Neuropeptide Y: a new mediator linking sympathetic nerves,blood vessels and immune system?

Neuropeptide Y (NPY(1-36)), a sympathetic cotransmitter and neurohormone, has pleiotropic activities ranging from the control of obesity to anxiolysis and cardiovascular function. Its actions are mediated by multiple Gi/o-coupled receptors (Y1-Y5) and modulated by dipeptidyl peptidase IV (DPPIV/cd26), which inactivates NPY's Y1-agonistic activity but generates the Y2 and Y5-agonist, NPY(3-36). Released by sympathetic activity, NPY is a major mediator of stress, responsible for prolonged vasoconstriction via Y1 receptors. Y1 receptors also mediate NPY's potent vascular growth-promoting activity leading in vivo in rodents to neointima formation. This and the association of a polymorphism of the NPY signal peptide with increased lipidemia and carotid artery thickening in humans strongly suggest NPY's role in atherosclerosis. NPY and DPPIV/cd26 are also coexpressed in the endothelium, where the peptide activates angiogenesis. A similar system exists in immune cells, where NPY and DPPIV/cd26 are coactivated and involved in the modulation of cytokine release and immune cell functions. Thus, NPY, both a messenger and a modulator for all three systems, is poised to play an important regulatory role facilitating interactions among sympathetic, vascular and immune systems in diverse pathophysiological conditions such as hypertension, atherosclerosis and stress-related alterations of immunity.     

Critical role of dipeptidyl peptidase IV in neuropeptide Y-mediated endothelial cell migration in response to wounding

Recently, we have discovered that neuropeptide Y (NPY), a sympathetic neurotransmitter, is also present in human umbilical endothelial cells (HUVECs), and is potently chemotactic and angiogenic by acting on one or several of Y1-Y5 receptors. In HUVECs, NPY is co-localized with dipeptidyl peptidase IV (DPPIV) which cleaves Tyr(1)-Pro(2) from NPY(1-36) to form NPY(3-36) resulting in the formation of a non-Y1 receptor agonist, which remains angiogenic. Presently we studied the effects of DPPIV's blockade using monoclonal antibodies (mAbs) on migration of HUVECs in response to NPY(1-36) or NPY(3-36) following cell wounding. Both peptides caused similar dose-dependent increases in cell migration (+80% at 0.1 nM) 12 h after wounding. DPPIV mAbs, E19 and E26, significantly reduced HUVEC's migration below that of the untreated cells, and blocked responses to NPY(1-36) but not NPY(3-36). Enhanced expression of DPPIV was found in the migrating cells and in cells with their protrusions at the edge of the wound (immunostaining and Western blot). Thus, DPPIV's expression is stimulated by endothelial wounding and its enzymatic activity is required for NPY-mediated chemotaxis. Furthermore, this suggests that non-Y1 receptors activated by NPY(3-36) (Y2, Y3 and/or Y5) mediate angiogenic effects of NPY.     

Interactions of multiple signaling pathways in neuropeptide Y-mediated bimodal vascular smooth muscle cell growth

Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.     

Stress‐induced changes in adrenal neuropeptide Y expression are regulated by a negative feedback loop

Neuropeptide Y is a co‐transmitter that is synthesized by chromaffin cells in the adrenal medulla. During the fight‐or‐flight response these cells release NPY in addition to epinephrine and norepinephrine. Following the stress‐induced reflex, the levels of NPY are increased as part of a homeostatic response that modulates catecholaminergic signaling. Here, we examined the control of NPY expression in mice after brief exposure to the cold water forced swim test. This treatment led to a shift in NPY expression between two populations of chromaffin cells that reversed over the course of 1 week. When NPY(GFP) BAC transgenic animals were exposed to stress, there was an increase in cytoplasmic, non‐secretable GFP, indicating that stress increased NPY promoter activity. In vivo blockage of Y2 (but not Y1 or Y5) receptors increased basal adrenal NPY expression and so modulated the effects of stress. We conclude that release of NPY mediates a negative feedback loop that inhibits its own expression. Thus, the levels of NPY are determined by a balance between the potentiating effects of stress and the tonic inhibitory actions of Y2 receptors. This may be an efficient way to ensure the levels of this modulator do not decline following intense sympathetic activity.     

Neuropeptide Y-induced angiogenesis in aging

Age-related changes in NPY-driven angiogenesis were investigated using Matrigel and aortic sprouting assays in young (2 months.) and aged (18 months.) mice. In both assays, NPY-induced vessel growth decreased significantly with age. In parallel, aged mice showed reduced expression (RT-PCR) of Y2 receptors and the NPY converting enzyme, dipeptidyl peptidase IV (DPPIV), in spleens. Aging of human microvascular endothelial cells in vitro led to a loss of their mitogenic responses to NPY accompanied by a lack of NPY receptor mRNAs. Thus, NPY-dependent angiogenesis is impaired with age, which is associated with a decreased expression of endothelial NPY receptors (Y2) and DPPIV.     

Stress hormone epinephrine enhances adipogenesis in murine embryonic stem cells by up-regulating the neuropeptide Y system

Prenatal stress, psychologically and metabolically, increases the risk of obesity and diabetes in the progeny. However, the mechanisms of the pathogenesis remain unknown. In adult mice, stress activates NPY and its Y2R in a glucocorticoid-dependent manner in the abdominal fat. This increased adipogenesis and angiogenesis, leading to abdominal obesity and metabolic syndrome which were inhibited by intra-fat Y2R inactivation. To determine whether stress elevates NPY system and accelerates adipogenic potential of embryo, here we "stressed" murine embryonic stem cells (mESCs) in vitro with epinephrine (EPI) during their adipogenic differentiation. EPI was added during the commitment stage together with insulin, and followed by dexamethasone in the standard adipogenic differentiation medium. Undifferentiated embryonic bodies (EBs) showed no detectable expression of NPY. EPI markedly up-regulated the expression NPY and the Y1R at the commitment stage, followed by increased Y2R mRNA at the late of the commitment stage and the differentiation stage. EPI significantly increased EB cells proliferation and expression of the preadipocyte marker Pref-1 at the commitment stage. EPI also accelerated and amplified adipogenic differentiation detected by increasing the adipocyte markers FABP4 and PPARγ mRNAs and Oil-red O-staining at the end of the differentiation stage. EPI-induced adipogenesis was completely prevented by antagonists of the NPY receptors (Y1R+Y2R+Y5R), indicating that it was mediated by the NPY system in mESC's. Taken together, these data suggest that stress may play an important role in programming ESCs for accelerated adipogenesis by altering the stress induced hormonal regulation of the NPY system.     

Dipeptidyl-peptidase-IV by cleaving neuropeptide Y induces lipid accumulation and PPAR-γ expression

We evaluated the effects of dipeptidyl peptidase-IV (DPPIV), and its inhibitor, vildagliptin, on adipogenesis and lipolysis in a pre-adipocyte murine cell line (3T3-L1). The exogenous rDPPIV increased lipid accumulation and PPAR-γ expression, whereas an inhibitor of DPPIV, the anti-diabetic drug vildagliptin, suppresses the stimulatory role of DPPIV on adipogenesis and lipid accumulation, but had no effect on lipolysis. NPY immunoneutralization or NPY Y(2) receptor blockage inhibited DPPIV stimulatory effects on lipid accumulation, collectively, indicating that DPPIV has an adipogenic effect through NPY cleavage and subsequent NPY Y(2) activation. Vildagliptin inhibits PPAR-γ expression and lipid accumulation without changing lipolysis, suggesting that this does not impair the ability of adipose tissue to store triglycerides inside lipid droplets. These data indicate that DPPIV and NPY interact on lipid metabolism to promote adipose tissue depot.     

Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo

Prior studies have revealed that the sympathetic nervous system regulates the clinical and pathological manifestations of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model mediated by Th1 T cells. Although the regulatory role of catecholamines has been indicated in the previous works, it remained possible that other sympathetic neurotransmitters like neuropeptide Y (NPY) may also be involved in the regulation of EAE. Here we examined the effect of NPY and NPY receptor subtype-specific compounds on EAE, actively induced with myelin oligodendrocyte glycoprotein 35-55 in C57BL/6 mice. Our results revealed that exogenous NPY as well as NPY Y(1) receptor agonists significantly inhibited the induction of EAE, whereas a Y(5) receptor agonist or a combined treatment of NPY with a Y(1) receptor antagonist did not inhibit signs of EAE. These results indicate that the suppression of EAE by NPY is mediated via Y(1) receptors. Furthermore, treatment with the Y(1) receptor antagonist induced a significantly earlier onset of EAE, indicating a protective role of endogenous NPY in the induction phase of EAE. We also revealed a significant inhibition of myelin oligodendrocyte glycoprotein 35-55-specific Th1 response as well as a Th2 bias of the autoimmune T cells in mice treated with the Y(1) receptor agonist. Ex vivo analysis further demonstrated that autoimmune T cells are directly affected by NPY via Y(1) receptors. Taken together, we conclude that NPY is a potent immunomodulator involved in the regulation of the Th1-mediated autoimmune disease EAE.     

Blockade of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors by agonist peptides is prevented by modulators of sodium transport. Implications for receptor signaling and regulation

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (IC50 300-400 pM). The cloned Y(4) receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y(4) sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

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.     

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.     

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.     

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.     

Modeling of Neuropeptide Receptors Y1, Y4, Y5, and Docking Studies with Neuropeptide Antagonist Analogues: Implications for Selectivity

Abstract Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y(1)-Y(5) and y(6). Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

PYY3-36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y2 receptors

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.     

Existence of both neuropeptide Y, Y1 and Y2 receptors in pig spleen: evidence using subtype-selective antagonists in vivo

The effects of the first selective, non-peptide, NPY Y2 receptor antagonist (S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamid (BIIE0246) were studied on splenic vascular responses evoked in the pig in vivo. BIIE0246 abolished the splenic vasoconstrictor response to the NPY Y2 receptor agonist N-acetyl[Leu25Leu31]NPY(24-36), but did not affect the response to the NPY Y1 receptor agonist [Leu31Pro34]NPY, which in turn was abolished by the selective NPY Y1 receptor antagonist (2R)-5-([amino(imino)methyl]amino)-2-[(2,2-diphenylacetyl)amino]-N-[(IR)-1-(4-hydroxyphenyl)ethyl]-pentanamide (H 409/22). Furthermore, the PYY-evoked splenic vasoconstrictor response was partially antagonized by BIIE0246 and subsequently almost abolished by the addition of H 409/22. It is concluded that BIIE0246 exerts selective (vs the NPY Y1 receptor) NPY Y2 receptor antagonism, and thus represents an interesting tool for classification of NPY receptors, in vivo. In addition, evidence for NPY Y2 receptor mediated vasoconstriction was presented. Furthermore, both NPY Y1 and Y2 receptors are involved in the splenic vasoconstrictor response to PYY.     

Unexpected high density of neuropeptide Y Y1 receptors in the guinea pig spleen

Receptors for neuropeptide Y (NPY) and peptide YY (PYY) have been extensively characterized in the brain. Less is known about NPY receptor subtypes in the spleen, though it is well established that NPY produces vascular contraction in this tissue. In the present study, we found an unusually high density of Y₁ receptors in the guinea pig spleen. These receptors are localized to the red pulp and exhibit a pharmacology that is consistent with the Y₁ receptor. On the other hand, only very low densities for Y₂ receptors were observed. Therefore, the guinea pig spleen may be a ideal tissue for further study of the role of Y₁ receptors in cardiovascular and immune function.