The neuropeptide Y (NPY) Y1 receptor subtype mediates NPY-induced antidepressant-like activity in the mouse forced swimming test.

The present study was undertaken to investigate the possible antidepressant-like effects of neuropeptide Y (NPY) in the mouse forced swimming test, an animal model widely used for the screening of potential antidepressant drugs. In addition, experiments were performed, using agonists and selective antagonists, to assess the potential role of NPY Y(1) and Y(2) receptor subtypes in this model. Complementary studies were performed in an open field apparatus to rule out any changes in locomotor activity that might have interfered with the interpretation of data from the mouse forced swimming test. Intracerebroventricular injections (0.03 nmole-3 nmole) of NPY, [Leu(31)Pro(34)]PYY (Y(1) agonist), NPY(13-36) (Y(2) agonist), BIBP3226, BIBO3304 (Y(1) antagonists) and BIIE0246 (Y(2) antagonist) were performed 30 min prior to testing in the mouse forced swimming test and open field. NPY administration significantly reduced immobility time in a dose dependent manner (p <.01 vs. control group), as did [Leu(31)Pro(34)]PYY (p <.01 vs. control group) and BIIE0246 (p <.05 vs. control group). In contrast, BIBO3304, BIBP3226 and NPY(13-36) did not display any activity at the doses tested. However, pretreatment with BIBO3304 or BIBP3226 significantly blocked the anti-immobility effects of NPY. Data from the open field demonstrated that BIIE0246 increased horizontal ambulation at the dose found to be active in the forced swimming test. Taken together, our results demonstrate that NPY displays antidepressant-like activity in the mouse forced swimming test, and suggest that this activity is mediated by the NPY Y(1) receptor subtype.     

The involvement of neuropeptide Y Y1 receptors in the blood pressure baroreflex: studies with BIBP 3226 and BIBO 3304.

To ascertain the role of the neuropeptide Y Y1 receptors in the vascular manifestations of the sympathetic baroreflex, 10-s bilateral carotid occlusions were performed in anesthetized cats; systemic blood pressure was monitored continually. This maneuver rose systolic blood pressure in 23 +/- 2 mmHg. Following 100 microg/kg BIBP 3226 or BIBO 3304 i.v., the increase in blood pressure elicited by the occlusions was only 14 +/- 1 and 15 mmHg, respectively. Both BIBP 3226 and BIBO 3304 displaced significantly 5.5 fold rightward the pressor dose-response curve elicited by exogenous neuropeptide Y, without altering the norepinephrine curve. Prazosin (10 microg/kg) reduced the pressor response elicited by the carotid occlusion to 12 +/- 4 mmHg. The simultaneous administration of BIBP 3226 plus prazosin rose the systemic blood pressure following the occlusion only 9 +/- 2 mmHg, supporting the involvement of neuropeptide Y in vascular sympathetic reflexes.     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors

GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. British Journal of Pharmacology (2000) 129, 37 - 46     

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.     

Antagonism of neuropeptide YY1 receptors does not inhibit ethanol's effects on cortical EEG and ERPs in Wistar rats

OBJECTIVE: Ethanol and neuropeptide Y (NPY) can have additive neurobehavioral effects. In the present study, the NPY Y1 receptor antagonist BIBP3226 was administered alone or in combination with a moderate dose of ethanol to determine whether it interacted with the neurobehavioral effects of ethanol. METHOD: Male Wistar rats were implanted with cortical recording electrodes and a lateral ventricular cannula. The effects of 1 nmol BIBP3226, 0.75 g/kg ethanol and the combination (BIBP3226 + EtOH) on neurophysiological activity and locomotion were then assessed. RESULTS: Ethanol significantly increased 1-2 Hz parietal cortical power and this effect was partially antagonized by BIBP3226. Peak frequencies in the parietal cortical 6-8 Hz and 8-16 Hz bands were also altered by ethanol, but these effects were not reversed by BIBP3226. BIBP3226 or ethanol, when administered alone, did not alter motor activity or cortical event-related potentials (ERPs) but administration of BIBP3226 + EtOH reduced motor activity, reduced parietal cortical N1 ERP amplitude and increased frontal cortical N1 ERP latency. CONCLUSIONS: In the present study, the most prominent effect of antagonizing central NPY Y1 receptors was a facilitation of the effects of ethanol. In particular, the effects of combined administration of BIBP3226 and ethanol are indicative of enhanced sedation and possibly cognitive impairment.     

Infusions of neuropeptide Y into the lateral septum reduce anxiety-related behaviors in the rat

Neuropeptide Y (NPY) is one of the most abundant peptides in mammalian brain and NPY-like-immunoreactivity is highly expressed in the lateral septum, an area extensively involved in anxiety regulation. NPY counteracts the neurochemical and behavioral responses to acute threat in animal models, and intracerebroventricular (i.c.v.) administration of NPY at low doses is anxiolytic. Less is known about the specific contributions of the lateral septum to NPY-mediated anxiety regulation. In Experiment 1, the effects of infusions of NPY (1.5 μg) into the lateral septum were investigated in three animal models of anxiety: the elevated plus-maze, novelty-induced suppression of feeding, and shock-probe burying tests. Experiment 2 examined the role of the NPY Y1 receptor in these models by co-infusing the Y1 antagonist BIBO 3304 (0.15 μg, 0.30 μg) with NPY into the lateral septum. In the elevated plus-maze, there were no changes in rats' open arm exploration, the index of anxiety reduction in this test. In the novelty-induced suppression of feeding test, rats infused with NPY showed decreases in the latency to consume a palatable snack in a novel (but not familiar) environment, suggesting a reduction in anxiety independent of increases in appetite. This anxiolysis was attenuated by co-infusion with BIBO 3304 (0.30 μg) in Experiment 2. Lastly, rats infused with NPY showed decreases in the duration of burying behavior in the shock-probe burying test, also indicative of anxiety reduction. However, unlike in the feeding test, BIBO 3304 did not attenuate the NPY-induced anxiolysis in the shock-probe test. It is concluded that NPY produces anxiolytic-like actions in the lateral septum in two animal models of anxiety: the novelty-induced suppression of feeding, and shock-probe burying tests, and that this anxiolysis is dependent on Y1 receptor activation in the feeding test.     

Effects of a selective neuropeptide Y Y(1) receptor antagonist BIBP 3226 on double peaked vasoconstrictor responses to periarterial nerve stimulation in canine splenic arteries

The periarterial electrical nerve stimulation (30 s trains of pulses at a frequency of 1, 4 or 10 Hz) induced a double peaked vasoconstriction consisting of an initial transient constriction (first peak) followed by a prolonged response (second peak) in the isolated, perfused canine splenic artery. At low frequencies (1 and 4 Hz), a neuropeptide Y (NPY) Y(1) receptor antagonist BIBP 3226 (0.1-1 microM) produced a dose-dependent inhibitory effect on the second peak, but did not modify the first peak. At a high frequency (10 Hz), 1 microM BIBP 3226 induced a slight, but significant inhibition on both the first and second peaked responses. At a low frequency (1 Hz), the first peak was not influenced by blockade of alpha(1)-adrenoceptors or NPY Y(1) receptors with prazosin (0.1 microM) or BIBP 3226 (1 microM), respectively, but abolished by P2X receptor desensitization with alpha,beta-methylene ATP (alphabeta-m ATP, 1 microM). At a high frequency (10 Hz), the first peak was mostly inhibited by alphabeta-m ATP and partially by prazosin and BIBP 3226. On the other hand, the second peak at a low frequency was largely decreased by BIBP 3226 and partially by prazosin and alphabeta-m ATP, whereas at a high frequency, it was largely attenuated by prazosin and partially by alphabeta-m ATP and BIBP 3226. The results suggest that at a low frequency, the firstly transient constriction of double peaked responses is mainly induced via an activation of P2X-receptors, whereas at a high frequency, it is mostly mediated by the P2X-receptors, and partially by alpha(1)-receptors and NPY Y(1)-receptors. The secondary prolonged vasoconstriction at frequencies used is predominantly mediated via both alpha(1)-receptor and NPY Y(1) receptor activations, and in part by P2X-receptors. Furthermore, an activation of NPY Y(1) receptors may play an important role in evoking the prolonged vasoconstrictor response to longer pulse trains of stimulation at a low frequency, whereas an alpha(1)-adrenoceptor activation exerts a main vasomotor effect for the prolonged response at a high frequency.     

In the amygdala anxiolytic action of mGlu5 receptors antagonist MPEP involves neuropeptide Y but not GABAA signaling.

Several lines of evidence indicate that inhibition of the metabotropic glutamate (mGlu) receptor 5 produces anxiolytic-like effects in rodents. Peptide neurotransmitter neuropeptide Y (NPY) produces an anxiolytic effect in rats after intraventricular or intra-amygdalar administration. Many classes of anxiolytic drugs exert their effect through the GABA-benzodiazepine (BZD) receptor complex. Therefore, in the present study we have investigated whether the anxiolytic action of MPEP (2-methyl-6-(phenylethynyl)pyridyne), an mGlu5 receptor antagonist, is mediated by a mechanism involving either the GABA-BZD receptor complex or NPY receptor. In the behavioral studies, the anxiolytic activity of MPEP (10 mg/kg, i.p.) was examined using plus-maze test. The BZD antagonist flumazenil (10 mg/kg, i.p.) was given to one group of rats and Y1 receptor antagonist BIBO 3304 (((R)-N-[[4-(aminocarbonylaminomethyl) phenyl] methyl]-N2-(diphenylacetyl)-argininamide trifluoroacetate)3304) (200 pmol/site, intra-amygdala) to the other. It was found that anxiolytic effects of MPEP were not changed by flumazenil, but were abolished by BIBO 3304. Immunohistochemical studies showed a high density of mGlu5 receptor immunoreactivity (IR) in the amygdala. The effect of MPEP on NPY expression in the amygdala was studied using immunohistochemistry (IH) and radioimmunoassay (RIA). Both methods showed a diminution of NPY IR expression, to about 43% (IH) or 81% (RIA) of the control level after multiple administrations, but we observed an increase up to 148% of the control after single MPEP administration. These effects may suggest a release of NPY from nerve terminals after MPEP administration. Our results indicate that the anxiolytic action of MPEP is conveyed through NPY neurons with the involvement of Y1 receptors in the amygdala and that BZD receptors do not significantly contribute to these effects.     

Effects of the Neuropeptide Y (NPY)-Receptor Antagonist BIBP3226 on Vascular NPY-Receptors with Different Ligand Requirements

Abstract: The aim was to examine effects of a newly developed neuropeptide Y (NPY)-receptor antagonist, BIBP3226 and to characterize NPY-receptors in the isolated guinea pig caval vein and human subcutaneous artery, respectively. BIBP3226 ≤1μM did not affect the basal tension. Pretreatment with increasing concentrations of BIBP3226 (10 nM - 1 μM) resulted in a progressive rightward shift of the concentration-response curve to the Y1-receptor selective agonist [Pro₃₄]NPY in the guinea pig caval vein. Regression analysis of the Schild plot gave a pA₂-value of 7.58 (7.20-8.33, 95% confidence interval), slope of regression line 0.96 (0.52-1.39, 95% confidence interval) and a correlation coefficient of 0.78. NPY and the C-terminal NPY 2-36 evoked equipotent concentration-dependent contractions, both of which were sensitive to BIBP3226. Although less potent than NPY 2-36, also the contraction induced by NPY 5-36 was antagonized by BIBP3226. In the human subcutaneous artery [Pro³⁴]NPY but not NPY 2-36 (≤0.3μM) evoked a concentration-dependent contraction. Pretreatment with BIBP3226 (0.1 μM) resulted in a rightward shift of the concentration-response curve to [Pro³⁴]NPY (from 7.38±0.10 to 6.95±0.16 (P<0.05, n=6). The present study has shown that the Y1-receptor-selective antagonist BIBP3226 potently antagonizes vascular NPY-receptors with different ligand requirements in the guinea pig caval vein and human subcutaneous artery, respectively. It appears that the guinea pig Y1-receptor is much less stringent in its demand on the N-terminal part of NPY than that of human Y1-receptors.     

Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y agonist responses in isolated human colon mucosa

1. The aim of this study was to provide a pharmacological characterization of the Y receptor types responsible for neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) effects upon electrogenic ion transport in isolated human colonic mucosa. 2. Preparations of descending colon were voltage-clamped at 0 mV in Ussing chambers and changes in short-circuit current (I(sc)) continuously recorded. Basolateral PYY, NPY, human PP (hPP), PYY(3 - 36), [Leu(31), Pro(34)]PYY (Pro(34)PYY) and [Leu(31), Pro(34)]-NPY (Pro(34)NPY) all reduced basal I(sc) in untreated colon. Of all the Y agonists tested PYY(3 - 36) responses were most sensitive to tetrodotoxin (TTX) pretreatment, indicating that Y(2)-receptors are located on intrinsic neurones as well as epithelia in this tissue. 3. The EC(50) values for Pro(34)PYY, PYY(3 - 36) and hPP were 9.7 nM (4.0 - 23.5), 11.4 nM (7.6 - 17.0) and 14.5 nM (10.2 - 20.5) and response curves exhibited similar efficacies. The novel Y(5) agonist [Ala(31), Aib(32)]-NPY had no effect at 100 nM. 4. Y(1) receptor antagonists, BIBP3226 and BIBO3304 both increased basal I(sc) levels per se and inhibited subsequent PYY and Pro(34)PYY but not hPP or PYY(3 - 36) responses. The Y(2) antagonist, BIIE0246 also raised basal I(sc) levels and attenuated subsequent PYY(3 - 36) but not Pro(34)PYY or hPP responses. 5. We conclude that Y(1) and Y(2) receptor-mediated inhibitory tone exists in human colon mucosa. PYY and NPY exert their effects via both Y(1) and Y(2) receptors, but the insensitivity of hPP responses to either Y(1) or Y(2) antagonism, or to TTX, indicates that Y(4) receptors are involved and that they are predominantly post-junctional in human colon.     

Neuropeptide Y regulates intracellular calcium through different signalling pathways linked to a Y(1)-receptor in rat mesenteric small arteries

Simultaneous measurements of intracellular calcium concentration ([Ca(2+)](i)) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)-elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca(2+)](i) and tension. These effects were antagonized by the selective Y(1) receptor antagonist, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-D-argininea mide (BIBP 3226) (apparent pK(B) values of 8.54+/-0.25 and 8.27+/-0.17, respectively). NPY (0.1 microM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension-[Ca(2+)](i) relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA(2) 7.87+/-0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 microM) induced a maximum relaxation and a return of [Ca(2+)](i) to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin-relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin-relaxed arteries with a pA(2) of 7.92+/-0.29. Electrical field stimulation (EFS) at 8-32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non-activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y(1) receptor in intact rat mesenteric small arteries, and provide little support for involvement of other postjunctional NPY receptors in the contractile responses to NPY. Neurally released NPY also seems to act through Y(1) receptors, and may serve primarily as an inhibitor of vasodilatation.     

Inhibition of sympathetic vasoconstriction in pigs in vivo by the neuropeptide Y-Y1 receptor antagonist BIBP 3226.

1. Recently, a potent non-peptide antagonist of neuropeptide Y (NPY)-Y1 receptors has been developed. In this study, the selectivity of this compound, BIBP 3226, as a functional Y1 receptor antagonist, and the possible role of endogenous NPY in sympathetic vasoconstriction in different vascular beds have been investigated in anaesthetized pigs. 2. BIBP 3226 specifically displaced [125I]-NPY binding with an IC50 value of 7 nM in membranes of pig renal arteries, which also were responsive to a Y1 receptor agonist, but had only minor effects in the pig spleen (IC50 55 microM), where instead [125I]-NPY binding was markedly inhibited by a Y2 receptor agonist. IC50 values in the same nM range for BIBP 3226 were also observed in rat and bovine cortex and dog spleen. 3. In anaesthetized control pigs in vivo BIBP 3226 (1 and 3 mg kg-1) markedly inhibited the vasoconstrictor effects of the Y1 receptor agonist [Leu31, Pro34] NPY(1-36), without influencing the responses to the Y2 receptor agonist N-acetyl [Leu28, Leu31] NPY(24-36), or to noradrenaline, phenylephrine, alpha,beta-methylene adenosine triphosphate or angiotensin II. 4. High frequency stimulation of the sympathetic trunk in control pigs caused a biphasic vasoconstrictor response in nasal mucosa, hind limb and skin: there was an immediate, peak response, followed by a long-lasting vasoconstriction. BIBP 3226 (1 and 3 mg kg-1) reduced the second phase by about 50% but had no effect on the peak response. In the spleen, kidney and mesenteric circulation (which lack the protracted response) BIBP 3226 was likewise without effect on the maximal vasoconstriction, and did not influence noradrenaline overflow from spleen and kidney. 5. The corresponding S-enantiomer BIBP 3435 had only marginal influence on [125I]-NPY binding (microM range) and did not inhibit the vasoconstrictor effects of any of the agonists used, including the Y1 receptor peptide agonist. Furthermore, BIBP 3435 did not affect the response to sympathetic nerve stimulation. Both BIBP 3435 and BIBP 3226 caused a slight transient decrease in mean arterial blood pressure (by about 5 and 15 mmHg at 1 mg kg-1 and 3 mg kg-1, respectively), accompanied by splenic and mesenteric vasodilatation, suggesting that this effect was unrelated to Y1 receptor blockade. 6. The peptide YY (PYY)- and NPY-evoked vasoconstriction in the kidney of reserpine-treated pigs was markedly reduced (by 95%) by BIBP 3226 while the vasoconstrictor effect in the spleen was attenuated by only 20%. BIBP 3226 did not influence stimulation-evoked NPY release. The vasoconstrictor response in reserpine-treated pigs to single impulse stimulation, which is observed only in nasal mucosa and hind limb, was unchanged regarding maximal amplitude and the integrated effect was only moderately reduced (by about 25%) in the presence of BIBP 3226 (1 mg kg-1). BIBP 3226 (1 mg kg-1) markedly reduced (by 55-70%) the long-lasting vascular response (total integrated blood flow reduction) evoked by sympathetic nerve stimulation at high frequency (40 impulses at 20 Hz) in spleen, kidney, nasal mucosa and hind limb. Furthermore, the maximal amplitude of the vasoconstriction was reduced mainly in the kidney (by 60%) and also in the spleen (by 40%). 7. It is concluded that BIBP 3226 can act as a selective Y1 receptor antagonist in the pig. Endogenous NPY via Y1 receptor activation may play a role in evoking the long-lasting vasoconstriction seen in nasal mucosa, hind limb and skin after high frequency stimulation of sympathetic nerves in control pigs. Furthermore, NPY via Y1 receptor mechanisms seems to be of major importance for the long-lasting component of the reserpine resistant sympathetic vasoconstriction in many vascular beds, and for the maximal vasoconstrictor response in the kidney. Circulating NPY and PYY induce splenic vasoconstriction via Y2-receptors in contrast to neuronally released NPY which mainly activates Y1 receptors.     

Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.     

The ability of neuropeptide Y to mediate responses in the murine cutaneous microvasculature: an analysis of the contribution of Y1 and Y2 receptors

1. The ability of neuropeptide Y (NPY) to modulate skin blood flow, oedema formation and neutrophil accumulation was investigated. Experiments were designed to examine the possible contribution of the Y2 receptor, in addition to the Y1 receptor, through use of Y2 receptor knockout mice (Y2-/-) and selective receptor antagonists. 2. The development of a 99mTc clearance technique for the measurement of microvascular blood flow changes in mouse dorsal skin revealed a dose-dependent ability of picomole amounts of NPY, and also of the Y1-preferred agonist Pro34NPY and the Y2-preferred agonist PYY(3-36) to decrease blood flow. 3. The Y1 receptor antagonist BIBO3304 blocked responses to the Y1 agonist at the lower doses, but only partially inhibited at the higher doses tested in Y2+/+. In Y2-/- receptor mice, the responses to the Y2 agonist were abolished at the lower doses and partially reduced at the highest dose tested, while those to the Y1 agonist were similar in both Y2+/+ and Y2-/-receptor mice. 4. In Y2+/+ receptor mice, the simultaneous injection of the Y2 antagonist BIIE0246 with BIBO3304 abolished Y2 agonist-induced decreases in blood flow over the dose range used (10-100 pmol). When the Y2 receptor antagonist BIIE0246 was given alone, it was not able to significantly affect the PYY(3-36)-induced response, whereas the Y1 receptor antagonist BIBO3304 partially (P<0.001) inhibited the decrease in blood flow evoked by PYY(3-36) at the highest dose. 5. NPY did not mediate either oedema formation, even when investigated in the presence of the vasodilator calcitonin gene-related peptide (CGRP), or neutrophil accumulation in murine skin. 6. We conclude that the major vasoactive activity of NPY in the cutaneous microvasculature is to act in a potent manner to decrease blood flow via Y1 receptors, with evidence for the additional involvement of postjunctional Y2 receptors. Our results do not provide evidence for a potent proinflammatory activity of NPY in the cutaneous microvasculature.     

Neuropeptide Y Y2 receptor-mediated attenuation of neurogenic plasma extravasation acting through pertussis toxin-sensitive mechanisms.

1. The effects of neuropeptide Y (NPY) receptor agonists (administered intravenously) were examined on plasma protein ([125I]-bovine serum albumin) leakage within dura mater evoked by unilateral trigeminal ganglion stimulation (0.6 mA, 5 ms, 5 Hz, 5 min), capsaicin (1 mumol kg-1, i.v.) or substance P (1 nmol kg-1, i.v.) in anaesthetized Sprague-Dawley rats. 2. NPY (EC50: 5.6 nmol kg-1) and NPY fragment 13-36 [NPY (13-36)] (ED50: 4.3 nmol kg-1), an NPY Y2 receptor agonist, dose-dependently attenuated [125I]-bovine serum albumin extravasation from meningeal vessels when administered 10 min prior to electrical stimulation. [Leu31, Pro34]-NPY, an NPY Y1 and Y3 receptor agonist, inhibited the response at a higher dose only (23 nmol kg-1) (P < 0.05). 3. NPY also significantly decreased plasma protein extravasation induced by capsaicin (1 mumol kg-1) but not by substance P (1 nmol kg-1). 4. Pertussis toxin (20 micrograms kg-1, administered intracisternally 48 h prior to stimulation) blocked completely the inhibitory effect of NPY and NPY (13-36) but did not inhibit extravasation alone. 5. We conclude that NPY inhibits neurogenically-mediated plasma protein extravasation acting through presynaptic pertussis toxin-sensitive NPY Y2 receptors, possibly by inhibition of neuropeptide release from perivascular trigeminovascular afferents.     

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.     

Neuropeptide Y modulates effects of bradykinin and prostaglandin E2 on trigeminal nociceptors via activation of the Y1 and Y2 receptors

BACKGROUND AND PURPOSE: Although previous studies have demonstrated that neuropeptide Y (NPY) modulates nociceptors, the relative contributions of the Y1 and Y2 receptors are unknown. Therefore, we evaluated the effect of Y1 and Y2 receptor activation on nociceptors stimulated by bradykinin (BK) and prostaglandin E2 (PGE2). EXPERIMENTAL APPROACH: Combined immunohistochemistry (IHC) with in situ hybridization (ISH) demonstrated that Y1- and Y2-receptors are collocated with bradykinin (2) (B2)-receptors in rat trigeminal ganglia (TG). The relative functions of the Y1 and Y2 receptors in modulating BK/PGE2-evoked CGRP release and increased intracellular calcium levels in cultured TG neurons were evaluated. KEY RESULTS: The Y1 and Y2 receptors are co-expressed with B2 in TG neurons, suggesting the potential for direct NPY modulation of BK responses. Pretreatment with the Y1 agonist [Leu31,Pro34]-NPY, inhibited BK/PGE2-evoked CGRP release. Conversely, pretreatment with PYY(3-36), a Y2 agonist, increased BK/PGE2 evoked CGRP release. Treatment with NPY evoked an overall inhibitory effect, although of lesser magnitude. Similarly, [Leu31,Pro34]-NPY inhibited BK/PGE2-evoked increases in intracellular calcium levels whereas PYY(3-36) increased responses. NPY inhibition of BK/PGE2-evoked release of CGRP was reversed by the Y1 receptor antagonist, BIBO3304, and higher concentrations of BIBO3304 significantly facilitated CGRP release. The Y2 receptor antagonist, BIIE0246, enhanced the inhibitory NPY effects. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that NPY modulation of peptidergic neurons is due to net activation of inhibitory Y1 and excitatory Y2 receptor systems. The relative expression or activity of these opposing receptor systems may mediate dynamic responses to injury and pain.     

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)     

Binding properties of three neuropeptide Y receptor subtypes from zebrafish: comparison with mammalian Y1 receptors

Neuropeptide Y (NPY) and peptide YY (PYY) are two related 36-amino-acid peptides found in all vertebrates and are involved in many physiological processes. Five receptor subtypes have been cloned in mammals (Y1, Y2, Y4, Y5, and y6). We have recently cloned three NPY/PYY receptor subtypes in zebrafish, called Ya, Yb, and Yc. Here we report on a direct comparison of the pharmacological properties of these three receptors in vitro using porcine NPY with alanine substitutions in positions 33–36 as ligands and three analogues with internal deletions: [Ahx^8–20]NPY, [Ahx^8–20, Pro³⁴]NPY, and [Ahx^5–24]NPY. In all cases, the zYc receptor was the most sensitive to the modifications of the NPY molecule and zYa was the least sensitive (except for the Arg → Ala replacement at position 33). Our data identified zYa as a receptor that can bind ligands specific for Y1, Y2, and Y4 receptors, while zYb and zYc were more Y1-like. All peptides with internal deletions bound to the zYa receptor with affinities similar to that of intact pNPY. Neither the Y1-selective antagonists BIBP3226 and SR120819A nor the Y2-selective BIIE0246 bound to any of the zebrafish receptors, although the amino acids identified as important for BIBP3226 binding were almost completely conserved. These results may prove helpful in molecular modeling of the three-dimensional receptor structure.