208
Inhibition of Sympathetic Neurotransmission in
Canine Blood Vessels by Adenosine and Adenine
Nucleotides
RAYMOND H .
VERHAEGHE, PAUL M. VANHOUTTE, AND JOHN T.
SHEPHERD
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SUMMARY Adenosine and the adenine nucleotides caused a
greater relaxation of strips of canine saphenous vein and tibial
artery when they had been contracted by nerve stimulation than
by exogenous norepinephrine. An infusion of adenosine into the
dogs' lateral saphenous vein, perfused at constant flow, caused a
greater relaxation of this vein when constricted by electrical
stimulation of the lumbar sympathetic chain than by exogenous
norepinephrine. That this difference was due to inhibition by
these compounds of the output of neurotransmirter from the sympathetic nerve endings was demonstrated by column chromatographic analysis of the radioactivity in the supervision fluid of
vein strips, previously incubated with tritiated norepinephrine.
Both adenosine and adenosine triphosphate (10"' M) reduced the
efflux of 3H-norepinephrine during nerve stimulation with electri-
cal impulses. Adenosine also reduced the efflux caused by potassium (30 mM), but not that caused by tyramine (6 x 10~6 M).
Theophylline antagonized the inhibitory effect of adenosine on
the sympathetic neurotransmission. We found that at 4 x 10 4 M
adenosine triphosphate still caused a decreased efflux of neurotransmitter during electrical stimulation, but with adenosine the
3
H-norepinephrine efflux no longer decreased and the overflow
of deaminated compounds increased. Furthermore, the same concentration of adenosine increased the efflux of 3H-norepinephrine and deaminated compounds in unstimulated strips,
and the increase of 3H-norepinephrine was enhanced after monoamine oxidase inhibition. Thus, we conclude that at higher
concentrations adenosine increases the intraneuronal leakage
of norepinephrine out of the storage vesicles.
ADENOSINE and the adenine nucleotides by a local
action on blood vessels cause their dilation. This has been
demonstrated both in vivo and in vitro.'"3 There is evidence that these substances can inhibit neuromuscular
transmission in the rat phrenic nerve-diaphragm and frog
sartorius preparations4'5 and cholinergic neurotransmission in the intestinal wall.6 The present experiments were
performed to determine whether adenosine and related
nucleotides inhibit adrenergic neurotransmission in canine
blood vessels.
allowing fine adjustments of the strip length and connected to a pen writing recorder (Gould Brush 220). For
electrical stimulation of the preparations, two rectangular
platinum electrodes (20- by 4-mm surface, 0.5 mm thick)
were placed parallel to vein strips. The electrical impulses
were of rectangular waves (0.2-20 Hz, 9 V, 2 msec) from
a direct current power supply and switching transistor
(RCA 2N-3034) triggered by a stimulator (Grass SM6C).
After an equilibration period (20-40 minutes) the length
of each strip was increased by 2-mm increments until the
contraction caused by a standard electrical stimulus (10
Hz for 10 seconds) reached a maximum.7 The length at
which this occurred was maintained throughout the experiment.
The following pharmacological agents were used: adenosine (Aldrich), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) (Sigma), /-norepinephrine bitartrate (Winthrop), tyramine hydrochloride (Abbott), pargyline hydrochloride (Abbott), and theophylline (Sigma).
All doses are expressed as final bath concentrations. The
drugs were removed from the bath solution by overflowing
the preparation with aerated Krebs-Ringer solution at
37°C.
When the effect of increasing concentrations of adenosine or nucleotides was investigated, sustained contractions were obtained with either electrical stimulation (2
Hz) or norepinephrine (6x10~ 7 M) . After the tension had
become maximal (control value), the selected concentration of adenosine or one of the nucleotides was added to
the bath. Usually 4 minutes was sufficient for the tension
to stabilize at its new level and this value was taken as the
response to adenosine or the nucleotide. Only one agent
was tested on each vascular strip. All strips were allowed
to recover for 10 minutes between two consecutive contractions.
Methods
STUDIES IN VITRO
Helical strips of lateral saphenous veins and anterior
tibial arteries, isolated from dogs (15-25 kg) anesthetized
with sodium pentobarbital (30 mg/kg, iv), were placed in
an organ bath filled with Krebs-Ringer solution (NaCl,
118.2 mM; KC1, 4.7 mM; MgSO4, 1.2 mM; KH2PO4, 1.2
mM; CaCl2, 2.5 mM; NaHCO3, 25 mM; calcium disodium
ethylenediaminetetraacetate (EDTA) 0.026 mM; and glucose, 5.5 mM) maintained at 37°C and aerated with a 95%
O2-5% CO2 mixture. The strips were attached to a force
transducer (Grass FT 03C) for isometric tension recording. The transducer was mounted on a movable support
From the Department of Physiology and Biophysics, Mayo Clinic and
Mayo Foundation, Rochester, Minnesota, and Department of Internal
Medicine, Universitaire Instelling, Antwerpen, Antwerp, Belgium.
Supported in part by Research Grant HL-5883 from the National Institues of Health, Public Health Service.
Dr. Verhaeghe is a recipient of a Public Health Service International
Research Fellowship (F05-TW-2167) on leave of absence from the University of Leuven, Belgium.
Address for reprints: Dr. John T. Shepherd, Mayo Clinic and Mayo
Foundation, 200 First Street, S.W., Rochester, Minnesota 55901.
Received June 9, 1976; accepted for publication September 8, 1976.
ADENINE COMPOUNDS AND SYMPATHETIC NERVES/Verhaeghe et al.
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Cumulative frequency-response curves to electrical
stimulation or dose-response curves to norepinephrine
were obtained first in control solution and repeated after
incubation for 10 minutes in solution containing adenosine
or one of the nucleotides. Repeated washings were made
and a second control curve was obtained after the experimental curve. The mean effective dose (ED50) and maximal response of the second control curve always differed
by less than 5% from those of the first. Both control
curves were averaged for plotting the results and for statistical calculation. Dose ratios were calculated as the displacement of the curve at 50% of the maximal response.
In some experiments, helical strips of saphenous veins
were incubated for 4 hours in Krebs-Ringer solution containing 1.25 x 10~7 M 7-3H-norepinephrine (specific activity, 9.8 Ci/mmol, New England Nuclear). At the end of
the incubation, the strips were rinsed in fresh KrebsRinger solution and mounted for superfusion. Each preparation was suspended in a moist tunnel-shaped chamber
maintained at 37°C and superfused at 3 ml/min by a
constant flow roller pump (Holter, model 911) with aerated (95% O2 and 5% CO2) Krebs-Ringer solution prewarmed to perfuse at 37°C. The strip was connected to a
force transducer (Grass FT 03C) for isometric tension
recording (Clevite Brush Mark 250). Two platinum wires
(10 cm long, 0.5 mm in diameter) were fixed parallel to
the strip. Both vein strip and electrodes were perfused
continuously. Electrical stimulation was similar to that in
the organ bath experiments. The original tension was set
at 2 g, and sampling at 2-minute intervals was started after
an equilibration period of 20 minutes. Insta-Gel emulsifier
(10 ml) (Packard) was added to 1-ml samples before
measurement of total radioactivity in a liquid scintillation
counter. Corrections for quenching were made with an
external standard. The counting efficiency was between
37% and 42.5%. Data are expressed as disintegrations
per minute (dpm) per minute of superfusion.
In some samples, norepinephrine was separated from its
metabolites in the superfusate by column chromatography. In a first step, catechol compounds were separated
from noncatechol metabolites and from tritiated water by
adsorption on alumina at pH 8.4 and subsequent elution
with acid. In a second step, norepinephrine was separated
from deaminated compounds in the alumina eluate and
normetanephrine from deaminated O-methylated metabolites in the alumina effluent by adsorption on a Dowex
50-X4 resin and subsequent elution with acid. The procedure is described in detail elsewhere.8-9 In a few experiments the deaminated compounds were further split by a
two-step elution from the alumina: 3,4-dihydroxyphenylglycol (DOPEG) and norepinephrine were first eluted
with acetic acid (0.2 N) and thereafter 3,4-dihydroxymandelic acid (DOMA) with hydrochloric acid (0.2 N and 1 N
in succession). Norepinephrine was subsequently separated from DOPEG on Dowex resin. Radioactivity was
measured in a liquid scintillation counter on 1-ml samples
of effluents and eluates from the columns.
STUDIES IN VIVO
The method used has been described.10' " Dogs (15-25
kg) were anesthetized with sodium pentobarbital (30 mg/
209
kg, iv). The 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. Because the blood flow through
the saphenous vein and the femoral vein pressure were
constant, the saphenous vein perfusion pressure was a
measure of the venous tone.
The lumbar sympathetic trunk was exposed, severed,
and stimulated (Grass SD 5 stimulator) through a platinum bipolar electrode (9 V, 2 msec, 2 and 10 Hz). Adenosine and /-norepinephrine bitartrate were infused at constant speed upstream from the roller pump.
ANALYSIS OF DATA
The number of strips reported for each group of experiments is also the number of dogs used. All data are
expressed as mean ± SE. For statistical analysis of the
data, Student's/-test was used.
Results
EFFECT OF ADENOSINE, ADP, AND ATP ON
RESPONSE OF VASCULAR STRIPS TO ELECTRICAL
STIMULATION AND NOREPINEPHRINE
The original record of an experiment in which a single
concentration of adenosine (10~4 M) was added during a
sustained contraction of a saphenous vein strip caused by
electrical stimulation (2 Hz) is shown in Figure 1. Adenosine induced an immediate relaxation which was maximal
within the 1st minute and was followed by a slight increase
in tension. The relaxation was rapidly and completely
reversible upon removal of adenosine. In identical experiments on six strips the depression of the response to
electrical stimulation (mean increase in tension, 1.30 ±
0.13 g) averaged 54.6 ± 4.1 % after 1 minute and 44.9 ±
4.1 % after 6 minutes. The tension returned to 100.7 ±
3.1 % of the control level after washing out the nucleoside.
The same concentration of adenosine had no effect on the
basal tension in these strips.
Twenty saphenous vein strips were contracted in random sequence either by electrical stimulation (2 Hz) or
norepinephrine (6 x 10~7 M); the tension increased by
2.99 ± 0.42 and 2.52 ± 0.27 g, respectively. Adenosine
(10~4 M) added to the bath during these contractions
caused only a moderate depression (average, 23.5 ±
2.6%) of the response to norepinephrine, but a signifiElectric
Stimulation,
Adenosine,
2 Hz
IO'4M
FIGURE 1 Effect of adenosine on a saphenous vein strip during
electrical stimulation.
CIRCULATION RESEARCH
210
cantly {P < 0.01) larger depression (average, 52.8 ±
8.3%) of the reaction to electrical stimulation.
Similar studies were carried out in three series of six
vein strips, using in random sequence several concentrations (10"6 to 5 x IO"4 M) of adenosine, ADP, and ATP,
respectively. The results are shown in Figure 2. In concentrations lower than 5 x 10"5 M, the three purine compounds depressed only the response to electrical stimulation; the responses to both electrical stimulation and norepinephrine were depressed with higher concentrations,
but the relaxation during the reaction to electrical stimulation always remained significantly greater than that during
the norepinephrine-induced contraction (P < 0.01).
In six other groups of six veins each, either frequencyresponse curves to electrical stimulation (0.5-15 Hz) or
VOL. 40, No. 2, FEBRUARY
Norepinephrine
Electric Stimulation
100 % • 8.2 * 0.66g
IO'7
IO'6
IO'5
1977
IOO % • 6.7 ± 0.48g
IO"4 0.5
I
Z
5
10 20
I
2
5
10 20
ADENOSINE (U)
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
_ IO-*
5XI0-
6
IO'a
SxlO'9
IO-*
5XIO-*
40
20
-H
40
IO'7
IO-6
I0'5
I0'4
0.5
60
%
80
o
ADP (u)
I0'6
5XI0'6
IO'S
5 X IO'a
I0'4
5xlO~*
to
O
0.
<o
Ui
20
<fc
4O
<O
10
Ul
60
f
L
ATP(M)
Ui
Q
FIGURE 3 Effect of the same concentration of adenosine, ADP,
and A TP on the reaction of saphenous veins to increasing concentrations of norepinephrine or to graded electrical stimulation. Data
are shown as mean ± SE for six veins in each group. The depression of the response to electrical impulses was significant for each
stimulation frequency. The response to norepinephrine was significantly depressed only for concentrations below 1.5 x 10~" M(P <
0.05).
cumulative dose-response curves to norepinephrine (6 x
IO"8 to 3 x IO"5 M) were obtained (Fig. 3). Adenosine,
ADP, and ATP (lO"4 M) significantly depressed the frequency-response curve to electrical impulses; the dose
ratios were 1.9, 1.9, and 2.0, respectively. In contrast, the
2O
depression of the dose-response curves to norepinephrine
by the same concentrations of the three adenine compounds was significant only for the lower doses of the
40
D Eltclric Stimulation, 2 Hz
catecholamine; the dose ratios were 1.3, 1.25, and 1.25,
100% • 3.42 ± O.ZOg
7
respectively.
• Norepinephrine, 6 X IO' M
100% • 3.43 * 0.19 g
Six other strips were treated with cocaine (3 x 10"5 M)
60
for 30 minutes before obtaining a frequency-response
FIGURE 2 Depression by adenosine, ADP, and ATP of the
curve to electrical stimulation (0.2-20 Hz). Adenosine
response to electrical stimulation and norepinephrine in three
(IO"4 M) depressed this curve to the same extent as in
groups of six saphenous veins. Data are expressed as mean ± SE.
untreated
strips, the dose ratio being 2.0.
* Difference between effect on electrical stimulation vs. norepinephA pair of helical strips was obtained from each of six
rine contraction is statistically significant (P < 0.01).
6
I0'
6
5xlO'
IO'S
WJ
IO-4
5XI0-4
ADENINE COMPOUNDS AND SYMPATHETIC NERVES/VWiaeg/ie et al.
tibial arteries. One member of each pair was contracted by
electrical stimulation, the other by norepinephrine. Adenosine (10~4 M) depressed the contractions caused by each
stimulation frequency significantly more than comparable
contractions caused by norepinephrine (Table 1).
TENSION,g
1.5 r
211
.
\. . 1 . . :
RADIOACTIVITY
dpm x IO3/min
7
3
EFFECT OF ADENOSINE AND ATP ON BASAL EFFLUX
OF 3H-NOREPINEPHRINE IN SAPHENOUS VEIN
STRIPS
TENSION
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The basal efflux of total radioactivity, ^-norepinephrine, and its metabolites was unaltered by adenosine
(10~5 M) in six unstimulated strips previously incubated
with labeled neurotransmitter. In contrast, in six other
strips, a higher concentration of adenosine (4 x 10~4 M)
resulted in a rise of the downward slope of the radioactivity efflux curve (Fig. 4). This was due mainly to an augmented outflow of deaminated metabolites, DOPEG
(23.8 ± 3.2%) and DOMA (20.8 ± 7.1 %), together with
a slight increase in 3H-norepinephrine (11.3 ± 3.2%).
Four additional strips were incubated for 30 minutes with
the monoamine oxidase inhibitor pargyline (1.5 x 10~ 4 M)
before superfusion; under these circumstances there was a
significantly (P < 0.01) larger increase in the efflux of
intact neurotransmitter (35.5 ± 3.9%) with the same
concentration of adenosine (Table 2).
ATP (10~5 M) caused a poorly sustained contraction of
five unstimulated strips as described by Furchgott,12 but
had no effect on the slope of the radioactivity efflux curve
or on the efflux of neurotransmitter or its metabolites.
ATP (4 x 10~4 M) caused a larger increase in tension in
seven other strips together with a slight depression of the
radioactivity efflux curve, because of a decrease in deaminated O-methylated metabolites (19.6 ± 3.9%) and normetanephrine (20.8 ± 4.8%) (Fig. 4 and Table 2).
RADIOACTIVITY
dpm X IO3/min
FIGURE 4 Effect of adenosine (left) and ATP (right) on tension
and efflux of total radioactivity in four series of unstimulated
saphenous vein strips. Data are shown as mean ± SE.
In 11 other strips, adenosine (4 x 10~4 M) depressed the
contraction obtained with electrical stimulation (2 Hz) by
41.8 ± 4.2%, but not the efflux of total radioactivity.
Column chromatographic analysis of the superfusate for
six of these strips showed that there was no decrease in the
3
H-norepinephrine; there was an increase in deaminated
metabolites (20.5 ± 3.7%) and a decrease in normetanephrine (8.2 ± 3%) (Fig. 6).
In eight strips ATP (10~5 M) depressed the contraction
by 17.3 ± 3.2% and the overflow of total radioactivity by
22.9 ± 2.4% during electrical stimulation at 1 Hz; a
decrease in 3H-norepinephrine (24.0 ± 4.0%) was found
by analysis of the superfusate of five of these strips. In nine
other stirps, ATP (4 x 10"4 M) depressed the contraction
by 20.9 ± 3.5% after an initial slight increase in tension
and the overflow of total radioactivity by 33.5 ± 3.7%.
After removal of the nucleotide there was a further transient decrease in tension (12.6 ± 1.2%). In the five strips
in which tests were made, ATP depressed the efflux of 3Hnorepinephrine (37.7 ± 4.7%), deaminated O-methylated metabolites (13.6 ± 3.9%), and normetanephrine
(14.8 ± 4.0%) (Fig. 7).
In five strips, a change in the K+ concentration of the
superfusion fluid from 5.9 to 30 min caused an increase in
tension, in radioactivity of the superfusate, and in 3Hnorepinephrine efflux. Adenosine (10~5 M) depressed the
contraction by 42.8 ± 5.1% and the increase in radioactivity by 45.3 ± 8.1% and in 3H-norepinephrine by 27.9
EFFECT OF ADENOSINE AND ATP ON OUTPUT OF
H-NOREPINEPHRINE DURING NERVE STIMULATION
IN SAPHENOUS VEIN STRIPS
3
In eight strips electrical stimulation (2 Hz) caused an
increase in tension together with an augmented efflux of
total radioactivity and 3H-norepinephrine into the superfusion fluid. Adenosine (10~5 M) depressed the contraction by33.0±4.3%, the overflow of total radioactivity by
19.9 ± 4.2%, and the overflow of 3H-norepinephrine by
26.7 ± 2.9%. All changes were reversible after removal
of adenosine. No significant changes occurred in the metabolite fractions (Fig. 5).
TABLE 1 Effect of Adenosine on Comparable Contractions Caused by Electrical Stimulation
and Norepinephrine in Canine Tibial Artery Strips
Norepinephrine
Electrical stimulation
Hz
0.5
2
10
15
Control'
(g)
1.75
3.46
7.2
8.21
±
±
±
±
0.46
0.73
1.01
1.03
Adenosine, 10"* int
96.7
87.0
52.5
50.0
±
±
±
±
1.4
2.4
5.0
3.2
Control*
(g)
1.5
3
1.5
3
x
x
x
x
10"7
10"7
10"6
10"6
1.86
3.21
7.20
8.70
±
±
±
±
0.52
0.43
1.22
1.33
Adenosine, 10"* Mt
86.2
68.2
14.9
10.4
±
±
±
±
2.2t
4.8$
5.5§
4.6§
Values shown as mean ± SE for six dogs.
* Increase in tension caused by stimulus in absence of adenosine.
t Percent depression of response by adenosine.
t, § Response to norepinephrine is signiflcantly less depressed than comparable response to electrical stimulation
(F < 0.01 and < 0.001, respectively).
212
CIRCULATION RESEARCH
VOL. 40, No. 2, FEBRUARY 1977
TABLE 2 Effect of Adenosine and ATP on Basal Efflux of 3H-norepinephrine and Metabolites in Unstimulated
Saphenous Vein Strips
Experimental condition
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n = 6
Control
Adenosine (10~5 M)
n = 6
Control
Adenosine (4 x 10~4 M)
n =4
Control
Adenosine (4 x 10~4 M) after pargyline
(1.5 x 10"4 M)
n = 5
Control
ATP (10"5 M)
n =7
Control
ATP (4 x 10-4 M)
Deaminated Omethylated metabolites
Norepinephrine
Deaminated metabolites
Normetanephrine
3.6 ± 1.1
3.7 ± 1.0
22.7 ± 6.3
24.0 ± 6.9
23.5 ± 2 . 3
23.5 ± 2.5
1.6 ±0.3
1.6 ±0.3
4.9 ± 0.8
5.5 ± 1.0*
15.8 ± 2.2* 11.7 s: l i t
19.3 ± 2.3*§ 14.0 i: 1.0ft
36.3 ± 6 . 3
37.6 ±7.3
3.7 + 0.6
3.4 ±0.5
7.8 ± 1.7
10.5 ± 2.2§
1.0 ± 0.6
1.4 ± 0.9
22.1 ± 1.6
21.4 ± 1.9
14.2 ±2.6
14.0 ± 3.0
2.9 ± 0.4
2.9 ± 0.4
27.3 ± 2.6
29.4 ± 2.5
29.7 ±2.4
28.0 ± 1.8
2.8 ±0.4
2.8 ± 0.5
4.8 ± 0.9
5.2 ± 1.0
25.5 ± 5.0
27.5 ± 5.5
20.2 ±3.5
16.2 ± 2.2§
2.6 ±0.3
2.1 ±0.3§
All data are expressed as dpm x 103 per minute of superfusion (mean ± SE); control = mean of value taken before and after exposure to adenosine or ATP
for each strip; n = number of strips.
* 3,4-Dihydroxyphenylglycol (DOPEG).
t 3,4-Dihydroxymandelic acid (DOMA).
X P < 0.05 (value significantly different from control).
§ P < 0.01 (value significantly different from control).
± 5.6%. In five other strips, adenosine (10~5 M) added
during contractions induced by tyramine (6 x 10"6 M)
caused a relaxation (24.5 ± 4.1%) but no change in
radioactivity and 3H-norepinephrine efflux (Fig. 8).
Five strips were superfused for 20 minutes with KrebsRinger solution containing theophylline (10~3 M). During
subsequent electrical stimulation (1 Hz), adenosine (10~5
M) depressed the contraction by 6.4 ± 1.0% and the
efflux of radioactivity by 7.7 ± 1.6%; the 3H-norepinephrine efflux was slightly but not significantly depressed (4.9
± 3.8%). Five other strips were used as a control and
stimulated at the same frequency. The same concentration
of adenosine depressed the contraction by 45.8 ± 7.2%,
the efflux of radioactivity by 41.4 ± 9.0%, and the efflux
of 3 H-norepinephrine by 50.0 ± 9 . 3 % . All differences
between both groups are significant (P < 0.01) (Fig. 9).
Stimulation. 2 Hz
Electric Stimulation, 2 Hi
MNE
a Dtominattd
o o-Uelh/lotid
3 Deaminattd
a NUN
FIGURE 5 Effect of adenosine (10~h M) on tension (top), total
radioactivity in the superfusate (middle), and efflux of 3H-norepinephrine (NE) and metabolites (bottom) in saphenous vein strips
during electrical stimulation. Data are shown as mean ± SE.
Arrows indicate time at which samples were collected for column
chromatography. NMN = normetanephrine.
FIGURE 6 Effect of adenosine (4 x 10~* u) on tension (top), total
radioactivity in the superfusate (middle), and efflux of zH-norepinephrine and metabolites (bottom) in saphenous vein strips during
electrical stimulation. Data are shown as mean ± SE. Arrows
indicate time at which samples were collected for column chromatography. NMN = normetanephrine.
ADENINE COMPOUNDS AND SYMPATHETIC NERVES/Ver/iaegfce et al.
Electric Stimulation. I Hi
213
3
Electric Stimulation. IHi
Theophylline. IO' M
Electric Stimulation, I Hi
2
O
2
Ul
/6
o o
Adenosine
IO'5M
11
Noreptnephrine
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FIGURE 7 Effect of ATP (10'h M) (left) and ATP (4 x 10-" M)
(right) on tension (top), total radioactivity in the superfusate (mid- FIGURE 9 Left panel: effect of adenosine (10 5 M) on tension
dle), and efflux of 3H-norepinephrine and metabolites (bottom)
(top), total radioactivity in the superfusate (middle), and efflux of
3
in saphenous vein strips during electrical stimulation. Data are
H-norepinephrine (bottom) during electrical stimulation of sapheshown as mean ± SE. Arrows indicate time at which samples
nous vein strips previously super fused with theophylline (W~3 u)
were collected for column chromatography.
for 20 minutes. Right panel: effect of the same concentration of
adenosine during electrical stimulation (ES) of saphenous vein
strips superfused with normal Krebs-Ringer solution.
IN VIVO EFFECT OF ADENOSINE ON RESPONSE OF
SAPHENOUS VEIN TO LUMBAR SYMPATHETIC
STIMULATION AND INFUSION OF NOREPINEPHRINE
In seven dogs, the lateral saphenous vein was perfused
at constant flow (100 ml/min) with autologous blood. The
infusion of adenosine (5 x 10~5 M) for 3 minutes did not
affect the perfusion pressure in the absence of stimulation.
Sustained venoconstrictions were obtained with two frequencies (2 and 10 Hz) of electrical stimulation of the
lumbar sympathetic chain; in each dog venoconstrictions
of comparable amplitude to those obtained with the two
stimulation frequencies also were evoked by selecting appropriate concentrations of norepinephrine (1 ± 0.4 x
10"6 M and 4 ± 0.9 x 10~6 M, respectively). Adenosine,
infused at 5 x 10"5 M for 3 minutes during these venoconstrictions, depressed the response to the two frequencies
Potassium. 30 mM
Tyramine. 6 X I0'6 M
FIGURE 8 Effect of adenosine (10 5 M) on tension (top), total
radioactivity in the superfusate (middle), and efflux of 3H-norepinephrine (bottom) in saphenous vein strips contracted by potassium (left) and tyramine (right). Data are shown as mean ± SE.
of lumbar sympathetic stimulation significantly more than
those caused by the corresponding concentrations of norepinephrine (Table 3).
Discussion
The comparison of the effect of adenosine during contractions of isolated blood vessels caused by electrical
stimulation and exogenously applied norepinephrine is a
first step to establish whether this nucleoside has an inhibiting effect on the sympathetic nerves. The interpretation of the results of this comparison depends on the
evidence that contractions of vascular strips caused by
electrical stimulation are due to release of norepinephrine
from the adrenergic nerve endings rather than to direct
excitation of the smooth muscle cells. The contractions of
isolated canine saphenous veins in response to electrical
field stimulation as used in the present experiments are
abolished by blockade of peripheral adrenergic transmission with bretylium tosylate or tetrodotoxin, by reserpine
treatment, by chronic sympathectomy, and by a-receptor
blockade."-13~15 Electrical stimulation augments the efflux
of 3H-norepinephrine in preparations previously incubated
with labeled transmitter.8 Thus, the contractions of the
isolated blood vessel strips caused by electrical stimulation
in the present experiments are due to evoked release of
catecholamines from the adrenergic nerve terminals.
Whether increasing concentrations of adenosine are
tested on the response to a constant stimulus, or vice
versa, the contractions induced by nerve stimulation are
more depressed than those caused by exogenous norepinephrine. This indicates that in the vein contracted by
nerve stimulation, part of the relaxation caused by adenosine is due to interference with sympathetic neurotransmission. This is confirmed by the fact that adenosine in a
concentration which has no effect on the basal efflux of
CIRCULATION RESEARCH
214
VOL. 40, No. 2, FEBRUARY
1977
TABLE 3 Effect of Adenosine on Comparable Venoconstriction Induced by Lumbar Chain
Stimulation and Norepinephrine in Canine Saphenous Veins Perfused with Aulologous Blood
Lumbar chain stimulation
Hz
Control*
(mm Hg)
2
10
71.5 ± 13 .7
101.9 ± 5 .4
Norepinephrine
Adenosine, 5 x
10"s Mt
Adenosine, 5 x
10"5 Mt
M X
61.8 ± 6.0
26.6 ± 5.4
10 - *
1 ±: 0.4
4 ±: 0 .9
Control* (mm Hg)
70.3 ± 16.5
102.6 ± 5.3
16.9 ± 6.3§
5.6 ± 5.1H
Values shown as mean ± SE for seven dogs.
* Increase in perfusion pressure caused by stimulus in absence of adenosine.
t Percent depression of response by adenosine.
t Final concentration in perfusing blood.
§, H Response to norepinephrine is significantly less depressed than comparable response to lumbar sympathetic
stimulation (P < 0.001 and P < 0.02, respectively).
3
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H-norepinephrine depresses the output of labeled neurotransmitter evoked by nerve stimulation. This decreased
efflux is due to an inhibition of norepinephrine release
rather than to enhanced neuronal reuptake, since there is
no increase in deaminated compounds. Also, the degree of
depression by adenosine of the contraction caused by
nerve stimulation is unaltered by cocaine. Adenosine
causes a similar difference in the degree of depression of
contractions of tibial artery strips caused by endogenous
vs. exogenous norepinephrine; thus the inhibitory effect of
the nucleoside on the sympathetic neurotransmission is
not restricted to the cutaneous veins.
Like adenosine, ADP and ATP also inhibit adrenergic
neurotransmission. This can be concluded from the experiments which demonstrate that both nucleotides depress
the response of saphenous veins to nerve stimulation more
than to exogenous norepinephrine. The decrease in the
efflux of labeled neurotransmitter by ATP during electrical stimulation supports this conclusion. The response to
electrical stimulation is depressed by concentrations of the
three compounds which do not affect the response to direct a-adrenergic activation of the smooth muscle cells.
When high concentrations of adenosine are used, the
greater depression of contractions to electrical stimulation
as compared to norepinephrine is still evident. However,
the inhibitory effect of the nucleoside on the sympathetic
neurotransmission can no longer be demonstrated by
measuring the outflow of labeled norepinephrine. In unstimulated strips, the same concentration of adenosine
causes an increased efflux of 3H-norepinephrine and of
deaminated metabolites; this suggests that more norepinephrine is leaking out of the storage vesicles into the
neuronal cytoplasm, where it is deaminated by monoamine oxidase. This interpretation is supported by the
experiments with pargyline, which further augments the
increase in 3H-norepinephrine caused by adenosine but
reduces drastically the deaminated metabolites. In strips
stimulated electrically, high concentrations of adenosine
also cause an increased efflux of deaminated compounds,
suggesting a similar increased leakage of norepinephrine
from the vesicles. No such effect is observed with high
concentrations of ATP; this may be related to the inability
of nucleotides to cross the neuronal membrane. In contrast, high concentrations of ATP decrease the metabolites
resulting from O-methylation, perhaps due to an inhibiting
effect either on catechol-o-methyltransferase or on the
extraneuronal binding or uptake of catecholamine.
Adenosine inhibits the release of 3H-norepinephrine
evoked by nerve depolarization both with electrical impulses and potassium but not the displacement caused by
tyramine. The liberation of norepinephrine by nerve depolarization is Ca2+-dependent, whereas that caused by
tyramine is not.16 However, whether adenosine acts by
hyperpolarizing the neuronal membrane, by directly inhibiting calcium influx, or by interfering in a further step in
the excitation-secretion coupling cannot be answered by
the present experiments. We can only speculate whether
the antagonism by theophylline of the prejunctional effect
of adenosine is an indication of the involvement of the
cyclic nucleotide system in the inhibition of the sympathetic neurotransmission. The answer to this problem can
only be provided by the development of methods to measure cyclic nucleotide levels in the sympathetic nerve endings in blood vessels.
The ability of adenosine and related nucleotides to inhibit release of norepinephrine in vascular strips implies
that at least part of the vasodilator properties of these
substances are due to such an effect on the sympathetic
nerves. The experiments performed in intact dogs in which
electrical stimulation was applied directly to the lumbar
sympathetic chain yield results similar to those obtained in
isolated blood vessels. These observations strengthen the
conclusion that adenosine can interfere with adrenergic
neurotransmission and indicate that this phenomenon can
occur in vivo.
Evidence has accumulated in recent years that inhibition
of adrenergic neurotransmission may represent a way of
vasodilation shared by several naturally occurring vasoactive substances. Acetylcholine,8' "• 17 histamine,18 and also
moderate increases in potassium concentrations19 have
been demonstrated to inhibit release of norepinephrine in
canine blood vessels during nerve stimulation. The present
studies extend this list of prejunctional vasodilators to
adenosine and the adenine nucleotides. These substances
are believed to play a role in the hyperemia of muscular
exercise,20-21 a condition associated with decreased responsiveness to sympathetic stimulation.22 Activation of
purinergic nerves has recently been considered to cause
dilation of several adrenergically innervated blood vessels.2'23 In these two situations the inhibitory effect of
ADENINE COMPOUNDS AND SYMPATHETIC NERVES/Verhaeghe et al.
purine compounds on adrenergic neurotransmission, as
demonstrated in the present study, could play a role.
Acknowledgments
It is a pleasure to acknowledge the assistance provided in various
portions of this study by R.R. Lorenz, M.D. Seppala, and J.Y. Troxell.
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Inhibition of sympathetic neurotransmission in canine blood vessels by adenosine and
adenine nucleotides.
R H Verhaeghe, P M Vanhoutte and J T Shepherd
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Circ Res. 1977;40:208-215
doi: 10.1161/01.RES.40.2.208
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