0022-3565/99/2893-1313$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 289:1313–1322, 1999
Vol. 289, No. 3
Printed in U.S.A.
Synergism between Neuropeptide Y and Norepinephrine
Highlights Sympathetic Cotransmission: Studies in Rat Arterial
Mesenteric Bed with Neuropeptide Y, Analogs, and
BIBP 32261
Vı́CTOR CORTÉS, M.VERÓNICA DONOSO, NELSON BROWN, RODRIGO FANJUL, CLAUDIA LÓPEZ, ALAIN FOURNIER,
and J. PABLO HUIDOBRO-TORO
Accepted for publication December 30, 1998
This paper is available online at http://www.jpet.org
ABSTRACT
Although abundant literature supports the notion that neuropeptide Y (NPY) synergizes in vivo and in vitro, the vasomotor
activity elicited by norepinephrine (NE), the converse interaction
(i.e., the adrenergic modulation of the NPY vasomotor response) has been less characterized. To assess whether NE
synergizes the vasomotor effect of NPY, the rat arterial mesenteric bed was chosen as a model experimental system. Mesenteries were precontracted with NE and few minutes later
were perfused with exogenous NPY. Under these conditions,
NPY contracted the arterial mesenteric bed with an EC50 value
of 0.72 6 0.06 nM. NPY was unable to contract this vascular
territory without an agonist-induced precontraction. Other agonists, such as endothelin-1, a synthetic analog of prostaglandin
F2a, or 5-hydroxytryptamine, also were effective primers because in their presence, NPY was a potent vasoconstrictor. In
contrast, mesenteries precontracted with KCl failed to evidence
the NPY-induced rise in perfusion pressure. Two structural
analogs of NPY, PYY and [Leu31,Pro34]NPY, mimicked the
activity of NPY. The NPY fragment 13-36 did not elicit such a
The abundance of immunoreactive neuropeptide Y (NPY)
surrounding blood vessels (Ekblad et al., 1984; Lundberg et
al., 1989; Donoso et al., 1997a) and the observation that
exogenous NPY increases blood pressure (Tatemoto et al.,
1982; Edvinsson et al., 1983; Mabe et al., 1985) were crucial
Received for publication August 12, 1997.
1
This work was supported by Cátedra Presidencial en Ciencias 1995 (to
J.P.H.-T.) and Fondo Nacional de Investigación Cientı́fica y Tecnologica Grant
1960502. V.C. was supported by a grant from CIM, Centro de Investigaciones
Médicas, Facultad de Medicina. V.C., N.B., R.F., and C.L. were supported by
intramural grants while residents at the Department of Physiology during
their training as medical students.
response. All NPY analogs exhibited less efficacy and potency
relative to NPY. The NPY- and related structural analog-induced vasoconstriction was competitively and reversibly antagonized by BIBP 3226; the pA2 of the NPY interaction was
7.0. The application of 0.1 to 1 mM BIBP 3226 or 0.1 to 10 nM
prazosin at the peak of the NPY vasomotor response elicited a
gradual blockade of the vasoconstriction. Although BIBP 3226
blocked the increase in perfusion pressure elicited by NPY,
leaving unaffected the NE-induced tone, 10 nM prazosin
blocked the full response, including the NE-induced component. Tissue preincubation with 200 nM nifedipine abolished
the NPY-induced vasoconstriction; likewise, the acute application of 10 to 100 nM nifedipine blocked gradually the maximal
NPY-induced contraction. Removal of the mesenteric endothelial layer increased the potency of NPY by 2-fold; it also slightly
potentiated the antagonist activity of BIBP 3226. The synergism
between NPY and NE backs the principle of sympathetic cotransmission.
in hypothesizing the involvement of NPY in vascular control.
Furthermore, the finding that most immunoreactive NPY
nerve fibers costore norepinephrine (NE; Ekblad et al., 1984;
Fried et al., 1986; Lundberg, 1996) indicated the stimulation
of the sympathetic neuroeffector junction might be a physiological mechanism for NPY. Three independent mechanisms
have been invoked to explain the increase in blood pressure
caused by the administration of exogenous NPY. The first
involves direct contraction of the smooth muscles in specific
vascular territories. In intact rings of most of the middlesized peripheral blood vessels, perfusion with NPY does not
ABBREVIATIONS: NPY, neuropeptide Y; PYY, peptide YY; NE, norepinephrine; PGF2a, 9,11-dideoxy-9a,11a-epoxymethano-prostaglandin F2a;
BIBP 3226, (R)-N2-(diphenacetyl)-N-(4-hydroxyphenyl)-methyl-D-arginine amide; ET-1, endothelin-1; 5-HT, 5-hydroxytryptamine; EC50, median
effective concentration.
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Unidad de Regulación Neurohumoral, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, P. Universidad Católica de
Chile, Santiago, Chile (V.C., M.V.D., N.B., R.F., C.L., J.P.H.-T.); and Université du Québec, Institut National Reserche Scientifique-Santé,
Pointe Claire, Montreal, Canada (A.F.)
1314
Synergism Between NPY and NE
emphasize the role of NPY in the regulation of the peripheral sympathetic vascular tone.
Materials and Methods
Perfusion of Isolated Rat Mesenteric Bed
At least 150 adult male Sprague-Dawley rats (250 –280 g), bred in
our Animal Reproduction Laboratories, were anesthetized with 50
mg/kg sodium pentobarbital. The abdominal cavity was incised at
the midline, and the superior mesenteric artery was cannulated with
polyethylene tubing. Perfusion with Krebs-Ringer buffer, bubbled
with 95% O2/5% CO2 and warmed to 37°C, was performed using a
peristaltic pump operating at a flow of 2 ml/min, as detailed by
Donoso et al. (1996). A pressure transducer was placed close to the
entrance of the artery (McGregor, 1965); any fluctuations in the
recorded perfusion pressure were interpreted as changes in the resistance of the mesenteric bed. The basal perfusion pressure of the
preparations oscillated between 10 and 30 mm Hg, with a mean
value of 23.7 6 5 mm Hg (n 5 67). All experiments were initiated
with a 3- to 4-min perfusion with 70 mM KCl. The preparations that
did not respond to the KCl challenge with an increase in the perfusion pressure of 30 to 60 mm Hg were discarded. Next, the tissues
were perfused with 3 mM NE to raise the vascular tone before
initiation of NPY perfusions.
Quantification of Results
Concentration-response experiments were routinely performed
with NPY and NPY structural analogs NE, endothelin (ET)-1, 5-HT,
and 9,11-dideoxy-9a,11a-epoxymethano-prostaglandin F2a (PGF2a).
In most cases, the potency of each agonist was expressed as the EC50
value, as calculated by interpolation from the respective concentration-response curve. The pA2 value was calculated according to the
method developed by Arunlakshana and Schild (1959). In some experiments, the concentration of the antagonist required to reduce the
vasomotor effect of 10 nM NPY by one half was likewise obtained by
interpolation from each concentration-response experiment.
Determination of Agonist Potencies
NPY and Related Structural Analogs. To estimate the potency
of NPY, mesenteric preparations were primed with 3 mM NE, a
concentration of the catecholamine that was added to the buffer
simultaneously with 0.1, 1, 10, 30, or, occasionally, 100 nM NPY or
with the structurally related analogs of the peptide. Each peptide
was tested in separate mesenteric preparations. In a parallel series
of experiments, the potency of NPY alone was compared with that
achieved by the equimolar mixture of [Leu31,Pro34]NPY, an NPY Y1
receptor agonist, and NPY 13-36, an NPY Y2 agonist.
Other Vasoconstrictors. NE, ET-1, 5-HT, and KCl evoke increases in perfusion pressure without the need of a precontraction;
therefore, concentration-response experiments were performed without precontracting the mesenteries. In one series of experiments, two
consecutive NE concentration-response experiments were performed
in a same preparation. The first NE concentration-response curve
was performed without a prior tone; 30 min later, the NE concentration-response curve was repeated after tissue precontraction with
3 mM NE.
Specificity of Several Agonists to Precontract
Mesenteries
Protocols were performed in which the rat arterial mesenteric was
precontracted with the following vasomotor agents: 2 to 10 nM ET-1,
10 mM 5-HT, 4 mM PGF2a, and 35 to 70 mM KCl. Each of these
compounds was perfused for 10 min before challenging the mesenteries with 0.1, 1, and 10 nM NPY. Separate preparations were used
to evaluate the effect of each vasoconstrictor. Care was taken to
choose agonist concentrations that raised the basal pressure 15 to 25
mm Hg and to ensure that the tone was well maintained. To evaluate
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elicit a direct contractile effect, except in cerebral arterial
rings, such as the basilar, pial, or cerebral medial vessels of
mammals, including humans (Edvinsson et al., 1983; Edvinsson, 1985; Abounader et al., 1995). Boric et al. (1995) observed in the hamster cheek microcirculation a direct contraction evoked by NPY mediated by a mixture of Y1 and Y2
NPY receptors. In support of this mechanism, binding experiments demonstrate the presence of a mixed population of Y1
and Y2 NPY receptors in several isolated blood vessels
(Zukowska-Grojec and Wahlestedt, 1993). The second mechanism is related to the potentiation of the vasomotor activity
of several endogenous vasoconstrictor agents such as NE,
5-hydroxytryptamine (5-HT), or angiotensin II (Edvinsson et
al., 1984; Wahlestedt et al., 1985; López et al., 1989). By far
the most thoroughly examined of NPY vascular effects is its
ability to potentiate the activity of several vasoconstrictors or
to inhibit vasorelaxatory responses such as those induced by
acetylcholine (Gulbenkian et al., 1992). This effect has been
demonstrated in practically all the blood vessels examined,
including in vivo studies with nonanesthetized experimental
animals (López et al., 1989; for a review, see Potter, 1991). A
third physiological explanation involves a presynaptic mechanism, mediated by neuronal Y2 NPY autoreceptors that
reduce the release of NE (Lundberg and Stjärne, 1984; Westfall et al., 1987; Lundberg et al., 1989).
The recent development of potent and highly selective NPY
antagonists has opened new avenues to the understanding of
the involvement of NPY in the physiology of the autonomic
nervous system, particularly its involvement in the control of
the vascular tone. The best studied of the NPY receptor
antagonists is BIBP 3226, a competitive nonpeptide molecule
that is characterized by its high affinity and specificity for
the NPY Y1 receptor (Rudolf et al., 1994; Doods et al., 1995).
In several in vitro assays, BIBP 3226 has an affinity ranging
between 0.5 and 5 nM (Rudolf et al., 1994) and displays a pA2
of 8.5 (Abounader et al., 1995). Although BIBP 3226 does not
essentially modify peripheral blood pressure, it significantly
reduces the NPY vasopressor response (Doods et al., 1995;
Mezzano et al., 1998) and that elicited by electrical stimulation of perivascular sympathetic nerves (Lundberg and Modin, 1995; Malmström and Lundberg, 1995; Racchi et al.,
1997).
Based on the fact that NPY nonspecifically potentiates
several vasoconstrictors, Itoi at al. (1986) and ZukowskaGrojec and Vaz (1988) made the original contribution that
after the infusion of NE to rats, the i.v. administration of
NPY caused a markedly potentiated pressor response.
These observations were confirmed by Wahlestedt et al.
(1990b) using isolated pulmonary artery rings. To expand
on these observations, our aim in the current study was to
characterize the NPY receptors and to explore the mechanism by which NE, and eventually other phospholipase
C-coupled vasoconstrictors, synergize the vascular smooth
muscle to the vasomotor action of NPY. The rat arterial
mesenteric bed seemed an appropriate model system for
this project because this vascular territory is essentially
refractile to the sole application of NPY (Donoso et al.,
1993, 1997b). The NE-NPY cooperation highlights cotransmission and is consistent with the notion that both neurochemicals are coreleased from peripheral sympathetic
nerve terminals (Donoso et al., 1997a). The present data
Vol. 289
1999
the influence of KCl on the NE responses, separate series of paired
NE concentration-response protocols were performed with and without a precontraction with 50 mM KCl.
Antagonism by BIBP 3226, an NPY Y1-Selective ReceptorBlocking Agent
NPY concentration-response experiments were performed before
and after 30 min of tissue perfusion with 0.1, 0.3, or 1 mM BIBP 3226.
The antagonist was maintained in the buffer system while the second NPY concentration-response protocol was performed. Each preparation was used to study the effect of a single antagonist concentration.
All NPY concentration-response experiments were performed in mesenteries precontracted with 3 mM NE. To test the specificity of the
antagonist, in a parallel set of experiments, NE concentration-response
experiments were performed before and 30 min after mesentery pretreatment with 1 mM BIBP 3226. Similar protocols were designed to
test whether BIBP 3226 antagonized the vasomotor responses elicited
by the structural analogs of NPY.
BIBP 3226. Mesenteries were contracted with 10 nM NPY; once
the maximal response developed, the preparation was perfused with
media that contained 10 nM NPY plus 0.01 mM BIBP 3226. After a
stable and notorious BIBP 3226 response ensued, the antagonist
concentration in the perfusion media was gradually increased from
0.01 to 10 mM. The buffer maintained 10 nM NPY during the performance of the full protocol. At the end of each experiment, the
mesenteries were perfused with drug-free Krebs-Ringer to determine the reversibility of the interaction and whether BIBP 3226 had
completely blocked the 10 nM NPY-induced vasomotor response.
Prazosin. To assess whether part of the NPY-induced vasomotor
response is due to the activation of an a1-adrenoceptor mechanism,
separate preparations were contracted with 10 nM NPY as detailed
above. Once the maximal NPY response was attained, tissues were
perfused with graded concentrations of prazosin (0.1, 1, and 10 nM).
At the end of each protocol, all preparations were additionally perfused with Krebs-Ringer buffer to determine the existence of a prazosin-resistant vasomotor component.
Nifedipine. To investigate the participation of L-type calcium
channels, in the 10 nM NPY-induced vasoconstriction, tissues were
perfused with 1, 10, and 50 nM nifedipine as detailed above. Nifedipine stock solution (10 mM) was prepared in ethanol and dissolved
thereafter in Krebs-Ringer buffer. Parallel experiments with vehicle
determined that ethanol did not interfere with the NPY vasomotor
activity or with the nifedipine-induced blockade.
Antagonism of NPY-Induced Vasomotor Response by
Nifedipine
For this protocol, preparations were perfused for 30 min with 200
nM nifedipine before the performance of an NPY concentrationresponse protocol. Because the NE-induced constriction is sensitive
to nifedipine, 10 mM NE was used to precontract the mesenteries by
20 to 30 mm Hg.
Removal of Endothelial Cell Layer
To assess the influence of the endothelial cell layer on the potency
of NPY and BIBP 3226, experiments were conducted in preparations
with and without the endothelial cell layer. The endothelium was
removed after tissue perfusion with buffer containing 0.1% saponin
for 55 s. This procedure has been previously used to remove or
destroy, at least partially, the endothelial cells (Donoso et al., 1996).
To visualize microscopically the destruction of the cell layer after
perfusion with saponin, preparations were fixed with Bouin’s solution for 24 h, dehydrated with alcohol, and embedded in paraplast.
Tissue slices (5 mm thick) were obtained from control and endothe-
1315
lium-denuded preparations that had been stained with hematoxilin
and eosin for light microscopy examination.
Parallel protocols were designed to evaluate the functional implications of the removal of the endothelial cell layer. Intact and endothelium-denuded mesenteries were precontracted with 3 to 10 mM
NE and dilated after perfusion with 0.1 or 1 mM acetylcholine.
Potency of NE and NPY. NE concentration-response protocols
were performed in endothelium-denuded preparations to study the
magnitude of the NE sensitization created by endothelium removal.
NPY concentration-response experiments were performed in tissues
primed with either 0.5 or 1 mM NE; otherwise, the NPY determination proceeded as usual.
BIBP 3226. Endothelium-denuded preparations were contracted
with 10 nM NPY as detailed above; tissues were next perfused with
buffer containing the NPY plus either 0.1, 0.3, or 1 mM BIBP 3226.
The protocol was identical to that outlined in tissues with intact
endothelium.
Peptides and Drug Sources
Porcine NPY and some of its structural analogs, such as PYY,
[Leu31,Pro34]NPY, and NPY 13-36, were synthesized using solid
phase by A. Fournier (INRS-Santé); ET-1 and some batches of NPY
were purchased from Peninsula Laboratories Inc. (Belmont, CA).
NE, 5-HT, PGF2a, prazosin, nifedipine, saponin, and other reagents
were purchased from Sigma Chemical Co. (St. Louis, MO). BIBP
3226 was kindly provided by Dr. K. Rudolf (Thomae GmbH, Germany). All drugs and peptides were dissolved in distilled water and
diluted accordingly in Krebs-Ringer buffer. Analytic-grade chemicals
from Merck (Darmstadt, Germany) were used to prepare buffer
solutions.
Statistical Analysis
ANCOVA was used to study the significance of the displacement of
concentration-response curves caused by several drug treatments or
endothelial cell removal. In all cases a value of P , .05 was considered statistically significant. The Dunnett’s tables for multiple comparisons with a common control were used when appropriate.
Results
Potency of NPY and Structurally Related Peptides
NPY and Related Structural Analogs. NPY and related
peptide analogs evoked concentration-dependent increases in
perfusion pressure in mesenteries precontracted with NE. In
the absence of an agonist-induced vasomotor tone, NPY
failed to increase the perfusion pressure (Fig. 1). The EC50
value of NPY was 0.72 6 0.06 nM (n 5 25); 10 nM NPY
elicited a sustained rise of approximately 100 mm Hg, equivalent to that attained with 10 mM NE. Perfusion with 3 mM
NE caused a well sustained increase in perfusion pressure of
about 30 mm Hg as long as the catecholamine is present in
the perfusion buffer (Fig. 1B). On abruptly changing of the
perfusion medium to a buffer containing only 10 nM NPY,
the pressure immediately dropped to its basal level (Fig. 1D).
The readdition of NE to the perfusion buffer caused an immediate restoration of the perfusion pressure to that attained
by NPY before the withdrawal of NE from the perfusion
medium. Most of the protocols were performed in mesenteries precontracted with 3 mM NE; larger concentrations of the
catecholamine were avoided because an excessive rise in the
perfusion pressure will introduce undesirable variables.
The vasomotor effect of NPY is mimicked by PYY and
[Leu31,Pro34]NPY but not by NPY 13-36 (Fig. 2). The potency
and efficacy of these peptides were less than those of NPY.
The median effective concentration of PYY is 3 nM; its effi-
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Acute Blockade of NPY-Induced Vasoconstriction by
Several Drugs
Cortés et al.
1316
Synergism Between NPY and NE
cacy was half of that attained with NPY. It was not possible
to estimate the EC50 value for [Leu31,Pro34]NPY because at
the concentrations used, this analog did not elicit a maximal
response. However, the approximate value is much larger
than that of NPY. The rise in perfusion pressure elicited by
simultaneous perfusion with an equimolar mixture of
[Leu31,Pro34]NPY and NPY 13-36 was inferior to that attained by NPY alone; the estimated EC50 was larger than
that of NPY (Fig. 2).
Other Vasocontractile Agents. In contrast to NPY,
agents such as ET-1, 5-HT, and eicosanoids do not require a
precontraction to elicit vasomotor activity. In the particular
case of NPY, provided the mesenteries are precontracted, the
peptide is as potent as ET-1 and 1000-fold more potent than
NE (Fig. 3). The efficacy of NPY is about 50% less than that
attained with either ET-1 or NE.
Specificity of Precontracting Agonist. The nature of
the precontraction required to elicit the vasomotor effect of
NPY is not receptor specific because several agonists, including ET-1, PGF2a, and 5-HT, mimicked the ability of NE to
synergize the NPY response (Table 1 and Fig. 4). Although
NE acted as the best agent, the effect of PGF2a was also
significant; ET-1 and 5-HT were less active.
Precontraction with 35 or 70 mM KCl was not able to elicit
NPY contractions, despite the marked increase in perfusion
pressure caused by the cation (Table 1). Precontraction with
50 mM KCl, which raised the perfusion pressure by 35 6 4.8
Fig. 2. Concentration-response curves for NPY and structurally related
peptides with selectivity for the Y1 and Y2 NPY receptor subtypes. Top,
vasomotor potency of NPY (n 5 12) and PYY (n 5 6). Bottom, studies with
[Leu31,Pro34]NPY (n 5 10), NPY 13-36 (n 5 3), and the equimolar mixture
of [Leu31,Pro34]NPY and NPY 13-36 (n 5 4). Symbols indicate mean
values; bars show the S.E.M.
mm Hg, did not modify the NE EC50 value. In a subset of four
parallel mesenteric preparations, the NE EC50 value before
the precontraction was 2.3 6 0.6 mM, a value that is not
different from that attained after mesenteric precontraction
with 50 mM KCl (2.4 6 0.12 mM). Furthermore, to rule the
influence of the tone on the vasomotor potency of physiological agents, the NE EC50 value was essentially unaltered by
mesenteric precontraction with NE. In fact, the NE EC50
value in a series of control protocols did not differ significantly from that attained in paired mesenteries precontracted with 3 mM NE (2.3 6 0.4 versus 2.7 6 0.4 mM, n 5 4).
Magnitude of NE Precontraction. During the summer
months, the 3 mM NE-induced vasomotor response exhibited
variability. The responses ranged from 8 to 80 mm Hg, with
a mean increase of 32 6 4 mm Hg (n 5 33). The responses
demonstrated, however, a normal, gaussian distribution. We
used these data to assess whether the magnitude of the rise
in tone elicited by this agent influenced the 10 nM NPY
response. For this purpose, these results were divided into
four arbitrary categories. The mean increase in perfusion
pressure averaged around 10 mm Hg for group I, 20 mm Hg
for group II, 40 mm Hg for group III, and 75 mm Hg for group
IV (Table 2). It is evident that in all groups, the effect of NPY
is concentration dependent; however, the magnitude of the
rise in perfusion pressure caused by the primer was not
paralleled by a proportional increase in the vasomotor effect
elicited by NPY.
Antagonism by BIBP 3226
BIBP 3226 caused a parallel, concentration-dependent
rightward displacement of the NPY concentration-response
curve (Fig. 5). The pA2 of the interaction was 7.0; the slope of
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Fig. 1. NPY needs an NE precontraction to evoke vasoconstriction. Polygraphic tracings show prototype protocols; each experiment was performed in a separate preparation. All preparations were calibrated at the
beginning of the protocol with a standard 4-min perfusion with 70 mM
KCl. Time scale in A is common to all four recordings. A, NPY vasoconstriction is not elicited in the absence of an NE precontraction tone. B,
perfusion pressure is maintained during prolonged perfusion with 3 mM
NE. C, concentration-dependent vasomotor effect of NPY is observed in
NE-precontracted mesenteries. D, the vasomotor effect of NPY is observed only when NE and NPY are coapplied simultaneously.
Vol. 289
1999
Cortés et al.
1317
Fig. 3. Potency of NPY and other vasomotor agonists in
the arterial mesenteric bed of the rat. To compare the
vasomotor potency of NPY, concentration-response studies were performed with ET-1, NE, 5-HT, and KCl. Mesenteries were precontracted with 3 mM NE only for the
performance of the NPY concentration-response protocols.
Separate mesenteric preparations were used to evaluate
the potency of each compound. Symbols refer to the mean
value; bars indicate the S.E.M. The median effective concentrations of the agonists examined were NPY, 0.72 6
0.06 nM (n 5 25); ET-1, 2.7 6 0.6 nM (n 5 8); NE, 2.3 6
0.6 mM (n 5 12); 5-HT, 2.0 6 0.3 mM (n 5 6); and KCl,
65 6 5 mM (n 5 18).
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TABLE 1
Lack of specificity of several primers used to elicit increase in perfusion
pressure caused by 10 nM NPY
Increase in Perfusion Pressure
Agonist-Induced
Precontraction
Vasomotor
Effect of 10 nM NPY
mm Hg
1 mM NE (4)
3 mM NE (12)
2 nM ET-1 (8)
10 mM 5-HT (4)
4 mM PGF2a (6)
35 mM KCl (6)
70 mM KCl (6)
12.5 6 2
38 6 7
15 6 2
13 6 2
18 6 4
14 6 3
63 6 12
25 6 5
87 6 10
40 6 5
28 6 3
70 6 10
863
10 6 2
Values are mean 6 S.E.M.
the plot was 1.52. The antagonism was partially reversible,
as demonstrated by the finding that at 30 to 60 min after
perfusion with drug-free buffer, 60% to 80% recovery of the
NPY responses was observed.
As with NPY, BIBP 3226 also blocked the increase in
perfusion pressure elicited by NPY structural analogs. The
mean increase in perfusion pressure elicited by applications
of 30 nM NPY, 100 nM PYY, 100 nM [Leu31,Pro34]NPY, and
an equimolar mixture of 30 nM [Leu31,Pro34]NPY plus NPY
13-36 was 101.0 6 11.8, 55.0 6 8.6, 58.76 14.6, and 55.0 6 27
mm Hg, respectively. In the presence of 0.1 mM BIBP, these
values were reduced to 35 6 6.7, 0 6 0, 210 6 7.6, and 6.3 6
4.7 mm Hg, respectively. Perfusion with 1 mM BIBP 3226
further antagonized the responses; these values were 3.3 6 1,
0 6 0, 28 6 0, and 0 6 0 mm Hg, respectively.
The BIBP 3226 antagonism was specific for NPY because it
did not affect the NE-induced vasoconstriction. The NE EC50
value in a control group of rats was 5.4 6 1.2 mM, a value that
did not differ significantly from the value of 3.0 6 0.4 mM
(n 5 5) obtained in mesenteries exposed to 1 mM BIBP 3226
for 30 min.
Blockade of NPY-Induced Increase in Motor Tone
Mesenteries contracted with 10 nM NPY generally experienced a rise in perfusion pressure that averaged 100 to 120
mm Hg. Under these experimental conditions, the vasomotor
effect elicited by 10 nM NPY could be antagonized by the
following drugs.
BIBP 3226. This nonpeptide NPY Y1 receptor antagonist
blocked, in a concentration-dependent manner, the increase
Fig. 4. Selectivity of the precontraction with several agonists. Representative tracings demonstrate the ability of several agonists to precontract
the mesenteries and evoke NPY vasoconstriction. Each agonist was examined in a separate mesenteric preparation. Every mesentery was calibrated before drug application with a 4-min exposure to 70 mM KCl.
Time scale (A) is common to all four recordings. A, lack of synergism due
to precontraction with 50 mM KCl, a cation concentration that consistently raised the mesenteric perfusion pressure by 50 mm Hg. Tracings
B–D show that precontraction with either 4 mM PGF2a or 10 mM 5-HT
and 10 nM ET-1, respectively, evokes NPY vasoconstriction.
in perfusion pressure elicited by NPY demonstrating a stepwise recording (see tracing in Fig. 5). Consistent with the
competitive nature of the antagonism and consonant with its
pA2, 100 nM BIBP 3226 reduced by one half the vasomotor
effect elicited by NPY (Fig. 6). Larger concentrations caused
a proportional effect. Experiments demonstrated, however, a
1318
Synergism Between NPY and NE
Vol. 289
TABLE 2
NPY concentration-response curves in 33 preparations primed with 3 mM NE
Increase in Perfusion Pressure
0.1 nM NPY
1 nM NPY
10 nM NPY
mm Hg
Group
Group
Group
Group
I (7), primer 8.57 6 0.92
II (12), primer 22.5 6 1.0
III (10), primer 40 6 3.3
IV (4), primer 76 6 6.6
17.1 6 10
12.1 6 5.7
29 6 9.3
32.5 6 13.6
48.6 6 11
67.5 6 10
77.5 6 10
59 6 10.7
93.6 6 13
111.3 6 9.1
116.5 6 9.4
66.3 6 11.4
Values are given as mean 6 S.E.M.
component resistant even to 10 mM BIBP 3226, which likely
is related to the NE precontraction tone.
Prazosin. This a1-adrenoceptor antagonist fully blocked
the increase in perfusion pressure evoked by NPY, also eliciting a stepwise polygraphic recording. Consistent with its
high affinity for a1-adrenoceptors, the prazosin concentration-response curve was parallel to that of BIBP 3226 but
displaced to the left at least 100-fold (Fig. 6). In contrast to
BIBP 3226, prazosin obliterated the NPY response, suggesting that in the absence of an NE tone, NPY is unable to
contract the mesentery. Prazosin failed to block the NPY
increase in mesenteric pressure in tissues precontracted with
PGF2a (Fig. 6), an indication that the prazosin blockade of
the NPY vasoconstriction is indirect and likely due only to its
a1-adrenoceptor-blocking properties. Prazosin did not block
the effect of NPY in mesenteries precontracted with 10 nM
ET-1 (n 5 2, data not shown), suggesting that in the absence
of a1-adrenoceptor activation, prazosin is unable to modify
NPY receptor activity.
Nifedipine. This L-type calcium channel blocker reduced
the NPY response in a stepwise, concentration-dependent
fashion (Fig. 7) with a potency intermediate between that of
prazosin and BIBP 3226 (Fig. 6). The relaxant action of
nifedipine developed slower than that developed by prazosin
or BIBP 3226 and apparently did not affect basal perfusion
pressure within 15 min. The vehicle alone did not interfere
with the NPY-induced contraction or modify the nifedipineinduced vasorelaxation.
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Fig. 5. BIBP 3226 antagonism of the NPY-induced vasomotor effect. Top,
tracing shows that the application of 0.1 and 1 mM BIBP 3226 gradually
blocks the NPY-induced vasoconstriction in a preparation precontracted
with 3 mM NE. The NE tone is resistant to BIBP 3226; the perfusion
pressure reaches baseline after mesenteric perfusion with drug-free
buffer. Bottom, the concentration-dependent vasomotor effect of NPY
(control, n 5 12) is antagonized in a concentration-dependent fashion by
pretreatment with 0.1 (n 5 4), 0.3 (n 5 4), and 1 (n 5 4) mM BIBP 3226.
Symbols indicate the mean values; bars indicate S.E.M. The pA2 is 7.0;
the slope of the Schild plot is 1.52 (r 5 0.997).
Fig. 6. Comparative potencies of prazosin, nifedipine, and BIBP 3226 to
block the NPY-induced vasoconstriction. Top, preparations were precontracted with 3 mM NE and coapplied with 0.1, 1, and 10 nM NPY. Once
10 nM NPY developed its maximal vasomotor response, separate preparations were perfused with several concentrations of either prazosin (n 5
10), nifedipine (n 5 4), or BIBP 3226 (n 5 10). Each drug was studied in
a separate group of rats. The magnitude of the rise in the perfusion
pressure caused by the application of 10 nM NPY is indicated by the
striped bar on the left and corresponds to the mean 6 S.E.M. of a set of
34 separate experiments. The dotted line indicates the basal perfusion
pressure elicited by NE before the coapplication of NPY. Values that
reach this line could be interpreted as antagonism of only the NPY
component of the rise in perfusion pressure. Only prazosin blocked both
the NPY-induced vasomotor response and the NE precontraction. The
graph shows the changes in pressure caused by each concentration of the
drugs examined. Symbols indicate mean values; bars indicate the S.E.M.
Bottom, lack of the NPY blockade by prazosin when the mesenteries were
precontracted with 4 mM concentration of the synthetic analog of prostaglandin F2a.
1999
Cortés et al.
1319
Nifedipine Antagonism
Preincubation of the mesenteries with nifedipine obliterated the NPY-induced vasomotor response displacing the
NPY concentration-response curve downward, suggestive of
a noncompetitive interaction (Fig. 7). In the presence of nifedipine, the concentration of NE was raised to 10 mM to
elicit a rise in perfusion pressure of 32.5 6 5 mm Hg (n 5 4)
approximating that obtained with 3 mM NE in control mesenteric preparations.
Vasomotor Response of NPY in Endothelium-Denuded
Mesenteries; Potency of BIBP 3226 to Block NPY-Induced
Contractions
The lack of the endothelium significantly augmented the
vasomotor response of NE. In endothelium-denuded mesenteric beds, the NE concentration-response curve was displaced to the left, decreasing the EC50 value compared with
intact preparations (0.69 6 0.3 versus 2.25 6 0.56 mM, n 5 4,
P , .01). The concentration of NE required to precontract the
mesenteries was reduced 3-fold to elicit an equivalent tone
(Fig. 8). The NPY EC50 value in the endothelium-denuded
preparations was 0.44 6 0.04 nM (n 5 4), a value that is
statistically different from that attained in intact preparations (0.72 6 0.06, P , .05). In the absence of the endothelial
cell layer, 100 nM BIBP 3226 completely blocked the vasoconstriction induced by 10 nM NPY. A 10-fold larger concentration of BIBP 3226 was required to evoke the same effect as
in intact preparations (Fig. 8).
Endothelium denudation after perfusion with saponin
Fig. 8. Removal of the endothelial cell layer increases the vasomotor
potency of NPY and the antagonist activity of BIBP 3226. Top, representative tracing from an endothelium-denuded preparation prepared after
perfusion with 1 mg/ml saponin for 55 s. Endothelium-denuded preparations were more sensitive to the vasomotor effect of NE and NPY than
were intact mesenteries. In these tissues, the application of 0.1 mM BIBP
3226 abolished the NPY vasomotor response. Middle, NPY concentrationresponse curves in eight intact and four endothelium-denuded mesenteries. Bottom, BIBP 3226 concentration-response curves to block the 10 nM
NPY vasomotor effect in intact and denuded preparations. Once the
maximal vasoconstriction elicited by 10 nM NPY developed, 0.1, 0.3, and
1 mM BIBP 3226 were cumulatively applied to all preparations. Shaded
column indicates the increase in perfusion pressure elicited by 10 nM
NPY in eight different intact arterial mesenteric preparations; open
column shows the increase in perfusion pressure caused by 10 nM NPY in
the four endothelium-denuded preparations. Symbols indicate the mean
values; bars denote the S.E.M. *P , .05.
caused a transient increase in the perfusion pressure (see
recording in Fig. 8) and a significant degree of endothelium
destruction as demonstrated by light microscopy examination. In separate but parallel protocols, we observed that on
the removal of the endothelial cells, the 0.1 mM acetylcholineinduced vasorelaxation decreased significantly from 42 6 5%
in control tissues to 10 6 2% (P , .01). Likewise in the same
tissues, 1 mM acetylcholine-induced vasorelaxation decreased from 73 6 4% in intact tissues to 29 6 2% (P , .01,
n 5 4) in the endothelium-denuded preparations.
Discussion
In contrast to other agonists, NPY requires a tone to contract the rat arterial mesenteric bed. Provided the mesenter-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Fig. 7. Blockade and antagonism of the NPY-induced vasomotor response
by nifedipine. Top, representative tracing shows an experiment designed
to show that nifedipine blocks, in a concentration-dependent manner, the
increase in perfusion pressure elicited by NPY in NE-precontracted mesenteric preparations. Its application caused a gradual blockade of the
NPY-induced vasomotor effect. Bottom, NPY concentration-response
curves were created in a set of control mesenteries and in four separate
tissues preincubated for 20 min with 200 nM nifedipine. Symbols refer to
the mean increase in perfusion pressure caused by each concentration of
NPY; bars denote the S.E.M.
1320
Synergism Between NPY and NE
Otherwise, KCl should promote the synergism. ZukowskaGrojec and Wahlestedt (1993) suggested the role of postreceptor transduction pathways, possibly phospholipase C, to
explain the in vivo and in vitro sensitization of NPY elicited
by NE. The present results further emphasize the involvement of L-type calcium channels, which are likely opened
after the release of a fraction of the intracellular calcium
stores. Future studies will examine whether protein kinase C
inhibition prevents the primary effect of NE and therefore
abolish the NPY vasoconstriction. We are in the process of
examining whether the NE-induced NPY facilitation occurs
in isolated rings of the rat mesenteric artery, a model system
that would greatly facilitate biochemical controls, necessary
for the development of this crucial protocol.
Considering that NE may act as a physiological cotransmitter together with NPY, the experiments using prazosin
were aimed at revealing the influence of the a1-adrenoceptor
in the interaction. Prazosin not only blocked the precontraction tone induced by NE but also completely blocked, in a
graded fashion, the NPY-induced vasoconstriction. To assess
whether prazosin elicits its antagonism by abolishing the
NE-induced precontraction or by directly modifying the NPY
component of the response, experiments were designed to
dissociate these two components of the response. In mesenteries precontracted with 4 mM PGF2a or 10 nM ET-1, the
NPY-induced vasoconstriction was not blocked by prazosin.
These results clearly support the notion that prazosin may
act essentially by abolishing the tone elicited by NE rather
than directly blocking the motor activity of NPY. This interpretation validates the intrinsic adrenergic nature of the
synergism, backing the importance of the cooperation between adrenergic and NPYergic mechanisms (Wahlestedt et
al., 1990b) in the physiology of sympathetic neurotransmission. It becomes impossible to dissociate whether NE sensitizes NPY or NPY synergizes NE. Both interactions likely
operate, highlighting the physiological cooperation between
this two cotransmitters in the vascular sympathetic neuroeffector junction.
This research opens an interesting opportunity to clarify
the NPY receptor subtype involved in the synergism with NE
and other contractile agonists. A simplistic analysis would
argue that the response is mediated by an NPY Y1 receptor
because the response is blocked in a competitive fashion by
BIBP 3226, the most selective Y1 antagonist available, and
mimicked by several NPY structural analogs with affinity for
this receptor. However, several findings suggest a more complex situation. First, the pA2 value of BIBP 3226 is significantly smaller than that reported for the competitive antagonism of direct NPY vasoconstriction in human cerebral
blood vessels (Abounader et al., 1995). Furthermore, the efficacy of PYY and [Leu31,Pro34]NPY was significantly lower
than that of NPY, contrasting markedly with the typical
activity of NPY Y1 receptor agonists. Third, it is clear that
the rat mesenteric bed does not significantly express Y2 receptors because the alleged NPY Y2 receptor agonist, NPY
13-36, is completely inactive. In contrast to observations in
the dog saphenous vein, a blood vessel that essentially contains Y2 receptors (Modin et al., 1991; Pheng et al., 1997), the
rat arterial mesenteric bed appears to be completely devoid of
a Y2 receptor population. Other vessels, such as the rat cava
vein, appear to express a mixed population of Y1 and Y2 NPY
receptors (Zukowska-Grojec et al., 1992). Fourth, coperfusion
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ies are precontracted with receptor agonists linked to phospholipase C, NPY behaves as a potent constrictor of the
arterial mesenteric territory. Among the primers tested, we
focused on NE, recognizing that NPY is commonly found
costored with the catecholamine. At the light of sympathetic
cotransmission (Burnstock, 1990), NE seemed to be a precontracting agent of physiological relevance.
Although the NE EC50 value was essentially unaltered by
its own precontraction, indicating that the affinity of the
tissue for this ligand is unchanged by its preexposure, this is
not the case with NPY. This peptide is unable to elicit per se
a vasomotor effect unless the mesenteric bed is precontracted
with a ligand coupled to a G protein-linked receptor. The
present findings favor the notion of a pharmacodynamic synergism, implying cooperation between the actions of both
ligands. The synergism is short lived, lasting essentially the
time both ligands are simultaneously in contact with the
mesentery. The precontraction requires a threshold, which in
the case of NE, oscillates between 5 and 8 mm Hg, rather
than being a concentration-dependent process. The intracellular signaling mechanism elicited by the a1-adrenoceptor
precontraction probably suffices to trigger the physiological
conditions that allow the NPY vasomotor effect to occur. A
precontraction causing a larger increase in the perfusion
pressure was avoided because it may limit the physiological
capacity of the vascular bed to contract with NPY. It appears
that the need of precontraction to evoke NPY contractions is
not an isolated observation. Leite et al. (1997) recently documented that angiotensin II also requires a precontraction to
elicit its characteristic vasomotor action in the rat arterial
mesenteric bed. This result illuminates a mechanistic link
between NPY and angiotensin II, a peptide with an accepted
role in vascular homeostasis. In the case of both peptides, the
precontraction must trigger a mechanism that facilitates or
initiates the intracellular machinery dealing with muscle
contraction.
The NE-induced precontraction shows seasonal variability
that markedly changes the magnitude of the 3 mM NEinduced precontraction. Efforts were made to reduce experimental variations by controlling the duration of the NE precontraction, time between the precontraction and the
application of NPY, the age of the animal, and other factors.
In this regard, Michel et al. (1992) reported that in SK-N-MC
cells, NPY elevates intracellular calcium, an effect that was
augmented by isoproterenol in a time-dependent fashion.
Our results are apparently independent of the duration of the
NE precontraction and of the interval between NE tone and
NPY application because in all cases, NPY was applied after
a 5- to 15-min period of mesenteric precontraction induced by
NE and other vasoconstrictors.
It is surprising to find that KCl, although promoting a
reasonable precontractile tone, was unable to synergize the
NPY response. Instead, vasoconstrictors as diverse as PGF2a,
ET-1, and 5-HT are all able to replace, to varying degrees, the
ability of NE to synergize NPY. All the agonists examined in
the mesentery precontraction act at specific ligand-activated
receptors and are all coupled to phospholipase C. Thereby,
they must all release intracellular calcium via the activation
of the inositol trisphosphate pathway and stimulate protein
kinase C activity. It is therefore likely that it is not the rise
in intracellular calcium that facilitates the NPY effect but
rather the activation of protein kinase C by diacylglycerol.
Vol. 289
1999
1321
cells, whereas other investigators have argued to the contrary (Huidobro-Toro et al., 1990; Potter, 1991). In an attempt to clarify the controversy, we compared the influence
of the endothelial cell layer in the vasomotor activity of both
NE and NPY in mesenteric preparations with and without
the endothelium. Results conclusively demonstrate that the
potency of both agents is increased 2- to 4-fold in the absence
of the endothelial cell layer. A previous study from our laboratory had similarly shown supersensitivity to NE in this
vascular bed after excision of the endothelium (Donoso et al.,
1996). We hypothesized that nitric oxide or other endothelial
vasodilatation factors may be responsible for this vasomotor
modulation, both in vivo as well as in isolated vessels. We
are, however, aware that the removal of the endothelial cell
layer may simply provide more auspicious pharmacokinetic
conditions favoring the transport of both the NPY and BIBP
3226 into the biophase or diminishing their metabolic degradation.
Recently, the activity of ATP in the physiology of sympathetic cotransmission was detailed in human blood vessels
(Racchi et al., 1999). Because NPY requires the blood vessels
to be precontracted and NE acts a model agonist, it is of
physiological significance that the sympathetic perivascular
nerves costore and corelease NE and NPY, providing a physiological scenario for sympathetic cotransmission (Burnstock, 1990; Wahlestedt et al., 1990b). Because the mesenteric bed is notoriously vasodilated under the present
experimental conditions, the agonist-induced precontraction,
with the exception of potassium, recreates a physiological
tone, favorable for the ensuing NPY contractile event.
In sum, the present findings add further support to the
notion that the sympathetic nervous system requires the
coordinated action of NE and NPY. Our results demonstrate
the need of a physiological precontractile tone triggered by
receptor ligand activation linked to phospholipase C activity.
Once a threshold level is attained, NPY receptor activation
sets off a series of intracellular mechanisms, among which
the activity of protein kinase C would seem to be crucial for
vasocontraction. The elucidation of the precise characteristics of the NPY receptors involved remains to be thoroughly
investigated once the appropriate pharmacological tools become available.
Acknowledgments
We thank Dr. K. Rudolf (K. Thomae GmbH) for providing a sample
of BIBP 3226, R. Miranda for the graphic material, and Drs. A.
Schliem and C. F. Valenzuela for editorial assistance.
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