The role of non-synonymous NPY gene polymorphism in the nitric oxide production in HUVECs

A Leu7Pro change in the signal peptide of preproNPY is a functional substitution, which changes the processing of NPY in cells and associates with several cardiovascular and metabolic conditions in humans. The current study investigates the effect of the P7 allele in endothelial cells, where decreased nitric oxide (NO) production is a promoting factor to endothelial dysfunction. The function of NO system was assessed in the human umbilical vein endothelial cells (HUVECs) with [p.L7]+[p.L7] or [p.L7]+[p.P7] genotype. NPY seems to have a significant influence on NO system in HUVECs, and the responses are time and genotype dependent. HUVECs with [p.L7]+[p.P7] genotype seem to have higher basal production of NO, but after a long term treatment with NPY these cells express less eNOS mRNA and overall eNOS protein levels are lower. These significant differences in the NO bioavailability may explain the association of the L7P polymorphism with several cardiovascular complications.     

The cardiovascular actions of centrally administered neuropeptide Y

The cardiovascular actions of intracerebroventricular (i.c.v.) administration of neuropeptide Y (NPY) were examined in conscious, unrestrained rats. A prolonged decrease in heart rate (HR) and a fall in mean arterial pressure (MAP) were obtained following i.c.v. administration of NPY (1 and 10 micrograms). Passive immunization with an antiserum directed against NPY confirmed that the slowing of HR following i.c.v. administration of NPY was mediated via a central nervous mechanism and not from leakage of NPY out of the brain. Administration of NPY into different brain parenchymal regions identified a putative site of action in the rostral region of the solitary tract. The mechanism of the decrease in HR caused by centrally administered NPY was investigated by i.c.v. administration of NPY to animals that were pretreated with agents that altered autonomic tone. Administration of NPY to atropine-treated animals produced a reversal of the atropine-induced tachycardia, suggesting that the NPY-induced decrease in HR was not due to augmented vagal tone. However, administration of NPY to animals pretreated with propranolol did not significantly lower HR below that obtained with propranolol alone. These data suggest that i.c.v. administration of NPY may cause a decrease in cardiac sympathetic outflow. The effects of centrally administered NPY on baroreflex function were studied. The changes in HR caused by NPY did not significantly alter baroreflex set-point or gain. These studies provide evidence that NPY acted within a brainstem region to decrease sympathetic nervous outflow, resulting in a decrease in HR and MAP.     

Cathepsin L participates in the production of neuropeptide Y in secretory vesicles, demonstrated by protease gene knockout and expression

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore , in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.     

Pressor effect of centrally administered neuropeptide Y in rats: role of sympathetic nervous system and vasopressin

The haemodynamic effects of intracerebroventricular (i.c.v.) administration of neuropeptide Y (NPY) in urethane-anaesthetized rats were studied. In Sprague-Dawley rats, NPY increased both blood pressure and heart rate in a dose-dependent manner. This response was unaffected by removal of the adrenal medullae or pretreatment with a specific vasopressin antagonist (180 ng/kg i.v.), but was abolished by phenoxybenzamine (1mg/kg i.v.). After pretreatment with propranolol (1mg/kg i.v.), the tachycardia was inhibited and the pressor response was of shorter duration than in controls. In 6-hydroxydopamine treated rats (two doses of 250 micrograms i.c.v., three days apart), NPY still elicited a pressor response and tachycardia, which were significantly higher than controls 15 minutes after the injection. Plasma levels of vasopressin were not altered by i.c.v. administration of NPY. However, in Brattleboro rats the peptide had no haemodynamic effects. Our results suggest that activation of sympathetic nervous system but not release of vasopressin or adrenal catecholamines into the bloodstream mediates the cardiovascular response to NPY. Central vasopressin pathways however may be involved.     

Adaptive responses in hypothalamic neuropeptide Y in the face of prolonged high-fat feeding in the rat

While a dysregulation in neuropeptide Y (NPY) signaling has been described in rodent models of obesity, few studies have investigated the time-course of changes in NPY content and responsiveness during development of diet-induced obesity. Therefore we investigated the effect of differing lengths (2-17 weeks) of high-fat diet on hypothalamic NPY peptide content, release and NPY-induced hyperphagia. Male Sprague-Dawley rats (211 +/- 3 g) were fed either a high-fat diet (30% fat) or laboratory chow (5% fat). Animals were implanted with intracerebroventricular cannulae to investigate feeding responses to NPY (0.5 nmol, 1 nmol) after 4 or 12 weeks of diet. At the earlier stage of obesity, NPY-induced hyperphagia was not altered; however, animals maintained on the high-fat diet for the longer duration were hyper-responsive to NPY, compared to chow-fed control rats (p < 0.05). Overall, hypothalamic NPY peptide content tended to be decreased from 9 to 17 weeks of diet (p < 0.05). Total hypothalamic NPY content was negatively correlated with plasma leptin concentration (p < 0.05), suggesting the hypothalamic NPY system remains responsive to leptin's inhibitory signal. In addition, hypothalamic NPY overflow was significantly reduced in high-fat fed animals (p < 0.05). Together these results suggest a reduction in hypothalamic NPY activity in high-fat fed animals, perhaps in an attempt to restore energy balance.     

Role of atrial natriuretic peptides and neuropeptide Y in blood pressure regulation

Atrial natriuretic peptides (ANP) are released into the circulation in response to enhanced atrial stretching. These peptides not only have diuretic and natriuretic properties, but also exert a relaxing effect on the vasculature. Moreover, they antagonize the contractions induced by norepinephrine and angiotensin II. Neuropeptide Y (NPY) is also a vasoactive peptide. It is widely distributed throughout the central and peripheral nervous systems. NPY is coreleased with norepinephrine by perivascular nerve endings. At high concentrations, this peptide has a direct vasoconstrictor effect. In addition, it enhances the vascular effect of various agonists, including norepinephrine and angiotensin II. Both ANP and NPY have an inhibitory effect on renin secretion. This effect may have important implications for the role of these peptides in cardiovascular regulation.     

Neuropeptide Y: a putative neurotransmitter

Since its isolation in 1982, neuropeptide Y (NPY) has received considerable interest. This 36 amino acid peptide has been identified widely throughout the central and peripheral nervous systems, and within the autonomic system it appears in close association but not exclusively within catecholamine containing nerves. NPY begins to meet some of the criteria required to be established as a neurotransmitter. Thus, the peptide has been localised exclusively within nerves, and electron microscopy has shown NPY within nerve terminals. High affinity, saturable binding sites for NPY have been demonstrated in rat brain membranes, and the peptide has been reported to be released into the circulation during sympathetic nerve stimulation. The peptide is pharmacologically active both within the central nervous system by altering blood pressure, feeding and anterior pituitary function and in the periphery where NPY acts as a vasoconstrictor.     

Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system

Neuropeptide Y (NYP) is a 36 amino acid peptide which shares considerable sequence homology with pancreatic polypeptide and peptide YY. NPY is widely distributed within neurons of the central and peripheral nervous systems, and occurs in mammalian brain in higher concentrations than all other peptides studied to date. Radioimmunoassay studies demonstrated high concentrations of NPY immunoreactivity within many regions of the hypothalamus and within the paraventricular thalamic nucleus, nucleus accumbens, the septum and medial amygdala. These findings correspond with the distribution of NPY containing terminals. Numerous cell bodies containing NPY are located within the cerebral cortex, caudate-putamen, hippocampus, hypothalamus, and nucleus tractus solitarius. Central administration of NPY causes a marked increase in ingestive behaviors, possibly related to the release of NPY from neurons in the arcuate nucleus that innervate the paraventricular nucleus of the hypothalamus. NPY projections from the arcuate nucleus to the medial preoptic area may be related to the central effects of NPY on luteinizing hormone release and sexual behavior. NPY immunoreactive terminals heavily innervated neurons within the amygdala and hypothalamus that are connected to the dorsal vagal complex, suggesting a role of NPY in central autonomic regulation. NPY terminals form a dense plexus around cerebral vessels and are probably responsible for NPY's potent vasoconstrictor effects in the cerebral cortex. Coronary vessels are also innervated heavily by NPY terminals, indicating a role for NPY in the pathogenesis of coronary vasospasm. NPY is present in pheochromocytomas and circulating levels of NPY may prove useful in the diagnosis of pheochromocytoma. Thus, anatomical and physiological studies suggest a varied, but important, function for NPY in mammalian nervous system.     

Comparison of the effects of neuropeptide Y (NPY) and 4-norleucine-NPY on isolated perfused rat hearts; effects of NPY on atrial and ventricular strips of rat heart and on rabbit heart mitochondria

Isolated perfused rat hearts were used to compare the effects of the synthetic neuropeptide Y (NPY) and 4-norleucine-NPY on cardiac function. Each peptide exhibited both negative inotropic and chronotropic effects, and also caused coronary vasoconstriction leading to a reduction in coronary flow. A comparison of the IC50 values from dose-response curves using 10(-14) to 10(-7) M peptides (IC50 is the peptide concentration that produced a 50% decrease of the maximal effect) indicated that NPY was more potent as inhibitor of contractility and less potently inhibited coronary flow and heart rate, whereas 4-norleucine-NPY had more inhibitory influence on coronary flow and heart rate and less on cardiac contractility. This difference in potencies suggests that the inhibitory effects of NPY on contractility, coronary flow and heart rate may be independent of each other. Since NPY also decreased the contractile force of isolated left atrial and right ventricular strips of the rat heart, the coronary flow decrease cannot be the cause of the negative inotropy of isolated heart. Pretreatment of atrial and ventricular strips with NPY did not influence the positive inotropic effect produced by the cardiac glycoside ouabain indicating that sarcolemmal Na+, K+-ATPase was not involved in the inhibitory inotropic effect of NPY. Further studies towards elucidating the mechanism of the negative inotropy of cardiac muscles using isolated heart mitochondria revealed that NPY uncoupled oxidative phosphorylation and blocked mitochondrial calcium uptake; the former event fosters negative inotropy. Since these effects on mitochondria occurred at concentrations 100-fold higher than those required for negative inotropy, the two effects of NPY may not be related.     

Stimulation of NPY Y2 receptors by PYY3-36 reveals divergent cardiovascular effects of endogenous NPY in rats on different dietary regimens

In the present experiments the gut hormone peptide YY3-36 (PYY3-36), which inhibits neuropeptide Y (NPY) release, was used as a tool to study the cardiovascular effects of endogenous NPY under different dietary regimens in rats instrumented with a telemetry transmitter. In a first experiment, rats were placed on a standard chow diet ad libitum and in a second experiment on a high-fat diet ad libitum. After 6 wk, PYY3-36 (300 microg/kg) or vehicle was injected intraperitoneally. In a third experiment, PYY3-36 or vehicle was administered after 14 days of 50% restriction of a standard chow diet. In food-restricted rats, PYY3-36 increased mean arterial pressure (7 +/- 1 mmHg, mean +/- SE, P < 0.001 vs. saline, 1-way repeated-measures ANOVA with Bonferroni t-test) and heart rate (22 +/- 4 beats/min, P < 0.001) during 3 h after administration. Conversely, PYY3-36 did not influence mean arterial pressure (0 +/- 1 mmHg) and heart rate (-8 +/- 5 beats/min) significantly in rats on a high-fat diet. Rats fed standard chow diet ad libitum showed an intermediate response (mean arterial pressure 4 +/- 1 mmHg, P < 0.05, and heart rate 5 +/- 2 beats/min, not significant). Thus, in our studies, divergent cardiovascular responses to PYY3-36 were observed in rats on different dietary regimens. These findings suggest that the cardiovascular effects of PYY3-36 depend on the hypothalamic NPY release, which is increased after chronic food restriction and decreased during a high-fat diet.     

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.     

Selection and characterization of a 7‐mer peptide binding to divalent cations

A 7‐mer peptide (S‐T‐L‐P‐L‐P‐P) that bound to various divalent cations was selected from a phage display peptide library. Isothermal calorimetric analysis revealed that the peptide bound to Pb²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. Through the use of CD studies, no secondary structural changes were observed for the peptide upon binding to divalent cations. Ala scanning mutant peptides bound to Hg²⁺ with a reduced affinity. However, no single substitution was shown to affect the overall affinity. We suggest that Pro residues chelate divalent cations, while the structure formed by the peptide is also important for the binding process. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON)

Neuropeptide tyrosine (NPY) is one of the most abundant and widespread peptides in the mammalian nervous system. Recent isolation and sequencing of the DNA encoding NPY has predicted the existence of a 97 amino acid precursor peptide. Proteolytic processing of this precursor could yield three separate peptide products, an N-terminal signal peptide, neuropeptide tyrosine and a 30 amino acid C-terminal flanking peptide (C-PON). Here, we present evidence that the predicted C-flanking peptide of NPY is widely distributed in both the central and peripheral nervous systems of several mammalian species including man, and has an identical distribution to NPY. It was also demonstrated, using correlative light microscopic immunostaining on serial sections and double electron microscopic immunocytochemistry, that C-PON and NPY immunoreactivities are co-localized in neuronal cell bodies of the brain cortex, sympathetic ganglion cells, norepinephrine-containing granules of the adrenal medulla and in human pheochromocytoma tumor cells.     

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes

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

Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries.

A sparse to moderate supply of nerve fibers containing neuropeptide Y-like immunoreactivity (NPY-LI), vasoactive intestinal polypeptide (VIP-LI), substance P (SP-LI), and calcitonin gene-related peptide (CGRP-LI) was demonstrated in the walls of human middle meningeal arteries. Comparison with similar studies on human cerebral and temporal arteries indicated a similar distribution and density. The immunoreactive material in all three arterial regions was characterized by reversed-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA). The major peak of NPY-LI, VIP-LI, SP-LI, and CGRP-LI in each extract eluted approximately with the same elution volume as that of the corresponding synthetic analogues. The concentration of NPY in the middle meningeal arteries was lower as compared to the temporal arteries. Low concentrations of SP-LI and CGRP-LI were found in the middle meningeal arteries as compared to the cerebral arteries. In isolated ring segments of human middle meningeal and cerebral arteries, NPY caused vasoconstriction but did not potentiate the contractile response of noradrenaline. In the temporal artery, NPY did not induce contraction but potentiated the vasoconstrictor response to noradrenaline. Vasoactive intestinal polypeptide, peptide histidine methionine-27, SP, neurokinin A, and CGRP relaxed all three types of cephalic arteries. The peptide effects were not antagonized by propranolol, atropine, or cimetidine. Comparison of the responses to VIP and SP of vessels from the different regions showed a similar pattern of reactivity. The response to SP was slightly (p less than 0.05) more potent, whereas the responses to CGRP were less potent in the middle meningeal as compared to that in cerebral (p less than 0.005) vessels.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS)

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations. We showed that R270C substitution strongly reduced β4GalT7 affinity towards xyloside acceptor, thus affecting GAG chains formation. This study establishes the molecular basis for β4GalT7 defects associated with altered GAG synthesis in EDS.