Introduction

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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 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 middle-sized peripheral blood vessels, perfusion with NPY does not 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 Zukowska-Grojec 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 emphasize the role of NPY in the regulation of the peripheral sympathetic vascular tone.

	Materials and Methods

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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 ± 5 mm Hg (n = 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 µM 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-9,11-epoxymethano-prostaglandin F₂ (PGF₂). 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 µM 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 µM 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 µM 5-HT, 4 µM PGF₂, 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 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 Receptor-Blocking Agent

NPY concentration-response experiments were performed before and after 30 min of tissue perfusion with 0.1, 0.3, or 1 µM 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 µM 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 µM BIBP 3226. Similar protocols were designed to test whether BIBP 3226 antagonized the vasomotor responses elicited by the structural analogs of NPY.

Acute Blockade of NPY-Induced Vasoconstriction by Several Drugs

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 µM 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 µM. 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 ₁-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 concentration-response protocol. Because the NE-induced constriction is sensitive to nifedipine, 10 µM 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 µm thick) were obtained from control and endothelium-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 µM NE and dilated after perfusion with 0.1 or 1 µM 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 µM 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 µM 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, PGF₂, 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

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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 ± 0.06 nM (n = 25); 10 nM NPY elicited a sustained rise of approximately 100 mm Hg, equivalent to that attained with 10 µM NE. Perfusion with 3 µM 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 µM NE; larger concentrations of the catecholamine were avoided because an excessive rise in the perfusion pressure will introduce undesirable variables.

View larger version (24K): [in this window] [in a new window]   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 µM 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.

View larger version (21K): [in this window] [in a new window]   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 = 12) and PYY (n = 6). Bottom, studies with [Leu31,Pro34]NPY (n = 10), NPY 13-36 (n = 3), and the equimolar mixture of [Leu31,Pro34]NPY and NPY 13-36 (n = 4). Symbols indicate mean values; bars show the S.E.M.

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.

View larger version (14K): [in this window] [in a new window]   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 µM 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 ± 0.06 nM (n = 25); ET-1, 2.7 ± 0.6 nM (n = 8); NE, 2.3 ± 0.6 µM (n = 12); 5-HT, 2.0 ± 0.3 µM (n = 6); and KCl, 65 ± 5 mM (n = 18).

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, PGF₂, 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 PGF₂ was also significant; ET-1 and 5-HT were less active.

View larger version (22K): [in this window] [in a new window]   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 µM PGF2 or 10 µM 5-HT and 10 nM ET-1, respectively, evokes NPY vasoconstriction.

Magnitude of NE Precontraction. During the summer months, the 3 µM NE-induced vasomotor response exhibited variability. The responses ranged from 8 to 80 mm Hg, with a mean increase of 32 ± 4 mm Hg (n = 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 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.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   BIBP 3226 antagonism of the NPY-induced vasomotor effect. Top, tracing shows that the application of 0.1 and 1 µM BIBP 3226 gradually blocks the NPY-induced vasoconstriction in a preparation precontracted with 3 µM 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 = 12) is antagonized in a concentration-dependent fashion by pretreatment with 0.1 (n = 4), 0.3 (n = 4), and 1 (n = 4) µM 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 = 0.997).

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 ± 11.8, 55.0 ± 8.6, 58.7± 14.6, and 55.0 ± 27 mm Hg, respectively. In the presence of 0.1 µM BIBP, these values were reduced to 35 ± 6.7, 0 ± 0, 10 ± 7.6, and 6.3 ± 4.7 mm Hg, respectively. Perfusion with 1 µM BIBP 3226 further antagonized the responses; these values were 3.3 ± 1, 0 ± 0, 8 ± 0, and 0 ± 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 ± 1.2 µM, a value that did not differ significantly from the value of 3.0 ± 0.4 µM (n = 5) obtained in mesenteries exposed to 1 µM 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 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 component resistant even to 10 µM BIBP 3226, which likely is related to the NE precontraction tone.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Comparative potencies of prazosin, nifedipine, and BIBP 3226 to block the NPY-induced vasoconstriction. Top, preparations were precontracted with 3 µM 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 = 10), nifedipine (n = 4), or BIBP 3226 (n = 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 ± 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 µM concentration of the synthetic analog of prostaglandin F2.

Prazosin. This ₁-adrenoceptor antagonist fully blocked the increase in perfusion pressure evoked by NPY, also eliciting a stepwise polygraphic recording. Consistent with its high affinity for ₁-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 PGF₂ (Fig. 6), an indication that the prazosin blockade of the NPY vasoconstriction is indirect and likely due only to its ₁-adrenoceptor-blocking properties. Prazosin did not block the effect of NPY in mesenteries precontracted with 10 nM ET-1 (n = 2, data not shown), suggesting that in the absence of ₁-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 nifedipine-induced vasorelaxation.

View larger version (27K): [in this window] [in a new window]   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.

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 µM to elicit a rise in perfusion pressure of 32.5 ± 5 mm Hg (n = 4) approximating that obtained with 3 µM 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 ± 0.3 versus 2.25 ± 0.56 µM, n = 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 ± 0.04 nM (n = 4), a value that is statistically different from that attained in intact preparations (0.72 ± 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).

View larger version (22K): [in this window] [in a new window]   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 µM BIBP 3226 abolished the NPY vasomotor response. Middle, NPY concentration-response 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 µM 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.

Endothelium denudation after perfusion with saponin 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 µM acetylcholine-induced vasorelaxation decreased significantly from 42 ± 5% in control tissues to 10 ± 2% (P < .01). Likewise in the same tissues, 1 µM acetylcholine-induced vasorelaxation decreased from 73 ± 4% in intact tissues to 29 ± 2% (P < .01, n = 4) in the endothelium-denuded preparations.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In contrast to other agonists, NPY requires a tone to contract the rat arterial mesenteric bed. Provided the mesenteries 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 ₁-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 µM NE-induced 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 PGF₂, 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. Otherwise, KCl should promote the synergism. Zukowska-Grojec 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 ₁-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 µM PGF₂ 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 with the Y₁ agonist [Leu³¹,Pro³⁴]NPY and the Y₂ agonist NPY 13-36 did not produce a concentration-response curve that would mimic more precisely that evoked by NPY, justifying the possible participation of other NPY receptor subtypes. We are aware that the ligand specificity is limited because [Leu³¹,Pro³⁴]NPY does activate Y₃ and Y₅ receptors, but so do NPY and PYY. In sum, our tentative conclusion does not favor the idea that the sole activation of Y₁ receptors accounts for the above-cited observations.

Although it is possible that a nonrecognized NPY receptor mechanism may be at work, various explanations could account for the observed effects. The receptors involved may either belong to a subclass of the NPY Y₁ receptor or correspond to a receptor subtype not fully characterized, perhaps one of the other four identified NPY receptor subtypes (Gerald et al., 1996; Gregor et al., 1996). The future availability of selective ligands for the NPY receptor subtypes will clarify this issue. An alternative explanative based on multiple receptors may also be invoked. Based on the studies by Wahlestedt et al. (1990a) and of Grundemar et al. (1993), it is also possible to hypothesize that multiple NPY receptors may be present in the rat arterial mesenteric bed. McAuley and Westfall (1992) have offered support for this notion. We have not ignored that the intracellular coupling mechanism of the NPY Y₁ receptor may differ from territory to territory. Herzog et al. (1992) documented that the NPY Y₁ receptor can be coupled to two different intracellular signaling systems. In view of the lack of pharmacological tools to more precisely elucidate the identity of the given NPY receptor in this territory, we conservatively interpreted the present findings suggesting a nonclassic Y₁ NPY receptor in the arterial mesenteric bed of the rat.

The NPY vasomotor response may have characteristics similar to those displayed in isolated blood vessels, particularly those present in the cerebral circulation, which are known to contract in response to NPY. We next investigated the importance of extracellular calcium. The current literature expresses conflicting views on the role of L-type calcium channels in the NPY pressor effect. In whole animals and in isolated blood vessels from the cerebral circulatory system, the contractile effect of NPY is reduced in the presence of dihydropyridines (Edvinsson, 1985; Mabe et al., 1985). However, there is no general agreement regarding the involvement of calcium channel sensitive to dihydropyridine in the NPY potentiation of the NE vasocontractile action (Potter, 1991). Present results clearly indicate that in this model system, nifedipine antagonizes and blocks noncompetitively the NPY effect. Parallel experiments using isolated rings from the dog basilar and middle cerebral arteries demonstrate that the direct contractile action of NPY was antagonized noncompetitively by nifedipine (R. Valenzuela and J. P. Huidobro-Toro, in preparation). These results lend support to our proposal that the vascular NPY receptors present in the mesenteric territory are linked, directly or indirectly, to L-type calcium channel activation.

The role of the endothelium in the NPY vasoconstriction was examined to fulfill two objectives. First, we sought to more definitively localize the NPY Y₁ receptors to the vascular smooth muscle. Second, we deemed it necessary to rule out the influence of the endothelial cell layer on the vasomotor activity of NPY. Hieble et al. (1988) reported that the contractile effect of NPY required undamaged endothelial 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.