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Venous Relaxation Caused by Acetylcholine
Acting on the Sympathetic Nerves
By Paul M. Vanhoutte and John T. Shepherd
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ABSTRACT
Experiments were performed to determine whether acetylcholine affects the sympathetic activation of the cutaneous veins of the dog. Changes in isometric tension of
saphenous vein strips were recorded at 37°C in an organ bath. Addition of acetylcholine at 1CH1 to 10~8 g/ml did not affect basal tension, but larger doses (5 X 1CH
to 5 X 1CH g/ml) caused a contraction of the strips which varied from slight to
marked. Acetylcholine at 1CH to 1CH g/ml caused a further increase in tension when
it was added to strips already contracted by norepinephrine, tyramine, KC1, or BaCl2;
in contrast, similar doses of acetylcholine caused relaxation of strips contracted by
liberation of norepinephrine from the nerve terminals by electrical stimulation (1—10 cps).
This relaxation was not influenced by propranolol or hexamethonium but was abolished
by atropine (10"8 g/ml). In intact dogs, the lateral saphenous vein was perfused
with autologous blood at constant flow. A sustained venoconstriction was induced
either by electrical stimulation of the lumbar sympathetic chain or by a continuous
infusion of norepinephrine. An infusion of acetylcholine (10~7 to l(h® g/ml min-1)
relaxed veins constricted by sympathetic stimulation but not those constricted by
norepinephrine. Thus, acetylcholine, in doses smaller than those known to have a
direct constrictor effect, causes relaxation of cutaneous veins, probably by inhibiting
the release of norepinephrine from nerve terminals.
KEY WORDS
cutaneous veins
cholinergic dilatation
vascular smooth muscle
norepinephrine
inhibition of adrenergic transmission
tyramine
atropine
• Acetylcholine, a potent vasodilator in vivo,
causes contraction in most isolated vascular smooth
muscle preparations (1, 2). In the venous system,
this direct constrictor effect has been observed in
isolated cutaneous (3-8), jugular (9), mesenteric
(9-12), portal (9, 13-16), pulmonary (17, 18), and
umbilical (19) veins. In the intact animal, application of acetylcholine causes splanchnic (20, 21) and
cutaneous (4, 22, 23) veins to contract.
Following monamine-induced constrictions, rabbit aorta (24) and ear artery (25) are relaxed by
acetylcholine, but arterioles from skin and mesentery are not (26). Reportedly, acetylcholine can
also depress the reaction of rat mesenteric (27) and
rabbit ear (28, 29) arteries to sympathetic stimulation.
Clement et al. (6) showed that, following
norepinephrine-induced contraction, the cutaneous
vein of the dog is not relaxed by acetylcholine. The
From the Mayo Clinic and Mayo Foundation, Rochester,
Minnesota 55901.
This investigation was supported in part by U. S. Public
Health Service Grant HL-5883 from the National Heart and
Lung Institute.
Received July 17, 1972. Accepted for publication
November 21, 1972.
CircuUlion Rtiurcb,
Vol. XXXII, Fiirutr,
197}
dog
tetrodotoxin
present experiments were designed to determine if
acetylcholine modifies the response of this vein to
sympathetic stimulation.
Methods
IN VITRO STUDIES
The experiments were performed on helical strips of
lateral saphenous veins isolated from dogs (15-25 kg)
anesthetized with sodium pentobarbital (30 mg/kg,
iv). The preparation was placed in a chamber filled
with Krebs-Ringer's bicarbonate solution (NaCl 118.2
mM, KC1 4.7 mM, MgSO4 1.2 ITIM, KH2PO4 1.2 mM,
CaCL 2.5 mM, NaHCOa 25.0 mM, calcium disodium
ethylenediaminetetraacetate, 0.026 mM, and glucose
11.1 mM) maintained at 37°C and aerated with a 20%
Oo-75% No-5% CO-, mixture. The strips were connected
to a strain gauge (Grass FT-03) for isometric tension
recording.
For electrical stimulation of the preparation, two
rectangular platinum electrodes were placed parallel to
the strips, as described previously (30, 31). Impulses
(1—10 cps) consisted of square waves (8 v, 2 msec)
provided by a d-c power supply and switching
transistor (RCA 2N-3034) triggered by a Grass S4
stimulator.
The following pharmacologic agents were used:
acetylcholine chloride, Z-norepinephrine bitartrate, tyramine hydrochloride, isoproterenol hydrochloride, propranolol hydrochloride, hexamethonium bromide, tetrodotoxin, and atropine sulfate. Each drug was dissolved
259
VANHOUTTE, SHEPHERD
260
in 0.1 ml of Krebs-Ringer's solution, which was the
volume added to the organ bath. The doses, except those
for norepinephrine and hexamethonium which are given
in terms of the free base, are expressed as final bath
concentrations of the salts. The drugs were removed
from the bath solution by overflowing the preparations
with aerated Krebs-Ringer's solution at 37°C.
Before the experiments were begun, the preparations
were placed at the optimal point of their length-tension
relationship (30). In each group of preparations, only
one strip was obtained from any one dog. The doses of
the drugs and the frequencies of the electrical impulses
were randomized. For the statistical analysis of the
data, Student's t-test for paired observations was used.
The data are expressed as means ± SE.
IN VIVO STUDIES
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The method used in this study has been described
(32). Dogs (16-20 kg) were anesthetized with sodium
thiopental (15 mg/kg, iv) and chloralose (80 mg/kg,
iv). Additional amounts of chloralose were given as
required to maintain the proper level of anesthesia. The
left lateral saphenous vein was cannulated at the ankle
and perfused at constant flow (100 ml/min) with
autologous blood from the terminal aorta. The perfusion
circuit consisted of a roller pump, a depulsator, and a
heat exchanger (37°C). The aortic pressure, the
saphenous vein perfusion pressure, and the femoral vein
pressure were continuously recorded. Since the blood
flow through the saphenous vein and the femoral vein
pressure were constant, the saphenous vein perfusion
pressure was a measure of the tone in the vein wall.
Sympathetic Stimulation.—The lumbar sympathetic
trunk was exposed, severed, and stimulated (Grass SD5
stimulator) via a platinum bipolar electrode placed
between the fifth and the sixth lumbar ganglion. Squarewave impulses (10 v, 2 msec) were applied at
frequencies of 1-10 cps.
Drugs.—Norepinephrine bitartrate and acetylcholine
chloride were infused at constant speed upstream from
the roller pump and the cannula, respectively.
Results
acetylcholine (5xlO~ u to 5 X 10"7 g/ml) was
investigated. The lower doses (5 X Kh11 to lf>8
g/ml) had no effect on base-line tension
(0.845 ± 0.480 g). A dose of 5 X 10-« g/ml caused a
slight contraction in seven of the nine strips (mean
increase in tension, 0.030±0.010 g); 10-7 g/ml
caused all of the strips to contract slightly (mean
increase in tension, 0.070 ±0.18 g). With the
highest dose tested, a marked increase in tension
(1.63 ±0.380 g) was obtained in all preparations.
Stimulated Preparations.—Figure 1 shows the
original record of an experiment in which a single
dose of acetylcholine (5xlO~ 8 g/ml) was given
during a sustained response to a 2-cps electrical
stimulation. Acetylcholine caused a marked relaxation of the strip, and this effect could be rapidly
reversed by washing out the drug. In identical
experiments on 15 preparations from different dogs,
the average response to electrical stimulation was
depressed to 9.1 ± 1.6% of the original reaction
(mean increase in tension, 1.540 ± 0.238 g).
In six saphenous vein strips, sustained contractions (mean increase in tension, 1.050 ±0.140 g)
were obtained by stimulation at 1 cps. While the
stimulation was continued, doses of acetylcholine
(5 X 10"9 to 5 X HH g/ml) were added to the bath
solution (Fig. 2, top). As the dose of acetylcholine
was increased, there was a progressive decrease in
the reaction to electrical stimulation; in five of the
six strips the decrease was maximal at a dose of
5 X 10"8 g/ml. At this dose the reaction to electrical
stimulation was decreased to 11.6 ± 3.9% of the
control contraction. Increasing the acetylcholine
concentration to 5 X 10~7 g/ml caused all of the
strips to contract.
IN VITRO STUDIES
Unstimulated Preparations.—In nine saphenous
vein strips, the effect of increasing doses of
Although acetylcholine decreased the contractions caused by electrical stimulation, it augmented
5 mln
i g
STIMULATION (2cps)
ACETYLCHOLINE (5x10-8 g/ml)
FIGURE 1
Isometric tension recording from a saphenous vein strip. Electrical stimulation caused an
increase in tension that was inhibited by acetylcholine.
CrcuUtwn R,s,.rcb, Vol. XXXII, Febrwtu 1973
261
VENOUS RELAXATION BY ACETYLCHOLINE
STIMULATION
( I cps )
I
A B O D E
NOREPINEPHRINE
( IO~* g/ml )
FIGURE 2
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Typical experiment showing the effect of increasing doses
of acetylcholine (A = 5 X 10-«. B = 10-*, C = 5 X JO-8,
D = JO-7, and E = 5 x 10~7 g/ml) on reactions of a saphenous vein strip to electrical stimulation (top) and norepinephrine (bottom). Acetylcholine was removed at the time
electrical stimulation ceased or together with the norepinephrine.
the contractions caused by the addition of norepinephrine to the bath solution (Fig. 2, bottom).
In six strips, a comparison was made of the effect
of increasing doses of acetylcholine (10~9, 5 X 10~9,
10-s, 5xlO- s , and 10~7 g/ml) on contractions
induced by electrical stimulation and by addition of
norepinephrine. Five stimulation frequencies (0.5,
1, 2, 5, and 10 cps) and four norepinephrine
concentrations (5X10" 9 , 10"*, 5x10-", and KH
g/ml) were selected to cause comparable changes
in tension (Fig. 3). During electrical stimulation,
the addition of acetylcholine decreased the contractions. When expressed as a percent of the control
increase in tension, this relaxation was directly
related to the dose of acetylcholine. In the presence
of norepinephrine, the addition of acetylcholine
caused a further increase in tension, and this
augmentation was also directly related to the dose
of acetylcholine.
Six strips were made to contract by applying
norepinephrine, tyramine, or electrical stimulation.
The frequency of stimulation and the dose of each
agent were selected to give comparable contractions. With acetylcholine (Fig. 4), the increases in
tension caused by norepinephrine (0.790 ± 0.085 g)
and by tyramine (0.730 ±0.100 g) were augmented to 117 ±4.19* and 124 ±7.45%, respectively, and the increase caused by electrical stimulation
(0.850 ±0.115 g) was decreased to 11.4 ±2.71%.
Six strips were made to contract by bathing them
in a solution containing 25 mM KCl (mean increase
100% • 2.26 g± 0.33
100% • 2.34 g ±0.23
120 -
o i
5
10
STIMULATION (cps)
NOREPINEPHRINE (g/ml)
FIGURE 3
Effect of increasing doses of acetylcholine on the reactions of dog saphenous vein strips to
electrical stimulation at five different frequencies (left) and to norepinephrine at four different
concentrations (right). Data are expressed as means for six strips, o = difference from control
value is statistically significant (P < 0.05), o — difference from control value and from next
lower acetylcholine dose is statistically significant (P < 0.05), and • = difference from control
value is not significant.
CtrcuUaio* Rtsurcb, Vol. XXXII, Ptiriurj 1973
262
VANHOUTTE, SHEPHERD
NOREPINEPHRINE
STIMULATION
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i g
5 min
ACETYLCHOLINE,
5xlO-8g/ml
FIGURE 4
Effects of acetylcholine on contractions of the same saphenous vein strip caused by norepinephrine (top), electrical
stimulation (middle), and tyramine (bottom). After stabilization of the effect, both acetylcholine and the agonist were
removed simultaneously. Tyramine was administered in
stepwise increases until the tension was similar to that with
electrical stimulation and norepinephrine.
in tension, 0.630 ±0.182 g); acetylcholine (5 X
g/ml) caused an additional increase in tension to
tOO % - 1.86
Q±
1.000 ± 0.275 g. Six other strips were made to
contract by adding BaCl2 at 0.25 mg/ml (mean
increase in tension, 0.542 ± 0.042 g); acetylcholine
caused a further increase in tension to 0.983 ± 0.172
g. In these 12 strips, the typical depression by
acetylcholine of the reaction to electrical stimulation was observed.
Pharmacologic Inhibitors.—With six strips, studies were made to determine if the relaxation
caused by acetylcholine during electrical stimulation was affected by beta-receptor blockade. Propranolol (lO^6 g/ml) prevented the relaxation
caused by isoproterenol (10~7 g/ml) given during
sustained 2-cps electrical stimulation.
The strips were made to contract by electrical
stimulation (2 cps), and acetylcholine (5 X HH
g/ml) was added to cause relaxation. Then the
experiment was repeated in the presence of
propranolol at 10~° g/ml. The acetylcholine-induced
relaxation was not influenced by beta-receptor
blockade.
With six strips, acetylcholine (5X10- 8 g/ml)
was added to the bath solution during a sustained
contraction caused by electrical stimulation (2 cps).
After stabilization of the relaxation induced by
acetylcholine, increasing amounts of hexamethonium were added (10"8 to 10~B g/ml). Hexamethonium was essentially without effect, although higher
doses did result in a slight increase in tension (Fig.
5, left).
100 % • 1.96
0.44
g ±
0.48
100
o
o
u.
o
Control
Acetylcholine
< 5 x IO'B
g/ml)
50
1-2 cps-l
10 " IO'r
10'" IO'S
g/ml
HEXAMETHONIUM
8
'
10'"
g/ml
ATROPINE
FIGURE 5
Left: Effect of increasing doses of hexamethonium on the relaxation of saphenous vein strips
caused by adding acetylcholine durihg electrical stimulation. Right: Effect of atropine on the
same strips. All values are means ± SE (N = 6).
Circulate*
Research, Vol. XXXII, February 1975
263
VENOUS RELAXATION BY ACETYLCHOLINE
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In the same strips, after the hexamethonium was
washed out, a second sustained contraction was
induced by electrical stimulation (2 cps), and then
relaxation was induced by acetylcholine (5 X 10"8
g/ml); the relaxation was allowed to stabilize.
Atropine (Kh8 g/ml) was added, and this drug
caused a rapid abolition of the relaxation due to
acetylcholine (Fig. 5, right). Atropine also caused
an abolition of the contraction due to acetylcholine
during norepinephrine-induced contractions (Fig.
6) and during contractions caused by KC1 and
BaCl2.
In five other strips, acetylcholine at 5 X 10"8 g/ml
caused a slight increase in tension (0.062 ± 0.025 g)
in resting preparations and depressed the reaction
to electrical stimulation (2 cps) to 13.5±5.9$ of
the original contractions (mean increase in tension,
0.518 ±0.118 g). The same dose of acetylcholine
augmented the reactions to norepinephrine at
2 X 10"8 g/ml (mean increase in tension,
0.769 ± 0.071 g) by 0.100 ± 0.020 g and the reactions
to BaClo at 0.25 mg/ml (mean increase in tension,
0.515 ±0.035 g) by 0.286 ±0.186 g. The addition
of tetrodotoxin (10"B g/ml) abolished the reactions
to electrical stimulation (1-15 cps) but did not
inhibit the reactions to norepinephrine (mean
increase in tension, 0.785 ± 0.090 g) or BaCl2
(mean increase in tension, 0.670 ±0.065 g).
In the presence of tetrodotoxin, acetylcholine still
caused an increase in tension (0.073 ± 0.033 g) in
resting preparations and further increased the
5 min
STIMULATION <2cps)
ACETYLCHOLINE (SxlO-'g/ml)
ATROPINE (IO-> g/ml)
reactions to norepinephrine and BaCl2 by
0.120 ±0.020 g and 0.395 ± 0.254 g, respectively
(Fig. 7).
IN VIVO STUDIES
Unstimulated Preparations.—In the five dogs
studied, acetylcholine infused for 2 minutes at a
dose of 10~7 g/ml min"1 had little or no effect on the
perfusion pressure in the saphenous vein. With a
higher dose (4xlO" T g/ml min"1), the vein constricted in four of the five dogs (mean increase in
perfusion pressure, 9.4 ± 5.7 mm Hg).
Lumbar Sympathetic Stimulation and Norepinephrine.—The infusion of acetylcholine during
venoconstriction induced by lumbar sympathetic
chain stimulation caused a decrease in perfusion
pressure. The decrease could be obtained with 10~7
g/ml min"1, and the maximal effects were obtained
with four times this dose (Fig. 8).
The results in the five dogs are summarized in
Table 1. In every dog, acetylcholine (4 X 10~7 g/ml
min"1) caused a marked inhibition of the response
to each frequency of stimulation. When the infusion
stopped, the perfusion pressure increased to the
control level. In two of these five dogs an additional
stimulation at 10 cps was used. The acetylcholine
infusion decreased the perfusion pressure from 130
to 55 mm Hg and from 115 to 60 mm Hg,
respectively; the pressure returned to control levels
when the infusion was stopped.
In the same dogs, the effects of infusing
acetylcholine (4 X 10"7 g/ml min"1) were investigated during a sustained venoconstriction caused by
the infusion of norepinephrine at 2 X10"7 and
4X10"7 g/ml min-1 (Table 1). Acetylcholine
caused either an additional increase or no change in
perfusion pressure; in no case was there a decrease
in perfusion pressure. After the infusion, the
perfusion pressure usually decreased, but in three
dogs it did not decrease to the level observed before
acetylcholine infusion; recirculation of the infused
norepinephrine may explain this finding.
Discussion
NOREPINEPHRINE
litlO-'
ACETYLCHOLINE ISilO''
ATROPINE (lO-'g/ml)
g/ml)
g/ml)
FIGURE 6
Effect of atropine on the reaction of a saphenous vein strip
to acetylcholine during electrical stimulation (top) and of
another strip during norepinephrine-induced contraction
(bottom).
GrcuUitm Reiarcb, Vol. XXX)I, Ptbrtury 1973
The purpose of these experiments was to examine
the influence of acetylcholine on veins constricted
by sympathetic nerve activity. A convenient approach to the problem was to study the changes in
isometric tension in the isolated dog saphenous vein
subjected to electrical stimulation. The interpretation of the results depends on the evidence that the
electrical stimulation caused contraction of the vein
VANHOUTTE, SHEPHERD
264
Te trodotoxin
Norepinephrine
Norepinephrine
5 mln
ig
I
Acetylcholine
t
t.* t
cps
BaCI2
1
t Acetylcholine
. .—
—
Acetylcholine
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Atropine
FIGURE 7
Inhibition by tetrodotoxin (1O~5 gjml) of the reaction of a saphenous vein strip to electrical
stimulation (8 v, 2 msec, 15 ens for 10 seconds) but not to norepinephrine (2 X iO""8 g/ml) or
BaClt (0-25 mg/ml). During reactions to norepinephrine or BaClt, further increases in tension
were obtained (left) with addition of acetylcholine (5 X 10-^ g/ml); these increases in tension
were not blocked by tetrodotoxin but were abolished by atropine at 10-* g/ml (right).
strips by stimulation of the sympathetic nerve
endings and not by direct activation of the smooth
muscle cells. The contractions of isolated dog
saphenous veins in response to electrical field
stimulation (identical to that used in the present
experiments) are abolished by blockade of postgangjionic adrenergic transmission with bretylium
tosylate (5, 8), by reserpine pretreatment (5), by
chronic sympathectomy (7, 8), and by alphareceptor blockade (5, 8). Thus, in the present
experiments, it can be concluded that the contractions of the isolated saphenous vein strips during
electrical stimulation are caused mainly by the
release of catecholamines from the adrenergic nerve
terminals; that tetrodotoxin abolished the response
to electrical stimulation supports this conclusion.
100
Ui
cc
to
to
Ui
ct ~^
"•?
li
to
u.
ct
UJ
Stimulus: ON
Acetylcholine
4 x 10' 7 g/ml / mln
OFF
FIGURE 8
Inhibition by acetylcholine infusion of the constriction of the lateral saphenous vein caused by
sustained electrical stimulation of the lumbar sympathetic chain (8 v, 2 msec, 5 cps in 18-kg
dog).
CircmUtie* Rtiurd,
Vol. XXX11, F,bru*n 1973
265
VENOUS RELAXATION BY ACETYLCHOLINE
TABLE 1
Effect of Acetylcholinc on Venoconslrictions Induced by Lumbar Sympathetic Slirmdation or Norepinephrine
Inf us ion in Intact Dogs
Before ACh infusion
Increases in driving pressure
(mm Hf)
During ACh infusion
After ACb infusion
1 cps
2 cps
5 cps
Lumbar Sympathetic Stimulation
18.1 =*= 4.5
4.4 ± 2.2
11.1 ± 5.6
40.7 =*= 6.5
29.5 ± 8.0
80.9 ± 8.2
21.5 ± 10.2
49.6 ± 4.9
84.1 ± 4.5
2 X 10"' g/ml min~l
4 X 10"' g/ml min"1
Norepinephrinc Infusion
49.8 ± 14.3
64.2 ± 20.9
76.3 ± 14.2
97.3 ± 15.3
55.3 ± 17.8
88.5 ± 17.6
Acetylcholine (ACh) at 4 X 10"' g/ml min" 1 was infused for 2 minutes.
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The appropriate control for the reactions obtained with electrical stimulation was the vein
constricted by exogenous norepinephrine. Strikingly, acetylcholine decreased the response to electrical
stimulation, but it augmented the reaction to
exogenous catecholamines. This finding strongly
suggests that acetylcholine has a dual action: at the
presynaptic level it inhibits the release of norepinephrine from the tissue stores leading to relaxation
of smooth muscle constricted by sympathetic
impulses, and at the smooth muscle cells acetylcholine causes contraction. Acetylcholine did not
inhibit the contractions caused by tyramine, suggesting that it cannot prevent the mobilization of
norepinephrine by that drug. These responses to
acetylcholine resemble those noted in isolated
arterial smooth muscle with guanethidine and
bretylium tosylate, because the latter drugs block
the reactions to nerve activation but not the
responses to norepinephrine and tyramine (27, 33,
34).
Since the relaxation caused by acetylcholine is
not influenced by beta-receptor blockade and
hexamethonium but is abolished by atropine, it
must be due to the muscarinic action of acetylcholine.
Our results in venous smooth muscle substantiate
the findings of Malik and Ling (27), Rand and
Varma (28), and Hume et al. (29), who worked on
isolated rat mesenteric and rabbit ear arteries and
reached similar conclusions on the mode of action
of acetylcholine.
In contrast, when contractions similar to those
caused by electrical stimulation were evoked by
norepinephrine, acetylcholine did not cause relaxation; in fact, a further increase in tension usually
resulted. An increase in tension was also obtained
CircuUiion RtUMtcb, Vol. XXXII, Fibrusry 1973
with acetylcholine in the vein strips made to
contract by addition of tyramine, KC1, or BaCl2.
These effects of acetylcholine were not affected by
tetrodotoxin and were blocked by atropine. They
can be explained by a direct action on the smooth
muscle cells, and this direct action can be obtained
in the resting strips (3, 5, 6, 8-10, 13, 16) but usually
only with larger doses than those necessary to
increase the tension in a strip already contracted.
Doses of acetylcholine which have no effect on
the tension of resting strips or strips contracted by
norepinephrine can cause marked relaxation of
strips contracted by electrical stimulation. This
finding suggests that the action on the sympathetic
nerve endings may occur with concentrations lower
than those necessary for the direct contracting
action of the drug.
In the in vivo studies, in which the electrical
stimulation was applied directly to the sympathetic
nerves, a striking difference was seen between the
depression by acetylcholine of the venoconstrictions
caused by lumbar sympathetic stimulation and the
augmentation by acetylcholine of the constrictions
caused by norepinephrine. The similarity of these
findings with the in vitro observations strengthens
the conclusion that, in the isolated venous strips,
acetylcholine is inhibiting the release of norepinephrine. In the arterial bed of the rabbit's ear, the
dilatation caused by acetylcholine is decreased by
reserpine (35). This effect, considered in conjunction with the present experiments, suggests that the
vasodilatation caused by acetylcholine in vivo may
be explained, at least in part, by the action of the
drug on sympathetic nerve endings.
Cholinergic vasodilator fibers in association with
sympathetic nerves have been described in different
species (36, 37). The transmitter released on
VANHOUTTE, SHEPHERD
266
stimulation of these fibers could act on a receptor in
the smooth muscle cells to cause the dilatation.
Another possibility, suggested by the present
experiments and by studies on isolated arteries
(27-29, 38,39) and on the heart (40, 41), is that the
cholinergic transmitter acts by inhibiting sympathetic adrenergic transmission.
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interactions
Venous Relaxation Caused by Acetylcholine Acting on the Sympathetic Nerves
PAUL M. VANHOUTTE and JOHN T. SHEPHERD
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Circ Res. 1973;32:259-267
doi: 10.1161/01.RES.32.2.259
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