Characterization of neuropeptide Y (NPY) receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226.

1. We have characterized pharmacologically the receptor subtype(s) responsible for the neuropeptide Y (NPY)-induced vasoconstriction in human cerebral arteries. NPY, PYY and several of their derivatives with well defined affinities at the known Y1 and Y2 receptor subtypes were used. Moreover, we tested the ability of the new Y1 receptor antagonist, BIBP 3226, to antagonize the NPY-induced cerebral vasoconstriction. 2. NPY, PYY and their agonists with high affinities at the Y1 receptor subtype ([Leu31-Pro34]-NPY and [Leu31-Pro34]-PYY) elicited strong, long lasting and concentration-dependent contractions of human cerebral arteries. Compounds with Y2 affinity such as PYY3-36 or NPY13-36 either elicited a submaximal contraction at high concentrations or failed to induce any significant vasomotor response. Also, the application of NPY or the specific Y1 agonist, [Leu31-Pro34]-NPY, to human cerebral vessels pretreated with the Y1 agonist, NPY13-36, resulted in contractile responses identical to those obtained when these compounds were tested without prior application of NPY13-36. 3. The order of agonist potency at the human cerebrovascular receptor was: [Leu31-Pro34]-NPY = [Leu31-Pro34]-PYY > or = NPY > PYY > PYY3-36 > > > NPY13-36, which corresponded to that reported previously at the neuronal and vascular Y1 receptors. 4. Increasing concentrations (10(-9)-10(-6) M) of the Y1 receptor antagonist, BIBP 3226, to human cerebral vessels caused a parallel and rightward shift in the NPY dose-response curves without any significant change in the maximal contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)     

NPY receptor subtype in the rabbit isolated ileum.

1. The purpose of this work was to verify the hypothesis that the rabbit ileum is a selective preparation for the NPY Y5 receptor by using new selective antagonists recently synthesized. Spontaneous contractions of the rabbit isolated ileum were recorded and binding experiments were performed in cells expressing the human NPY Y1, Y2, Y4 or Y5 receptor subtype. 2. NPY analogues produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum with the following order of potency hPP > rPP > PYY > or = [Leu31,-Pro34]-NPY > NPY > NPY13-36. Pre-exposure to rPP, PYY, [Leu31,Pro34]-NPY or NPY (but not NPY13-36) inhibited the effect of subsequent administration of hPP suggesting cross-desensitization of the preparation. The apparent affinity of the various agonists studied was correlated to the affinity reported for the human Y4 receptor subtype (and to a lesser extent for the rat Y4 subtype) but not to the affinity for the Y5 receptor subtype. 3. BIBO 3304, a selective NPY Y1 receptor antagonist, and CGP 71683A, a selective NPY Y5 receptor antagonist, did not affect the response to hPP. JCF 109, another NPY Y5 receptor antagonist, produced an inhibition of the response to hPP but only at the highest dose tested (10 microM) which also, by itself, produced intrinsic inhibitory effects. 4. 1229U91, a non-selective ligand for Y1, Y2, Y4 and Y5 receptors with high affinity toward the Y1 and Y4 receptor subtypes, produced a concentration-dependent transient inhibition of the spontaneous contractions of the rabbit ileum and a dose-dependent inhibition of the response to hPP (apparent pKB: 7.2). 5. These results suggest that in the rabbit ileum, the NPY receptor involved in the inhibition of the spontaneous contractile activity is a NPY Y4 receptor subtype.     

Neuropeptide Y (NPY)-induced suppression of activity in the rat: evidence for NPY receptor heterogeneity and for interaction with alpha-adrenoceptors

The receptor mechanisms mediating the neuropeptide Y (NPY)-induced suppression of behavioural activity have been examined in the rat. The interaction of NPY with central noradrenergic mechanisms was also studied. The non-selective alpha-adrenoceptor antagonist, phentolamine (15-60 nmol intracerebroventricularly, i.c.v.), caused a dose-related antagonism (up to 50%) of the NPY-induced suppression of activity. The selective alpha 2-adrenoceptor antagonist, idazoxan (0.125 mg/kg intraperitoneally, i.p.), was even more effective, while the selective alpha 1-adrenoceptor antagonist, prazosin, was without effect. In addition, we examined whether the recently postulated subdivision of peripheral NPY receptors was also applicable to the brain. The ability of the C-terminal 13-36 amino acid fragment of NPY (postulated to activate NPY-Y2 receptors) to reproduce the effects of the full molecule (postulated to activate both NPY-Y1 and -Y2 receptors) was tested. NPY-(13-36) (0.4-10.0 nmol i.c.v.) failed to produce any suppression of activity. On the contrary, it produced an increase in locomotor activity and rearings at low doses. This effect was not blocked by phentolamine. We conclude that the NPY-induced suppression of activity is produced to a large extent by modulation of alpha 2-adrenergic transmission. Our results also provide evidence for heterogeneity among the central NPY receptors, with the NPY-induced suppression of activity being mediated by the NPY-Y1 receptor subtype.     

Neuropeptide Y induced attenuation of catecholamine synthesis in the rat mesenteric arterial bed

The effect of neuropeptide Y (NPY) on the basal and nerve stimulation-induced increase in norepinephrine synthesis was studied in the isolated and perfused mesenteric arterial bed of the rat. Tyrosine hydroxylation, the rate-limiting step in catecholamine (CA) biosynthesis, was assessed by measuring the accumulation of DOPA in the perfusate/superfusate overflow after perfusion of the mesenteric arterial bed with the decarboxylase inhibitor m-hydroxybenzyl hydralazine (NSD-1015). Treatment with NDS-1015 resulted in a time-dependent increase in DOPA production and nerve stimulation (8 Hz, supramaximal voltage, 2 ms duration) increased DOPA production even further. NPY 1 to 100 nM was observed to produce a concentration-dependent attenuation in both the basal and nerve stimulation-induced increase in DOPA formation.To come to an understanding of the NPY receptor subtype mediating the inhibition of CA synthesis, the rank order of potency of a series of NPY analogs with varying selectivity for NPY receptor subtypes including intestinal polypeptide (PYY), PYY 13-36, Leu36 Pro34 NPY, human pancreatic polypeptide (h-PP), and rat pancreatic polypeptide (r-PP) were determined. In addition, the effect of various selective NPY antagonists on the inhibitory effect of NPY was also examined. These included the Y1 antagonist BIB03304, the Y2 antagonist BIIE0246, and the Y5 antagonist CGP71683. The IC50's for NPY, PYY, PYY13-36, Leu31 Pro34 NPY, and hPP in inhibiting CA synthesis were 5, 7, 15, 30, and 33 nM respectively. rPP failed to inhibit CA synthesis. All 3 of the NPY antagonists produced attenuation of the NPY-induced inhibition of CA synthesis, but it took a combination of all 3 to completely block the effect of a maximal inhibitory concentration of NPY.These results demonstrate that NPY inhibits CA synthesis in the perfused mesenteric arterial bed and can do so by activation of a variety of receptors including the Y1, Y2, and Y5.     

Neuropeptide Y-induced potentiation of noradrenergic vasoconstriction in the human saphenous vein: involvement of endothelium generated thromboxane

1. We investigated the potentiating effect of low concentrations of neuropeptide Y (NPY) on the vasoconstriction induced by transmural nerve stimulation (TNS) and noradrenaline (NA) in human saphenous veins. The effects of (i) endothelium removal; (ii) the addition of the NO pathway precursor L-arginine; (iii) the ET_A/ET_B endothelin receptor antagonist Ro 47-0203; (iv) the cyclo-oxygenase inhibitor, indomethacin; (v) the selective thromboxane A₂ (TxA₂) receptor antagonists Bay u3405 and ifetroban, and (vi) the TxA₂ synthase inhibitor, UK 38485, were studied in order to gain information about the mechanisms of NPY-induced potentiation.
2. Contractile response curves for TNS (0.5–8 Hz) and for exogenously administered NA (0.1–3 μM) were obtained in superfused saphenous vein rings. The contractions induced by both TNS and NA at all tested frequencies and concentrations, respectively, were significantly potentiated by 50 nM NPY in endothelium intact veins. Conversely, in endothelium-denuded vessel rings the contractile-response curves to TNS and NA overlapped both in the absence and presence of NPY, thus suggesting that a release of vasoactive substances from endothelial cells could account for the noradrenergic NPY-induced potentiation.
3. In vessels with intact endothelium, the potentiating action of NPY on TNS and NA was unaffected by the presence of high concentrations of the NO precursor L-arginine (3–10 mM) or the non-selective ET_A/ET_B endothelin receptor antagonist, Ro 47-0203 (10 μM). These data indicate that the NPY-induced effect does not involve either the endothelium-derived vasodilator nitric oxide or the vasoconstrictor endothelin. Conversely, in the presence of the cyclo-oxygenase inhibitor, indomethacin (30 μM), NPY failed to potentiate the vasoconstrictions produced by either nerve stimulation or by exogenous NA, thus providing evidence that arachidonic acid metabolites through the cyclo-oxygenase pathway are mainly responsible for the potentiation evoked by NPY.
4. When the TxA₂ receptor antagonists, Bay u 3405 (1 μM) and ifetroban (1 μM) were added to the superfusing medium, NPY did not alter either the frequency- or the concentration-response curves for either TNS or NA. Accordingly, both TNS- and NA-induced contractions were not potentiated by NPY in the presence of the TxA₂ synthase inhibitor, UK 38485 (10 μM). This clearly demonstrates the pivotal role of TxA₂ in NPY-induced potentiation.
5. In superfused vein rings with endothelium, a subthreshold concentration (0.2 nM) of the TxA₂ mimetic U 46619 potentiated both TNS- and NA-induced vasoconstrictions. This potentiation was higher at low stimulation frequencies and low NA concentrations, and resembled that produced by NPY.
6. Our results indicate that in the human saphenous vein NPY potentiates the contractions produced by sympathetic nerve stimulation acting at the postjunctional level, primarily on endothelial cells. In particular, the NPY-induced release of a cyclo-oxygenase metabolite, namely TxA₂, may have a synergistic effect on the vasoconstriction induced by the noradrenergic mediator. Thus, such a mechanism may play a key role in the maintenance of the sympathetic tone of large human capacitance vessels.

     

Inhibition of neuropeptide Y-induced potentiation of noradrenaline-induced vasoconstriction by PP56 (D-myo-inositol 1,2,6-tris-phosphate).

1. Although neuropeptide Y (NPY) is a potent vasoconstrictor in many vascular beds, nanomolar concentrations of this peptide potentiate the noradrenaline-induced contractions in rabbit gastroepiploic and femoral arteries, and guinea-pig mesenteric and uterine arteries. 2. The potentiating effect of NPY on noradrenaline-induced contraction was present in endothelium-denuded femoral arteries. 3. The potentiating effect of NPY on noradrenaline-induced contraction was antagonized by PP56 (D-myo-inositol 1,2,6-trisphosphate) in low concentrations (down to 0.1 nM). This antagonistic effect was observed in all four types of vessels studied. Contractions induced by noradrenaline, histamine, endothelin-1 and potassium were not altered by PP56 in concentrations upto 1 microM in femoral artery of rabbit. 4. We provide evidence that a non-peptide (PP56) can selectively antagonize NPY-induced effects in rabbit and guinea-pig peripheral arteries without affecting the vasoconstrictor response to noradrenaline.     

Role of ARC NPY neurons in energy homeostasis

Neuropeptide Y (NPY) is one of the most abundant and widely distributed neurotransmitters in the mammalian brain and appears to be an important regulatory peptide in both the central and peripheral nervous systems. The arcuate nucleus (ARC) is the major site of expression for NPY within neurons in the hypothalamus. The most noticeable effect of NPY is the stimulation of feeding. To date, six NPY receptor subtypes have been cloned and pharmacologically characterized, and there is evidence for further NPY receptor subtypes. The recent isolation and cloning of a rat NPY receptor with the required pharmacology for the feeding response, called the Y(5) receptor, has led to the suggestion that this is the "feeding" receptor. There is, however, still controversy as to whether the Y(5) receptor represents the true "feeding" receptor, since selective Y(1) receptor antagonists are capable of antagonizing NPY-induced hyperphagia. It also is possible that another, as yet unidentified, NPY receptor subtype(s) may mediate the effects of NPY on energy balance. The potency and behavioral specificity of NPY on feeding and its ability to induce obesity, together with the evidence that ARC-NPY neurons operate homeostatically to counteract energy deficits, all suggest that the ARC-PVN projection is important in regulating energy balance. The next few years will tell us whether or not these important physiological lessons will be successfully translated into a safe and effective form of therapy for human obesity.     

Receptor subtypes Y1 and Y5 mediate neuropeptide Y induced feeding in the guinea-pig

1. Neuropeptide Y (NPY) is one of the most potent stimulants of food intake. It has been debated which receptor subtype mediates this response. Initially Y(1) was proposed, but later Y(5) was announced as a 'feeding' receptor in rats and mice. Very little is known regarding other mammals. The present study attempts to characterize the role of NPY in feeding behaviour in the distantly related guinea-pig. When infused intracerebroventricularly, NPY dose-dependently increased food intake. 2. PYY, (Leu(31),Pro(34))NPY and NPY(2 - 36) stimulated feeding, whereas NPY(13 - 36) had no effect. These data suggest that either Y(1) or Y(5) receptors or both may mediate NPY induced food intake in guinea-pigs. 3. The Y(1) receptor antagonists, BIBO 3304 and H 409/22 displayed nanomolar affinity for the Y(1) receptor (K(i) values 1.1+/-0.2 nM and 5.6+/-0.9 nM, respectively), but low affinity for the Y(2) or Y(5) receptors. When guinea-pigs were pretreated with BIBO 3304 and H 409/22, the response to NPY was inhibited. 4. The Y(5) antagonist, CGP 71683A had high affinity for the Y(5) receptor (K(i) 1.3+/-0.05 nM) without having any significant activities at the Y(1) and Y(2) receptors. When CGP 71683A was infused into brain ventricles, the feeding response to NPY was attenuated. 5. The present study shows that NPY stimulates feeding in guinea-pigs through Y(1) and Y(5) receptors. As the guinea-pig is very distantly related to the rat and mouse, this suggests that both Y(1) and Y(5) receptors may mediate NPY-induced hyperphagia also in other orders of mammals.     

Evidence for two neuropeptide Y receptors mediating vasoconstriction.

The presence of receptor subtypes mediating the vascular and prejunctional effects of neuropeptide Y (NPY) was investigated using the Y2 receptor agonist, NPY-(13-36), and the Y1 agonist, [Leu31,Pro34]NPY. NPY-(1-36) and [Leu31,Pro34]NPY administered i.v. to anesthetized pigs evoked dose-dependent increases in mean arterial blood pressure and splenic and renal vascular resistance, and a decrease in heart rate. The potency of [Leu31,Pro34]NPY was 10-30% that of NPY-(1-36). In the spleen, NPY-(13-36) evoked vasoconstriction similar to that evoked by [Leu31,Pro34]NPY, but did not significantly increase renal vascular resistance or mean arterial blood pressure. Local intra-arterial administration of [Leu31,Pro34]NPY caused an increase in nasal mucosal vascular resistance with a potency similar to that of NPY-(13-36) evoked only a minor (17%) increase in nasal mucosal vascular resistance. The NPY analogues were further characterized in receptor binding studies on pig spleen membranes. Compared to NPY-(1-36), 800 times higher concentrations of [Leu31,Pro34]NPY, and 7 times higher concentrations of NPY-(13-36) were required to achieve the same 50% displacement of [125I]NPY-(1-36). Electrically evoked contractions in rat vas deferens were inhibited by 50% by 0.05 microM NPY-(1-36) and 0.3 microM NPY-(13-36), while [Leu31,Pro34]NPY only slightly attenuated the contractions (by 24% at 1 microM). The present data suggest the existence of subtypes of NPY receptors mediating vasoconstriction. Thus, the splenic vascular bed of the pig contains both Y1 and Y2 receptors while the Y1 subtype predominates in the kidney, nasal mucosa and for blood pressure control. The prejunctional receptor in rat vas deferens seems to be of the Y2 subtype.     

Discrimination between neuropeptide Y and peptide YY in the rat tail artery by the neuropeptide Y1-selective antagonist, BIBP 3226.

1. The ability of the novel, nonpeptide, neuropeptide Y (NPY) Y1-selective antagonist, BIBP 3226 ¿(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide¿, to antagonize the increase in perfusion pressure induced by NPY and peptide Y (PYY) was tested in the perfused rat tail artery, a postjunctional Y1-receptor bioassay, precontracted by 1 microM phenylephrine. 2. NPY and PYY produced a concentration-dependent enhancement of the vasoconstrictor response evoked by 1 microM phenylephrine. Although NPY and PYY are roughly equipotent, the maximal contractile response elicited by PYY was about twice that elicited by NPY. 3. Increasing concentrations of BIBP 3226 caused a parallel and rightward shift in the NPY concentration-response curve without depressing the maximal response. The contractile effect of NPY was potently inhibited in a competitive manner. The pA2 value for BIBP 3226 was 7.01 +/- 0.08, a value equivalent to that observed in the rabbit saphenous vein. Although increasing concentrations of BIBP 3226 shifted the concentration-response curve of PYY to the right without any significant decrease in the maximal vasoconstrictor response, the antagonism appeared non-competitive as the slope of the Schild plot was significantly different from unity (0.58 +/- 0.04). 4. In conclusion, these data confirm that BIBP 3226 is a potent and selective nonpeptide Y1 receptor antagonist. Moreover, they show that complex interactions occur between BIBP 3226 and postjunctional receptors activated by PYY. We postulate that BIBP 3226 might discriminate between the effects of NPY and PYY at the postjunctional level in the rat tail artery. It may be that distinct receptors for NPY and PYY exist; these may or may not allosterically interact with each other. Another working hypothesis would be that there is a single receptor complex with allosterically interacting binding sites for the two peptides.     

Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors.

1. A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens. 2. Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13-36) each caused concentration-related contractions with the order of potency PYY > or = Leu31Pro34NPY = NPY > NPY (13-36), consistent with the Y1 receptor subtype. 3. 1229U91 had no agonist activity in the arteries but caused a concentration-related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration-response curves. The antagonism was competitive with pKBS of 7.69 +/- 0.15 and 7.47 +/- 0.13 in the mouse and rat arteries, respectively. 4. Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13-36) inhibited the twitches with the order of potency PYY > NPY > NPY(13-36) >> Leu31Pro34NPY, consistent with the Y2 receptor subtype. 5. 1229U91 inhibited the vas deferens twitch with a shallow concentration-response curve and a time-course of inhibition distinct from that of NPY. 1229U91 (30 microM) did not cause a rightward shift of the NPY concentration-response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different. 6. It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.     

Actions of calcium antagonists on pre- and postjunctional effects of neuropeptide Y on human peripheral blood vessels in vitro

The mechanisms underlying the contractile effects of neuropeptide Y (NPY) in relation to those of noradrenaline (NA) on small human blood vessels were studied in vitro. NPY caused contractions of mesenteric veins, renal and skeletal muscle arteries but not of mesenteric arteries. NPY was about 5- to 10-fold more potent than NA. The maximal contractile responses to NPY (5 X 10(-7) M) were 38 +/- 4, 37 +/- 8 and 95 +/- 16% of the response evoked by NA 10(-5) M in the mesenteric vein, renal and skeletal muscle arteries respectively. The NPY effects were resistant to adrenoceptor antagonists. The calcium antagonist nifedipine reduced the effect of NA but not the contractile response to NPY on mesenteric veins. Nifedipine and felodipine reduced the contractile response to both NA and NPY on renal and skeletal muscle arteries. In contrast to the contractile effects of K+, the responses to NPY and NA were largely uninfluenced by changes in extracellular Ca2+ concentrations. Nifedipine still inhibited the NPY contractions in a Ca2+-free medium while high extracellular Ca2+ (7.5 mM) partly reduced the nifedipine effect. NPY reduced the nerve stimulation-evoked [3H]NA overflow from the mesenteric veins via a nifedipine resistant mechanism. The stable analogue alpha, beta-methylene adenosine triphosphate (mATP) was more potent than ATP and had nifedipine-sensitive contractile effects similar to those of NA on the human blood vessels without influencing the nerve-evoked [3H]NA efflux. In conclusion, NPY exerts a potent nifedipine-sensitive vasoconstrictor activity, especially on human skeletal muscle arteries in vitro, although the influx of extracellular calcium may not be a crucial mechanism. The NPY-induced contractions of mesenteric veins and the inhibition of nerve-evoked [3H]NA efflux seem to be mediated via nifedipine resistant messenger systems.     

Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels.

The contractile effect of neuropeptide Y (NPY) was tested on isolated segments of basilar artery, central ear artery, gastro-epiploic artery and vein, and femoral artery and vein from the rabbit. At 30 nM NPY did not evoke vasoconstriction; at 300 nM NPY evoked a weak and variable response. NPY greatly potentiated the response of the gastro-epiploic and femoral arteries to noradrenaline without affecting the maximum response. As tested on the gastro-epiploic artery NPY was effective at concentrations of 1 nM and higher. As tested on the femoral artery the potentiating effect of 30 nM NPY on noradrenaline-evoked contractions was apparent immediately and 30 min after the application of NPY, but not after one hour. NPY (30 nM) potentiated the contractile response to noradrenaline and histamine but not to 5-hydroxytryptamine or high K+. The response to histamine was augmented in both arteries and veins, whereas the response to noradrenaline was enhanced in arteries but not in veins. NPY failed to potentiate the prostaglandin F2 alpha-evoked contraction except in the gastro-epiploic vein.     

Neuropeptide Y inhibits double peaked vasoconstrictor responses to periarterial nerve stimulation primarily through prejunctional Y2 receptor subtype in canine splenic arteries

1 The effects of BIIE 0246, a novel and non-peptide neuropeptide Y (NPY) Y2 receptor antagonist on sympathetic vasoconstriction of the canine splenic artery were investigated. 2 The vasoconstrictor response to periarterial electrical nerve stimulation was described to be a double peaked vasoconstriction consisting of an initial transient, dominantly P2X purinoceptor-mediated constriction followed by a prolonged, mainly alpha1 adrenoceptor-induced response. 3 BIIE 0246 at a concentration of 0.1-1 microM dose-dependently potentiated double peaked constrictions at low frequencies (1 and 4 Hz), whereas at high frequency (10 Hz), it failed to affect these responses. BIIE 0246 (1 microM) also enhanced double peaked responses even in the presence of rauwolscine (0.1 microM). NPY (13-36) (1-100 nM), a selective Y2 receptor agonist reduced these two peaked responses in a dose-related manner. The vasoconstriction to noradrenaline (0.1-10 nmol) or adenosine triphosphate (0.01-1 micromol) was not significantly influenced by either 1 microM BIIE 0246 or 100 nM NPY (13-36). Exposure of tissues to 1 microM BIIE 0246 almost completely prevented the suppression of double peaked constrictions by NPY (13-36) (10 nM) or by NPY (10 nM). 4 We conclude that NPY inhibits sympathetic purinergic and adrenergic vasoconstrictions through an activation of prejunctional Y2 receptor subtype in the neurovascular junction of the canine splenic artery.     

Regional heterogeneity in the contractile and potentiating effects of neuropeptide Y in rat isolated coronary arteries: modulatory action of the endothelium.

1. Neuropeptide Y (NPY) induced a concentration-dependent contraction of isolated rings of proximal epicardial (PC) and distal intramural (DC) coronary arteries of the rat, with an EC50 of ca. 1 x 10(-7) M. The NPY-induced contraction at 3 x 10(-7) M was significantly smaller in PC than DC arteries: 34% vs. 55% of the 125 mM K(+)-induced response, respectively. 2. NPY (2 x 10(-8) M) increased the sensitivity to noradrenaline (NA) and 5-hydroxytryptamine (5-HT) more in PC (4.2 and 2.8 fold, respectively) than in DC arteries (2.2 and 1.4 fold, respectively). The maximal contractile response to NA and 5-HT was increased more in DC (43% and 29%, respectively) than in PC arteries (20% and 12%, respectively). 3. Removal of the endothelium increased the sensitivity and maximal response to NPY as well as the spontaneous myogenic tone in PC but not in DC arteries. NPY had no relaxing effect on PC and DC arteries submaximally contracted with 10(-6) M prostaglandin F2 alpha, suggesting that spontaneous rather than stimulated release of endothelium-derived relaxing factor (EDRF) depresses the contractile action of NPY in PC arteries. 4. The results indicate a heterogeneity in the contractile and potentiating action of NPY in rat coronary arteries depending on size or location in the coronary circulation.     

Neuropeptide Y2 receptors are involved in enhanced neurogenic vasoconstriction in spontaneously hypertensive rats

1. The present study addressed the role of neuropeptide (NPY) Y2 receptors in neurogenic contraction of mesenteric resistance arteries from female spontaneously hypertensive rats (SHR). Arteries were suspended in microvascular myographs, electrical field stimulation (EFS) was performed, and protein evaluated by Western blotting and immunohistochemistry. 2. In vasopressin-activated endothelium-intact arteries, NPY and fragments with selectivity for Y1 receptors, [Leu31,Pro34]NPY, Y2 receptors, NPY(13-36), and rat pancreatic polypeptide evoked more pronounced contractions in segments from SHR than in Wistar Kyoto (WKY) arteries, even in the presence of the Y1 receptor antagonist, BIBP3226 (0.3 microM, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]D-arginineamide). 3. In the presence of prazosin and during vasopressin activation, EFS-evoked contractions were larger in arteries from SHR compared to WKY. EFS contractions were enhanced by the Y2 receptor selective antagonist BIIE0246TF (0.5 microM, (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-y1]-1-piperazinyl]-2-oxoethyl]cyclo-pentyl-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide), reduced by BIBP3226, and abolished by the combination of BIBP3226 and BIIE0246TF. 4. Immunoblotting showed NPY Y1 and Y2 receptor expression to be similar in arteries from WKY and SHR, although a specific Y2 receptor band at 80 kDa was detected only in arteries from WKY. 5. Immunoreaction for NPY was enhanced in arteries from SHR. In contrast to arteries from WKY, BIIE0246TF increased NPY immunoreactivity in EFS-stimulated arteries from SHR. 6. The present results suggest that postjunctional neuropeptide Y1 and Y2 receptors contribute to neurogenic contraction of mesenteric small arteries. Moreover, both enhanced NPY content and altered neuropeptide Y1 and Y2 receptor activation apparently contribute to the enhanced neurogenic contraction of arteries from SHR.     

Heterogeneity of the neuropeptide Y (NPY) contractile and relaxing receptors in horse penile small arteries

The distribution of neuropeptide Y (NPY)-immunorective nerves and the receptors involved in the effects of NPY upon electrical field stimulation (EFS)- and noradrenaline (NA)-elicited contractions were investigated in horse penile small arteries. NPY-immunoreactive nerves were widely distributed in the erectile tissues with a particularly high density around penile intracavernous small arteries. In small arteries isolated from the proximal part of the corpora cavernosa, NPY (30 nM) produced a variable modest enhancement of the contractions elicited by both EFS and NA. At the same concentration, the NPY Y(1) receptor agonist, [Leu(31), Pro(34)]NPY, markedly potentiated responses to EFS and NA, whereas the NPY Y(2) receptor agonist, NPY(13-36), enhanced exogenous NA-induced contractions. In arteries precontracted with NA, NPY, peptide YY (PYY), [Leu(31), Pro(34)]NPY and the NPY Y(2) receptor agonists, N-acetyl[Leu(28,31)]NPY (24-36) and NPY(13-36), elicited concentration-dependent contractile responses. Human pancreatic polypeptide (hPP) evoked a biphasic response consisting of a relaxation followed by contraction. NPY(3-36), the compound 1229U91 (Ile-Glu-Pro-Dapa-Tyr-Arg-Leu-Arg-Tyr-NH2, cyclic(2,4')diamide) and eventually NPY(13-36) relaxed penile small arteries. The selective NPY Y(1) receptor antagonist BIBP3226 ((R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]D-arginineamide) (0.3 microM) shifted to the right the concentration-response curves to both NPY and [Leu(31), Pro(34)]NPY and inhibited the contractions induced by the highest concentrations of hPP but not the relaxations observed at lower doses. In the presence of the selective NPY Y(2) receptor antagonist BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-y1]-1-piperazinyl]-2-oxoethyl]cyclo-pentyl-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2, 4-triazol-4-yl]ethyl]-argininamide) (0.3 microM), the Y(2) receptor agonists NPY(13-36) and N-acetyl[Leu(28,31)]NPY (24-36) evoked potent slow relaxations in NA-precontracted arteries, under conditions of nitric oxide (NO) synthase blockade. Mechanical removal of the endothelium markedly enhanced contractions of NPY on NA-precontracted arteries, whereas blockade of the neuronal voltage-dependent Ca(2+) channels did not alter NPY responses. These results demonstrate that NPY can elicit dual contractile/relaxing responses in penile small arteries through a heterogeneous population of postjunctional NPY receptors. Potentiation of the contractions evoked by NA involve both NPY Y(1) and NPY Y(2) receptors. An NO-independent relaxation probably mediated by an atypical endothelial NPY receptor is also shown and unmasked in the presence of selective antagonists of the NPY contractile receptors.     

Dissociation of potentiation of Leu31 Pro34 neuropeptide Y on adrenergic and purinergic transmission in isolated canine splenic artery

The present study observed the effects of an activation of neuropeptide Y (NPY) Y1 receptors on adrenergic and purinergic components of double-peaked vasoconstrictor responses to periarterial nerve stimulation in the isolated, perfused canine splenic arteries. The results showed that 3-30 nM Leu31 Pro34 neuropeptide Y (LP-NPY) produced a dose-dependent potentiation of double-peaked vasoconstrictor responses to trains of 30-s pulses at 1, 4 or 10 Hz of stimulation. The potentiation of LP-NPY of the nerve-stimulated vasoconstrictions were completely inhibited by subsequent blockade of alpha1-adrenoceptors or Y1 receptors with 0.1 microM prazosin or with 1 microM BIBP 3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininami de), respectively. The remaining responses in the presence of LP-NPY and prazosin were abolished by P2X receptor desensitization with 1 microM alpha,beta-methylene ATP. Moreover, 30 nM LP-NPY failed to modify the vasoconstrictor responses to nerve stimulation after treatment with prazosin. A subsequent administration of alpha,beta-methylene ATP completely suppressed the remaining responses after prazosin and LP-NPY. The vasoconstrictions induced by 0.003-1 nmol noradrenaline and 0.003-1 micromol ATP were slightly, but not significantly enhanced by 30 nM LP-NPY. The observations indicated that activation of postjunctional NPY Y1 receptors may have an important role in the modulation of adrenergic rather than purinergic transmission of the sympathetic co-transmission.