Neuropeptide Y (NPY) and the pig heart: release and coronary vasoconstrictor effects

The effects of electrical stimulation of the stellate ganglia on the arterio-venous concentration differences of neuropeptide Y (NPY)-like immunoreactivity (LI) over the pig heart were studied in vivo in relation to changes in heart rate and left ventricular pressure. Furthermore, the effects of NPY on coronary vascular tone were analysed in vivo and in vitro. Stellate ganglion stimulation at a high frequency (10 Hz) caused a clear-cut, long lasting increase in plasma levels of NPY-LI in the coronary sinus compared to the aorta, suggesting release of this peptide from sympathetic terminals within the heart. The stimulation-evoked overflow of NPY-LI from the heart was enhanced about 3-fold by alpha-adrenoceptor blockade using phenoxybenzamine, suggesting that NPY release is under prejunctional inhibitory control by noradrenaline (NA). Combined alpha- and beta-adrenoceptor blockade abolished most of the positive inotropic response of the heart upon stellate ganglion stimulation, while a considerable positive chronotropic effect remained. After guanethidine treatment, stellate ganglion stimulation still produced a small positive inotropic and chronotropic effect on the heart. The stimulation evoked NPY overflow was markedly reduced by guanethidine indicating an origin from sympathetic nerve terminals. Injection of NPY into the constantly perfused left anterior descending artery in vivo caused a long lasting, adrenoceptor antagonist resistant increase in perfusion pressure, suggesting coronary vasoconstriction. NPY contracted coronary arteries in vitro via a nifedipine-sensitive mechanism. NA dilated coronary vessels both in vivo and in vitro via beta-adrenoceptor activation. It is concluded that sympathetic nerve stimulation increases overflow of NPY-LI from the heart suggesting release from cardiac nerves in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Neuropeptide Y (NPY) on the release of anterior pituitary hormones in the rat

Neuropeptide Y (NPY) has been recently localized in several hypothalamic nuclei in the mammalian brain. In order to investigate the possible role of NPY on neuroendocrine function, we have investigated the effects of the peptide on the release of anterior pituitary hormones in the rat. Both intravenous (300 μg) or intraventricular (2 to 15 μg) injection of NPY produced in gonadectomized male rats a significant and long-lasting decrease of plasma LH levels. A short duration stimulating effect on prolactin plasma levels was also observed after the intravenous but not after the intraventricular injection of NPY. Plasma levels of the other pituitary hormones were not significantly modified after NPY injection. When incubated in vitro with anterior pituitary cells in monolayer culture, NPY produced no significant change in release of pituitary hormones. Thus NPY seems to exert a selective effect on LH release. Since this effect can be observed after both intravenous and intraventricular injection, it might be hypothesized that NPY could affect LHRH release in two areas which lack blood-brain barrier: the organum vasculosum of the lamina terminalis (OVLT) which contains LHRH cell bodies and NPY fibers and the median eminence which contains both LHRH and NPY fibers. The effect on prolactin release needs to be carefully evaluated in different experimental conditions.     

Neuropeptide Y (NPY) in alcohol intake and dependence

Neuropeptide Y has a role in alcohol intake and dependence. NPY's effect on alcohol intake appears to be in part dependent on the individual's history of alcohol dependence. In models of high intake such as alcohol-preferring, selectively bred rat lines (e.g., the P-line and the HAD line), as well as in ethanol-vapor-exposed subjects, NPY modulates alcohol intake while leaving it unaffected during baseline conditions. The primary receptor subtype mediating NPY's effect on ethanol intake remains in question. The Y2-antagonist BIIE0246 significantly suppresses ethanol intake in an operant paradigm with a sensitization to the effect of BIIE0246 in vapor-exposed subjects. We propose the NPY system to be one of the most interesting target systems for the development of treatments for alcohol abuse and dependence.     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen, progesterone-treated rats

We studied the effects on plasma LH levels of intracerebroventricular (ICV) administration of neuropeptide Y (NPY), NPY analog (NPY-A), galanin (GAL) and neuropeptide K (NPK) in ovariectomized (ovx) and in ovx rats pretreated with estradiol benzoate (EB) and progesterone (P). Plasma LH levels were estimated in blood drawn from an intrajugular cannula before (0 min) and at 10, 20, 30 and 60 min after the ICV injection of either saline (3 microliter) or one of the neuropeptides in saline. The three classes of peptides elicited different LH responses in the two experimental paradigms. NPY and NPY-A (0.5 or 2 micrograms) decreased LH release in ovx rats and stimulated LH release in EBP ovx rats. However, GAL (0.5, 2 or 10 micrograms) failed to suppress LH release in ovx rats, but it readily increased plasma LH levels in a dose-related fashion in EBP ovx rats. In contrast, NPK readily decreased LH release in ovx rats in a time-related fashion for up to 60 min, but was mildly effective in EBP ovx rats as only a high dose of 10 micrograms produced a small significant increase. Collectively, our results show that (1) NPY can differentially effect LH release in ovx and EBP ovx rats but this property is not equally shared by the neuropeptides that have a similar anatomical disposition in the hypothalamus and (2) the excitatory effects of GAL are demonstrable in the steroid-primed rats and the inhibitory effects of NPK are apparent in the steroid-unprimed ovx rats. Since NPK induced a long-lasting marked suppression with little evidence of LH excitation at low doses, we speculate that either NPK alone or in conjunction with other peptides may mediate the suppression of LH release induced by gonadal steroids.     

Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells

The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y(2)) and hPP (Y(4)), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R + G?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?36)) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.     

Neuropeptide Y (NPY) and peptide YY (PYY) receptors in rat brain

1. Specific binding sites for neuropeptide Y (NPY) and peptide YY (PYY) were investigated in rat brain areas using quantitative receptor autoradiography with 125I-Bolton-Hunter NPY (125I-BH-NPY) and 125I-PYY, radioligands for PP-fold family peptides receptors. 2. There were no differences between localization of 125I-BH-NPY and 125I-PYY binding sites in the rat brain. High densities of the binding sites were present in the anterior olfactory nucleus, lateral septal nucleus, stratum radiatum of the hippocampus, posteromedial cortical amygdaloid nucleus, and area postrema. 3. In cold ligand-saturation experiments done in the presence of increasing concentrations of unlabeled NPY and PYY, 125I-BH-NPY and 125I-PYY binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, molecular layer of the cerebellum, and area postrema was single and of a high affinity. There was a significant difference between the affinities of 125I-BH-NPY (Kd = 0.96 nM) and 125I-PYY binding (Kd = 0.05 nM) to the molecular layer of the cerebellum. The binding of the two radioligands to the other areas examined had the same affinities. 4. When comparing the potency of unlabeled rat pancreatic polypeptide (rPP), a family peptide of NPY and PYY, to inhibit the binding to the areas examined, rPP displaced 125I-BH-NPY and 125I-PYY binding to the area postrema more potently than it did the binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, and molecular layer of the cerebellum. 5. Thus, the quantitative receptor autoradiographic method with 125I-BH-NPY and 125I-PYY revealed differences in binding characteristics of specific NPY and PYY binding sites in different areas of the rat brain. The results provide further evidence for the existence of multiple NPY-PYY receptors in the central nervous system.     

Pancreatic hormone response to neuropeptide Y (NPY) perifusion in vitro

Available data on the effect of neuropeptide Y (NPY) on insulin release are conflicting and little data exist regarding the effect of NPY on glucagon secretion. The purpose of the present study, therefore, was to characterize the direct effect of NPY on the release of these pancreatic hormones and to examine the role of glucose on these interactions. Using a perifused mouse islet system, we found that NPY suppressed both basal and glucose-stimulated insulin secretion. Thus, basal insulin release assessed as mean integrated area under the curve/20 min (AUC/20 min) decreased from 1446 +/- 143 pg to 651 +/- 112 pg (P less than 0.05) with the addition of 2 x 10(-8) M NPY and the AUC/20 min for glucose stimulated insulin output decreased from 1973 +/- 248 pg to 1426 +/- 199 pg (P less than 0.05). In both cases, this inhibitory effect was followed after removing NPY by a stimulation of insulin secretion which was typical of a 'rebound off-response'. In contrast, NPY exerted a stimulatory effect on basal glucagon release and significantly reversed the suppressive effect of high glucose on glucagon output. The basal glucagon AUC/20 min increased from 212 +/- 103 pg to 579 +/- 316 pg (P less than 0.05), while glucagon secretion in the presence of 27.7 mM glucose increased from 75 +/- 26 pg to 255 +/- 28 pg (P less than 0.01). In conclusion, we have shown that the direct effect of NPY on the endocrine pancreas is to suppress insulin but stimulate glucagon secretion. These data are compatible with a role for NPY in the regulation of pancreatic hormone output.     

Immunocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus

Summary In order to study the distribution of neuropeptide Y-like immunoreactivity in the human hypothalamus, an immunocytochemical localization of this peptide was performed. Using antibodies developed against synthetic porcine neuropeptide Y (NPY), we have been able to localize immunoreactivity in neuronal cell bodies located exclusively in the infundibular nucleus. Immunostained fibers were found in several regions in the hypothalamus with a high concentration in the periventricular areas. Fibers were also found in the neurovascular zone of the median eminence, the pituitary stalk and the posterior pituitary. These results suggest that immunoreactive material related to porcine NPY is present in the human hypothalamus, with a distribution similar to that observed in the rat.     

Norepinephrine-like effects of neuropeptide Y on LH release in the rat

Neuropeptide Y, a recently isolated neuropeptide exhibited norepinephrine-like effects on LH release after intracerebroventricular administration at doses from 0.5 to 10 micrograms. While it promptly suppressed LH release in ovariectomized rats, there was a dose-related stimulation of LH secretion in ovarian steroid primed-ovariectomized rats. In view of the evidence that neuropeptide Y may coexist with adrenergic neurotransmitters, these findings suggest that it may play a role in regulation of LH release in the rat, either independently or in concert with catecholamines.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

神经肽Y(NPY)的生理功能研究进展

神经肽Y(NPY)是机体内的一种重要且保守的神经递质,一般以前体形式存在,释放的有活性的NPY主要通过与其受体结合发挥作用。NPY受体包含了亚型Y1、Y2、Y3、Y4、Y5、Y6、Y7、Y8。Y1和Y2是NPY发挥收缩血管作用的关键受体;Y1、Y2和Y5是NPY调节动物摄食行为的关键受体;Y1、Y2和Y4是NPY调控动物焦虑、沮丧行为的必要受体。着重对NPY与其各种受体结合后如何行使动物的相关生理功能的情况进行了阐述。     

Neuropeptide Y (NPY)-induced feeding behavior in female rats: comparison with human NPY ([Met17]NPY), NPY analog ([norLeu4]NPY) and peptide YY

Porcine neuropeptide Y (pNPY) administered into the third ventricle of the brain is known to elicit a powerful feeding response in steroid-treated ovariectomized and intact male rats. The present study compared the effects of pNPY and 3 structurally related peptides, human NPY (hNPY), an analog of NPY (NPY-A, [norLeu4]NPY) and peptide YY (PYY) on feeding behavior in intact female rats. Intraventricular administration of pNPY, hNPY, NPY-A and PYY over a dose range of 0.5 to 10 micrograms evoked feeding behavior to a varying extent. Cumulative food intake during 60 and 120 min was increased in a dose-related fashion at 0.5 and 2.0 microgram for the 4 peptides. Whereas the 10-micrograms dose of pNPY evoked a feeding response smaller than that seen after 2 micrograms, the responses to either 10 micrograms hNPY or 10 micrograms PYY were similar to that seen after 2 micrograms. The effects of these peptides on the time spent eating were quite different: while pNPY increased the time spent eating, this effect was not dose-related, whereas hNPY, NPY-A and PYY produced dose-related increments in the time spent eating. The most dramatic increment in local eating rate was observed after 2.0 micrograms pNPY, with lesser increments seen after 2.0 microgram hNPY and NPY-A. This increased local eating was apparently responsible for the highest cumulative food intake observed. These results demonstrate that (a) 2 micrograms pNPY is equally effective in stimulating feeding behavior in intact female rats as it is in steroid-primed ovariectomized female and intact male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Summary Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Neuropeptide Y (NPY) in neuroblastoma: effect on growth and vascularization

Neuroblastomas are pediatric tumors of sympathetic origin, expressing neuronal markers, such as NPY and its receptors. Due to this, neuroblastomas are often associated with elevated plasma levels of NPY, which correlates with poor clinical outcome of the disease. This clinical data corroborates the recent discovery of growth-promoting actions of NPY in neuroblastomas. The peptide has been shown to stimulate proliferation of neuroblastoma cells in an autocrine manner and induce tumor vascularization. Since both processes are mediated by the same Y2 and Y5 receptors, targeting this pathway may be a potential bidirectional therapy for these children's tumors.     

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.     

Neuropeptide Y (NPY) and NPY receptors in the cardiovascular system: implication in the regulation of intracellular calcium

The 3-dimensional confocal microscopy technique has allowed us to identify the presence of yet another cardioactive factor and its receptor, namely neuropeptide Y (NPY) and its Y1 receptor, at the level of vascular smooth muscle cells and heart cells including endocardial endothelial cells (EECs). Using this technique, we also demonstrated that NPY is able to induce an increase in both cytosolic and nuclear calcium in all these cell types. Furthermore, besides being expressed at the level of EECs, NPY is also released from these cells following a sustained increase of intracellular Ca2+. This suggests the ability of NPY to contribute to the regulation of the excitation-secretion coupling of EECs and the excitation-contraction coupling of cardiomyocytes and vascular smooth muscle cells.