Cloning and characterization of the guinea pig neuropeptide Y receptor Y5

The Y5 receptor has been postulated to be the main receptor mediating NPY-induced food intake in rats, based on its pharmacological profile and mRNA distribution. To further characterize this important receptor subtype, we isolated the Y5 gene in the guinea pig, a widely used laboratory animal in which all other known NPY receptors (Y1, Y2, Y4, y6) [2,13,33,37] have recently been cloned by our group. Our results show that the Y5 receptor is well conserved between species; guinea pig Y5 displays 96% overall amino acid sequence identity to human Y5, the highest identity reported for any non-primate NPY receptor orthologue, regardless of subtype. Thirteen of the twenty substitutions occur in the large third cytoplasmic loop. The identities between the guinea pig Y5 receptor and the dog, rat, and mouse Y5 receptors are 93%, 89%, and 89% respectively. When transiently expressed in EBNA cells, the guinea pig Y5 receptor showed a high binding affinity to iodinated porcine PYY with a dissociation constant of 0.41 nM. Competition experiments showed that the rank order of potency for NPY-analogues was PYY = NPY = NPY2-36 > gpPP > rPP > NPY 22-36. Thus the pharmacological profile of the guinea pig Y5 receptor agrees well with that reported for the Y5 receptor from other cloned species.     

A Y2 receptor mimetic aptamer directed against neuropeptide Y.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 by l-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala(31),Aib(32)]NPY and the Y1/Y5 receptor-binding peptide [Leu(31),Pro(34)]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [(3)H]NPY with high affinity, a low affinity displacement of [(3)H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower K(i) value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors.     

Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors

It is well documented that neuropeptide Y (NPY) exerts a wide range of biological functions through at least five NPY Y receptor subtypes (Y1-Y5), but its immunological effects only recently came into focus. Using NPY family peptides and NPY-related receptor-specific peptides as well as Y1 and Y2 receptor antagonists, we have tested which NPY Y receptors are involved in NPY-induced modulation of rat peritoneal macrophage function in vitro. NPY and PYY increased oxidative burst in phorbol myristate acetate (PMA)-stimulated macrophages involving activation of protein kinase C (PKC), and decreased it in zymosan-stimulated cells resembling inhibition of signaling pathways subsequent to binding of zymosan particles for the iC3b fragment receptor on macrophages. The combined treatment with NPY and NPY Y receptor antagonists revealed that NPY-induced potentiation of oxidative burst in PMA-stimulated cells is mediated through Y1 and Y2 receptors, while NPY-induced suppression in zymosan-stimulated cells is mediated through Y2 receptors only. NPY-related peptides differently modulated macrophage function, confirming involvement of NPY Y2 receptor in both potentiation and suppression of oxidative burst in these cells. Additionally, it was shown that NPY Y5 receptor mediated suppression of oxidative burst in PMA- and zymosan-stimulated macrophages. Taken together, the present data reveal an NPY Y1 and Y2/Y5 receptor interaction in NPY-induced modulation of macrophage functions related to inflammation.     

Energy Metabolic Profile of Mice after Chronic Activation of Central NPY Y1, Y2, or Y5 Receptors**

Objective: Neuropeptide Y (NPY), a 36‐amino acid peptide with orexigenic properties, is expressed abundantly in the central nervous system and binds to several NPY receptor subtypes. This study examines the roles of the NPY Y1, Y2, and Y5 receptor(s) in energy homeostasis. Research Methods and Procedures: We administered intracerebroventricular NPY (3 μg/d) or selective peptide agonists for the Y1, Y2, and Y5 receptor subtypes to C57Bl/6 mice for 6 days by mini‐osmotic pumps to assess the role of each receptor subtype in NPY‐induced obesity. Energy expenditure (EE) and respiratory quotient (RQ) were studied using indirect calorimetry. Adiposity was measured by DXA scanning and fat pad dissection. Insulin sensitivity was tested by whole‐blood glucose measurement after an insulin challenge. Results: Central administration of the selective Y1 agonist, Y5 agonist, or NPY for 6 days in mice significantly increased body weight, adiposity, and RQ, with significant hyperphagia in the Y5 agonist‐ and NPY‐treated groups but not in the Y1 agonist‐treated group. The NPY, Y1, or Y5 agonist‐treated mice had little change in total EE during ad libitum and pair‐feeding conditions. Conversely, selective activation of the Y2 receptor reduced feeding and resulted in a significant, but transient, weight loss. Discussion: Central activation of both Y1 and Y5 receptors increases RQ and adiposity, whereas only Y5 receptor activation reduces energy expended per energy ingested. Selective activation of Y2 autoreceptors leads to hypophagia and transient weight loss, with little effect on total EE. Our study indicates that all three NPY receptor subtypes may play a role in regulating energy homeostasis in mice.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Selective activation of central NPY Y1 vs. Y5 receptor elicits hyperinsulinemia via distinct mechanisms

Central administration of neuropeptide Y (NPY) stimulates hyperphagia and hyperinsulinemia. Recent evidence has suggested that the Y1 and Y5 receptor subtypes may both mediate NPY-stimulated feeding. The present study attempts to further characterize the role of central NPY receptor subtypes involved in hyperinsulinemia. NPY and peptide analogs of NPY that selectively activated the NPY Y1 or Y5 receptor subtype induced feeding and hyperinsulinemia in satiated Long Evans rats, whereas NPY analogs that selectively activated the NPY Y2 or Y4 receptor subtype did not. To determine whether NPY-induced hyperinsulinemia is secondary to its hyperphagic effect, we compared the plasma insulin levels in the presence and absence of food after a 1-min central infusion of NPY and its analogs at 15, 60, and 120 min postinfusion. Our data suggest that selective activation of central NPY Y1 receptor subtype induced hyperinsulinemia independent of food ingestion, whereas the NPY Y5 receptor-induced hyperinsulinemia was dependent on food ingestion. Central administration of the selective Y1 receptor agonist D-Arg25 NPY eventually decreased plasma glucose levels 2 h postinfusion in Long Evans rats.     

Neuropeptide Y (NPY) modulates oxidative burst and nitric oxide production in carrageenan-elicited granulocytes from rat air pouch

We studied the effects of neuropeptide Y (NPY) and NPY-related receptor specific peptides on functions of carrageenan-elicited granulocytes in vitro and ability of NPY to modulate carrageenan-induced air pouch inflammation in rats in vivo. Anti-inflammatory effect of NPY comprises reduced granulocyte accumulation into the air pouch, to some extent attenuation of phagocytosis, attained via Y1 receptor, and considerable decrease in peroxide production, albeit mediated via Y2 and Y5 receptors activation. Conversely, NPY increases nitric oxide production and this potentiation is mediated via Y1 receptor. It is concluded that NPY Y1 and Y2/Y5 receptors’ interaction participates in NPY-induced modulation of granulocyte functions related to inflammation.     

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.     

Novel potent antagonists of human neuropeptide Y Y5 receptors. Part 2: substituted benzo[a]cycloheptene derivatives

Novel benzo[a]cycloheptene derivatives were prepared for the purpose of searching new neuropeptide Y-Y5 (NPY-Y5) receptor antagonists. The structure-activity relationships are described and compound 2o (FR226928) showed the most potent affinity for Y5 receptor of all we prepared and was found to have higher potency and better selectivity for Y5 over Y1 receptor affinities when compared with the known lead compound 1.     

Cloning and characterization of a zebrafish Y2 receptor

The NPY receptors belong to the superfamily of G-protein coupled receptors and in mammals this family has five members, named Y1, Y2, Y4, Y5, and Y6. In bony fish, four receptors have been identified, named Ya, Yb, Yc and Y7. Yb and Y7 arose prior to the split between ray-fined fishes and tetrapods and have been lost in mammals. Yc appeared as a copy of Yb in teleost fishes. Ya may be an ortholog of Y4, but surprisingly no unambiguous receptor ortholog to any of the mammalian subtypes has yet been identified in bony fishes. Here we present the cloning and pharmacological characterization of a Y2 receptor in zebrafish, Danio rerio. To date, this is the first Y2 receptor outside mammals and birds that has been characterized pharmacologically. Phylogenetic analysis and synteny confirmed that this receptor is orthologous to mammalian Y2. We show that the receptor is pharmacologically most similar to chicken Y2 which leads to the conclusion that Y2 has acquired several novel characteristics in mammals. Y2 from zebrafish binds very poorly to the Y2-specific antagonist BIIE0246. Our pharmacological characterization supports our previous conclusions regarding the binding pocket of BIIE0246 in the human Y2 receptor.     

NPY, NPY receptors, and DPP IV activity are modulated by LPS, TNF-alpha and IFN-gamma in HUVEC

Since NPY increases endothelial cell (EC) stickiness for leukocytes, we studied the effects of LPS, TNF-alpha and IFN-gamma on its expression and action in HUVEC. Cytokines raised NPY and pro-NPY intracellular content and dipeptidyl peptidase IV (DPP IV) activity. Y1 and Y2 receptors were expressed in basal conditions, and LPS, TNF-alpha and IFN-gamma induced Y5 receptor expression with a concomitant extinction of Y2 receptor expression. NPY induced an intracellular calcium increase mainly mediated by Y2 and Y5 receptors in basal conditions. After stimulation with LPS, TNF-alpha and IFN-gamma, calcium increase was mainly caused by Y5 receptor. The modulation of the NPY system by LPS, TNF-alpha and IFN-gamma, and the NPY-induced calcium signaling suggest a role for NPY during the inflammatory response.     

The neuropeptide secretoneurin stimulates adhesion of human monocytes to arterial and venous endothelial cells in vitro

Neuropeptide Y (NPY), 36-amino acid amidated peptide expressed in central and peripheral neurons, regulates a variety of physiological activities, including food intake, energy expenditure, vasoconstriction, anxiolysis, nociception and ethanol consumption. NPY binds to a family of G-protein coupled receptors whose activation results in inhibition of adenylyl cyclase activity. To more fully characterize the signal transduction pathways utilized by the NPY receptor subtypes, the pathways leading to phosphorylation of the extracellular signal regulated protein kinases 1 and 2 (ERK) have been compared in CHO cells expressing each of the four cloned human NPY receptor subtypes, Y(1), Y(2), Y(4) and Y(5). NPY Y(1), Y(2), Y(4) and Y(5) receptor-mediated ERK phosphorylation was blocked by pertussis toxin (PTX) exposure, indicating that all four receptors are coupled to inhibitory G(i/o) proteins. Exposure to the protein kinase C (PKC) inhibitor GF109203X diminished Y(1), Y(2) and Y(4) receptor-mediated ERK phosphorylation but completely blocked Y(5) receptor-mediated ERK phosphorylation. Additionally, Y(5) receptor-mediated ERK phosphorylation was inhibited by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin to a greater extent than was Y(1)-mediated ERK phosphorylation. These results demonstrate that in CHO cells, the Y(5) receptor and the Y(1), Y(2) and Y(4) receptors utilize different pathways to activate ERK.     

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.     

Synthesis of new thiophene and benzo[b]thiophene hydrazide derivatives as human NPY Y(5) antagonists

Neuropeptide Y is one of the most potent appetite stimulating hormones known. Novel thiophene and benzo[b]thiophene hydrazide derivatives were synthetized and evaluated biologically as NPY Y(1) and Y(5) receptor subtype antagonists. They were found to have nanomolar binding affinities for human NPY Y(5) receptor, obtaining the lead compound, trans-N-4-[N'-(thiophene-2-carbonyl)hydrazinocarbonyl]cyclohexylmethyl-4-bromobenzenesulfonamide, which binds with a 7.70 nM IC(50) to the hY(5) receptor.     

Bis(31/31')[[Cys(31), Nva(34)]NPY(27-36)-NH(2)]: a neuropeptide Y (NPY) Y(5) receptor selective agonist with a latent stimulatory effect on food intake in rats

The actions of neuropeptide Y (NPY) are mediated by at least six G-protein coupled receptors denoted as Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). Investigations using receptor selective ligands and receptor knock-out mice suggest that NPY effects on feeding are mediated by both Y(1) and Y(5) receptors. We have previously shown that Cys-dimers of NPY C-terminal peptides exhibit Y(1) selectivity relative to Y(2) receptors. Re-investigation of their selectivity with respect to the newly cloned receptors, has identified bis(31/31') [[Cys(31), Nva(34)]NPY(27-36)-NH(2)] (BWX-46) as a Y(5) receptor selective agonist. BWX-46 selectively bound Y(5) receptors, and inhibited cAMP synthesis by Y(5) cells with potencies comparable to that of NPY. Moreover, BWX-46 (10 microM) exhibited no significant effect on the cAMP synthesis by Y(1), Y(2), and Y(4) cells. Thus, BWX-46 constitutes the lowest molecular weight Y(5) selective agonist reported to date. Intrahypothalamic (i.h.t)-injection of 30 and 40 microg of BWX-46 stimulated the food intake by rats in a gradual manner, reaching maximal level 8 h after injection. This response was similar to that exhibited by other Y(5) selective agonists, but differed from that of NPY, which exhibited a rapid orexigenic stimulus within 1 h. It is suggested that the differences in the orexigenic stimuli of NPY and Y(5) agonists may be due to their differences in the signal transduction mechanisms.     

Distinct Changes in Peptide YY Binding to, and mRNA Levels of, Y1 and Y2 Receptors in the Rat Hippocampus Associated with Kindling Epileptogenesis

Abstract: Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of ¹²⁵I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% ( p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36–87% ( p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50–64% ( p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% ( p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.     

Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5)

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.