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.     

The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age

Neuropeptide Y (NPY)-induced modulation of the immune and inflammatory responses is regulated by tissue-specific expression of different receptor subtypes (Y1–Y6) and the activity of the enzyme dipeptidyl peptidase 4 (DP4, CD26) which terminates the action of NPY on Y1 receptor subtype. The present study investigated the age-dependent effect of NPY on inflammatory paw edema and macrophage nitric oxide production in Dark Agouti rats exhibiting a high-plasma DP4 activity, as acknowledged earlier. The results showed that NPY suppressed paw edema in adult and aged, but not in young rats. Furthermore, plasma DP4 activity decreased, while macrophage DP4 activity, as well as macrophage CD26 expression increased with aging. The use of NPY-related peptides and Y receptor-specific antagonists revealed that anti-inflammatory effect of NPY is mediated via Y1 and Y5 receptors. NPY-induced suppression of paw edema in young rats following inhibition of DP4 additionally emphasized the role for Y1 receptor in the anti-inflammatory action of NPY. In contrast to the in vivo situation, NPY stimulated macrophage nitric oxide production in vitro only in young rats, and this effect was mediated via Y1 and Y2 receptors. It can be concluded that age-dependant modulation of inflammatory reactions by NPY is determined by plasma, but not macrophage DP4 activity at different ages.     

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.     

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.     

Neuropeptide Y receptor in cultured vascular smooth muscle cells: ligand binding and increase in cytosolic free Ca2+

We have previously shown that neuropeptide Y (NPY) increases cytosolic free Ca2+ concentration [( Ca2+]i) in porcine aortic smooth muscle cells. In this study, specific NPY receptor binding sites were identified in the cells by use of [125I]Bolton-Hunter NPY [( 125I]BH-NPY). Binding was to a single population of the sites with a Kd of 1.1 +/- 0.2 nM and a Bmax of 0.68 +/- 0.10 pmol/mg protein. [125I]BH-NPY binding was displaced by NPY-related peptides including members of the pancreatic polypeptide (PP) family. The potency of these peptides other than human PP for displacing [125I]BH-NPY binding was substantially consistent with their potency for increasing [Ca2+]i. Human PP had no effect on [Ca2+]i even at 10(-5) M, but it inhibited the NPY-induced increase in [Ca2+]i with a potency comparable to that for displacing [125I]BH-NPY binding. NPY(13-36) was about 500 and 300 times less effective than porcine NPY in increasing [Ca2+]i and in displacing [125I]BH-NPY binding, respectively, showing that the NPY receptor in cultured vascular smooth muscle cells is of the Y1-type.     

Neuropeptide Y receptor(s) mediating feeding in the rat: characterization with antagonists

The present study evaluated the effect of the neuropeptide Y (NPY) Y1 receptor antagonists BIBO 3304 and SR 120562A and of the Y5 receptor antagonists JCF 104, JCF 109, and CGP 71683A on feeding induced either by NPY or food deprivation. In a preliminary experiment, NPY was injected into the third cerebroventricle (3V) at doses of 0.07, 0.15, 0.3, or 0.6 nmol/rat. The dose of 0.3 nmol/rat, which produced a cumulative 2-h food intake of 11.2 +/- 1.9 g/kg body weight, was chosen for the following experiments. The antagonists were injected in the 3V 1 min before NPY. The Y1 receptor antagonist BIBO 3304 significantly inhibited NPY-induced feeding at doses of 1 or 10 nmol/rat. The Y1 receptor antagonist SR 120562A, at the dose of 10 but not of 1 nmol/rat, significantly reduced the hyperphagic effect of NPY, 0.3 nmol/rat. The Y5 receptor antagonists JCF 104 and JCF 109 (1 or 10 nmol/rat) and CGP 71683A (10 or 100 nmol/rat) did not significantly modify the effect of NPY, 0.3 nmol/rat. However, JCF 104 (10 nmol/rat) and CGP 71683A (100 nmol/rat), but not JCF 109 (10 nmol/rat), significantly reduced food intake during the interval from 2 to 4 h after injection of a higher dose, 0.6 nmol/rat, of NPY. Feeding induced by 16 h of food deprivation was significantly reduced by the Y1 receptor antagonist BIBO 3304 (10 nmol/rat), but it was not significantly modified by the same dose of SR 120562A or JCF 104. These findings support the idea that the hyperphagic effect of NPY is mainly mediated by Y1 receptors. The results obtained with JCF 104 and CGP 71683A suggest that Y5 receptors may have a modulatory role in the maintenance of feeding induced by rather high doses of NPY after the main initial feeding response.     

Neuropeptide Y enhances permeability across a rat aortic endothelial cell monolayer

Previously, in vivo studies showed that neuropeptide Y (NPY) elevates vascular permeability in isolated lung perfusion preparations, possibly through binding to the NPY Y(3) receptor. The present study used monolayers in a double-chamber culture method under conditions of normoxia (5% CO(2)-20% O(2)-75% N(2)) or hypoxia (5% CO(2)-5% O(2)-90% N(2)) to test the hypothesis that NPY directly affects rat aortic endothelial cells (RAECs). RAECs were cultured on the base of the upper chamber, into which FITC-labeled albumin was introduced, and permeation into the lower chamber was measured. The RAEC monolayer was treated with 10(-8)-3 x 10(-7) M NPY for 2 h in normoxia or hypoxia. In hypoxia, NPY concentration dependently increased the permeability of the RAEC monolayer, whereas in normoxia no significant change was observed. Peptide YY, NPY Y(1), and NPY Y(2) receptor agonists and NPY Y(1) receptor antagonist exerted no significant effects under hypoxic conditions. NPY-(18-36), an NPY Y(3) receptor antagonist, elicited an inhibitory action on the NPY-induced increase in monolayer permeability. Furthermore, neither N-monomethyl-l-arginine, a nitric oxide synthase inhibitor, the bradykinin B(2) receptor antagonist FK-3657, nor the vascular endothelial growth factor receptor-coupled tyrosine kinase inhibitor tyrphostin SU-1498, injected into the medium of the upper chamber, affected the NPY-induced permeability changes under hypoxic conditions. The results suggest that the NPY-induced increase in permeability across the RAEC monolayer is closely related to low O(2) tension, possibly mediated by direct action on the NPY Y(3) receptor expressed on the endothelial cell membrane. Furthermore, this NPY-induced increase is not likely due to nitric oxide, bradykinin, or vascular endothelial growth factor.     

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.     

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.     

Synthesis and evaluation of new hydrazide derivatives as neuropeptide Y Y5 receptor antagonists for the treatment of obesity

NPY is the most potent orexigenic agent known to man, with NPY Y1 and NPY Y5 being the receptor subtypes that are most likely responsible for centrally-mediated NPY-induced feeding responses. Based on the aforementioned, novel hydrazide derivatives were prepared for the purpose of searching new NPY Y5 receptor antagonists. Many of the compounds exhibited nanomolar binding affinity for this receptor, affording trans-N-(4-[N'-(3,4-dichlorophenyl)hydrazinocarbonyl]cyclohexylmethyl)-4-fluorobenzenesulfonamide, which showed the best activity (IC(50)=0.43nM).     

Food intake regulation in rodents: Y5 or Y1 NPY receptors or both?

Neuropeptide Y (NPY), one of the most abundant peptides in rat and human brains, appears to act in the hypothalamus to stimulate feeding. It was first suggested that the NPY Y1 receptor (Y1R) was involved in feeding stimulated by NPY. More recently a novel NPY receptor subtype (Y5R) was identified in rat and human as the NPY feeding receptor subtype. There is, however, no absolute consensus since selective Y1R antagonists also antagonize NPY-induced hyperphagia. Nevertheless, new anti-obesity drugs may emerge from further pharmacological characterization of the NPY receptors and their antagonists. A large panel of Y1R and Y5R antagonists (such as CGP71683A, BIBO3304, BIBP3226, 1229U91, and SYNAPTIC and BANYU derivatives but also patentable in-house-synthesized compounds) have been evaluated through in vitro and in vivo tests in an attempt to establish a predictive relationship between the binding selectivity for human receptors, the potency in isolated organs assays, and the inhibitory effect on food intake in both normal and obese hyperphagic rodents. Although these results do not allow one to conclude on the implication of a single receptor subtype at the molecular level, this approach is crucial for the design of novel NPY receptor antagonists with potential use as anti-obesity drugs and for evaluation of their possible adverse peripheral side effects, such as hypotension.     

Possible roles of neuropeptide Y Y3-receptor subtype in rat aortic endothelial cell proliferation under hypoxia, and its specific signal transduction

The present study was undertaken to determine whether neuropeptide Y (NPY) induces proliferation of rat aortic endothelial cells (RAECs). Since NPY increased the permeability of RAEC monolayers to large molecules via the NPY Y(3) receptor, RAEC proliferation has been evaluated in terms of NPY-receptor subtypes and also intracellular mechanisms. RAECs were incubated with gases containing 20, 15, or 10% O(2) and a certain amount of N(2), depending on the O(2) content in 5% CO(2) incubators. NPY (10(-9)-10(-6) M) increased the RAEC numbers under hypoxic conditions, such as 15 or 10% O(2). Peptide YY elicited no proliferative effect on RAEC, and NPY-(18-36) inhibited the NPY-induced increase in cell number, suggesting that NPY increases the RAEC count through the NPY Y(3) receptor. Pertussis toxin, U-73122, GF-109203X, myristorylated autocamtide-2-related inhibitory peptide, and wortmannin inhibited the NPY-induced proliferation of RAEC concentration dependently. DY9760e little affected the proliferation caused by NPY. ML-9 and imatinib actually enhanced the NPY-induced proliferation of cells. These results indicated that the NPY Y(3) receptor is coupled with G(i) protein, and that NPY-induced increases in RAEC proliferation are mediated by phospholipase C-protein kinase C and/or phosphatidylinositol 3-kinase pathways. In intracellular Ca(2+)-calmodulin-dependent pathways, calmodulin-dependent protein kinase II partly participates in the NPY-induced cell proliferation. Regarding the previously reported effect of NPY on the permeability of RAEC monolayers to large molecules, it is probable that protein kinase C and phosphatidylinositol 3-kinase pathways are activated for both permeability and cell proliferation induced by NPY under hypoxia, relevant to new insights into the roles of NPY in ischemia-hypoxia.     

Modulation of intracellular calcium changes and glutamate release by neuropeptide Y1 and Y2 receptors in the rat hippocampus: differential effects in CA1, CA3 and dentate gyrus

In the present work, we investigated the role of pre- and post-synaptic neuropeptide Y1 (NPY1) and Y2 receptors on the calcium responses and on glutamate release in the rat hippocampus. In cultured hippocampal neurones, we observed that only NPY1 receptors are involved in the modulation of intracellular free calcium concentration ([Ca(2+)](i)). In 88% of the neurones analysed, the increase in the [Ca(2+)](i), in response to depolarization with 50 mM KCl, was inhibited by 1 microM [Leu31,Pro34]NPY, whereas 300 nM NPY13-36 was without effect. However, studies with hippocampal synaptosomes showed that both NPY1 and Y2 receptors can modulate the [Ca(2+)](i) and glutamate release. The pharmacological characterization of the NPY-induced inhibition of glutamate release indicated that Y2 receptors play a predominant role, both in the modulation of Ca(2+)-dependent and -independent glutamate release. However, we could distinguish between Y1 and Y2 receptors by using [Leu31,Pro34]NPY and NPY13-36. Active pre-synaptic Y1 receptors are present in the dentate gyrus (DG) as well as in the CA3 subregion, but its activity was not revealed by using the endogenous agonist, NPY. Concerning the Y2 receptors, they are present in the three subregions (CA1, CA3 and DG) and were activated by either NPY13-36 or NPY. The present data support a predominant role for NPY2 receptors in mediating NPY-induced inhibition of glutamate release in the hippocampus, but the physiological relevance of the presently described DG and CA3 pre-synaptic NPY1 receptors remains to be clarified.     

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice

Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.     

NPY signaling through Y1 receptors modulates thalamic oscillations

Neuropeptide Y is the ligand of a family of G-protein coupled receptors (Y(1) to Y(6)). In the thalamus, exogenous and endogenously released NPY can shorten the duration of thalamic oscillations in brain slices from P13 to P15 rats, an in vitro model of absence seizures. Here, we examine which Y receptors are involved in this modulation. Application of the Y(1) receptor agonist Leu(31)Pro(34)NPY caused a reversible reduction in the duration of thalamic oscillations (-26.6+/-7.8%), while the Y(2) receptor agonist peptideYY((3-36)) and the Y(5) receptor agonist BWX-46 did not exert a significant effect. No Y receptor agonist affected oscillation period. Application of antagonists of Y(1), Y(2) and Y(5) receptors (BIBP3226, BIIE0246 and L152,806, respectively) produced results consistent with those obtained from agonists. BIBP3226 caused a reversible disinhibition, an effect that increases oscillation duration (18.2+/-9.7%) while BIIE0246 and L152,806 had no significant effect. Expression of NPY is limited to neurons in the reticular thalamic nucleus (nRt), but Y(1) receptors are expressed in both nRt and adjacent thalamic relay nuclei. Thus, intra-nRt or nRt to relay nucleus NPY release could cause Y(1) receptor mediated inhibition of thalamic oscillations.     

Self-regulation of agonist activity at the Y receptors

Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY]>[peptide YY] (PYY) for the Y1, and [human PP]>[PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.     

Role of neuropeptide Y and its receptors in the progression of endocrine-related cancer

The neuropeptide Y (NPY) family of peptides, in addition to its many physiological actions, has also been involved in the modulation of tumor progression, with specific reference to endocrine-related cancers such as neuroendocrine tumors, breast and prostate cancers. These have been found either to express NPY receptors, or to secrete NPY-related peptides, or both. The study of the role of the NPY family of peptides in the biology of endocrine-related tumors, specifically concerning cell proliferation, angiogenesis, invasion and metastatization, may help to clarify some aspects of tumor pathophysiology, as well as to indicate novel diagnostic markers and therapeutical approaches.     

神经肽Y及其受体Y1、Y2对痛觉调制的研究进展

神经肽Y（neuropeptide Y,NPY）是一种由36个氨基酸残基组成的肽类激素,属胰多肽家族,广泛分布于中枢及外周神经组织的神经元中。NPY主要参与摄食行为、心血管活动、垂体分泌等生理功能的调节。NPY还参与了痛觉调制。NPY受体有Y1、Y2、Y3、Y4、Y5和Y6六种亚型。目前对Y1受体和Y2受体的研究较多,显示Y1受体和Y2受体参与痛觉调制。但现在对NPY在痛觉中的具体作用机制还不清楚。该文对NPY及其Y1受体、Y2受体在痛觉调制中的作用作一概述。     

Two different signal transductions of neuropeptide Y1 receptor in SK-N-MC cells.

The signal transduction systems of the neuropeptide Y (NPY) Y1 receptor were studied in SK-N-MC human neuroblastoma cells. NPY induced an increase in intracellular calcium ion concentration ([Ca2+]i) and inhibition of forskolin-stimulated cyclic AMP accumulation, which were mediated through Y1 receptors. One-min preincubation of cells with phorbol 12-myristate 13-acetate (PMA) inhibited both signal transductions dose-dependently, but its effect on [Ca2+]i was about 100-fold more potent than that on cyclic AMP. PMA had no effect on [125I]BH-NPY binding in SK-N-MC cells and hardly inhibited the endothelin-1-induced increase in [Ca2+]i. Pertussis toxin also inhibited the NPY-induced [Ca2+]i increase 30-fold more effectively than the NPY-mediated inhibition of cyclic AMP accumulation. These results indicate that Y1 receptors in SK-N-MC cells couple to two signal transduction systems that have different sensitivities to phorbol ester and pertussis toxin treatments.     

中脑导水管周围灰质内微量注射神经肽γ减弱慢性神经痛大鼠对伤害性刺激的反应

探讨神经肽Y(neuropeptide Y,NPY)在SD大鼠中脑导水管周围灰质(periaqueductal grey,PAG)对伤害性刺激反应的作用。应用热板和机械压力实验法,以大鼠后爪缩爪反应潜伏期(paw withdrawal latency,PWL)为痛阈指标。观察PAG内微量注射NPY对PWLS的影响。PAG内注射0．05、0．1、0．2nmol NPY均显著地增加慢性神经痛大鼠的双侧PWLS,且呈量效关系。NPY引起的PWLs增加可被Y1受体拮抗剂和阿片受体拮抗剂所阻断。结果提示,在大鼠PAG微量注射NPY可产生明显的镇痛作用。