Neuromodulation of the cardiac vagus: comparison of neuropeptide Y and related peptides

Neuropeptide Y (NPY), a putative co-transmitter in noradrenergic sympathetic nerves of the cardiovascular system, inhibits the negative chronotropic action of the cardiac vagus. In the present study, peptides related to NPY were tested for potency in producing this effect. In bilaterally vagotomized, anaesthetised dogs, the increase in pulse interval caused by electrical stimulation of the peripheral stump of the right vagus was measured before and after intravenous administration of peptide. The effects of doses of NPY were compared with those of equimolar doses of peptide YY (PYY), and of avian and human pancreatic polypeptides (APP and HPP). PYY inhibited the vagal action more effectively than did NPY. APP and HPP, however, caused no change in strength of vagal action at the doses used. The response to a second injection of NPY, given soon after the injection of APP or HPP, was not significantly different from the original. Thus no evidence was obtained for a competitive inhibition of the action of NPY by either pancreatic polypeptide. A C-terminal hexapeptide fragment of human pancreatic polypeptide was also tested. Like APP and HPP, it neither inhibited the cardiac vagus nor blocked the action of NPY. The order of potency obtained here (PYY greater than NPY much greater than APP, HPP, CFPP) can be expected to be of use in efforts to distinguish the active site(s) of the NPY molecule, and to characterise the receptors involved in these modulatory effects.     

Structural similarities of micelle-bound peptide YY (PYY) and neuropeptide Y (NPY) are related to their affinity profiles at the Y receptors

Here, we investigate the structure of porcine peptide YY (pPYY) both when unligated in solution at pH 4.2 and when bound to dodecylphosphocholine (DPC) micelles at pH 5.5. pPYY in solution displays the PP-fold, with the N-terminal segment being back-folded onto the C-terminal alpha-helix, which extends from residue 17 to 31. In contrast to the solution structure of Keire et al. published in the year 2000 the C-terminal helix does not display a kink around residue 23-25. The root mean square deviation (RMSD) for backbone atoms of the NMR ensemble of conformers to the mean structure is 0.99(+/-0.35) Angstrom for residues 14-31. The back-fold is supported by values of 0.60+/-0.1 for the (15)N(1)H-NOE and by generalized order parameters S(2) of 0.74+/-0.1 for residues 5-31 which indicate that the peptide is folded in that segment. We have additionally used DPC micelles as a membrane model and determined the structure of pPYY when bound to it. Therein, an alpha-helix occurs in the segment comprising residues 17-31 and the N terminus freely diffuses in solution. The hydrophobic side of the amphipathic helix forms the micelle-binding interface and hydrophobic side-chains extend into the micelle interior. A significant stabilization of helical conformation occurs in the C-terminal pentapeptide, which is important for receptor binding. The latter is supported by positive values of the heteronuclear NOE in that segment (0.52+/-0.1 compared to 0.08+/-0.4 for the unligated form) and by values of S(2) of 0.6+/-0.2 (versus 0.38+/-0.2 for the unligated form). The structures of micelle-bound pPYY and pNPY are much more similar than those of pPYY and bPP with pairwise RMSDs of 1.23(+/-0.21)A or 3.21(+/-0.39) Angstrom, respectively. In contrast to the conformational similarities in the DPC-bound state their structures in solution are very different. In fact pPYY is more similar to bPP, which with its strong preference for the Y(4) receptor displays a completely different binding profile. Considering the high degree of sequence homology of pNPY and pPYY (>80%) and the fact, that their binding affinities at all receptor subtypes are high and, more importantly, rather similar, it is much more likely that PYY and NPY are recognized by the Y receptors from the membrane-bound state. As a consequence of the latter the PP-fold is not important for recognition of PYY or NPY at the Y receptors. To our knowledge this work provides for the first time strong arguments derived from structural data that support a membrane-bound receptor recognition pathway.     

Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion

Neuropeptide Y (NPY) injected into the paraventricular nucleus (PVN) is known to elicit a powerful feeding response in satiated, brain-cannulated rats [41, 42, 43]. The present experiment investigates the effect of peptide YY (PYY), a structurally-related peptide, on feeding behavior and, in addition, the effects of both PYY and NPY on the pattern of macronutrient selection. Injection of PYY directly into the PVN, in doses ranging from 7.8 to 235 pmol/0.3 μl, caused a strong, dose-dependent stimulation of feeding behavior, as well as a small stimulation of drinking behavior, in satiated rats. The mean latency to eat was 9.3 min, with substantial feeding occurring within 30 min of the injection. At low doses, the increase in feeding was seen predominantly during the first hr. At the highest dose, in contrast, food intake continued to increase progressively over the next few hr, such that by 4 hr postinjection food intake was more than 20 g over vehicle baseline. In 1 hr tests with 3 pure macronutrient (protein, fat and carbohydrate) diets simulataneously available, PYY and NPY (78 pmol/0.3 μl) both elicited a strong and selective increase in carbohydrate consumption, with little or no effect on protein or fat consumption. These results suggest that hypothalamic receptors sensitive to PYY and NPY may participate in the control of carbohydrate consumption.     

Immunohistochemical distribution of neuropeptide Y,peptide YY,pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women

Few studies have suggested that neuropeptide Y (NPY) could play an important role in skin functions. However, the expression of NPY, the related peptides, peptide YY (PYY) and pancreatic polypeptide (PP) and their receptors have not been investigated in human skin. Using specific antisera directed against NPY, PYY, PP and the Y₁, Y₂, Y₄ and Y₅ receptor subtypes, we investigated here the expression of these markers. NPY-like immunoreactivity (ir) in the epidermal skin could not be detected. For the first time we report the presence of positive PP-like ir immunofluorescent signals in epidermal cells, i.e. keratinocytes of skin from three areas (abdomen, breast and face) obtained as surgical left-overs. The immunofluorescent signal of PP-like ir varies from very low to high level in all three areas. In contrast, PYY-like ir is only expressed in some cells and with varied level of intensity. Furthermore and for the first time we observed specific Y₁ and Y₄ receptor-like ir in all epidermal layers, while the Y₂ and Y₅ subtypes were absent. Interestingly, as seen in human epidermis, in Episkin, a reconstituted human epidermal layer, we detected the presence of PP-like as well as Y₁-like and Y₄-like ir. These data have shown the presence and distribution of PYY, PP and Y₁ and Y₄ receptors in the human skin and Episkin, suggesting possible novel roles of NPY related peptides and their receptors in skin homeostasis.     

Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling

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Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

Corticosterone regulates the expression of neuropeptide Y and reelin in MLO-Y4 cells

Osteocytes that have a dendritic appearance are widely believed to form a complex cellular network system and play crucial roles in mechanotransduction as a principal bone mechanosensor, which is the basis of their neuronallike biology, as previously reported. Neuropeptide Y (NPY) and reelin mRNA, which are brain-specific neurogenic markers, have been identified in osteocytes. However, changes in the production of NPY and reelin in response to specific biochemical stimulation are unknown. In this study, we investigated the in vitro effect of corticosterone, one of the endogenous glucocorticoids, on the expression of NPY and reelin in the MLO-Y4 osteocyte cell line. Cells were treated with corticosterone at different concentrations (10(-9) M-10(-5) M) for 1, 3, 6, 12 and 24 h. As revealed, corticosterone reduced the MLO-Y4 cell viability and proliferation in a dose- and time-dependent manner based on an MTT assay and a Vi-CELL analyzer. The cells were then incubated with corticosterone (10(-6) μM), and the NPY and reelin expression levels were detected at 1, 3, 6, 12 and 24 h using real-time PCR and Western blot analysis. These results demonstrated that at the gene and the protein levels, corticosterone significantly upregulated the NPY and reelin expression in a time-dependent manner. The application of a glucocorticoid receptor antagonist, RU486, reversed the reduced cell viability and the increased expression of NPY and reelin that were caused by corticosterone. To the best of our knowledge, this is the first report to verify that corticosterone regulates the NPY and reelin expression in osteocytes.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of neuropeptide Y gene expression and genotyping with hypertension in chronic kidney disease

ObjectivesThe prognosis of high-risk patients might be greatly ameliorated using genetic predisposition risk factors. Sympathetic activity and innate immunity related to neuropeptide Y function may be related to dyslipidemia and atherosclerosis. The aim of this study is to detect the correlation between Neuropeptide Y (NPY) SNP rs16147 and its gene expression in chronic kidney disease with and without hypertension.MethodsThis study carried out on 150 subjects who were divided into 3 main groups group (I) 50 CKD patients with hypertension, group (II) 50 CKD patients without hypertension and group (III) 50 healthy individuals. Carotid intima media thickness (CIMT) was measured by Ultrasound. Kidney function test and lipid profile were performed. Genotyping and gene expression of neuropeptide Y (NPY) were performed using real time PCR.ResultsThere was a significant increase in number and percentage of CC genotype and C allele of NPY SNP distribution in CKD patients with and without hypertension when compared to controls. A significant association was found between CC genotype and C allele and the risk of CKD with hypertension with odd ratio 3.26 and 1.77, respectively. There is a significant positive correlation between NPY gene expression level and CIMT among chronic kidney disease patients with highest level of TC, LDLc and CIMT among CC genotype of NPY gene.ConclusionA significant association was found between CC genotype and C allele of NPY at rs16147 with increase NPY gene expression and risk of developing hypertension in CKD.     

Anxiolytic-like effect of the selective neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze

The involvement of Neuropeptide Y (NPY) in the pathophysiology of mood disorders has been suggested by clinical and preclinical evidence. NPY Y1 and Y2 receptors have been proposed to mediate the NPY modulation of stress responses and anxiety related behaviors. To further investigate the role of Y2 receptors in anxiety we studied the effect of BIIE0246, a selective Y2 receptor antagonist, in the elevated plus-maze test. Rats treated with 1.0 nmol BIIE0246 showed an increase in the time spent on the open arm of the maze. In addition, to study the effects of the Y2 antagonism on NPY protein level, NPY-like immunoreactivity was measured in different brain regions following treatment with BIIE0246, but no statistically significant effects were observed. These results suggest that BIIE0246 has an anxiolytic-like profile in the elevated plus-maze.     

Effect of high dietary copper on weight gain and neuropeptide Y level in the hypothalamus of pigs

An experiment was performed to examine the effect of dietary copper supplementation on weight gain, neuropeptide Y (NPY) concentration and NPY mRNA expression level in the hypothalamus of pigs. Forty-five crossbred pigs were randomly assigned to three groups of 15 pigs, each comprising five replicates of 3 animals. Pigs were allocated to diets that contained 10 mg/kg (as a control), 125 and 250 mg/kg copper as CuSO₄. Live weight gain and feed conversion efficiency was determined at the end of the experiment and five pigs, selected at random from each group, were slaughtered and the hypothalami collected for determination of NPY concentration and NPY mRNA expression level. The results showed that average daily gain (ADG) and average daily feed intake (ADFI) were higher and feed:gain (F:G) ratio was lower in pigs fed the diets with 125 and 250 mg/kg copper (P<0.05), respectively, than in pigs fed a diet with 10 mg/kg copper, but that there was no statistically significant difference in growth performance between animals of the 125 mg/kg and the 250 mg/kg copper groups. Furthermore, pigs fed diets with 125 and 250 mg/kg copper had higher NPY concentrations and NPY mRNA expression levels in their hypothalamus than control animals. The data indicated that 125 and 250 mg/kg copper gave similar responses in terms of weight gain, whilst high dietary copper could enhance NPY concentration and NPY mRNA expression level in the hypothalamus of pigs. High dietary copper appears to increase feed intake and promote weight gain by enhancing NPY concentration and NPY mRNA expression level in the hypothalamus of pigs.     

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.     

Distinct roles of the Y1 and Y2 receptors on neuropeptide Y-induced sensitization to sedation

Intracranial injection of neuropeptide Y (NPY) increases the sensitivity to sodium pentobarbital and ketamin sedation and has similar properties as GABA agonists on sleep. Mice sensitive to sedation have increased levels of NPY in many brain regions and Y1(-/-) mice show a marked resistance to barbiturates. Here we characterized the role of the NPY Y receptors in anesthetic-induced sedation. We show that Y1 and Y2, but not Y5, receptors participate in the modulation of sedation. Administration of a Y1 agonist increased the sodium pentobarbital-induced sedation and Y1(-/-) mice were less sensitive to this anesthetic. However, Y2(-/-) mice display increased sensitivity, showing that Y2 modulates GABAergic induced sedation both pharmacologically and physiologically and has a functionally opposing role to the Y1 receptor. Analysis of Y1(-/-)/Y2(-/-) double mutant mice show that increased sensitivity by Y1 occurs independent of the Y2 receptor, while the decreased sensitivity mediated by Y2 depend on an intact Y1 receptor. In contrast to sodium pentobarbital, both Y1 and Y2 receptors increase the sensitivity in a collaborative fashion to NMDA antagonist-induced sedation. These data demonstrate the physiological and pharmacological impact of the Y1 and Y2 receptors on sedation.     

Effect of neuropeptide Y microinjected into the hypothalamus on ethanol consumption

Guide cannula were implanted in rats aimed at the paraventricular nucleus (PVN) of the hypothalamus for microinjection of neuropeptide Y (NPY), D-NPY_27–36, or vehicle. In the Wistar rat, there was no significant effect on the consumption of ethanol. In Myers’ high ethanol preferring (mHEP) rats, D-NPY_27–36 caused a significant 54% decrease in ethanol consumption from baseline, but the response was not different from vehicle. NPY-induced feeding in satiated Wistar rats, was blocked by a Y₁ receptor antagonist, D-NPY_27–36. D-NPY_27–36 decreased 78% feeding in food-deprived rats. Thus, neither the Wistar nor the mHEP rat perceives ethanol as a source of calories comparable to food.     

Circulating neuropeptide Y in dog plasma consists of multiple peptide fragments

Neuropeptide Y-like immunoreactivity (NPY-LI) in dog plasma was characterized and quantified using three extraction methods (Sep-Pak:acetonitrile, HCl:ethanol, and ethanol). Sep-Pak extraction yields the best recovery and preserves the integrity of the peptide. Oxidized NPY is not generated during blood collection. Using two antisera of different specificities, at least three peptide forms in normal dog arterial and venous plasma were detected. A peptide with retention times similar to oxidized NPY or peptide YY is the major component of plasma NPY-LI under basal conditions, but NPY(1–36) predominates during sympathetic stimulation. The mature peptide in dog plasma is similar to human NPY. The antiserum ABII provides a more accurate measure of circulating NPY(1–36) and its oxidized form. The antiserum ABI is useful for detecting NPY-like fragments.     

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。     

Structural dynamics of neuropeptide hPYY

We have employed a combination of experiment and simulation to characterize the ensemble of structures sampled by human Peptide YY (hPYY), an important member of the neuropeptide Y family. Experimental structural characterization carried out with far UV circular dichroism spectroscopy and Fourier Transform-Infrared measurements confirmed that the major feature of the secondary structure of hPYY is the α-helix, encompassing about half the peptide residues, with smaller contributions from turn and β-sheet like structures. The peptide undergoes thermal denaturation characterized by a melting temperature of 48°C with an enthalpy change of -24.5 kcal/mol and entropy change of -76.2 cal/(mol K). In our computational studies, based on a 4-μsec MD trajectory generated with the AMBER03 potential, we found excellent agreement of the predicted features with experimental data, including a stable C-terminal helix, a central turn and conservation of about 80% of measured long-range NOE contacts. The main structural fluctuations involved partial helix unwinding and large-scale motions of the N-terminal. Our joint experimental/computational approach leads to several insights into the biological function of PYY. We conclude that the C-terminal helix is crucial for the structural integrity of PYY. The structures and motions found in the simulations suggest microscopic explanations for observed changes in biological activity of the peptide upon mutation and truncation. We also performed microsecond-length MD and replica-exchange simulations of hPYY with the OPLS-AA force field, for which computed structures did not agree well with experimental data, predicting significant loss of helicity and NOE contacts.     

Localization of neuropeptide Y and its C-terminal flanking peptide in human renal tissue

We produced and characterized three anti-C-flanking peptides of neuropeptide Y (CPON) monoclonal antibodies. The K_a for these antibodies ranged from 0.4 to 0.8 × 10⁸ l/mol with an IC₅₀ for CPON(1–30) at about 20 nM as determined by ELISA. All these antibodies are IgG1 and recognize the 16–30 part of CPON. These antibodies and a specific anti-NPY monoclonal antibody were used to study the localization of CPON and NPY in the human kidney. The avidin-biotin technique was employed. NPY and CPON immunoreactivities were present in large amount in the renal tubules of the human kidney but not in the glomeruli. No labeling was found within the renal arterioles and veins, but some immunoreactivity was evidenced in the perivascular area. Because no specific receptor for CPON has been described to date, the presence of this peptide in the tubules may be due to a tubular reabsorption or perhaps to a local synthesis of pro-NPY.     

Increased food intake by neuropeptide Y is due to an increased motivation to eat

Neuropeptide Y (NPY), administered intracerebroventricularly, is a potent orexigenic agent. To determine if NPY-induced eating represented an increase in motivation to eat (e.g., hunger) rather than pathological or stimulus-bound eating, we determined its effect on eating in three paradigms, including lever press, appetitive passive avoidance and quinine-adulterated milk. NPY-injected mice consumed more milk when required to work for it in a lever press apparatus and tolerated shock to the tongue for drinking milk. Increasing the dose of NPY also allowed mice to overcome a taste aversion for quinine-adulterated milk. Overall, these studies support the hypothesis that NPY causes a specific increase in the motivation to eat, rather than nonspecific or stimulus-bound behavior.