropath. Expo Neurol. 31 (1972) 464-488.
23. Perez, V.J. and Olney, J.W., Accumulation of glutamic acid in
the arcuate nucleus following subcutaneous administration of the
amino acid. J. Neurochem. 19 (1972) 1777-1781.
24. Pollin, W., Cardon, P. and Kety, S., Schizophrenic patients fed
amino acid loads with Iproniazid. Science 175 (1961) 1365-1366.
25. Spaide, J., Davis, J. and Himwich, J., Plasma amino acids in
schizophrenic patients fed methionine or cysteine loads with a
monoamine oxidase inhibitor. Am. I. C1in. Nutr. 24 (1971) 1053-
1059.
26. Stegink, L., Filer, L., Baker, G., Glutamate metabolism in the
neonatal pig. J. Nutr. 103 (1973) 1138-1145.
27. Stegink, L., FiIer~ and Baker, G. Glutamate effect on plasma
and breast milk amino acid levels in lactating women. Proc. Soc.
Exp. !!£!. Med. 149 (1972) 838-841. --
28. Stegink, L., Pitkin, R., Reynolds, W., Filer, L., Boaz, D.,
Brummel, M., Placental transfer of glutamate and its metabolites
in the primate. Am. I. Obstet. i Gyn. 122 (1975) 70-78.
29. Van Harreveld, A. and Fifkova, E., Light and electronmicroscopic
changes in central nervous tissue after electrophoretic injection
of glutamate. Exp. Mo1ec. Patho1. 15 (1971) 61-81.
PHYSIOPATHOLOGY OF THE BLOOD-BRAIN BARRIER

Michael W. Bradbury

Department of Physiology
King's College
London WC2R 2LS

CEREBRAL OEDEMA AND FLUID MOVEMENT ACROSS CEREBRAL CAPILLARIES

Cerebral oedema is an abnormal accumulation of fluid in the

brain. In gray matter, this excess fluid appears in electron
micrographs to be mainly contained in astrocytes. In white matter
it is largely in the extracellular spaces 17 Since the volume of
the interstitial fluid in brain must necessarily depend on a
balance of fluid exchanges across the cerebral capillaries and
since the astrocytes are part of the blood-brain barrier, cerebral
oedema must be related to a disturbance of fluid exchange at this
interphase. An understanding of this disturbance presupposes a
knowledge of the factors normally determining fluid distribution
across the blood-brain barrier.

In the capillaries of most regions of the body, net fluid

movement has been considered since the classical studies of
Starling and of Landis to depend on the difference between the
hydrostatic pressure gradient favouring ultrafiltration from blood
and the colloid osmotic pressure gradient favouring reabsorption.
Solutes of small molecular weight are likely to have little
influence. They cross the capillary wall freely through water-
filled channels and thus cannot maintain effective osmotic gradients.

The corresponding situation at the blood-brain barrier may be

inferred from three sets of general observations: -

1. The blood-brain barrier is freely permeable to water molecules.

Deuterium oxide administered into the bloodstream equilibrates in

507
508 M.W. BRADBURY

brain in a few seconds 4 • Tritiated water injected into the carotid

artery is 1arge1y21, but not totally extracted during a single
capillary pass through brainS Net move~ent of water across the
blood-brain barrier in response to an induced osmotic gradient also
occurs moderately rapidly, the half time for exchange being about
10-15 min. 13

2. The osmolality of cerebrospinal fluid and of brain tissue is

the same or similar to that of blood, both under normal conditions
and after an osmotic disturbance has been induced in the b100d. 2 ,7

3. Movement of ions and other small polar molecules across the

blood-brain barrier is very restricted, the half times for exchange
of the major ions being several hours 9.

Thus since the blood-brain barrier is almost a perfect semi-

permeable membrane with respect to water on one hand and to polar
solutes on the other, it may be anticipated that the net movement
of water will depend not only on the 'colloid' osmotic pressure
difference ~cross it, but on the total osmolality of the fluids on
either sid. O)f the barriers. Whatever processes active or passive
determine d ltribution of polar solutes, especially the major mono-
valent ions chloride, sodium and potassium across the blood-brain
barrier will secondarily determine the volume of the cerebral fluids.
Hydrostatic pressure gradients across the blood-brain barrier are not
likely to be effective in moving fluid, since they will not provide
enough energy to separate water from its crystalloid solutes. They
might playa role in moving interstitial fluid into CSF spaces or
vice versa.

VOLUME CONTROL OF CEREBRAL FLUIDS

If the blood of an animal is rendered acutely hyponatraemic or

hypernatraemic by the intravenous infusion of appropriate solutions,
the gain or loss of water by brain is about that predicted. Water
shifts across the barrier in amounts sufficient to again make the
cerebral fluids isotonic to blood, the brain swelling in hypotonic
states and shrinking in hypertonic states 11 ,lS.

If hypo- or hypertonicity is maintained in the blood for

several hours, an interesting phenomenon occurs. The water content
of the brain returns towards and may reach its control value despite
a continuing osmotic disturbance in the blood. Yannet 20 subjected
cats to peritoneal lavage with 5% glucose and thus maintained a
considerable hyponatraemia for 22-24 hours. The total water content
of brain was quite unchanged, even in animals in which the combined
concentration of sodium and chloride in serum was reduced by 28%,
Firure 1. Similar observations have been made in various species
7, 8,19. Estimates of the chloride space suggest that not only the
FLUID MOVEMENT AND THE BLOOD BRAIN BARRIER 509

total water, but also the individual volumes of extracellular

fluid and of intracellular fluid are controlled.

90
TOTAL WATER
I + t

"i
a;
~

ICF

..
OJ 60
~ ......
:§
Cl
0

-------.
~
ECF
"-
E 30 .........
a:
w
f-
«
:;:

0
180 210 240 270

SERUM [Na + CI] mM/I.

Figure 1. Total water, extracellular fluid (chloride space)

and intracellular fluid (by difference) in the brains of
cats rendered hyponatraemic for 22-24 hours by peritoneal
lavage with 5% glucose. Drawn from results of Yannet 20

In chronic hypernatraemia, brain water is similarly controlled.

Thus Jelsma and McQueen 16 deprived dogs of water for up to four
days. At the end of this period, skeletal muscle had on the basis
of its dry weight shrunk in volume by 21%, whereas brain on a
similar basis had only shrunk by 10%. The total situation in the
rat is well represented by the comprehensive study of Holliday,
Kalayci and Harrah 15 • These authors produced the four 'corner-stone'
conditions of acute hyponatraemia, acute hypernatraemia, chronic
hyponatraemia and chronic hypernatraemia. In the acute disturbances
the water shifts were about 75% of that predicted from the brain as
an ideal osmometer. The changes in the chronic disturbances were
in marked contrast, little swelling occurring in hyponatraemia and
little shrinkage in hypernatraemia.
510 M.W. BRADBURY

SOLUTE MOVEMENT AND ACCUMULATION AS VOLUME DETERMINANTS

Most workers who have studied the water content of brain during
osmotic disturbances have also estimated the content of the monovalent
ions, chloride, sodium and potassium. As might be expected there is
little net loss or gain of electrolyte during either acute hypona-
traemia or hypernatraemia ll ,15. During the chronic disturbances
there are, however, marked shifts of some or all of the major
electrolytes and no doubt these shifts playa part in volume control.

Yannet 20 demonstrated a considerable loss of both chloride and

sodium from the brain of his hyponatraemic cats. As with water
similar findings have been reported by other authors 18 ,19,7 It
seems likely that the loss of these two ions stabilizes the volume
of the extracellular fluid of brain. Certainly the chloride space
of brain remains constant, although this parameter may overestimate
the true extracellular space.

1
In chronic h pernatraemia sodium and chloride correspondingly
increase in brain 5,7. Again the gain seems just about adequate to
maintain the chloride space constant.

Since potassium is the main intracellular cation in brain, it

might be anticipated that cell volume would be maintained by net
movement of this ion out of or into brain. This is certainly true
of chronic hyponatraemia when big losses of potassium occur from
brain 20 The results of Dila and PappiuslO, Table 1, particularly
clearly show the swelling and lack of potassium change in skeletal
muscle, contrasting with a constancy of volume and large potassium
loss from brain. Presumably there must be an equivalent loss of
mobile anions from the brain cells or else a decrease in the
amount of negative charge on fixed anions; no information to the
author's knowledge exists on this point.

In hypernatraemia, there does not appear to be any net gain

in potassium corresponding to loss of hyponatraemia 14 ,15,7. In
Table 2,7 the gain of sodium and chloride in brain contrasts
markedly with the stability of its potassium content. Finberg,
Luttrell and Redd 14 have postulated that cell volume is maintained
by the creation of 'idiogenic osmoles'. The possible identity of
these will be discussed in the next section.

PRIMARY MECHANISMS OF VOLUME CONTROL

Little is known about the mechanism which causes the shifts

of sodium and chloride and which stabilizes the extracellular fluid
of brain. A first question might be to ask whether the net exchange
initially occurs directly between brain and blood or whether it
first occurs between brain and CSF. It is conceivable that the
FLUID MOVEMENT AND THE BLOOD BRAIN BARRIER 511

TABLE 1

Hyponatraemia in Rats

NORMAL CHANGE %

SERUM
(mM!l or mOsm)

Na 141 + 2 102 + 8 -28

Osm. 328 + 19 254 17 ± -22

SKELETAL MUSCLE
(mM!Kg dry wt)

Na 98 + 8 67 ± 9 -32
K 459 ±29 441 ± 18 -4
Swelling +12

BRAIN

Na 218 + 8 173 + 14 -21

K 490 +
17 401 + 16 -18
Swelling +3

Effect of 2-3 days severe hyponatraemia, induced by ADH 10

injection and 2.5% glucose infusion. From Dila and Pappius.

changes in intracranial pressure induced by an osmotic disturbance

might be accompanied by hydrostatic pressure gradients which
favoured the appropriate movement of either interstitial fluid into
the CSF spaces or CSF into the interstitial fluid spaces. Elliott
and Jasper 12 estimated the change of brain bulk in rabbits after
infusions of hypotonic or hypertonic solutions of glucose. The
swelling and shrinkage of the brain were 2-3 times greater in
animals with the brain exposed than in animals with the skull intact.
Some mechanical effect of the skull is thus suggested.

The loss of potassium from brain cells which occurs in sub-

acute and chronic hyponatremia may be due to mechanisms operating
either at the plasma membranes of the brain cells themselves or at
the blood-brain barrier or at both sites. When mammalian retina is
incubated ~ vitro, a hypotonic medium will induce some net loss of
potassium, particularly if ~he osmolality is 212 mOsm or belowl •
Bradbury, et al. 6 observed little loss of potassium from frog brain
incubated in solutions of less extreme hypotonicity. Estimations
of potassium in the CSF of hyponatremic animals might discriminate
between the effect of a process occurring mainly at brain cell
512 M.W. BRADBURY

TABLE 2

Hypernatraemia in Cats

NORMAL CHANGE '70

PLASMA (mM/ 1) :

Na 144.7 + 3.0 162.2 ± 2.9 12.1

SKELETAL MUSCLE
(mM/Kg dry st)

Cl 50 + 2 46 ±
2 -8.0
Na 75 + 3 74 ± 6 -1.3
K 433 + 14 420 + 5 -3.0
Swelling -13.6

BRAIN

Cl 176 ± 5 206 ± 11 +17.0

Na 250 ± 6 275 + 13 +10.0
K 415 + 8 422 + 6 +0.2
Swelling -4.4

Effect of 48 hours hypernatraemia, induced by peritoneal

lavage with 4% NaCl. From Bradbury and Kleeman 7 •

membranes on one hand and at the barriers between cerebral extra-

cellular fluid on the other. Thus, if hyponatremia were primarily
causing potassium ions to move from cells to extracellular fluid,
the potassium concentration in CSF should increase. On the other
hand, if it were mainly stimulating movement of potassium from
extracellular fluid to blood across one or other of the barriers,
the concentration of potassium in CSF should decrease. The only
observations on this point known to the author are those of Bradbury
and Kleeman 7 • In 6 hyponatremic cats in which sodium concentration
in plasma had been decreased by 18 mM and 15 mM at 3 days and 5 days
respectively, the potassium concentration in cisternal CSF fell
from a mean of 2.63 ± 0.04 mM to 2.31 ± 0.07 mM and 2.37 ± 0.06 mM
respectively.

The decrement in potassium, though small, was highly consistent,

Fig. 2. The potassium concentration in the CSF of hypernatraemic cats
rose slightly and in a less consistent fashion. Possibly a fall in the
concentration of sodium in cerebral extracellular fluids potentiates
potassium pumping across one or other of the major barriers into blood.
Certainly active transport of potassium by ouabain-sensitive pumping
is known to be favoured by a reduction of external sodium in other
systems. Also ventriculocisternal perfusion of a low sodium fluid
FLUID MOVEMENT AND THE BLOOD BRAIN BARRIER 513

will cause a reduction in the potassium concentration in the out-

flowing CSF at the cisterna magna (Fig. 3)8. Whatever the nature of
the mechanism it is a logical major process leading to potassium loss
from brain at the blood-brain of blood-CSF barriers.

3·0

2.0 '---_ _ _- - L_ _ _ _- ' -_ _ _ _- ' - - -

125 150 175 200
Na in c.sJ. (m-equiv/I.)

Figure 2. Variation of potassium concentration (K) with

sodium concentration (Na) in cisternal CSF from osmotically
normal, hyponatraemic (2 and 5 days) and hypernatraemic
(2 days) cats. From Bradbury and Kleeman 7 •

Little is known about the solute or solutes which may increase

in concentration in brain cells in hypernatremia and this may perhaps
be largely responsible for the cell volume adjustment in this
condition. Baxter 3 has analysed a number of amino-acids in the
brain of a toad, Bufo boreus, which is capable of living in both fresh
and brackish water, its main body fluids following the change in the
external osmolality. In 50% sea-water there were up to 150% increases
514 M.W. BRADBURY

in the concentration of certain amino-acids in brain as compared

with their concentrations in the brains of toads kept in fresh
water.

Figure 3. The potassium concentration in the effluent

from bilateral ventricular cisternal perfusion in three
rabbits. The inflowing fluid contained potassium at
2.9 mM (interrupted line). At the arrow a low sodium
fluid (Na 24 roM) was substituted for the normal fluid.
From Bradbury and Stulcova 8 •

CONCLUSION

The volumes of both the extracellular and intracellular fluids

of the brain are well controlled during chronic osmotic disturbances.
The control of extracellular volume is related to shifts of sodium
and chloride ions in and out of brain. The mechanism of these
shifts is largely unknown.

Cellular volume of brain is maintained in hypotonic states by

the loss of potassium from brain. The mechanism is probably
increased by pumping of potassium ions from cerebral extracellular
fluids to blood rather than a primary loss of potassium from the
cells to the interstitial fluid. Control of cell volume of brain
in hypernatraemia is not based on an uptake of potassium into brain.
It may be mediated by the metabolic creation or accumulation of
FLUID MOVEMENT AND THE BLOOD BRAIN BARRIER 515

'idiogenic osmoles'. The identity of such solutes is unknown, but

there is indirect evidence that amino-acids might be involved. A
full understanding of the mechanisms determining net fluid movement
across the blood-brain barrier would appear to be a prerequisite
for the elucidation of the pathophysiology of cerebral oedema.

REFERENCES

1. Ames, A., Isom, J.B., and Nesbett, F.B., Effect of osmotic

changes on water and electrolytes in nervous tissue, J. Physiol.,
177 (1965) 246-262.
2. Arieff, A.I., Kleeman, C.R., Keushkerian, A., and Bagdoyan, H.,
Brain tissue osmolality: method of determination and variations
in hyper- and hypo-osmolar states, J. Lab. Clin. Med. 79
(1972) 334-343.
3. Baxter, C.F., Intrinsic amino acid levels and the blood-brain
barrier. In Progress in Brain Research, 29 (1967) 429-444 ••
4. Bering, E.A., Water exchange of central nervous system and
cerebrospinal fluid, J. Neurosurg. 9 (1952) 275-287.
5. Bolwig, T.G., and Lassen, N.A., The diffusion permeability to
water of the rat blood-brain barrier, Acta Physiol. Scand., 93
(1975) 415-417.
6. Bradbury, M.W.B., Bagdoyan, H., Berberian, A., and Kleeman, C.R.,
Effect of osmolarity on cell water and electrolytes in the
isolated frog brain, Amer. J. Physiol. 215 (1968) 730-735.
7. Bradbury, M.W.B., and Kleeman, C.R., The effect of chronic osmotic
disturbance on the concentrations of cations in cerebrospinal
fluid, J. Physiol. 204 (1969) 181-193.
8. Bradbury, M.W.B., and Stulcova, B., Efflux mechanism contributing
to the stability of the potassium concentration in cerebrospinal
fluid, J. Physiol. 208 (1970) 415-430.
9. Davson, H., Physiology of the cerebrospinal fluid. Churchill,
London, 1967.
10. Dila, C.J., and Pappius, H.M., Cerebral water and electrolytes.
An experimental model of inappropriate secretion of anti-diuretic
hormone, Arch. Neurol. 26 (1972) 85-90.
11. Eichelberger, L., and Roma, M., Water and electrolyte distribution
in blood and tissue in normal dogs following hypotonic saline
injections, Amer. J. Physiol. 159 (1949) 57-66.
12. Elliot, K.A.C., and Jasper, H., Measurement of experimentally
induced brain swelling and shrinkage, Amer. J. Physiol. 157
(1949) 122-129.
13. Fenstermacher, J.D., and Johnson, J.A., Filtration and reflection
coefficients of the rabbit blood-brain barrier. Amer. J. Physiol.
211 (1966) 341-346.
14. Finberg, L., Luttrell, C., and Redd, H., Pathogenesis of lesions
in the nervous system in hypernatremic states. II. Experimental
studies of gross anatomic changes and alterations of chemical
composition of the tissues, Pediatrics, 23 (1959) 46-53 ••
516 M.W. BRADBURY

15. Holliday, M.A., Kalayci, M.N., and Harrah, J., Factors that
limit brain volume changes in response to acute and sustained
hyper- and hyponatremia, J. Clin. Invest., 47 (1968) 1916-1928.
16. Jelsma, L.F., and Mcqueen, J.D., Effect of water restriction
on brain water, J. Neurosurg.,26 (1967) 35-40.
17. Klatzo, I., Neuropathological aspects of brain oedema,
J. Neuropath. Exp. Neurol., 26 (1967) 1-14.
18. Swinyard, E.A., Effect of extracellular electrolyte depletion
on brain electrolyte pattern and electro-shock seizure threshold.
Amer. J. Physiol., 156 (1949) 163-169.
19. Woodbury, D.M., Effect of acute hyponatremia on distribution
of water and electrolytes in various tissues of the rat,
Amer. J. Physiol., 185 (1956) 281-286.
2.0. Yannet, H., Changes in brain resulting from depletion of
extracellular electrolyte, Amer. J. Physiol., 128 (1940)
683-689.
21. Yudilevich, D.L., and Rose, N. De, Blood-brain transfer of
glucose and other molecules measured by rapid indicator
dilution, Amer. J. Physiol., 220 (1971) 841-846.
BRAIN BARRIER PATHOLOGY IN ACUTE ARTERIAL HYPERTENSION

Barbro Johansson

Department of Neurology, University of Goteborg

Sahlgren Hospital, S-4l3 45 Goteborg, Sweden

ACUTE HYPERTENSION INDUCED BY VASOACTIVE SUBSTANCES

Acute hypertension induced by various vasoactive substances

can damage the blood-brain barrier (BBB) to protein in experimental
animals. Thus, the brains from 27 out of 30 cats showed areas of
extravasation of Evans blue-albumin after a systolic blood pressure
increase exceeding 80 mm Hg induced by intravenous injection of
metaraminol 28. Similar lesions are seen in acute hypertension
induced by angiotensin and noradrenaline. Approximately 70 per
cent of rats developed BBB lesions after angiotension-provoked rise
of the mean arterial blood pressure of 50-80 mm Hg 25 whereas a
lower incidence has been reported in rabbits with the same vaso-
active substance 6 The incidence of BBB lesions can be modif-
ied by several factors as will be discussed later. This might help
to explain that some studies have failed to show permeability
increase in acute hypertension (for a review of the literature see
Johansson 1974 22

ACUTE HYPERTENSION INDUCED BY CLAMPING OF THE THORACIC AORTA

Although some earlier studies failed to show increased perm-

eability to protein in hypertension induced by compression of the
thoracic aorta we found rather extensive BBB lesions in 10 out of
13 dogs 29 • Recently we have demonstrated such lesions also in
rats.
RENAL HYPERTENSION

The blood-brain barrier lesions induced by acute hypertension

are similar to those described by Byrom 4 in an extensive study

517
518 B. JOHANSSON

on renal hypertensive rats. He noted focal areas of tryptan blue

extravasation in 87 per cent of rats with what he called '~yper­
tensive crises", Le. rapid increase of the blood pressure com-
bined with clinical symptoms such as epileptic fits. The hyper-
tensive rats with gradual pressure increase and no symptoms did
not show similar permeability disturbances.

THE LOCATION OF BBB LESIONS IN DIFFERENT SPECIES

The distribution of the BBB lesions varies with the species.

Some workers have reported that the protein extravasation occurs
predominently in the arterial boundary zones between the main
cerebral arteries 6,16. Although it is true, particularly in
the rabbit, that there is a tendency for lesions to accumulate in
these areas, they are by no means confined to them. In cats the
most common sites of lesions are the cerebral cortex close to the
midline of the fronto-parieto-occipital lobes but lesions may be
seen in all areas of the cortex and in the basal ganglia and
somewhat less frequentl~ in the cerebellum, pons and the medulla
oblongata. The lesions are usually symmetrical (Fig. 1). In rats
with angiotensin-induced hypertension the most common findings are
small dots evenly distributed over the hemispheres, but more than
one third of the rats have lesions in the cerebellum also. In
most species the primary lesions are practically always confined to
the gray matter although the extravasated tracer can spread to
underlying white matter, particularly when the blood pressure is
kept high for a long period of time. However, rats often have
small lesions in one or both of the olfactory tracts and, occasion-
ally, small spots are seen in the white matter of the cerebellum
and the brain stem. After aortic compression in dogs the most
extensive lesions were seen within the parieto-occipital lobes and
the next in the cerebellum and brain stem. Some edematous spread
occurred to the white matter in most of the brains, probably re-
flecting the longer duration of hypertension in this series.

THE PATHOPHYSIOLOGY OF THE PERMEABILITY DISTURBANCE

The influence of vasoactive substances. Byrom 5 thoroughly

discussed and finally refuted a role for renin in the causation of
the hypertensive BBB lesions. Angiotensin has been claimed to have
a direct effect on arterial and capil~ary permeability as a conse-
quence of endothelial cell contraction34 • While the occurrence of
endothelial contraction has been denied by others, metaraminol and
noradrenaline have been re~orted to increase the vesicular transport
in cerebral vessels lO ,19,4. Even if vasoactive substances
have a permeability increasing effect per se it is hard to explain
the focal character of the BBB lesions in this way. An increased
number of pinocytotic vesicles are found in acute hypertenSion also
in areas with no protein extravasation 19 and large doses of meta-
ALTERATIONS OF THE BARRIER IN HYPERTENSION 519

raminol do not result in BBB lesions if a rapid blood pressure

increase is prevented 21. Moreover, unilateral ligation of one
common carotid artery in rats which does not prevent the blood from
reaching the homolateral hemisphere, prevents BBB lesions on that
same side in angiotensin-induced acute hypertension 26

Fig.l. Coronal section of the brain from a cat killed 30 minutes

after a metaraminol-induced increase of the mean arterial blood
pressure of 80 mm Hg. The extravasation of Evans blue-albumin
usually is less extensive. This cat was pretreated with papaverine,
and vasodilation increases the vulnerability of the cerebral vessels
to acute hypertension 24

Mechanical effects of high intraluminal pressure. Most workers

now agree that it is the increased intraluminal pressure that is the
most important factor. The controversial question has been whether
the lesions are caused by vasospasm or by vasodilation and over-
stretching of the vessels.

A rise in intravascular pressure increases the tone of the

vascular smooth muscles in the resistance vessels and causes
arterial vasoconstriction while a decrease of pressure results in
vasodilatation l • This so-called Bayliss reflex provides the
basis for the myogenic theory of blood flow autoregulation, i.e.
the tendency to keep blood flow constant independent of the perfus-
520 B. JOHANSSON

ion pressure 11 •

Byrom 4 studied the pial arteries under a cranial window in

renal hypertensive rats. He noticed that arteries in rats with
"Hypertensive crises" showed a striking picture with severe con-
strictions alternating with dilatations. He suggested that the
physiological autoregulatory vasoconstrictioIl went out of hand and
resulted in an uncontrolled vasospasm in hypertensive crises and
that this vasospasm led to ischemia and focal edema. Byrom's
hypothesis has been widely quoted 6,35 but he himself has later
in an extensive review discussed the possibility that the dilated
parts of the vessels might be leaking as has been shown to
be the case in mesenteric and retinal vessels 5. The permeability
disturbances occur very rapidly and the tracer Evans blue albumin
can be seen in the vessel walls, particularly in arterioles and
small arteries but also in capillaries, in animals killed by per-
fusion 30 seconds after the rise of the blood pressure 22. As
a short duration ischemia does not damage the blood brain barrier
to protein 2,20 it seems unlikely that vasospasm and ischemia
can be of pathogenic importance for the permeability disturbances
in the early stage of acute hypertension.

A study was conducted to differentiate between the vasoconstr-

ictor effect of metaraminol and the mechanical effect of the intra-
luminal pressure • The effect of a single rapid pressure rise
was compared to that of a graduated (step-wise) rise in cats 2l
Animals exposed to a rapid blood pressure increase showed BBB lesions
in accordance with earlier studies 28. With the stepwise proced-
ure an autoregulatory vasoconstriction presumably took place after
each step. No protein extravasation was seen in the brains from
these animals. In experiments with abrupt blood pressure increase
and blood-brain barrier lesions the sagittal sinus venous pressure
increased in the initial stage when it could not be explained by
cerebral edema. This finding suggests a lack of autoregulation with
high blood flow rather than low. Fog 11 who studied the auto-
regulatory response of pial arteries, noted that a sudden high blood
pressure increase occasionally resulted in vasodilation. He comment-
ed that a large rise in pressure might overcome the power of the
artery to constrict and cause passive dilatation. Giese 17 later
also suggested that these observations indicated a breakdown of the
autoregulation of the cerebral circulation. A number of recent
studies both in animals and in humans have shown that there is an
upper limit of autoregulation, i.e. that the cerebral blood flow
increases at high blood pressure levels 8,36,39,40.

In a study in dogs made hypertensive by clamping of the

thoracic aorta no consistent correlation between BBB lesions and
abolished autoregulation (increase in CBF) was found 19. The CBF
was determined with the radioactive gas elimination method and the
ALTERATIONS OF THE BARRIER IN HYPERTENSION 521

gamma activity registered externally with a single scintillation

detector; the CBF measured thus represented both damaged and non-
damaged areas of the brain. To compare the blood flow in areas
with and without extravasation of sodium fluorescein, 3H- ethanol,
a freely diffusable substance that distributes in the brain in
proportion to blood flow, was infused in cats with acute hypertens-
ion. The animals were killed at the end of the infusion and the
activity of the tracer determined in samples from areas of the cortex
showing BBB lesions and in samples from unstained areas. In cats
killed within the first few minutes after a blood pressure increase
the highest activity, indicating highest blood flow, was found in
BBB damaged areas (Fig.2).

At a later stage of acute hypertension the situation is more

complex; circulatory disturbances may develop due to edema. The
cerebral blood flow in hypertension may show marked regional differ-
ences 7 ,15,30. Although the initial permeability disturbances
are not caused by ischemia, metabolic studies are needed to evaluate
the relative importance of areas with high and low flow for the
total damaging effect on the brain.

C Br

300 % I-

200 % I-

100 % I-

Exp. No . I

Fig.2. Activity of 3H-ethanol in control areas (open columns)

compared to areas with BBB lesions (dotted columns) in cats with
acute hypertension. Each pair of columns represents one animal.
12-22 cortical samples were taken from each brain. Mean value
+ SEM. The mean of control areas in each brain is arbitrarily set
to 100 per cent 23
522 B. JOHANSSON

The studies discussed so far suggest that rapid dilation and

overstretching of the resistance vessels causes the BBB damage.
Further support for this hypothesis was obtained by the following
experimental approach. The tension in the vessel wall is given by
the product of pressure and internal radius divided by the thick-
ness of the vessel wall 13,14 and dilated resistance vessels are
thus exposed to higher tension than non-dilated vessels at rapid
increase of blood pressure. If mechanical stretching were the main
BBB damaging factor, dilated vessels would be more liable to damage
in acute hypertension. Vasodilatation, induced by papaverine in-
jection, in fact increased the vulnerability of the cerebral vessels
to acute hypertension. The BBB lesions were more severe and the
tendency for edema much more pronounced 24. A similar aggravating
effect could be obtained by carbon dioxide-induced vasodilation.
Moreover, vasoconstriction resulting in a smaller radius and thicker
vessel wall, had a preventing effect. Thus none of six hyperventilated
cats with PC02 17-25 mm Hg showed any BBB lesions after a comparable
blood pressure increase (unpublished observations).

The reported results show that the vascular distension caused

by the high intraluminal pressure is the crucial factor in the
pathogenesis of BBB lesions in acute hypertension.

FLUORESCENCE MICROSCOPICAL AND ULTRASTRUCTURAL STUDIES OF THE

BBB LESIONS. LOCATION AND MECHANISMS OF THE VASCULAR DAMAGE

Tryptan blue stained arterioles have been observed in brains

from renal hypertensive monkeys 35 and Giocamelli et al. 18 claim-
ed that only small arteries and arterioles showed increased perme-
ability to peroxidase in renal hypertensive rats.

Evans blue binds to serum albumin in vivo and the dye-protein

complex can be traced in fluorescence microscopy due to its red
fluorescence 38. With this technique the walls of pial and cort-
ical arteries and arterioles were seen to contain Evans blue-albumin
when the animals were killed by perfusion starting as early as 30
seconds after the blood pressure increase 22 Also capillaries
and occasionally venules could be seen to contain the fluorescent
tracer but apparently less frequently particularly in animals with
slight damage. In a few animals with moderate blood pressure increase
and no focal extravasation of Evans blue albumin in gross inspection
some arterioles were fluorescent. This was not seen in the control
animals. The extravasation of the tracer into the brain parenchyma
could either be seen as a diffuse fluorescence of the parenchyma or
as accumulation of the tracer in neurons 22,28,29.

The endothelial cells of the cerebral vessels are normally

joined to one another by tight junctions which constitute the BBB
ALTERATIONS OF THE BARRIER IN HYPERTENSION 523

to protein 2,33. Some vesicular transport of intravenously

injected peroxidase has however been shown to occur in the normal
mouse brain across segments of arterioles in some parts of the
brain 41.

Under pathological conditions the entry of protein could be

either through the junctions or the endothelial cells. In the
latter case it could theoretically take place by pinocytosis, by
diffuse entry into the cells or by focal breaks in the endothelium.
Several studies on different kinds of hypertension have demonstrated
increased pinocytotic activity. However, Giocamelli et al
18 also observed peroxidase in the junctions in the arterioles
and small arteries; they thought that this constituted the main
route of passage for this protein.

Our own studies on rabbits after metaraminol-induced hyper-

tension suggested that peroxidase might pass the endothelial cells
in three ways, i.e. through channels in the cytoplasm, through
transendothelial pinocytosis or through split or incomplete
junctional complexes between endothelial cells 19. The tracer
was seen in junctions only in animals killed within two minutes
after the blood pressure increase and it was not a frequent finding.
Later studies have suggested that passage through channels might be
the most important way as the largest distension of the basement
membrane and the most heavy staining were noted in areas with trans-
endothelial channels. The described pattern was seen in arterioles,
capillaries and, occasionally, in venules. The basement membrane
showed an increased electron capacity particularly in arterioles.
Diffuse staining of the cytoplasm of endothelial cells but not of
the smooth muscle cells was seen in a few arterioles.

THE PROTECTIVE EFFECT OF STRUCTURAL ADAPTATION IN THE RESISTANCE

VESSELS IN ESSENTIAL HYPERTENSION

In essential hypertension adaptive structural changes in the

resistance vessels, mainly in the form of an increased thickness of
the media, result in a decreased lumen/wall ratio 12. This would
be expected to incr~ase the resistance to high intraluminal pressure
unless aneurysms or other wall defects are present. Strains of
spontaneously hypertensive rats (SHR) have been developed in New
Zealand and Japan 31,37. These particular rats are probably the
most appropriate animal models available of essential hypertension
in man.

SHR of a strain with a low incidence of spontaneous cerebral

lesions were compared to normotensive control rats (NCR) after an
angiotensin-induced blood pressure increase 25 Young SHR did not
differ from NCR but SHR older than 5 months rarely developed BBB
lesions.
524 B. JOHANSSON

The cerebral vessels of SHR thus had a higher resistance to

acute hypertens ion as regards BBB damage. The abrupt blood pres·sure
increase did not induce any intracerebral hemorrhages but in another
experimental series one SHR sustained a subarachnoidal hemorrhage
after an angiotensin injection. Some recent substrains of SHR have
a high incidence of cerebral hemorrhage and infarct 32. Studies of
these strains may help to throw some light on the controversial
question of the pathogenesis of intracerebral hemorrhages in
essential hypertension.

THE RELEVANCE OF ANIMAL STUDIES ON ACUTE HYPERTENSION TO THE

CLINICAL SYNDROME OF HYPERTENSIVE ENCEPHALOPATHY

BBB dysfunction to protein tracers in experimental acute

hypertension has been demonstrated in many species e.g. rats,
rabbits, cats, dogs and monkeys. It seems very likely that similar
lesions occur in humans in acute hypertension. In Byrom's renal
hypertensive rats tryptan blue extravasation was found only in
brains from rats with acute pressure rise and with clinical symptoms.
Other investigators using the same experimental model have reported
similar findings 10,18

The clinical syndrome of hypertensive encephalopathy in man

occurs predominantly when normal vessels are exposed to high pressure
such as in eclampsia and with pheochromocytoma or in rapidly devel-
oping renal hypertension. It is, however, rather uncommon in patients
with benign essential hypertension and it is in this group of patients
that adaptive structural changes of resistance vessels can be expected
to be most pronounced.

As mentioned, spontaneously hypertensive rats (SHR) were less

prone to develop BBB lesions at acute pressure rise than control rats.
In an attempt to correlate clinical symptoms with BBB lesions the blood
pressure was increased by angiotensin in unanesthetized animals (12
NCR and 10 SHR) 27 Neither neurological symptoms nor BBB lesions
were seen in the SHR. Six of the NCR developed generalized convulsions
and their brains all showed BBB lesions. Three normotensive rats without
convulsions showed discrete BBB lesions. Convulsions were never ob-
served in animals whose brains did not show tracer extravasation.
These observations are in agreement with the hypothesis that acute
hypertensive crises in persons with non-adapted blood vessels are
connected with permeability changes in the brain vessels.

CONCLUSIONS

According to a number of recent investigations the pathomecha-

nism of the BBB lesions in acute hypertension is rapid distension -
over-stretching of the vessels. Small arteries and arterioles, but
also capillaries, are promptly damaged. The vascular tone of the
ALTERATIONS OF THE BARRIER IN HYPERTENSION 525

resistance vessels is of importance; vasodilation has an aggrava-

ting and vasoconstriction a preventing effect. The cerebral
vessels of spontaneously hypertensive rats have .an increased
tolerance to acute pressure increase, presumably due to structural
adaptation. This is in line with the clinical observation that
acute hypertensive crises mainly occur when normal vessels are
exposed to high pressure such as in eclampsia or when the structural
adapt ion may be less developed such as in malignant hypertension of
short duration.

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526 B. JOHANSSON

12. Folkow, B., Hallback, M., Lundgren, Y., Sivertsson, R., and
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15. Gannushkina, I.V., Shafranova, V.P., Dad iany , L.N., and Ga1ayda,
T.V., Mechanisms related to decrease of CBF during acute in-
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17. Giese, J., The pathogenesis of hypertensive vascular disease,
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19. Hansson, H.A., Johansson, B., and Blomstrand, C., Ultrastructural
studies on cerebrovascular permeability in acute hhpertension,
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20. Hossman~K.-A., and Olsson, Y., Influence of ischemia on the

passage of protein tracers across capillaries in certain blood-
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in rats (to be published).
ALTERATIONS OF THE BARRIER IN HYPERTENSION 527

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stroke-prone spontaneously hypertensive rat (SHR), Circulat. Res.
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Autoregulation of brain circulation in severe arterial hyper-
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 AUTHOR INDEX
Abbott, N.J., 151-164
Banay-Schwartz, M., 349-370;
415-434
Bass, N.H., 31-40
Battistin, L., 465-477
Berger, B., 337-345
Berto11ini, A., 319-335
Betz, A.L., 133-149
Blanc, G., 337-345
Bogdanski, D.F., 291-305
Bradbury, M.W., 507-516
Bradford, H.F., 395-404
Brightman, M.W., 41-54
Broadwell, R.D., 41-54
Christensen, H.N., 3-12
Cremer, J.E., 95-102
Cutler, R.W.P., 435-446
de Be11eroche, J.S., 395-404
del Carmine, R., 319-335
DeMontis, M.G., 89-94
Drewes, L.R., 133-149
Gessa, G.S., ?-9-94
Gi1boe, D.D., 133-149
G1owinski, J., 337-345
Haber, B., 179-198
Hamberger, A., 221-236
Hertz, L., 371-383
Hutchison, H.T., 179-198
Johansson, B., 517-527
Kjeldsen, C.S., 265-272
K1atzo, I., 479-495
Kontro, P., 237-252
Lahdesmaki, P., 237-252
Lajtha, A., 349-370; 415-434
Levi, G., 273-289; 319-335
529
Levin, E., 111-129
Lorenzo, A.V., 447-461
Lund-Andersen, H., 265-272
Lundborg, P., 31-40
Mandel, P., 199-209
McIlwain, H., 253-264
Massare11i, R., 199-209
Neal, M.J., 211-220
Nystrom, B., 221-236
Oja, S.S., 237-252
01dendorf, W.H., 103-109
01ianas, M., 89-94
Olney, J.W., 497-506
Ona1i, P.L., 89-94
Orlowski, M., 13-28
Piccoli, F., 405-411
Pichon, Y., 151-164
Poce, U., 273-289
Pratt, O.E., 55-75
Raiteri, M., 273-289; 319-335
Roberts, P.J., 165-178
Se11strom, A., 221-236
Sepulveda, F.V., 77-87
Spatz, M., 479-495
Spector, R., 447-461
Stinus, L., 337-345
Tag1iamonte, A., 89-94
Takagaki, G., 307-317
Tapia, R., 385-394
Tassin, J.P., 337-345
Teller, D.N., 349-370
Thierry, A.M., 337-345
Tradatti, C.E., 111-129
Woi1er, C.T., 221-236
Yudi1evich, D.L., 77-87
 SUBJECT INDEX
ACETIC ACID
radioactive, brain metabolism
in rats with porto-caval
anastomosis, 98, 99
ACETYLCHOLINE
newly synthesized, preferential
release, 390
ACETYLCHOLINESTERASE
inhibition, and choline
uptake, 202, 205, 208
ADENINE
derivatives, movements and
actions in the CNS, 261, 262
output from cerebral
tissues, 258, 260
release from nerve
terminals, 261, 262
transport in tissues of the
brain, 253, 262
entry into brain in vitro, 255
ADENOSINE
axonal transport, 260, 261
central actions, 253
entry into brain in vitro, 256
mononucleotides, entry into
brain, 257, 258
output from cerebral tissues
in vitro, 259, 260
ADENOSINE MONOPHOSPHATE
cyclic, entry into brain, 257
theophylline-opposed
formation, 259, 260
ADENOSINE TRIPHOSPHATASE
and amino acid uptake by brain
slices, 363, 366
and transport of biogenic
amines, 294, 297, 300
in different neuronal and glial
preparations, 377, 378
531
ADENOSINE TRIPHOSPHATE
levels in brain slices, 355
relation to amino acid
uptake, 350-352, 355, 363, 366
AMINES, see biogenic amines
AMINO ACID
effects neurotransmitters, 67,
68
high affinity uptake and
transmitter inactivation, 225,
273, 274
levels after convulsions, 467
release, from ganglia, 173, 174
from neurons and glia, 226-
228
from spinal cord, 439-444
438
from spinal cord slices and
synaptosomes, 436, 437
uptake in slices, energetics,
349-367
transport, energy dependence, 424
transpor·t, function of '(-
glutamyl cycle, 18
in neurons and glia, 221-232
in spinal and sympathetic
ganglia, 165-17 5
in synaptosomes, 273-286
into the living brain, in
relation to their meta-
bolism, 61-63
ion dependence, 423
regional after convulsions,
470
substrate specificity in
vivo and in vitro, 421
through brain capillaries,
79-81
uptake, in slices, dissociation
from glycolysis, 352
effects of Na+ and K+ levels
and fluxes, 358-365
possible energy sources,
350, 351
532
sensitivity to different
inhibitors, 355-357
inhibition by maleate, in
kidney, 20
in spinal and sympathetic
ganglia, 167-168
AMINO ACIDS
fractional extraction from
brain, 80
labelling with precursors in
brain, 96-98
mutual inhibition of transport
from blood to brain, 63-65
neurotoxic, 497-504
neurotransmitter, reuptake and
exchange, 285-286
release by high K+, 372-375
neutral, species differences in
passage through capillaries, 81
passage from blood, effect of
hypoxia and pC02 tension, 486-
489
radioactive, accumulation into
neurons, glia and synaptosomes,
223, 224
AMINOACIDURIAS
transport of amino acids into
the brain, 67
r-AMINOBUTYRIC ACID
exchange in synaptosomes,
concentration dependence, 374, d-AMPHETAMINE
375
possible role, 385, 386
sodium-dependence, 378, 379
substrate specificity, 379,380
fluxes in synaptosomes, effects
of ouabain and A23l87, 382-385
homo- and heteroexchange in
ganglia, 174
localization of uptake by auto-
radiography, in ganglia, 169,170
in retina, 216
neurotransmitter role in spinal
cord, 435
newly synthesized, functional
significance, 389-391
INDEX
parallel decrease of synapto-
somal uptake and exchange, 281
release, from ganglia, 173, 174
from incubated nervous
tissues, effect of K+, 372-
375
from isolated neurons and
glia, 226-228
from retina, 218
role in retina, 211
synthesis-dependent release,
in vitro, 388
in vivo, 385-387
uptake and exchange in synapto-
somes, 273-286
uptake, effect of Ca 2+ in glia
and synaptosomes, 226
in ganglia, substrate
specificity, 171, 172
in retina, effect of amino-
oxyacetic acid, 215
kinetics, 212
subcellular locali-
zation, 215-216
substrate specificity,
215
in spinal and sympathetic
ganglia, 167-169
p-AMINOHIPPURIC ACID
elimination from brain and
CSF,33-35
effect on doapmine fluxes in
synaptosomes, 398-401
on 3H-dopamine release from
synaptosomes, 323-326
on 3H-5HT release from
synaptosomes, 325-327
on 3H-norepinephrine release
from synaptosomes, 323, 324,
326-328
ASPARTIC ACID
neurotoxic effects, 499-501
BARBITURATES
lipid-water partition coefficient
arid penetration into brain, 108
INDEX
BIOGENIC AMINES
characteristics of transport at
nerve endings, 291, 292
effect of Na+ on synaptosomal
transport, 294, 297-299
evidence for outward transport
from nerve endings, 299-302
release from synaptosomes,
effects of various agents, 321-
333
release versus reuptake inhibit-
ion, 319
role of outward transport in
synaptic transmission, 301
storage, effects of electro-
lytes, 299
transport, at nerve endings,
effect of ouabain, 294, 297,298
the ion gradient hypothesis,
293-297
in nerve tissue cultures,
189
BLOOD-BRAIN BARRIER
brain areas where it is absent,
47-52
dysfunction and glutamate neuro-
toxici ty , 503
effects of convulsions 465-475
effects of high intraluminal
pressure on, 519-522
effects of ischemia on, 483, 485
functions, 425
lesions, in essential hyper-
tension, 523
in hypertension, lo~ation, 518
maturation, and glutamate neuro-
toxicity, 503
methods for bypassing it, 41
movement of fluids across the,
507-515
opening, hyperosmotic, 42-45,481
482, 484
hypertensive, 45-47,517-525
in tumors, 42
pathological aspects, 479-490
120
permeability, key elements, 104,
105
533
influence of vasoactive
substances, 518
to amino acids, 61-66
to amino acids and sugars,
77-85
to drugs, lipid-water partition
coefficient, 108-109
to Fab, pharmacological
alterations, 123-127
to glucose, 56-59, 133-145
during anoxia, 136-138
during hypoglycemia, 136
effect of mercury, 'f80
to glucose and maino acids,
effects of 02, pC02 tension,
484, 486-489
to proteins, 114-116
in pathological conditions,
120
pharmacological alterations,
122-127
to proteins and peptides, 41-52
to tyrosine and tryptophan,
effect of insulin, 89-93
vascular damage in arterial
hypertension, 522, 523
BLOOD FLOW
cerebral, in hypertensive
animals, 520, 521
BRAIN
amino acids, labelling with
precursors, 96-98
capillaries, fluid movement
across, 507, 508
permeability in cerebral
tumors, 42
presence of carriers, 77-85
uptake of 2-deoxy-glucose
in vitro, 489, 490
versus general capillaries,
103, 104
wall of, 41
damage and oral amino acid
intake, 497-504
fluids, solute movement and
accumulation, 510
volume control, 508~5l0
534
uptake index, for amino acids
and sugars, 484, 486-489
in rats with porto-caval
anastomosis, 100
utilization of precursors in
pathology, 98-100
BRAIN SLICES
amino acid transport as related
to ATP and ATPase, 350, 351,
353,364,367
functional aspects, 425
regional distribution, 426
as a two compartment serial
system, 266, 267
effects, of glucose deprivation
on amino acid uptake, 354, 355,
358
of potassium, on amino acid
release and uptake, 272-275
on transport of ions and
water, 276
of slicing on amino acid
transport, 418
energetics of amino acid trans-
port in, 349; 424
influx of taurine into, 239-245
ionic requirements for amino
acid transport in, 358-365; 423
protein synthesis, comparison
to in vivo rates, 416
sensitivity of amino acid uptake
to inhibitors, 355-357
swelling and amino acid trans-
port, 419
taurine efflux and exchange in,
245-246
uptake and release of glutamic
acid in, 312, 313
uptake of glucose analogs, 268-
271
BUTYRIC ACID
radioactive, brain metabolism in
porto-caval anastomosis, 98, 99
CALCIUM
effect on release,
of amino acids from ganglia,
171
INDEX
of catecholamines from
nerve endings, 299
of glycine from spinal
cord, 436-438, 443
of 3H-norepinephrine from
synaptosomes, 321, 322
ionophore, effects on GABA
and glutamate fluxes in
synaptosomes, 282-285
CAPILLARIES
amino acid transport, 421
of brain, altered uptake of
2-deoxy-glucose in vitro, 489,
490
permeability alterations in
cerebral tumors, 42
presence of carriers for
amino acids and sugars,
77-85, 421
transport substrate
specificity, 422
versus general capillaries,
103-104
wall of, 41
CATECHOLAMINES (see also biogenic
amines)
newly synthesized, relation
to synaptic transmission, 390
uptake in vitro as an index
of catecholaminergic innervat-
ion, 338
CEREBROSPINAL FLUID
bulk-flow, and elimination of
acid metabolites, 32-34, 37, 38
development of removing acid
metabolites, 31-40
formation in developing rats, 32
penetration of proteins into,
112-114
proteins, origin, 112-114
sink action of the, and role in
the removal of 5-HIAA from
brain, 37
drug distribution 447-461
INDEX
CHLORIDE
control of brain fluid volume,
510-514
CHOLINE
high affinity uptake, 199, 205
uptake kinetics in nerve cell
cultures, 185, 201-204
COMPARTMENTS
exchange in vivo and in
vitro, 402 - - - in enzyme patterns,
CONVULSIONS
amino acid uptake 446-449
CORPUS STRIATUM
distribution of dopamine uptake
in, 339, 340
synaptosomes, dopamine fluxes
in, 397-402
release of amines from, 323-
332
CORTEX
cerebral, demonstration of
dopaminergic terminals, 337
distribution of dopamine
uptake, 341
synaptosomes, release of
amines, 323-327
CRAYFISH
CNS connective, movements of
Na+ and K+, 155-161
CYSTEINE
brain damage on oral intake,
499, 502
2-DEOXY-GLUCOSE
abnormal transport through the
barrier, 480, 481, 483-485
altered capillary uptake in
vitro, 489, 490 -
uptake in synaptosomes from
ischemic brains, 485, 486
535
DESIPRAMINE
effect on norepinephrine
efflux, 300-302
DESMETHYLIMIPRAMINE
effects on induced release
of biogenic amines, 327-329
DEVELOPMENT
changes in amino acid transport,
408-409
405-406
in ions, spaces, 407
DIAMINOBUTYRIC ACID
effect on GABA exchange, 280
effect on glial transport of
GABA, 229
DOPAMINE
distribution of uptake, in brain,
339-341
fluxes in synaptosome "beds",
effects of amphetamine, 398-401
newly synthesized, release from
synaptosome "beds", 398
release from synaptosomes
effects of amphetamine, 398,
400, 323, 326
effect of K+, 398, 401
effect of 8-phenylethylamine
derivatives, 330, 331
uptake in cortex, 338, 339
uptake and release in synapto-
somes, 398, 401
uptake as an index of dopaminer-
gic innervation, 338, 341, 342
DRUGS
distribution between tissues and
CNS and partition coefficients,
106-107, 447-461
uptake mechanisms 447-461
536
EDEMA
abnormal penetration of proteins,
120, 121
water and electrolyte movements,
507-514
ENZYME
deficiencies of the r-glutamyl
cycle, 21
developmental changes, 405-406
ETHANOL
partition coefficient and
penetration, 108
EVANS BLUE
abnormal penetration into
brain, 42-47
penetration into brain vessels
during hypertension, 522
EXTRACELLULAR SPACE
in brain slices, 266, 267,419
after convulsions, 468
GANGLIA
release and exchange of amino
acids in, 171, 172
structure and function, 166
uptake of GABA and glutamate
into, 167-172
GLIA
buffer for K+, crayfish CNS, 159-
161
vertebrate studies, 152-155
function in ganglia, 175
isolated, accumulation of radio-
active amino acids, 223, 224
homoexchange and hetero-
exchange of GABA, 229, 230
metabolic and ionic requir~
ments for amino acid uptake,
226
release of GABA from, 228
transport of amino acids, 222-
230
responses to high K+ concentrat-
ions, 378
INDEX
retinal, GABA uptake into, 212
role, in re~ulation of extra-
cellular K , 151-162
in transmitter inactivation,
226
site of GABA and glutamate
uptake in ganglia, 168, 169
tissue culture, GABA uptake,
181
amino acid uptake, 184
choline transport, 185
GLUCOSE
analogs, extracellular diffus-
ion, membrane transport, 269
passage, effect of hypoxia
and pC0 2 tension, 484,
486-488
uptake in brain slices, 268,
269
uptake in slices from
ischemic brains, 270
depletion, and amino acid
transport, 377, 378, 381
influx and efflux in the living
brain, 55-59, 69
kinetics of transport, 134, 135
during hypoglycemia, 136
during anoxia, 136-138
relation between transport and
utilization in vivo, 56-59
transport across the blood-
brain barrier, chemiostatic
model, 141
comparison with erythrocyte,
141-144
mechanisms, 138-141
model for translocation, 144,
145
structure-activity relations,
141-144
transport, in perfused brain, 133-
145
effect of insulin, 69
through brain capillaries,
81-83
GLUTAMATE DECARBOXYLASE
inhibition, relationship to the
onset of convulsions, 386
INDEX
GLUTAMIC ACID
analogs and neurotoxicity, 498,
499
brain damage on oral intake,
497-504
compartmentation in ganglia, 171,
172
exchange in synaptosomes,
concentration dependence, 275,
276
possible role, 285, 286
sodium dependence, 279
fluxes in synaptosomes, effects
of ouabain and A23l87, 282-285
formation from glutamine in
fractions, 231
hypothalamic lesions after s.c.
injections of, 498-500
foods, neurotoxicity in 504
labelling from different
urecursors, 97, 98
metabolism of isomers in
slices, 309, 310, 311
neurotransmitter role in spinal
cord, 435
release, induced by K+, 372-375
from ganglia, 173, 174
substrate specificity of uptake
in ganglia, 172
transuort in cortical slices and
synaptosomes, 312-315
uptake and exchange in synapto-
somes, 273-286
uptake in slices and synapto-
somes, effect of ouabain, 313,
314
uptake in spinal and sympathetic
ganglia, 167-169
GLUTAMINE
comoartmentation in brain cells,
231
labelling from different
precursors in brain, 97, 98
1-GLUTAMYL CYCLE
analysis of the various compon-
ents, 15-18
537
enzyme deficiencies in heritable
disorders of metabolism, 21
function in amino acid trans-
port, 18-21
1-GLUTAMYL TRANSPEPTIDASE, 16-18
GLUTATHIONE
role in transport of amino
acids, 13-23
synthesis and degradation, 13
GLYCINE
neurotransmitter role in spinal
cord, 435
release, from intact spinal
cord, 438-444
from spinal cord slices and
synaptosomes, 436, 437
GLYCOLYSIS
as energy source for amino
acid uptake in slices, 351,
363, 364
dissociation from transport in
slices, 352
HEMICHOLINIUM-3
effect on choline uotake in
cell cultures, 202, 203, 206
HORSE RADISH PEROXIDASE
abnormal penetration from
blood, 43-47
penetration during arterial
hypertension, 522, 523
retrograde axonal transport, 52
HYDROGEN
ion, role in transport, 10
5-HYDROXYINDOLEACETIC ACID
elimination from brain and
CSF, 33-38
mechanisms for removal from
brain, 36
5-HYDROXYTRYPTMlINE
release from synaptosomes,
effect of amphetamine, 325-327
538 INDEX

effect of e-phenylethylamine MALEATE

derivatives, 331, 332 inhibition of amino acid
synthesis related to tryptophan uptake, 20
influx, 68, 89
METABOLITES
HYPERTENSION mediation of apparently spon-
acute, relevance of animal taneous migrations of, 4
studies, 524 transport at cell membranes,
arterial, and blood-brain barrier 3-11
lesions, 517-525
essential, blood-brain barrier METHIONINE
lesions in, 523 neurotoxicity in schizophrenics,
503
HYPOTHALAMUS
lesions following glutamate MONOSACCHARIDES
administration, 498 as inhibitors of glucose trans-
at different ages, 500 port, 142
synaptosomes, release of 3H-
dopamine from, 325 NERVE CELL
release of 3H-norepinephrine cultures, choline uptake in,
from, 323, 324, 330 185-189, 201-206
amino acid uptake in, 180-185
HYPOXANTHINE
entry into brain tissue in NEUROBLASTOMA
vitro, 257 clones, uptake of choline into,
185; 201-204
INSULIN GABA, 182
effect of glucose trans~ort, 69 amino acids, 184
role in the transport of tyro-
sine and tryptophan, 89-93 NEURONS
accumulation of amino acids,
INULIN 223, 224
clearance from CSF, 32 metabolic and ionic require-
ments for amino acid uptake,
ISCHEMIA 226
effects on blood-brain barrier, transport of amino acids,
483, 485 221-232
lack of effect on sugar trans- necrosis, following oral intake
port in slices, 270 of glutamate, 497-504
retinal, GABA uptake into, 217
KETONE BODIES
influx and metabolism, 59, 60 NEUROTOXICITY
of glutamate and other amino
LACTIC ACID acids, 497-504
influx and efflux, 59, 60
NEUROTRANSMITTERS
LIVER model for continuous membrane
by-pass, effects on metabolite flux, 401, 402
transport and compartmentation,
95-102
INDEX
NOREPINEPHRINE
cotransport with Na+, model, 295
mechanisms of uptake into nerve
endings, 292-299
release by outward transport,
299-302
release from synaptosomes,
effect of amphetamine, 323,
324, 326, 327, 328
effect of Ca 2 +, 321-322
effect of 8-pheny1ethy1amine
derivatives, 329, 330
effect of reserpine, 322-323
uptake as index of noradrenergic
innervation, 338
ONTOGENESIS
of K+-induced amino acid release,
373, 374
OUABAIN
effect on biogenic amines trans-
port at nerve endings, 230, 294,
297, 298
on dopamine release from
synaptosomes, 396
on GABA and glutamate fluxes
in synaptosomes, 282-284
on glutamate uptake in slices
and synaptosomes, 313, 314
radioactive, lack of passage
from blood to brain, 84
8-PHENYLETHYLAMINE
derivatives, effects on amine
release from synaptosomes, 328-
333
structural requirements for
releasing activity, 331-333
POTASSIUM
and the control of brain fluid
volume, 386-390
concentration in synaptosomes,
297
effects on amino acid release,
372-374
on biogenic amine transport,
294, 297, 300
539
on energy metabolism and
Na+-K+-ATPase, 377
on GABA and glutamate
release, 373
on GABA and glutamate
uptake, 376
on transport of inorganic
ions and water, 376
extracellular, mechanisms for
the regulation of, 151-162
movements in crayfish CNS,
155-161
removal from extracellular
space, 152-155
requirements for amino acid
transport in slices, 358,
359, 360, 364, 365
PROTEINS
penetration into brain,
from blood, 114-116
from cerebrospinal fluid,
116-118
from extranervous tissues,
118-120
in pathological conditions,
120
pharmacological studies,
122-127
penetration into the cerebro-
spinal fluid, 112-114
PYRUVIC ACID
influx and efflux in the living
brain, 59, 60
RESERPINE
effect on 3H-norepinephrine
release from synaptosomes, 322,
323
RETINA
autoradiographic localization
of 3H-GABA, 216
kinetics of 3H-GABA uptake, 212
release of 3H-GABA from, 218
subcellular localization of 3H-
GABA uptake, 215
taurine efflux and exchange in,
245-246
540
taurine uptake into, 239-243
SODIUM
and the control of brain fluid
volume, 508-514
concentration in synaptosomes,
297
cotransport with substrate, 292, TAURINE
293
with biogenic amines in
nerve endings, 295, 296
effect, on GABA and glutamate
exchange in synaptosomes, 278,
279
on glutamate uptake and
release, 314, 315
on synaptosomal transport of
biogenic amines, 294, 297-299
on taurine transport, 243, 244
lack of effect on blood-brain
sugar transport, 82
levels and fluxes in slices,
relation to amino acid trans-
port, 358-364
movements in crayfish CNS, 155-
161
SPINAL CORD
neurotransmitter role of glycine,
GABA and glutamate in, 435
release of amino acids from,
in vitro, 436, 437
in vivo, 438-444
STRYCHNINE
effect on glycine release from
spinal cord, 441
SYNAPTOSOMES
apparatus for superfusing, 221,
320
from ischemic brain, uptake of
2-deoxy-glucose, 485, 486
release of biogenic amines,
effects of drugs, 322-333
subpopulations accumulating
taurine, 241
taurine efflux, 245, 246
transmitter release and reuptake
in, 396, 401, 402
INDEX
uptake and exchange of GABA
and glutamate, 273-286
uptake of choline, 201-204
uptake of glutamate, 312-315
uptake of taurine into, kinetics,
239-245
binding to synaptosomal membranes,
246-248
efflux from brain and retina,
245, 246
from retina, effect of
light, 245
synthesis in brain, 237
transport, in vivo, 238, 239
uptake in vitro,
energy dependence, 243, 244
kinetics, 239-241
sodium dependence, 243-245
substrate specificity, 243
TETRAHYDROCANNABINOL
retention in the body, 109
THYROID HORMONES
effects on amino acid influx,
69, 70
TRANSPORT
axonal, of adenosine, 260
retrograde, 119
differentiation of distinct
systems, 8
direction of spontaneity, 4
duality or multiplicity of
systems, 6
in vivo and in vitro, 415-429
mechanisms, the ion gradient
hypothesis, 292-297
in the cerebrospinal fluid,
31-38
net uptake versus exodus, 7, 8
of acid metabolites in the
CSF, 31-38
of adenine derivatives in brain,
253-262
of amino acids and sugars across
capillaries, 77-85
INDEX 541

of amino acids in slices, TUMORS

effects of K+, 371-379 altered capillary permeability,
effects of swelling, 420 42
energetics, 349-367, 424
substrate specificity, 421 UREA
of amino acids in neurons and hypertonic, and opening of the
glia, 179, 221-232 blood-brain barrier, 42-45
of biogenic amines at nerve
endings, 291-302 WATER
of choline in nerve cell cultur-- movement across the blood-
es, 185, 199-206 brain barrier, 507-515
of dopamine in discrete brain
areas, 337-343
in synaptosomes, 397-402
of GABA and glutamate, in
ganglia, 165-175
in synaptosomes, 273-286
of GABA in retina, 211
of glucose across the blood-
brain barrier, 133-145
of glucose analogs in brain
slices, 265-271
of glutamic acid in synaptosomes,
307-316
of glycine and other amino acids
in spinal cord, 435-444
of metabolites, pathological
aspects, 479-490
of metabolizable substances in
the living brain, 55-71
of taurine in the eNS, 237-248
of tyrosine and tryptophan from
blood to brain, 89-92
reverse phases, 5
role of glutathione in, 13-23
role of the hydrogen ion in, 10
studies, rational for using
isolated neurons and glia, 222

TRYPTOPHAN
transport, effect of insulin,
89-93

p-TYRAMINE
effects on the release of amines
from synaptosomes, 329-333

TYROSINE
transport from blood to brain,
effect of insulin, 89-93