REACTIONS

OF STRIPS OF RABBIT AORTA TO ISOPROPYLARTERENOL, SODIUM NITRITE OTHER DRUGS

ROBERT F. FURCHGOTT
Washington for publication
AND

EPINEPHRIXE, AND

SUCHIN
School December

BHADRAKOM
of 29, Medicine, 1952 SI. Louis,

Department

of

Pharmacology, Received

University

Mo.

The

in vitro

responses

of rings,

strips

or segments

of arteries

to various

drugs

and physical changes have been the subject of numerous investigations since the pioneer work of 0. B. Meyer (1905). In most cases the source of arteries has been slaughterhouse animals. However, in the course of studies in this laboratory was for on found both the relation that qualitative smooth of such spirally and between cut metabolism strips quantitative of rabbit paper and and contraction aorta of the of smooth were admirably effects primarily general muscles suited on of drugs it thoracic

investigations

arterial response tics as

METHODS.

muscle. The present strips to various drugs, test

experiment aorta (Krebs were trimmed which gently moved The contained 15 degrees held and incision. long, and about cutting, was and off. was between forward resulting smooth relative were cut

is concerned also with their

with the characteris-

pharmacological
For solution tissue During of the the aorta, rotated spiral 20 cm. 4 cm. length at each thoracic

objects.
a rabbit removed Hetiseleit, The done the whole with thumb the was its use kept long in strip muscle to for toward weighing and 1932) length a small, and scissors usually fibers axis. (the 2.5 placed at of to 3.5 in room aorta fingers kgm. a Petri was of the in such about circular From with working at in solution fold. this and on the to The solution reservoir adjusted strips. In of friction continuousvibration to the iron stand this Krebs volume During was dish then rapidly containing Excess cut scissors, operators a manner 0.4 mm. fibers strip the solution. with of the was per give between cent giving 9 fold experiments writing of supporting small along the free thick, of the shorter whole the the aid type was Krebs02-5

decapitated. Krebsfat and uncut hand, 2 mm. intact strips proof a close

The

descending

bicarbonate connective spiral. portion was wide aorta) of 2 to cedure The stainless previously accomplished flask per to cent The to and dilute CO2 bicarbonate of 7.4. plification, in order points amplitude them. This Fund. been
2

temperature.

sharp-pointed

gradually and

as to permit

a continuous oriented

(unstretched)

experiments.

of preparing strips were steel described by any solution was isotonic and minimize kymograph by means

the strips, the tissue was mounted in muscle chambers The (Furchgott, the over-flow containing bubbled, levers both were lag paper, of a small previously added 0.01 in of the to in response the levers vibrating the exert muscle bath 1950). principle, drug 3! at muscle ink-writing 4 gm. due were

moistened of 20 ml.

S-hooks.

assembly, Replacement with least glucose. enough

maintained of solution fresh a thousand Through chambers gravity-type,

37.5#{176},was a muscle from solution 95 flask, to many

chamber reservoir

a pH am-

counterweighted the

tension small subjected

to the motor

amount

connected

investigation Two made preliminary (Furchgott, address: Thailand.

was

supported on of

by some

a grant of the

from work

the

Life

Insurance in this Hospital

Medical paper have Medical

Research already School,

reports 1952a,b). Department

included

Present

Physiology,

Chulalongkorn

Bangkok,

129 -rhi-6
On

E2H9_GT0

130

ROBERT

F.

FLRCHGOTF

AND

SUCHIN

BHADRAKOM

Stock solutions of l-epinephrine bitartrate trate (Winthrop-Stearns) were kept frozen terenol hydrochloride (Winthrop-Stearns diphosphate (van Camp) and acetylcholine

and

monohydrated

l-norepinephrine

bitar-

prior to use. Stock Isuprel containing bromide (Eastman)

solutions of isopropylaranti-oxidants), histamine were kept refrigerated,

and

in the

case

of the

last

two
In

drugs,
for
experiments

were

made
with

up freshly
use
the

about
made

every

week.
drugs the

Working
stock solusaline

solutions of desired tions with isotonic

contained ment muscle and 0.01 kept chamber M in an were

concentration
saline.

experimental solutions
Individual

were freshly

by diluting

sympathomimetic additions

HCI,

and
ice-bath 0.2

the
ml.

working
throughout.

were

refer

to the

concentrations

or less. The concentration of the salt rather than the

prepared before each experiof solutions of drugs to a figures used in this paper always free base. In kymograph records,

-0 z w
0
LU 0

z.
I
0

z
LU
-j

z
0

F
FIG. 1.

2 TIME
in length
was initial were attached length constructed showing and to under

IN
exerting

3 HOURS of
rabbit 4 (following

4
aortic immediate made under tension. strips

5
with stretching on original B. Typical time.

6
Abscissa is

Change
strip of Curves experiments

sensitivity
lever from gradual tension

time in A. per

after cent

gm. tension.

Ordinate

is change
on kymograph

in length
attachment records. experiment to 3 X

to lever). Three

measurements elongation

showing gradual increase in sensitivity to epinephrine mal sensitivity in 2 to 3 hours. At each horizontal l-epinephrine bitartrate. the
drug

with
bar, the

time,
strip

with
was

attainment
exposed

of maxi10

concentration
after RESULTS. the

figure
particulara

at the
ddition.

point

of addition

represents

the

total

concentration

of the

strips about 1A),

Changes in Length and Drug Sensitivity with Time. When aortic are first attached to levers under 4 gm. tension they immediately stretch 40 per cent. They then undergo a further gradual increase in length (fig. which usually amounts to about 5 per cent, but varies from 2 to 10 per follows elongation an exponential is essentially curve complete with a in 1.25

cent. This gradual increase approximately half-time of 10 to 25 minutes, so that to 3 hours.

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

131 with a gradual in a tone

To loss later

determine of spontaneous section

whether tone,3 paper, Additions produced of this

the use

gradual was made

elongation of NaNO2,

is associated which, as will low completely

be shown

is able

to suppress

to moderate

of aortic strips. during elongation

of this drug at concentrations only slight relaxation and

of 10 to 10 did not alter

to strips the time-

characteristics of the gradual elongation itself (fig. 7C). It is therefore concluded that the gradual elongation does not result from a gradual loss of spontaneous tone, but rather that it results from a physical process in which certain undefined structural elements of the If a standard small concentration intervals progressively 1B). Once after mounting a strip reaches has increases and maximal sensitivity of epinephrine proper type aortic strips of epinephrine a maximum been attained, remains the to be rather slowly (10 lengthen to the
10-8)

under tension. is tested at response

in a muscle

chamber,

contractile

in 2.5 hours the response essentially progressive

to 3 hours (fig. to a small standfor 4 to in sensitivity 6

ard test dose hours. By the of strips incubation to

usually of experiments

constant increase

epinephrine was shown at 37.5#{176} Krebs in solution, or on any increase

simply than

dependent on on any changes

the period of related to the

gradual elongation, rine. The progressive also occurs with

sensitizing effects of successive in sensitivity is not peculiar drugs of time. with times the such as norepinephrine, and

additions of epinephto epinephrine, but histamine Relaxation concentration after and ite of 6

other

stimulating same period of Contraction of 10 to 100

acetylcholine over the Time Characteristics Removal. epinephrine minutes.4 begins even (fig. On On rapidly 5 minutes 2A). From addition shortening washing during after this

Epinephrine minimal

effective

begins within out epinephrine the washing but changing time on solution relaxation

5 seconds from gradually

and is completed within 2 to the muscle chamber, relaxation becomes to much slower, so that 90 per cent complete follows an exponential

it is only 75 approximately

curve with a half-time of 10 to 15 minutes. On addition of high concentrations of capable of producing maximal contraction, completion than with after washing
In the present

epinephrine (of the order of 10-s), about 10 minutes are required for after Indeed, washing is much slower over the first 5 minutes then
a state and on that the

of

moderate no

shortening. However, concentrations relaxation

paper the

(fig.

relaxation 2B). detectable.

has
that

is usually
term We tone
believe

Relaxation used
a state muscle, depends to

sets
of on such maintenance

in at

been
such

define
is

maintained continuous activity of

contraction
activity

(or contracture?).

dependent

requires The
the the
heavy was after

of the contractile elements of arterial energy derived from metabolic processes. maintenance of tone after epinephrine
concentration water and reagents
which that arterial addition. might strips This

smooth addition

epinephrine distilled
metals found epinephrine

in the used
in one was

solution bathing the arterial in making the solution need

the oxidation to of muscle corrosion added chamber of the epinephrine. particular traced

strip. For to be free

could not stock

this reason of traces of

it tone hypo-

catalyze

Occasionally, maintain of the

metal

dermic corrected

needle the

used as a bubbler situation.

in the

muscle

chamber

in question.

Changing

the

needle

132 rate which

ROBERT

F.

FURCHGOPT

AND

SUCHIN

BHADRAKOM

gradually gradually.

increases Additional

over

a period within

of about the required complete

5 minutes first 5 minutes for 50 per relaxation.

and cent

then do not The

begins hasten

to decrease the onset and about

washes

of relaxation. About another 60 minutes

15 minutes are for essentially an exponential delayed onset

relaxation, last half

of the relaxation time of about epinephrine time required

is approximately 10 minutes. The

function of relaxation

of time, after due

with a halfflushing out to the in the

solution giving maximal response is probably for outward diffusion to reduce the concentration

in part of the drug

(B)

jX1O94CC

I-

Response to varying concentrations addition of drug and W indicates change A. Contraction on addition and immediate
FIG. 2.

of l-epinephrine bitartrate. Arrow indicates of fluid. Time marks are at 5 minute intervals. relaxation on washout of low concentration of
was of approximately maximal about high 6 cm. concentration 27 per cent. in length. of C. B. Graded was Contraction Strip contraction on epinephrine.

epinephrine. addition was and

Strip delayed in 3 cm.

(under length

tension and

of on

lever) washout

relaxation

about

shortened

resulting from progressive increase in concentration tion series). Strip was about 3 cm. in length, and
22 per cent.

of epinephrine
shortening

(epinephrine

concentraapproximately

region sentially The

of the

smooth

muscle

receptors of relaxation

below after

a level the

capable washout of

of producing both moderate

es-

maximal contraction. final gradual phase

and high sustained

concentrations of epinephrine tone or spontaneous tone. This

is not due is indicated

to a gradual by the fact

loss of drugthat the addi-

tion of NaNO2 (10 to 10-s) to strips during this phase extra relaxation, which is of similar magnitude to that which have first been allowed to complete their relaxation

produces only a slight produced with strips (fig. 7D).

Relation of Response to Concentration of Epinephrine. The minimal effective concentration of epinephrine (that concentration giving a 1 to 2 mm. rise on the kymograph record) was determined on 32 aortic strips which had first been

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

133 values ranged

allowed

to

attain

maximal

sensitivity X the
10-10

(table minimal

1).

The

observed

from 1 to 20 X 10-#{176} (mean: 5.9 tration range up to about 5 times of contraction rifle added; tion than per at the

0.9 X 10-10 (SEM)). effective concentration, to the greater upper half amount degree

In the concenthe degree of epinephof contraclow range

is usually approximately however, with many strips of epinephrine effective minimal concentration

proportional a significantly occurs in the itself.

microgram

of this

z z
LU I0 I U) -J

4
L&. 0 I-

z
LU C-) LU 0

876
FIG.

A and
strips.

curves
on and was at curves. tively. the B. used lower

-LOG OF EPINEPHRINE CONCENTRATION 3. Typical concentration-activity curves obtained with l-epinephrine bitartrate. B. Plot of data from first epinephrine concentration series with fully sensitized Note deviation of actual (solid) curves through points from theoretical (dotted) at intermediate higher levels of contraction. Theoretical curves were constructed basis of Equation 1 using K values of 1.4 X 108 and 1.9 X 10 respectively for
C and for levels Theoretical D. C as of Plot for of A, data and in for and C and from D second as the D for have B. close epinephrine Note K values in each of 4.2 approximation X concentration case of 10 the theoretical and 1.4 series. decreased and X 10 Same sensitivity observed respecstrip

contraction, curves

The

responses

of strips with

to progressively

higher

concentrations

of epinephrine, constrips in the con-

added in series centration-activity half-maximal range 0.5 traction of 10-i,

no washout curves (fig.

(fig. 2), were utilized to obtain complete 3). In 35 experiments with fully sensitized at

contraction to 5 X 10-8 (15 and to 30 per occasionally

was obtained (mean: 3.4 X 10-8 cent shortening) at a concentration

epinephrine 0.3 X 10-8 of 10-i.

concentrations (SEM)). Maximal

is generally

achieved

at a concentration

134 The generally actual deviate

ROBERT

F.

FURCHGOTT

AND

SUCHIN

BHADRAKOM

concentration-activity considerably from

curves curves

obtained predicted

with

fully

sensitized equation (Equation

strips (1937), 1)

by Clarks

C
where contractile

CMS K +
drug concentration complex.5 arterial strips 8, and This at
CM

C is the

contractile (at

response very high

at drug

is the

maximal apparent results high are less

response

concentration),

K is the
deviation intermediate

dissociation constant for the drug-receptor from relatively smaller responses of the concentrations introduced than If than based on

would be predicted from the the responses to epinephrine response (figs. 3A and after being subjected

equation when concentrations epinephrine

K values producing
concentration

one-half maximal an arterial strip,

3B). to one

series, is washed and allowed to relax fully (over a period of 1 to 1.5 hours), and is then subjected to a second concentration series, there is generally no significant difference between the absolute heights of maximal contraction in the two series (0 5 per cent). However, nephrine in a second series is always series. This contraction, required At higher As a result tion series Clarks 3D). The reduction is most with anywhere second series of contraction the sensitivity reduced below its of a strip sensitivity toward in a epifirst

marked at contraction levels below half-maximal from two to five times more epinephrine being than the in the first to produce difference in sensitivity equivalent becomes less responses. marked.

in the levels of this much

differential change in sensitivity, curves more closely approximate theoretical than sensitivity do curves from first during concentration a second

from second concentracurves predicted by series concentration (figs. 3C and with

equation reduced

of a strip

series

epinephrine is the result of changes concentrations of epinephrine during indicated by the finding that a 5 to strip to a single traction, reduces of epinephrine not limited high dose sensitivity

brought about during exposure to high the first concentration series. This is 10 minute exposure of a fully sensitized maximal to graded condoses

of epinephrine, capable of producing just as effectively as does exposure series exposure (table 2). The decrease to a high concentration to norepinephrine,

in a concentration to epinephrine. After decrease

in sensitivity is of this drug, acetylcholine, and

there is a similar histamine.

The equation as

in sensitivity

given

is simply

an

algebraic

rearrangement

of

Clarks

equation,

100
KX
=
-

where X is the drug concentration, K is the apparent association receptor, and V is the response in per cent of the maximal text is preferred because of the common use of an analogous and because it has been found more convenient in a further drug-receptor interaction (Furchgott, to be published).

constant between drug and response. The formula in the formula in enzyme chemistry development of the theory of

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

135 drugs following ex-

The posure reported (Cantoni

reduction to a dose previously and

in sensitivity

to

low

doses essentially artery

of stimulating

of a drug causing for sheep carotid 1946) and increasing this reduction with solution a number
in

maximal contraction (Wilkie, 1928), guinea uterus (Daniel, 1952).

has been pig ileum According

Eastman, and Eastman, counteracted

guinea

pig

to Cantoni several fold ileum. In our

the K+ concentration in sensitivity in the rabbit aorta increasing

of their medium case of guinea pig the K+ concentra-

own

experiments Krebs were

tion of the regular Equally ineffective tion. to high The only epinephrine

up to 2.5 times was of other alterations the decrease could

TABLE

ineffective in this regard. of the regular Krebs solufollowing was exposure suffito allow

manner

which

in sensitivity partly
1

concentrations

be

reversed

Comparison

of

effects

of epinephrine

and

other

drugs

on
REQUIRED

strips
FOR

of

rabbit

aorta
RELATIVE HEIGHT RESPONSE OF

CONCENTRATION DRUG RESPONSE

Minirn:1

detectable

Maximal

response

1-epinephrine l-norepinephrine histamine acetylcholine isopropylarterenol

C C C C R5 C

1-20 1-20 2-20 1-50 1-10

X
X X X

10-10 100 10

1-10 1-10

X
X

10
10

100 100
75-97

-40 1-10 1-10 X X

10
10

10
10

10-45
5-10 72-80 required and represents for that

X 10-6

5
relaxation. on fully The sensitized

x
strips

I0

In minimal

this

table detectable

C giving

denotes response

contraction was determined

arid

concentration

concentration

concentrations

a 1 to 2 mm. displacement of the lever point. All concentrations are of the salts, not of the free bases. (See section on METHODS for salts used.) * Relaxation with isopropylarterenol was determined on strips brought to a state of moderate tone by prior addition of one of the other drugs listed in table. Relative height of maximal response in case of relaxation is based on comparison of absolute increase in length of strip on relaxation with absolute decrease in length on maximal contraction with

epinephrine. cient time (2 to 3 hours) to elapse after dose of that drug (table 2). Norepinephrine and Isopropylarlerenol. trate arterial of the (SEM). and monohydrated at norepinephrine concentration relative greater strip response Except a similar low to norepinephrine for this the In washout six bitartrate (10 to that of the large de-sensitizing bitarthe same ratio .05

experiments were to 10-8). of epinephrine at

epinephrine tested The low on average was 1.18 concentrations,

somewhat

sensitivity

the general behavior toward epinephrine

of the strips toward norepinephrine is the same (table 1). If maximal contraction is produced by the necessary and and

as that addition

of one of these drugs at the drug at the same concentration, no additional contraction. In contrast to epinephrine

high concentration, addition of the other in the presence of the first drug, produces norepinephrine, isopropylarterenol gives no

136 contractions concentration 4A). Further in

ROBERT

F.

FURCHGOPT

AND

SUCHIN

BHADRAKOM

the

concentration order of 10_s in concentration

range

between

10-s

and

10-v.

Only

when occur with (fig. the

of the increase

is reached leads

does contraction first to greater contraction,

maximal response being attained at about 5 X 10. This maximal contractile response was found in five experiments to be about 75 per cent of that obtained with epinephrine. Both the rate of shortening on addition of a stimulating dose TABLE
Sen8itivity to low concentrations of causing epinephrine

2
before and after exposure to a concentration contraction
CONTRACTION BlIGHT

maximal

#{163}XP. NO.

EPI

NIPS.

CONCN.

10

Before Epineph.
mm.

After 30-40 mm.

Washout 6-70 mm.

of 10 Epinephrine 120 mm. 180 mm.

-

1 2

3 X 10- 3 X10-
10-8

27 3.2
17

4*
-

8
1.5 9

12 2.5
13

3
4

3 X10-
3 X10

17 7.5

2*

5 4

6 5.5

8 6

Strips in all experiments were approximately 3 cm. long when fully stretched under tension of lever. Contraction height is in mm. rise of lever point on kymograph record. * Strip had not yet completed relaxation following washout of 10- epinephrine.

ii
(A)

___

FIG. 4. Responses to isopropylarterenol arterial strip with high concentrations washout (W). B. and C. Relaxation with initially brought to a state of moderate and acetylcholine (AC).

hydrochloride. of isopropylarterenol

A.

Contraction (I), and addition

of untreated relaxation after

low concentrations
tone by the previous

of isopropylarterenol
of epinephrine

of strips
(E)

of isopropylarterenol case of epinephrine In order to show

strips,

and

the

rate

of relaxation 2A). effect

on washout

are

slower

than on

in the aortic

(compare clearly

figs. 4A and the relaxing

of isopropylarterenol

with

it is first a stimulating other

necessary drug.

to bring the The stimulating such

strips to a state of low to moderate tone drug may be acetylcholine, histamine, as epinephrine and norepinephrine. Sub-

or even

sympathomimetics

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

137 range of

sequent 10- to relaxing creased of

additions

of

isopropylarterenol

over

the

low

concentration

10-v now produce significant relaxation (figs. 4B and 4C). The maximal effect occurs between 10-i and 10-6. When the concentration is infurther, relaxation gives way to contraction. Indeed, the concentration causing relaxation of absolute is usually brought initially maximal being changes between to a state relaxation frequently has a biphasic the initial In terms followed by a small, slowly developed in length the maximal relaxing effect 7 and 15 per cent rather of its than maximal low conto mod-

isopropylarterenol

effect, with contraction.

of isopropylarterenol tracting effect. If strips are erate, cause results

of maximal,

tone with epinephrine or norepinephrine, isopropylarterenol relaxation. In experiments with maximal tone due have not been consistent. In three such experiments, degree of relaxation, is very by epinephrine but similar on in three both arterial

can no longer to histamine, our isopropylarterenol others it produced and previously unlike to a is the

was able to produce a small no detectable relaxation. Relaxation qualitatively treated with isopropylarterenol, state case not only but also with Acetyli.holine histamine with to that

isopropylarterenol produced (Furchgott, gives relaxation by the addition

quantitatively strips

Dibenamine never tone

1952a,b). However, epinephrine, of untreated strips first brought of other stimulating drugs. This

of moderate

with concentrations of epinephrine which alone give contraction much lower concentrations (10- to 10#{176}). and Histamine. Certain characteristics of acetylcholine and of aortic strips are listed in table 1. The usual minimal

as stimulants

effective concentration of acetylcholine is about 5 X 10-i (although it varies markedly in different strips), and that of histamine about 5 X 1O_8. The quantitative relation of sensitivities to epinephrine, to histamine, and to acetylcholine varies about tional an the greatly in different strips. With concentrations of acetyicholine up proporis usually above for of histo five times the to concentration. large minimal effective level, response However, in the case of response as the (fig. 5A and of response to those with concentration B). for low to acetyicholine described moderate for and epinephrine. concentrations frequently However, is approximately histamine there is raised

inordinately minimal The time are half

2 to 5 times

effective level characteristics very of the similar

histamine in about

already

experiments

tamine a gradual rise initial rapid contraction (4 out of 10 experiments) of maximal Relaxation contraction was never

in tone occurred for as long as 10 minutes following the (fig. 5B). In contrast to this, there sometimes occurred a gradual loss of tone of strips following attainment with high obtained concentrations either with of histamine. acetylcholine or histamine (over

the range 10b0 to 10-i), regardless of whether the strip beingtested was initially at rest or in a state of moderate to high tone due to the previous addition of another stimulating drug. Sodium Nitrite and Spontaneous Tone. When NaNO2 is added at a concentration of 10-i to strips of rabbit aorta in a state of elevated tone due to the prey-

138 ous addition of relaxation initial tonus 30 per

tion of

ROBERT

F.

FURCHGOTT

AND

SUCHIN

BHADRAKOM

of a stimulating drug, relaxation always occurs does not depend on the particular stimulating level. At low to moderate levels (contraction produced Indeed, by epinephrine), in many experiments, the

6A). The degree drug but on the heights up to about relaxation the strips on addiactually

(fig.

cent of the maximum NaNO2 is complete.

(A)

5Xt0

-.

.013 FIG.
5.

M responses to acetyicholine bromide (AC), histamine diphosphate (H),

Typical

and KCI. Arrow indicates of KC1 is in excess of that

addition in regular

of drug and W indicates Krebs solution.

change

of fluid.

Concentration

(A

(
1 -I

S.

NQP4O2.)O
-

-3

(C)

(D)
-\

Nc*N01

i
FIG. 6. Responses to sodium nitrite. Record A was obtained with strip approximately 3 cm. in length (under tension of lever), while all other records were obtained with strips approximately 6 cm. in length. Vertical bar in record B is 1 cm. on original record. A. Relaxation by NaNO2 of strip initially brought to a state of moderate tone with epinephrine. B. Effect of NaNO2 on fully stretched strips. Extra elongation after NaNO2 in lower tracing was greatest obtained in present experiments. C. Effect of NaNO2 added to strip undergoing gradual elongation five minutes after attachment to weighted lever. Two lower curves are from same record one hour and two hours later. D. Effect of NaNO2 added at beginning of gradual phase of relaxation following initial rapid phase of relaxation on washout of

stimulating

dose

of epinephrine.

relax

to slightly

greater

lengths

than

they

had

before

the

elevation

of tone

with of the 6A the with

the stimulating drug. Relaxation begins within a few seconds NaNO2 and is initially even more rapid than relaxation removal of the stimulating drug by rapid change of solution and
fast

of the addition resulting from (compare figs.

6D).
initial

However,
phase of

as in the
relaxation

case
after

of the removal of such drug by washing, NaNO2 is followed by a slow phase,

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

139 along an exponential tone addition due of to 10-i

the

lever

approaching

the

base

line

of complete

relaxation

curve with a half-time of about 10 to 15 minutes. The finding that a strip initially in a state of low the previous to
which

to moderate on

addition

of a stimulating greater that many by with in reflecting

of a per

drug

often

relaxes

NaNO2
stimulating tone

a length slightly drug suggested

can be suppressed of experiments

than it had aortic strips This which was had

prior to the addition of the possess a slight spontaneous shown been drugs. elongation to be allowed Such the to strips (fig. 6B). case attain on in a esthe The

large sentially addition

number of

nitrite. strips underwent the

cent

constant 10-i apparently

to only

length NaNO2
a fraction

absence

of stimulating a slight suppression of the

usually

elongation,
at most

of spontaneous tone, amounted initial length. It was not increased

Lii

a-

LOG

EPI

CONCENTRATION

7. Effect of presence of various concentrations of NaNO2 on response to progressive increases in concentration of l-epinephrine bitartrate. The strips used were all from the same aorta. The level of maximal response at 10-i epinephrine with each concentration of NaNO2 is based on the effect of the addition of each concentration of NaNO2 to the control strip in the presence of 10-i epinephrine. NaNO2 concentrations were as follows: none 0 (control), 10- X, 10L, 10 #{149}.
FIG.

by raising the dividual strip in an

ment.

nitrate treated

concentration with NaNO2 state or in a state

to 10. was the of tone

of the

The same

final relaxed length whether the strip tone prior

of an inhad been to treatwith nitrite

It also

unstimulated small
despite in

low which

drug-induced can

The
persists persists

amount
numerous

of spontaneous
changes

be suppressed
aortic

solution

bathing

strips.

the presence of Dibenamine, atropine or diphenhydramine, so that seems unlikely that it is due to a continuous release of epinephrine, norepinephrine, acetylcholine or histamine within the strips. On the other hand, interfering with the energy metabolism of arterial strips by shifting from aerobic to anaerobic conditions in the absence of glucose does suppress spontaneous on tone just effectively as nitrite (Furchgott, The results of a typical
antagonism are

it

as

to be published). quantitative experiment fig. 7. At any given epinephrine

NaNO2-epinephrine concentration the

plotted

in

140 degree
10-6

ROBERT

F.

FURCIIGOTT

AND

SUCHIN

BHADRAKOM

of to 10.

depression At any

increases given nitrite

as

the

NaNO2 increases. completely

concentration the degree

increases of depression

from deof epito

concentration

creases fig. 7,

as the epinephrine 10-i NaNO2 is able

concentration to suppress
10-8,

Thus, in the experiment the response to epinephrine the response to 10 in high concentration does not vascular

up to a concentration nephrine only 6 per break through nitrite on a direct interference

muscle.

approaching cent. This inhibition with the the an K

ability

yet it lowers of epinephrine

indicates contractile concentration arterial strip

that the inhibition mechanism of the (0.0058 is increased

depend smooth

Potassium carbonate

Chloride. If solution bathing KC1,

addition

N) of the Krebs-bi3 to 4 times by the 5C, both the washout of KCI which on rate are no

addition of extra of shortening on very much slower

the

than

strip contracts. As shown in fig. of KCI and the rate of relaxation in the case of epinephrine. Additions

more than double the K+ concentration of the Krebs solution are usually incapable of causing visible contraction of arterial strips. However, such subthreshold additions definitely potentiate the contractile response produced by low concentrations of epinephrine. If strips are brought to a state of moderate tone by the addition of KC1, they are usually more resistant to the relaxing
effects of

isopropylarterenol

and

NaNO2

than

strips or histamine. of arteries who used

brought for pig

to in vitro carotid

a comparable studies arteries was as a

state first

of tone described

DIsCUssIoN.

with epinephrine, acetylcholine The use of spirally cut strips by Lewis and Koessler (1927), case of arteries of about cut strips are preferable with larger arteries), only moderate reports on the
arterial smooth

source. In the aorta, spirally used frequently records with any previous
actions vantages. of Its

2 to 3 mm. diameter, such as the rabbit to rings or transected rings (such as because they give adequate kymograph We have been unable to find aorta as a test object for the redistinct spirally adcut

lever amplification. use of the rabbit

muscle, yet

source

is a common

it appears to have certain laboratory animal. Preparing

strips from it is not difficult. Such strips respond enough to permit relatively rapid diffusion of drugs into their deeper layers. Also when such strips are solution, as there
frequently

well to drugs and are thin from the surrounding medium suspended in well oxygenated prevailing
thicker

there might

is no danger
be with from rings

of anaerobic
or strips slaughterhouse

conditions
from much animals.

in deeper
arteries of the

layers
type

obtained

Another advantage of strips of rabbit aorta under our experimental conditions is that they possess almost negligible spontaneous tone and never exhibit rhythmic contraction. This makes them especially well suited for quantitative studies on drugs
response

from

causing contraction, since the variable influence of these factors on the to such drugs does not have to be evaluated. In this regard they differ certain preparations of arteries from larger animals which may develop spontaneous (e.g., Rothlin, of strips of tone and rhythmic contraction 1920; Weiss, 1920; Ducret, rabbit aorta to epinephrine, during 1931). histamine, the course acetyl-

both considerable of an experiment The responses

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

141 responses of rings animals, or such

choline, strips

and

NaNO2

are

similar

in many

respects arteries

to

the

of various

principal

distributing

of slaughterhouse

as studied by numerous see papers by Rothlin we have been unable preparations of arteries. it can is unique in that high concentrations. Ahlquist (1949), propylarterenol Returning be noted after they to that are

earlier (1920) to find Among

workers. (For omprehensive reviews of literature and Monnier (1943)). As for isopropylarterenol, any previous reports as to its effects on isolated the drugs which we have so far to with investigated it at of isorelaxation at low concentrations finding gives direct support blood pressure experiments and contractions the conclusion rabbits, that

cause This based on cause

can

vasoconstriction

as

well

as

vasodilation. of rabbit sensitivity to aorta, it should of such strips increases observed (e.g., Rothincrease in similar phase

the more the gradual mounted

general properties of strips increases in length and in muscle chambers are

similar

by others with preparations of isolated arteries from larger animasi un, 1920, and Ducret, 1931). In our own experiments the gradual length was approximately an exponential function of time. A very

of gradual elongation was found to follow the initial rapid phase of relaxation of rabbit aortic strips after the washout of stimulating drugs. Although we are iiot prepared to speculate as to what elements in the strips are gradually lengthening under such out the possibility loss of spontaneous The
stimulating

circumstances, our that the phenomenon tone. in sensitivity

represent may

experiments of gradual of freshly

with NaNO2 appear to rule elongation is due to a gradual mounted

from

gradual
drugs

increase brought during

which exposure poor have to

strips
a state

to low

doses the strips that

of

a gradual

recovery

of relatively

poor are
when traction state

sensitivity subjected
strips by of

on by their
become a high sensitivity

the
fully

trauma This
sensitized

and

stimulation
are of stimulated epinephrine, of

to which with the

to they stimulating

preparation.
concentration to

is consonant

finding
maximal revert

conto drugs. a

relatively

low

concentrations

Following such The reduction

an exposure in sensitivity

sensitivity resulting

again increases from exposure

as a function of time. to high concentrations of

epinephrine accounts for the finding that concentration-activity curves obtained in second epinephrine concentration series deviate less from theoretical curves than do concentration-activity curves obtained in first series. If it is assumed sensitivity would series that changes in the state develop at high levels be expected would follow that the a theoretical of the smooth of contraction muscle during which lead to reduced a first series, then it levels curve from of contraction such but a

actual concentration-activity curve up to moderate

fall below it at high might be represented levels of contraction.6

On sociation indicates another the that function basis constant K of for is not which Clarks the the

levels of contraction. In terms of Equation 1 this effect by a progressive increase in the magnitude of K at high Furthermore, if the changes leading to reduced sensitivity
original drug-receptor actual dissociation on the formulation, complex. number the constant, of total K in but equation recent probably receptors, work that the

1 should
in this state

be the
laboratory

distimes

However,

constant

is dependent

of metabo-

142

ROBERT

F.

FLJRCHGOTF

AND

SUCHIN

BHADRAKOM

are largely unreversed by further changes developing are likely contractions

the time a second concentration at high levels of contraction during the responses obtained be expected to fall fairly

series is run, any the second series at all levels of close to a theo-

to be less marked. Theiefore, in a second series would curve. be higher constant

retical concentration-activity of a second series would would remain more nearly series.

In terms of Equation 1, K at the beginning than at the beginning of a first series, but at all contraction levels during the second

SUMMARY

1. The cological amount freshly gradual to three temporary

general test objects

properties have

of spirally been described.

cut

strips Such strips

of rabbit possess

aorta only

as

pharmasmall When and two by

a very

of spontaneous tone and never exhibit rhythmic contractions. mounted in muscle chambers they undergo a gradual elongation increase hours. exposure in sensitivity If a fully to a high were found to stimulating sensitized strip concentration to give only drugs is caused over to a period contract it reverts of aortic epinephrine strips initially of about maximally

of epinephrine, contraction and of addition

to a state strips > fall in

of low sensitivity. 2. Drugs which the following > acetyicholine. 3. Sodium state of low order nitrite

as to potency: causes tone complete by

norepinephrine relaxation previous

histamine in drug. a It

placed

to moderate

the

of a stimulating

also appears to be able to completely However, in the face of maximal tone it causes only slight 4. Isopropylarterenol relaxation. causes

suppress spontaneous tone due to high concentrations at low on other concentrations. addition, have and been

of aortic strips. of epinephrine, However, of relaxation The equaat

relaxation contraction. of contraction and certain

high concentrations it causes 5. The time-characteristics after washout, of epinephrine curves 6. Concentration-activity relation of the observed tion has been discussed.

drugs

described.

curves for epinephrine to theoretical curves

have been presented. predicted by Clarks

REFERENCES
AHLQUIST, CANTON!, CLARK,

R. P.: Am. J. Physiol., 153: 586, 1948. G. L., AND EASTMAN, G.: THIS JOURNAL, 87: 392, 1946. A. J.: in Heffters Handbuch der Experementellen Pharmacologie,
Berlin,
E. S.: E.: Arch.

vol.

4, J.

Sprin-

ger,
DANIEL,

1947.
Fed. Proc., f. d. ges. THIS 11: 335, 1952.

DUCRET, FURCHGOrr,

Physiol.,

227:

753,
1950.

1931.

R. F.:

JOURNAL,

99: 1,
shown

lism of the muscle,

of the
constant

etc. As will be
by Clarks
an experiment.

in a later

publication,

concentration-activity

curves

type

predicted
throughout

equation

can

only

be expected

when

this

function

remains

REACTIONS

OF

AORTIC

STRIPS

TO

DRUGS

143

FURCHGOTT,

FIJRCHGOTF, ItREBS, LEWIS, MEYER, MONNIER, ROTHLIN,

R. F.: R. F.: H. A., AND

Fed.
THIS

Proc.,

JOURNAL,

HENSELEIT,

J. H., AND KOESSLER, 0. B.: Z. f. Biol., 48: 352, 1905. M.: Helv. Physiol. Acta, 2: 279, 1943. E.: Biochem. Z., 111: 219, 1920.
S.: Arch. f. d. ges. D.: THIS JOURNAL, Physiol., 181:
1928.

217, 1952a. 106: 387, 1952b. K.: Z. f. physiol. Chem., 210: 33, 1932. K. K.: Arch. Int. Med., 39: 182, 1927.

11:

WEiss,
\V!!JuE,

213,

1920.

34: 1,