774

Interaction of Neuropeptide Y and the

Sympathetic Nervous System in
Vascular Control in Man
John Clarke, MRCPI; Nigel Benjamin, MRCP; Simon Larkin, BSc; David Webb, MRCP;
Attilio Maseri, MD, FACC; and Graham Davies, MD, FRCP

Background. There is increasing evidence that neuropeptide Y (NPY) contributes to the

autonomic control of the circulation. NPY coexists with noradrenaline in perivascular nerve
terminals, may be released during sympathetic stimulation, and is a potent constrictor of the
human coronary circulation and other vascular beds. In vitro studies show that NPY can act
either directly on vascular smooth muscle or indirectly by modulation of the presynaptic release
or the postsynaptic actions of noradrenaline. It is unclear to what extent these mechanisms
operate in vivo.
Methods and Results. The effect on forearm blood flow of intra-arterial NPY was studied in six
volunteers during coinfusion of noradrenaline and during reflex sympathetic stimulation
induced by lower-body negative pressure. NPY alone induced a dose-dependent reduction of
forearm blood flow in all subjects studied, to a maximum of 49+6.1%. The reduction of flow
during infusion of noradrenaline alone was 42±8%. The response to noradrenaline was
unaffected by coinfusion of a threshold constrictor dose of NPY (50 pmol/min). Furthermore,
the reflex sympathetic vasoconstrictor response to 20 cm H20 of lower-body negative pressure
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was similar in both the infused and control arms during the infusion of 50 pmol/min NPY. The
response to noradrenaline was abolished by a-blockade with phentolamine, but the flow
reduction induced by NPY was unaffected by a-blockade.
Conclusions. NPY is a potent constrictor of human forearm resistance vessels and has a direct
effect independent of a- receptors. NPY has no detectable modulating effect in vivo on the
action of endogenous or infused noradrenaline. (Circulation 1991;83:774-777)

F ollowing the demonstration by Lundberg et al'
that sympathetic nerve terminals contain a
peptide showing an immunoreactivity similar
to that of avian polypeptide, Tatemoto et at2 isolated
the structurally related peptide, neuropeptide Y
(NPY), which was subsequently shown to be colocal-
ized with noradrenaline in sympathetic nerve end-
ings.3 Evidence has since accumulated implicating
NPY in autonomic control of the circulation. NPY is
a potent constrictor of human vascular tissue4 and is
released with noradrenaline during sympathetic
nerve stimulation.5 In vitro studies have shown that
From the Department of Cardiovascular Medicine (J.C., S.L.,
A.M., G.D.), Royal Postgraduate Medical School, Hammersmith
Hospital and the Department of Pharmacology (N.B., D.W.), St.
George's Hospital, London, UK.
N.B. is a Wellcome Research Fellow. A.M. is British Heart
Foundation Professor of Cardiovascular Medicine.
Address for correspondence: Dr. John Clarke, Department of
Cardiovascular Medicine, Hammersmith Hospital, DuCane Road,
London W12 OHS, UK.
Received November 16, 1988; revision accepted October 16,
1990.
NPY acts directly, presumably through its own recep-
tor, on vascular smooth muscle by a mechanism de-
pendent on extracellular calcium.6 Experimental evi-
dence suggests that NPY also has an indirect action at
the neuroeffector junction. In a way similar to that of
noradrenaline itself,7 NPY has been shown to inhibit
the presynaptic release of noradrenaline4 and to po-
tentiate the postsynaptic action of noradrenaline.8 We
examined the effect of locally administered NPY on
forearm blood flow and defined a dose that is just
above the constrictor threshold. We then investigated
whether this dose of NPY influences vasoconstriction
resulting from reflex sympathetic activity and from
exogenous noradrenaline infusion.
Methods
Six healthy male volunteers between 25 and 40
years of age participated in each of these studies,
which were approved by the Ethics Committee of St.
George's Hospital. Informed consent was given by all
volunteers. Forearm blood flow was measured in
both arms simultaneously using venous occlusion
 Clarke et al NPY and Sympathetic Control of Vasculature
plethysmography with temperature-compensated
mercury-in-Silastic strain gauges.9 During measure-
ment, blood flow to the hands was prevented by wrist
cuffs inflated to 200 mm Hg. A collecting cuff pres-
sure of 40 mm Hg was used. Flows were recorded for
10 of every 15 seconds. The mean of the final five
measurements of each recording period was used for
subsequent analysis.
A 27-gauge unmounted steel cannula was inserted
into the nondominant (left) brachial artery using
local anesthesia (1% lidocaine hydrochloride). Solu-
tions were infused at a constant rate of either 0.5 or
1.0 ml/min throughout the experiment by means of a
constant-rate infusion pump (944A, Harvard Appa-
ratus, South Natick, Mass.). When two drugs were
infused simultaneously, a Y-connector delayed mix-
ing until the solutions entered the cannula. The
dominant arm was not cannulated and served as a
control. Each study took place in the morning, after
the volunteers had rested supine in a quiet clinical
laboratory for a minimum of 30 minutes at a room
temperature maintained between 260 and 28°C. Sa-
line (0.9% NaCl, Travenol Laboratories, Deerfield,
Ill.) was infused for 10 minutes, followed by infusion
of three incremental doses of freshly reconstituted
NPY (50, 200, and 1,000 pmol/min, Peninsula Labo-
ratories, Inc., Belmont, Calif.), each given for 6
minutes. Forearm blood flow was measured during
the last 3 minutes of each infusion, and measure-
ments continued at intervals for up to 20 minutes
after stopping the NPY infusion. The 10-minute
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saline infusion was repeated, followed by infusion of
incremental doses of noradrenaline (125, 250, and
500 pmol/min), each given for 6 minutes. Forearm
blood flow was measured during the final 3 minutes
of each infusion. After 20 minutes, the infusions of
noradrenaline were repeated during coinfusion of 50
pmol/min NPY.
In each subject a lower-body negative-pressure
chamber constructed of polyethylene sheeting over a
reinforced mesh wire framework was placed around
the lower portion of the body from the iliac crest
down.10 A seal was formed between the walls of the
chamber and the subject using adhesive tape. An
adjustable vacuum pump was connected to the sealed
chamber, and the negative pressure achieved was
measured continuously using a water manometer.
Saline, 50 pmol/min NPY, and saline were sequen-
tially infused during three consecutive 12-minute
periods. During each period, forearm blood flow was
measured continuously after the third minute. From
the sixth to the ninth minutes lower-body negative
pressure was applied to a degree (20 cm H2O) that
has been shown to have no effect on blood pressure
and heart rate.10
In four subjects 500 pmol/min noradrenaline and
then 1,000 pmol/min NPY were infused for 6 minutes
each, followed by an interval of 10 minutes to allow
blood flow to return to baseline. Phentolamine (300
nM/min) alone was then infused for 20 minutes, at
which time noradrenaline was added to the phentol-
775
3^ 4-
X +1
o c
_.0 3.
r E
0
2-
oE
L_
1
saline 50 200 1000
Log dose NPY (pmol/min)
FIGURE 1. Infusion of incremental doses of neuropeptide Y
(NPY) into left (nondominant) brachial artery of six men
resulted in dose-dependent reduction in mean+SEM forearm
blood flow. *p<0.05, **p<0.01 different from saline by
Student's paired t test.
amine infusion. Following cessation of the noradren-
aline infusion, NPY was infused during continued
infusion of phentolamine. Blood flow was measured
during the last 3 minutes of each infusion.
Student's t test for paired observations was used
for statistical analysis, and the data are presented
as mean±SEM.
Results
NPY reduced forearm blood flow in a dose-depen-
dent fashion in all six subjects (Figure 1). The lowest
dose (50 pmol/min) induced slight reduction in some
subjects, but the overall effect was not significant. At
the peak dose, blood flow was reduced from 3.8±0.6
to 1.8±0.2 ml/100 ml/min. At this dose, the effect of
NPY was detectable within 2 minutes of commencing
the infusion and persisted for up to 15 minutes after
stopping the infusion. Blood flow in the control arm
showed no consistent change during the study.
Noradrenaline also reduced forearm blood flow in
all subjects in a dose-dependent fashion (Figure 2,
left), from 4.8±1.1 to 2.5±0.4 ml/100 ml/min. This
response was unaffected by coinfusion of 50 pmol/
min NPY (Figure 2, right), blood flow falling from
5.2±1.2 to 2.3±0.4 ml/100 ml/min. The onset of the
response to noradrenaline occurred in <1 minute,
6
0
C5.
Q0 E 4
0
cp C
3. **
o 2
U-
1'
SAL 125 250 500 SAL 125 250 500
Dose noradrenaline (pmol/min)
FIGURE 2. Left: Mean-+SEMforearm bloodflow response
to infused noradrenaline in six subjects. Right: Coinfusion of
50 pmol/min neuropeptide Y failed to significantly alter re-
sponse to noradrenaline. *p<0.1, **p<0.05 different from
saline (SAL) by Student's paired t test.
 776 Circulation Vol 83, No 3 March 1991

LBNP LBNP LBNP Discussion

4.0 -
We examined the effects on the human forearm
.-
0-
^
3.5 - vascular bed of intra-arterial administration of NPY.
At low concentrations, this peptide caused a large
-E dose-dependent reduction in blood flow very similar
3.0 -
'
to that demonstrated by Pernow et al.11 Previous
0
E 2.5 - studies have shown three possibly distinct actions of
o
NPY in vitro using animal tissue: presynaptic inhibi-
2.0 - tion of noradrenaline release, postsynaptic potentia-
L saline | NPY | saline | tion of noradrenaline's action, and a direct action
FIGURE 3. Effect of neuropeptide Y (NPY) on mean-+SEM independent of a-adrenergic receptors. The failure of
constrictor response to 20 cm H20 lower-body negative pres- the nonselective a-blocking agent phentolamine in
sure (LBNP) in six men. LBNP produced similar reductions in these circumstances to diminish or abolish the con-
forearm blood flow in both arms durting saline infusion. strictor action of NPY is clear evidence of the exis-
Infusion of 50 pmol/min NPYfailed to alter ratio of constric- tence of a direct mechanism of action in vivo in man.
tion in the two arms, as shown by ratio of infused arm While no significant enhancement of reflex con-
flowkcontrol arn flow. ni, control ann; , infused (nondom- striction to lower-body negative pressure was found
inant) ann. during NPY infusion, a small decrease in blood flow
was observed in both arms, suggestive of a central
effect of the small increment in the circulating NPY
and blood flow returned to basal values within 5 concentration.
minutes of stopping the infusion. The similarity of the pressor responses in the two
Lower-body negative pressure reduced forearm arms to lower-body negative pressure suggests that
blood flow in both arms, by 14.0±3% during saline the much higher local concentration of NPY was
infusions and by 20.3±3.0% during NPY infusion unable to directly modify sympathetic constriction in
(differences not significant, Figure 3). While there the peripheral circulation. This lack of potentiation
was a trend toward enhanced reflex flow reduction of constriction, induced by either reflex or by infusion
during NPY infusion in both arms, this did not reach of noradrenaline, suggests that in the human forearm
the level of significance. NPY acts directly rather than through presynaptic or
A single dose of 1,000 pmol/min NPY resulted in a postsynaptic augmentation of noradrenergic sympa-
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reduction in blood flow similar to that induced by 500 thetic function. This is in contrast to many in vitro
pmol/min noradrenaline. Coinfusion of phentol- studies that clearly demonstrate an indirect constric-
amine caused an increase in basal forearm blood flow tor effect of NPY.6,8 It is possible that with intralu-
and abolished the response to noradrenaline, but the minal administration, the concentration of the pep-
response to NPY remained unchanged and was un- tide is relatively less on the adventitial side of the
diminished (Figure 4). vessel, where the majority of the neuroeffector junc-
tions are located. However, an enhanced sympathetic
response to lower-body negative pressure has been
14
documented with intraluminal administration of an-
giotension II and, therefore, accessibility of binding
.
_. 12 -

sites does not appear to be a limiting factor.12

;=U) Compared with noradrenaline, NPY is slower to
X0 +1 10 act but the constriction is longer-lasting. The re-
o c
8- sponse in the forearm was quite reproducible and
similar in all subjects studied. This is in contrast to
E E 6- our'3 and other8 observations in the coronary bed
1_._

u 0
4- and in vitro, where responses are variable and tend to
0 E be "all or nothing."
L-- 2-
phentoamnei The reduction in forearm blood flow demonstrates
01
j the ability of NPY to produce constriction of resis-
saline saline | saline saline saline | saline tance vessels, as has been demonstrated in the coro-
I NA I INPYI I NA EIINPYI nary circulation in dogs13 and humans14 and is con-
sistent with the observation of more NPY-containing
FIGURE 4. Effect ofphentolamine on mean +SEM constric- nerve vesicles in smaller arteries.15
tor effect of noradrenaline(NA) and neuropeptide Y (NPY) in The observation that phentolamine fails to abolish
four men. Infusion of 500 pmol/min NA and 100 pmol/min the constrictor response to NPY is consistent with
NPYresult in similar reductions in forearm blood flow. During the documented failure of a-blockade to completely
coinfusion of phentolamine, which increases resting blood abolish the vascular effects of sympathetic stimula-
flow, response to NA is abolished while maximum response to tion in other models.'6 Indeed, a-blockade actually
NPY is unaltered. increases the overflow of NPY during nerve stimula-
 Clarke et al NPY and Sympathetic Control of Vasculature
tion.17 In view of the fact that varying stimulation
frequencies of sympathetic nerves may cause a pref-
erential release of NPY from the nerveterminal,'8 it
is conceivable that there are conditions in which
sympathetic stimulation (central or otherwise) may
provoke NPY release alone. It is therefore possible
that this peptide plays an important part in constric-
tor responses resulting from sympathetic stimulation.
References
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777
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KEY WORDS * neuropeptide Y * sympathetic nervous system
vascular control