Cellular and Molecular Neurobiology, Vol. 9, No.

2, 1989

Review

Adrenocorticoid Action in the Spinal Cord:

Some Unique Molecular Properties of
Glucocorticoid Receptors
Alejandro F. D e Nicola, ~ Daniel F. Moses, ~ Susana
Gonz~lez, ~ and Eduardo Orti 1

Received October 17, 1988; accepted December 15, 1988

KEY WORDS: corticosterone; dexamethasone; central nervous system; rat; stress; hippocampus.

SUMMARY

1. Glucocorticoid hormones affect several functions of the spinal cord, such

as synaptic transmission, biogenic amine content, lipid metabolism, and the
activity of some enzymes (ornithine decarboxylase, glycerolphosphate de-
hydrogenase), indicating that this tissue is a target of adrenal hormones.
2. Corticosterone, the main glucocorticoid of the rat, is detected at all
regional levels of the spinal cord, and cold stress increases this steroid,
predominantly in the cervical regions.
3. Intracellular glucocorticoid receptors have been found in the spinal cord,
with higher concentrations in the cervical and lumbar enlargements. Prima facie,
these receptors presented biochemical, stereospecifical, and physicochemical
properties similar to those of receptors found in other regions of the nervous
system. The prevalent form in the spinal cord is the type II receptor, although
type I is also present in small amounts.
4. The type II glucocorticoid receptor of the spinal cord shows an affinity
lower (Kd 3.5 nM) than that of the hippocampal type II site (Kd 0.7 nM) when
incubated with [3H]dexamethasone. This condition may impair the nuclear
translocation of the spinal cord receptor.
5. Another peculiar property of spinal cord type II site is a greater affinity
for DNA-cellulose binding than the hippocampal receptor during heat-induced

1Laboratorio de Bioquimica Neuroend6crina, Instituto de Biologia y Medicina Experimental,

Obligado 2490, 1428 Buenos Aires, and Departamento de Bioquimica, Facultad de Medicina,
Universidad de Buenos Aires, Argentina.
179
0272-4340/89/0600-0179506.00/0~ 1989 Plenum Publishing Corporation
180 De Nicola, Moses, Gonzalez,and Orti

transformation. Also, the spinal cord receptor shows resistance to the action of
RNAse A, an enzyme which increases DNA-cellulose binding of the hippocampal
receptor, indicating that both receptors may be structurally different.
6. Therefore, it is possible that a different subclass of type II, or "classical
glucocorticoid receptor," is present in the spinal cord. This possibility makes the
cord a useful system for studying diversity of glucocorticoid receptors of the
nervous system, especially the relationship between receptor structure and
function.

INTRODUCTION

The central nervous system (CNS) is an important target of the steroids produced
by the adrenal cortex. A complex relationship exists between the brain, as
modulator of the stress response, which leads to the stimulation of adrenocortical
secretion and the brain as modulator of adrenal hormones, which regulate several
activities Of the CNS. Although a large number of adrenal steroid effects in the
CNS have been described, they can be summarized into neuroendocrine
mechanisms of control of pituitary-adrenal activity, metabolic changes, and
behavioral control. A wide spectrum of biochemical and physiological mechan-
isms has been described, such as actions on electrical activity, neurotransmitter
receptor function, neurotransmitter metabolism, neuropeptide and second mes-
senger actions, regulation of gene transcription, enzyme induction and activity,
etc. (for references see McEwen et al., 1986b).
It is accepted that the actions of adrenocortical steroids are mediated by
interaction with intracellular receptors, which upon hormone binding are con-
verted into DNA-binding proteins (Yamamoto, 1985). Thus, in CNS as well as in
peripheral tissues, the horm0ne-receptor complex regulates--positively or
negatively--transcription of specific genes into mRNA (Etgen et al., 1980). There
is a growing amount of information reporting the regional distribution, pro-
perties, a n d tentative functions o f glucocorticoid (GC) receptors. Early work
suggested some differences in receptor properties, when binding of the natural
GC of the rat, corticosterone (CORT), was compared to that of the synthetic
hormone dexamethasone (DEX). Heterogeneity of receptors in CNS has received
considerable attention, leading to the recognition that two to three receptor
systems may coexist in brain cells (McEwen et al., 1986b). In the case of CORT it
has been shown to bind to at least two sites, called types I and II in analogy with
the kidney (Marver, 1980). Type I shows a high affinity for CORT and
aldosterone (ALDO), and it has also been called the "CORT-preferring" species.
Type II binds synthetic steroids with a high affinity, and it shows a low preference
for CORT and ALDO; it has also been called the "classical GC receptor," as it
resembles receptors extracted from peripheral tissues such as liver, kidney,
muscle, or thymus (Reul and De Kloet, 1985). Furthermore, based on physiolog-
ical evidence showing that ALDO but not CORT or DEX reduces salt appetite
developing in adrenalectomized rats (McEwen et al., 1986a) or biochemical and
physicochemical evidences (Coirini et al., 1983, 1985; Wrange and Yu, 1983), and
The SpinalCord as the Target of GlucocorticoidHormones 181

on autoradiographic localization of putative receptors (Birmingham et al., 1984),

a third type may also be present in CNS, mediating specific mineralocorticoid
actions (De Nicola et al., 1981).
GC receptors are found in high quantities in hippocampus and associated
areas of the limbic system (McEwen et al., 1986b), although other areas contain
binding sites as well. For instance, the arcuate, ventromedial, and paraventricular
nucleus of the hypothalamus (Magarifios et al., 1988; Reul and De Kloet, 1985);
amygdala (Coirini et al., 1983; Warembourg, 1985), caudate-putamen (De Fiore
and Turner, 1983), cerebral cortex (Meaney and Aitken, 1985), optic nerve
(Meyer et al., 1982), superior cervical ganglion (Towle and Sze, 1982), locus
coeruleus, n. of the solitary tract, and dorsal raphe of the brain stem (Reul and
De Kloet, 1985) have been shown to contain GC receptors, suggesting their
widespread distribution in regions subserving different functions. Whereas type I
GC receptors are found mostly in hippocampus, septum, and amygdala, type II
receptors are present in almost every CNS region analyzed (Fuxe et al., 1985;
Reul and De Kloet, 1985; McEwen et al., 1986b).

GLUCOCORTICOID ACTION IN THE SPINAL CORD

We became interested in studying the spinal cord for several reasons. First,
we thought that a considerable amount of information on the mechanism of
hormonal action could be gained from studying a tissue organizationally simpler
than the brain: the spinal cord conveys information to the brain, and information
flows from the brain down to the spinal cord neurons; additionally, some local
pathways exist, restricted to the tissue. Second, the spinal cord concentrates
binding sites for other classes of steroid hormones. Thus, estrogen-concentrating
neurons were anatomically defined by Keefer et al. (1973) and Morrel et al.
(1982), whereas androgen target sites were specified in mature animals by Sar and
Stumpf (1977) and by MacLusky et al. (1987) and in developing spinal cord by
Reid et al. (1981). Mineralocorticoid binding sites were mapped autoradiographi-
cally (Stumpf and Sar, 1979) and characterized biochemically in our laboratory
(Orti et al., 1986). Therefore, the spinal cord makes an ideal tissue for studying
interrelationship in hormonal action.
Third, corticoids have a direct role in spinal cord physiology. For instance,
GC administration regulates in the cord synaptic transmission and neuronal
excitability (Hall, 1982), rapidly increases the dopamine content (Hall and
McGinley, 1982), reduces lipid peroxidation (Hall and Braughler, 1981), shortens
recovery time after injury (Hall, 1982), and increases the activity of certain
enzymes such as glycerolphosphate dehydrogenase and ornithine decarboxylase
(Orti et al., 1987). Fourth, cytosolic binding sites for [3H]DEX (Clark et al., 1981;
Duncan and Stumpf, 1984; Well, 1986) and neuronal concentration of systemati-
cally injected [3H]CORT (Duncan and Stumpf, 1984) were described in other
laboratories, giving support to our own results, demonstrating a direct role of
adrenal steroids as modulators of spinal cord function.
182 De Nicola, Moses, Gonz~llez, and Orti

ID

i
O
H .

~D

t-
O

CI-C2 C3-C 7 T1-T8 Tg-L3 L4 L6 -

Fig. 1. Concentration of corticosterone in the spinal cord

from intact rats (filled columns) and rats subjected to cold
stress (4°C during 8 hr; cross-hatched columns). The content
of corticosterone was determined by RIA in the sections
corresponding to numbered vertebrae under the columns.
Stress elevated hormone levels significantly in all regions
(P<0.01). (*) P < 0 . 0 5 vs levels in L4-L6 of the stressed
group; (**) P < 0.05 vs levels in T1-T8, T9-L3, and L4-L6
of the intact group; (***) P < 0 . 0 1 vs levels in all other
regions of intact group. Reproduced from Orti et al.
(1985a).

Glucocorticoid Content of the Spinal Cord

An important step showing that the spinal cord was a GC-responsive tissue
was the demonstration that CORT was present in tissue from adrenally intact rats
and that it responded to manipulations that decrease or increase plasmal levels of
CORT. We found substantial amounts of CORT, detected by radioimmunoassay
in spinal cord of resting rats, with maximal quantities in the upper cervical regions
(vertebrae C1-C2, 1.5 ng/100 mg tissue), with a downward reduction to less than
0.1 ng/100 mg tissue in the lumbar regions (Fig. 1). CORT levels in five regions
macrodissected from spinal cords (C1-C2, C3-C7, T1-T8, T9-L3, and L4-L6)
increased after rats were subjected to cold stress, with higher levels still
predominating in the upper regions (Fig. 1). We also observed that if adrenalec-
tomized rats received a single injection of 1 mg CORT, it accumulated in cytosol
and nuclear fractions prepared from spinal cord homogenates. This uptake was
markedly suppressed if the animals were pretreated with DEX for 3 days (Orti et
al., 1985a). These studies suggested that the spinal cord concentrates endogenous
and exogenously given GC and that the prevalent retention mechanism (receptor)
is probably the so-called type II, in view of the displacement exerted by DEX, a
suitable ligand for type II sites (Orti et al., 1985a).
Glucocorticoid Receptors in the Spinal Cord
GC receptors we assayed in the cytosolic fraction of spinal cords from
adrenalectomized rats (Orti et al., 1985b). We observed that binding presented a
The Spinal Cord as the Target of Glucocorticoid Hormones 183

.... J~.._
~T

Fig. 2. Regional distribution of binding sites for

[3H]dexamethasone in the spinal cord. Cords were dissected
into sections corresponding to the following vertebrae: A,
C1-C2; B, C3-C7; C, T1-T8; D, T9-L3; E, L4-L6. B and
D contained the cervical and lumbar enlargements, whereas
E contained the horse-tail and ilium terminale. Results are
expressed as percentage binding with respect to D, which
was taken as 100. (*) P < 0.05 vs E. Modified from Orti et al. 6

(1985b). ,;o s0 '; g l:~

*/,Relative Binding

high affinity for [3H]DEX (Ka, 3.2nM), low capacity (Bmax, 160fmol/mg
protein), and good specifity: relative binding affinity was high for GC (triam-
cinolone, DEX) and anti-GC (RU 26988), somewhat lower for CORT and
progesterone, and very low for ALDO and sex hormones. That [3H]DEX-binding
molecules of the spinal cord resembled the "classical GC receptor" was further
evidenced by measuring the sedimentation coefficient in glycerol gradientes
containing molybdate. The values obtained--9.6 to 9.8ss--suggested the pre-
sence of undegraded holoreceptors, similar to those found in our laboratory for
other GC target tissues such as hippocampus and placenta (Coirini et al., 1983;
Heller et al., 1984).
Regional distribution of binding sites showed a tendency for higher con-
centrations in cervical (C3-C7) and lumbar (T9-L3) enlargements (Fig. 2), with
lower amounts in horse-tail and ilium terminale. In vivo labeling of receptors was
also attempted, after administration of a tracer dose of [3H]CORT i.v. into
adrenalectomized rats. Using this procedure, the hormone occupies pre-
dominantly type I sites, since more hormone should be given to occupy type II

Table I. Uptake of [3H]Corticosterone into Whole Homogenate and Purified Cell

Nuclear Fractions of the Spinal Cord and Hippocampus a

Purified nuclei
Whole homogenate (fmol/mg % of whole
Region (fmol/mg of DNA) of DNA) homogenate
Hippocampus 1613 + 278 72.8 + 10.5" 4.51
C1-C2 714 + 192 3.9 + 0.9 0.55
C3-C7 934 + 309 3.8 + 0.4 0.41
T1-T8 975 + 258 3.0 + 0.7 0.31
T9-L 3 738 + 223 5.2 + 1.3 0.70
L4-L6 857 5:199 1.6 + 0.5 0.19

Fifty microcuries of [3H]corticosterone was injected i.v. ito adrenalectomized rats.

Homogenates were prepared from spinal cord and hippocampus, and a nuclear
fraction was purified as described by Orti et al. (1985a). Radioactivity was
determined in these fractions and converted into fmol/mg DNA. Reproduced from
Ortl et al. (1985a).
* P < 0.01 vs values in all spinal cord regions.
184 De Nicola, Moses, Gonzfilez, and Orti

sites (Reul et al., 1987). Cell nuclear localization of injected [3H]CORT was
analyzed in five macrodissected regions of the spinal cord, areas previously used
for measuring binding of [3H]DEX in vitro. In these studies, we found that the
whole homogenate uptake amounted to 50% of that found in hippocampus,
which compared favorably with the proportion of cytosolic receptors in the two
tissues (Orti et al., 1985b). Nearly 5% of the whole homogenate radioactivity
remained associated with the purified nuclear fractions prepared from hippocam-
pus (Table I), in contrast to levels in the cord, which were less than 1%.
In summary, we demonstrated that high-affinity, low-capacity binding sites,
with specificity and some physicochemical properties of GC receptors, existed in
the spinal cord. However, the low degree of competition by CORT in vitro,
coupled with the high displacement of injected CORT by concomitantly given
DEX, plus the poor cell nuclear localization of [3H]CORT given systemically (in
spite of the sizable amounts of cytoplasmic receptors), suggested again that the
bulk of GC receptors of the spinal cord belonged to the type II class.

Heterogeneity of Steroid Receptors

As the above-mentioned results suggested more than one receptor type for
GC in the spinal cord, we determined the concentration of types I and II
receptors employing a single radioactive ligand displaced from each one of the
sites by competitors with specificity as receptor markers. Therefore, we incubated
the synthetic hormone [3H]DEX with cytosol from the spinal cord of adrenalec-
tomized rats. Specific binding to type II sites was considered that competed by

(3H)- D EX
IHIPPOCAMPOSI ISPINAL CORD t l/II
50- ,2.O
CL
U~
E 40-
-6
T ,1.5
30-
"u
C ,1.0
o 20-
El

10-
.L
~ o II [ II HIPPO SCORD

Fig. 3. Binding of [3H]dexamethasone to receptor types

in cytosol of spinal cord and hippocampus. Cytosol from
nervous tissues were incubated with 20 nM of tritiated
dexamethasone (a) plus 2/~M RU 28362 (b) or with 2/~M
of both corticosterone and RU 28362 (c). It was assumed
that binding to type II sites resulted from a minus b, and
binding to type I from b minus c. (*) Significantly lower
than hippocampal type I sites (P<0.02); (**) sig-
nificantly lower than I/II ratio of hippocampus (P <
0.01). Reproduced from Moses et aL (1988).
The Spinal Cord as the Target of Glucocorticoid Hormones 185

10o

Fig. 4. Displacement of [3H]dexamethasone incub- ,tu 80

c~ mpus
ated with cytosol from hippocampus or spinal cord, by
! 6O
the antiglucucocorticoid RU 28362. The competitor
was far more effective in spinal cord than in hip-
pocampus, demonstrating that type II sites (i.e., those "~'-~ 20'40~ o r d
suppressed by RU 28362) were more abundant in the
spinal cord. IC50 values, calculated by logit-log trans- m
formation of displacement curves, were 0.1/zM for 'J- .
spinal cord and 1.7/*M for hippocampus. o51 I~ lao
RU 28362 (ford-excess)

2 #M R U 28362, a pure anti-GC which blocks type II sites but without affinity for
type I sites (Coirini et al., 1985), whereas binding of [3H]DEX to type I sites was
considered that displaced by 2/~M CORT in the presence of 2/,M R U 28362
(Moses et al., 1988). The hippocampus was always included in our assays, as this
region of the CNS contains substantial amounts of both type I and type II sites
(McEwen, 1988). In our hands, as shown in Fig. 3, the ratio I/II in hippocampus
was 1.2. In the cord, however, while type II sites were in the range of
hippocampal receptors, a marked reduction was obtained for type I sites.
Consequently, the I/II ratio was very low, an index of receptor heterogeneity in
the two regions (Fig. 1). Scatchard analysis disclosed that type I had a lower
affinity for [3H]DEX (Kd, 5 nM) in comparison to type II (Kd, 2 nM). It remained
that for CORT the affinity of these receptors is the opposite, i.e., higher for type
I than for type II. In another series of experiments, we used [3H]CORT to label
type I and II sites; the results were similar to those seen with [3H]DEX in that
more type I sites were present in hippocampus than in spinal cord, but in this case
limited binding to type II occurred upon the addition of [3H]CORT to the cytosol
of both tissues. Therefore, [3H]DEX seems a ligand of choice to visualize both
receptor classes in the CNS.
Further support for the heterogeneous nature of GC receptors in spinal cord
and hippocampus was obtained after competition with 0.5 to 100-fold increasing
concentrations of R U 28362. As shown in Fig. 4, the anti-GC effectively displaced
[3H]DEX from spinal cord cytosol, whereas 50% inhibition was not reached in
hippocampus, even at the highest competitor concentration.

DNA Cellulose Binding of Glucorticoid Receptors Reveals

Further Receptor Heterogeneity

The accepted theory of steroid hormone action establishes that GC hor-

mones control the expression of specific genes in the periphery as well as in CNS.
In its native state, the receptor localizes in cytoplasm, and after hormone binding
the GC-receptor complex translocates into the cell nuclei (Etgen et al., 1980;
McEwen et al., 1986b; Yamamoto, i985). Binding of hormone promotes changes
in the structure and function of the receptor, and the process by which the
receptor acquires affinity for nuclear components has been called "transforma-
tion". This mechanism implies a change in receptor charge from being a neutral
or acidic protein into being a basic protein, capable of binding to D N A and other
186 De Nicola, Moses, Gonz~ilez, and Orti

negatively charged polyanions (Schmidt and Litwack, 1982). Cell-free GC

receptor transformation is usually achieved by increasing the incubation tempera-
ture and can be tested in vitro by measuring its binding to DNA-ceUulose (Beato
et aL, 1973; Simons et aI., 1976).
We have studied the transformation of GC receptors extracted from spinal
cord and hippocampus, by charging the receptor with [3H]DEX and heating the
mixture to a form which binds to DNA-cellulose. In preliminary experiments,
binding of the GC-receptor complex of both tissues was compared under
transforming conditions, i.e., after heating for 45 min at 20°C in the absence of
molybdate, an inhibitor of transformation. Total binding of ligand to receptor in
the spinal cord was half that in the hippocampus, and in terms of femtomoles per
milligram of protein, it was slightly lower with regard to DNA-cellulose binding
(hippocampus, 26.4 + 2.9; spinal cord, 20.5 + 1.6; N = 4). The percentage of total
binding bound to DNA-cellulose was, however, higher for the spinal cord
(17.0 + 0.7 vs 11.6 + 1.0; p < 0.005).
This unexpected finding prompted us to analyze the transformation pro-
perties in greater detail. One factor that modulates GC receptor movement into
the nuclei and its binding to DNA-cellulose is RNA. Several groups have
suggested that RNA is associated with the cytoplasmic from of the GC receptor in
diverse tissues such as liver, mammary gland, MCF cells, and AtT-20 pituitary
tumor cells (Chong and Lippman, 1982; Hutchens et al., 1982; Tymoczko and
Phillips, 1983). The complex formed between RNA and receptors behaves like a
ribonucleoprotein upon ultracentrifugation (Economidis and Rosseau, 1985).

5O ,~e p h i d e x LH-20
A DNA-CelIuIose B 50
Q. i i i li

Ot

40
t

I,o 0~
O

S
0
¢.9

•vC 30 -} 3o Z
t'~

0 O
m

-t':o
x 20 20 t--
.h
a
! <
i
o

c ~

0 Hippo-
Control RNAse Control RNAse S.Cord
A A campus

Fig. 5. Effect of RNAse A on binding properties of glucocorticoid receptors in

tOSol of spinal acord and hippocampus. Cytosol was charged with
]dexamethasone, then transformed by heating (20°C, 30 min) in the presence or
absence of RNAse A . Aliquots were then applied to Sephadex LH 20 columns to
measure total binding or were treated with dextran-charcoal to adsorb unbound
hormone. An aliquot of the supernatant obtained after centrifugation was applied
to DNA-ceUulose columns to measure transformation. (A) Sephadex LH 20 and
DNA-cellulose binding of total binding and transformed receptor, respectively.
(B) Percentage of total receptor bound which was eluted with 0.3 M NaCI from
DNA-cellulose columns. (*) P < 0.05; (**) P < 0.01 vs control (no RNAse added).
Reproduced from Moses et al. (1987).
The Spinal Cord as the Target of Glucocorticoid Hormones 187

Application of ribonuclease (RNAse) A increased binding of the GC receptor to

DNA-cellulose, suggesting that RNA-bound hormone receptor complex is
inhibiting DNA binding and that transformation of the receptor to the DNA-
binding state may involve hydrolysis of associated RNA (Rossini, 1985). In our
subsequent experiments, we observed that [3H]DEX binding to cytosolic recep-
tor, measured by Sephadex LH 20 chromatography, was increased after RNAse
A treatment of hippocampal cytosol, but it remained unchanged in the spinal
cord (Fig. 5). The degree of DNA-cellulose binding was stimulated by RNAse A
in the hippocampus only (Fig. 5). Thus, whereas 45% of the total GC receptors
of the spinal cord showed an affinity towards DNA-cellulose in the presence or
without RNAse A, enzyme treatment of hippocampal cytosol stimulated associa-
tion with this polyanion from 30 to 43% (P < 0.01) (Moses et al., 1987). The
spinal cord remained insensitive to all concentrations of RNAse A tested,
although the hippocampus responded to the three concentrations of the enzyme
added (1.5, 3, and 6 mg/ml) in terms of increased affinity toward DNA-ceUulose.
However, the experiments described so far were performed with [3H]DEX
labeling of "total" receptors, without taking into consideration that the spinal
cord and the hippocampus show a pronounced receptor heterogeneity. Therefore,
in another series of experiments, we measured binding to DNA-cellulose
employing conditions that allowed separate binding of type I and II receptors.
Interestingly, the type I sites detected in both tissues (although with much higher
quantities in hippocampus), plus the type II sites of the hippocampus, responded
to RNAse A treatment by increasing its affinity for DNA-cellulose (Fig. 6). Type

RNAsE EFFECT ON RECEPTOR TRANSFORMATION

Type I Receptors Type II Receptors Total Receptors

30 iO- O.
I
M

I II

"I
20 ~
z

t--

t--

rn
:o- i I t °1
Hippocarnpus S. C o r d HippocarnpusS . C o r d Hippocampus S. Cord

Fig. 6. Binding of [3H]dexamethasone-receptor complexes to DNA-cellulose in the

absence (open columns) or presence of RNASe A (3mg/ml) (cross-hatched
columns) during the transformation process. Cytosol was incubated with radioactive
hormone for labeling total receptors (right-hand graph), for type II sites (middle
graph), and for type I sites (left-hand graph). RNAse A increased the percentage
binding to DNA-cellulose of type I sites in hippocampus [(***) P <0.02] and spinal
cord [(**) P<0.01], of type II sites in hippocampus [(**) P<0.01] and of total
receptors in hippocampus [(*) P < 0.05]. No effects of RNAse A were seen for type
II or total receptors in the spinal cord. Reproduced from Moses et al. (1988).
188 De Nicola, Moses, Gonz~lez, and Orti

II sites of the spinal cord, however, remained insensitive to the enzyme. In order
to explain this unusual behavior, we have hypothesized that RNAse A-sensitive
receptors (i.e., type I in both tissues, type II in hippocampus) are ribonucleopro-
teins, whereas the insensitive ones (i.e., type II in the spinal cord) do not contain
RNA. In other words, the differential enzyme effect between the spinal cord and
the hippocampus may be ascribed to further heterogeneity of type II receptors
themselves, although other explanations can be considered (Moses et al., 1988).

Determination of Glucocorticoid Receptors in Microdissected

Regions of the Spinal Cord
The spinal cord constitutes a good example of a histologically complex tissue,
in that it is composed of white and gray matters, the two showing an almost
perfect anatomical resolution. Due to the fact that the synthetic steroid [3H]DEX
binds to two receptor types, we hypothesized that these sites may not be evenly
distributed within the spinal cord. Previous data (Fig. 2) showed higher binding in
regions corresponding to the cervical (vertebrae C3-C7) and lumbar (T9-L3)
enlargements, areas rich in gray matter containing large motoneurons. Autoradi-
ographic data also showed high radioactive [3H]CORT incorporation into spinal
motoneurons (Duncan and Stumpf, 1984). By means of the punch-out technique
of Palkovits (1973), we observed that both gray and white matter contained GC
receptors. After resolution between type I and type II sites by differential
competition (see above), we found that type I sites were more abundant in gray
matter (5.2 fmol/mg protein) than in white matter (2.4 fmol/mg protein), whereas
type II sites were found in roughly similar quantities in anterior and posterior
horns and anterior, posterior, and lateral columns (20-30fmol/mg protein).
However, we have yet to obtain data concerning the biochemical characterization
of type II receptors of gray and white matters, in order to ascertain any possible
differences regarding their binding to DNA-cellulose in the basal state as well as
after RNAse A treatment.

CONCLUSION

Our studies demonstrate that the spinal cord GC receptors share some
properties with GC receptors found in hippocampus, a well-studied target of
hormonal action in the CNS. However, some differences emerged at the level of
nuclear binding in vivo, which was substantially lower in the spinal cord. In this
sense, GC uptake into subcellular fractions of the cord resembled the profile
found in the optic nerve (Meyer et al., 1982) rather than that in the hippocampus.
These differences were ascribed to the heterogenous nature of receptor sites,
assuming that the high-affinity form or type I receptor was almost exclusive of the
hippocampus. This form would be saturated at the low levels o f [3H]CORT
administered in vivo and, hence, would easily translocate into nuclei. The
low-affinity or type II receptor, which was prevalent in the spinal cord, would not
be saturated and its nuclear localization was poor. While this reasoning may
The Spinal Cord as the Target of Glucocorticoid Hormones 189

partly account for the results shown in Table I of reduced nuclear localization of
[3H]CORT in the spinal cord, a second explanation seems feasible. It is possible
that type II receptors of the cord are prevented from translocation due to its
affinity being still lower than the hippocampal type II receptors, considering that
some proportion of the latter could travel to the nuclear fraction. That this may
be the case was shown by results of Scatchard analysis, in which the Kd for type II
receptors using [3H]DEX as ligand were higher for spinal cord (Kd, 3.5 nM) than
for hippocampus (Kd, 0.7 nM) (Moses et al., 1988).
Therefore, it appears that the spinal cord expresses a peculiar form of GC
receptor. Unexpectedly, some other unique properties of type II sites came to
light after discovering their high association with DNA-cellulose in the basal state
and their refractoriness to RNAse A action. The results indicate that the
dynamics of transformation are different among receptors showing affinity and
competition typical of the "classical GC receptor," suggesting further receptor
heterogeneity. It is possible that the spinal cord expresses a type IIb receptor,
which differs structurally from the type IIa receptor of the hippocampus. Further
experimentation is necessary to disclose whether the spinal cord receptor contains
RNA or not. It also remains to be established if these peculiar biochemical
properties of type II receptors of the spinal cord have evolved to fulfill a
particular function in this tissue.
It should be mentioned that the appearance of more than one class of GC
receptors has been repeatedly recognized in normal and abnormal tissues. Thus,
distinct molecular forms of GC receptors (with type II characteristics) have been
identified in melanoma (Hutchens et al., 1981), several bovine tissues (Do et al.,
1979), rat liver nuclei (Kaufmann and Shaper, 1984), and Hela $3 cells (Currie
and Cidlowsky, 1982). Corticosteroid binder Ib is another example of receptor
diversity (Mayer and Litwack, 1983). Recently, Luttge and Emadian (1988)
identified in mouse brain cytosol a new class of GC receptor. Their relationship to
the type II receptor of the spinal cord is hypothetical but further investigation
seems worthwhile.
In summary, the spinal cord seems an ideal tissue to study a new class of GC
receptor, due to its quantitative availability. It also offers possibility for use as
a model system for employing biochemical and molecular biology techniques to
characterize diversity in GC receptors.

ACKNOWLEDGMENTS

This work was supported by grants from the NIH (NS 20866-03A1), the
National Research Council of Argentina (PID 3089300/85), and the University of
Buenos Aires.

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