Effect of ganglion blockade with pentolinium on circulating neuropeptide Y levels in conscious rats.

The vasoconstrictor peptide, neuropeptide Y (NPY), is present in perivascular noradrenergic neurons of all mammals studied and may be important in the regulation of blood pressure. High plasma levels of NPY have been measured in the rat. To investigate partially the source and factors controlling the release of the circulating peptide, the effect of pentolinium tartrate administration has been studied in conscious rats. Pentolinium given as a bolus (5 mg/kg) followed by an infusion of a further 5 mg/kg/30 min produced a highly significant reduction in blood pressure of more than 40 mm Hg, when compared to either basal values or control animals treated with saline. Pentolinium treatment resulted in significantly lower plasma neuropeptide Y levels (31.0 +/- 6.7 fmol/ml) compared with those of control animals (78.6 +/- 8.2 fmol/ml). Circulating catecholamines were also significantly reduced in those animals receiving pentolinium. These results are compatible with circulating NPY arising from the sympathetic nervous system, with release being controlled by the mechanisms already established for catecholamines.

Body Mass Index Increase, Serum Leptin, Adiponectin, Neuropeptide Y and Lipid Levels during Treatment with Olanzapine and Haloperidol

Introduction: Body mass index (BMI) increase is an undesired effect associated with antipsychotics, and crucial for patients` global health and treatment compliance. We aimed to investigate the relation between BMI during olanzapine or halopericlol treatments and leptin, neuropeptide Y (NPY), adiponectin and lipid serum levels. Methods: In this 9-month, randomized and naturalist study, 34 male patients, 18 on olanzapine and 16 on haloperidol group were enrolled, all were under monotherapy. Patient outcome was evaluated with positive and negative syndrome scale (PANSS) at every 3-month period. In each visit, BMI, leptin, NPY, lipid, olanzapine or haloperidol levels were also monitored. Results and Discussion: Leptin levels positively correlated with BMI in olanzapine (r = 0.64, p < 0.001) and haloperidol (r = 0.73, p < 0.001) groups; only in olanzapine patients, the former also correlated with PANSS score (r = 0.54, p < 0.05). NPY levels negatively correlated with olanzapine levels (r = -0.65, p < 0.01). Adiponectin levels had not significantly varied. Conclusion: Antipsychotics probably interfere on leptin and NPY signalling ways and disturb these hormones in eating behaviour control.

Influence of sodium intake on circulating levels of neuropeptide Y.

Neuropeptide Y (NPY) is present in the adrenal medulla, in sympathetic neurons as well as in the circulation. This peptide not only exerts a direct vasoconstrictor effect, but also potentiates the vasoconstriction evoked by norepinephrine and sympathetic nerve stimulation. The vasoconstrictor effect of norepinephrine is also enhanced by salt loading and reduced by salt depletion. The purpose of this study was therefore to assess whether there exists a relationship between dietary sodium intake and the levels of circulating NPY. Uninephrectomized normotensive rats were maintained for 3 weeks either on a low, a regular or a high sodium intake. On the day of the experiment, plasma levels of NPY and catecholamines were measured in the unanesthetized animals. There was no significant difference in plasma norepinephrine and epinephrine levels between the 3 groups of rats. Plasma NPY levels were the lowest (65.4 +/- 8.8 fmol/ml, n-10, Mean +/- SEM) in salt-restricted and the highest (151.2 +/- 25 fmol/ml, n-14, p less than 0.02) in salt-loaded animals. Intermediate values were obtained in rats kept on a regular sodium intake (117.6 +/- 20.1 fmol/ml). These findings are therefore compatible with the hypothesis that sodium balance might to some extent influence blood pressure regulation via changes in circulating NPY levels which in turn modify blood pressure responsiveness.

Effects of a nonpressor dose of neuropeptide Y on cardiac output, regional blood flow distribution and plasma renin, vasopressin and catecholamine levels.

Neuropeptide Y (NPY) is a peptide with vasoconstrictor properties known to be present in the central nervous system as well as in sympathetic nerve endings and the adrenal medulla. The purposes of this study were to investigate in normotensive conscious rats the effects of nonpressor doses of NPY on cardiac output and regional blood flow distribution (using radiolabeled microspheres) as well as on plasma renin activity, plasma catecholamine and vasopressin levels. NPY (0.1 microgram/min) infused i.v. for 30 min modified neither blood pressure nor heart rate. Cardiac index was at comparable levels in NPY- as in vehicle-treated rats (17.7 +/- 1.6, n = 8, vs. 21.3 +/- 0.9 ml/min/100 g, n = 8, mean +/- S.E.M.). There was no significant difference in regional blood flow distribution between the two groups of rats, except for the large intestine (0.42 +/- 0.06 vs. 0.71 +/- 0.1 ml/min/g in NPY- and vehicle-treated rats, respectively, P less than .05). Basal plasma renin activity and catecholamine levels were not modified by NPY whereas plasma vasopressin levels were lower (P less than .05) in rats given NPY (0.76 +/- 0.3 pg/ml, n = 8) than in those having received the vehicle (2.2 +/- 0.4 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Brain levels of neuropeptide Y in experimental pneumococcal meningitis

Neuropeptide Y (NPY), which is found in high concentrations in several regions of the brain including nuclei of the brain stem and in nerve fibers surrounding cerebral vessels, has been proposed to play a role in regulating cerebral blood flow (CBF) and systemic vegetative functions. Since CBF is altered during meningitis, we examined whether NPY concentrations changed in various regions of the rabbit brain in response to experimental pneumococcal meningitis. Changes were most pronounced in the medulla, where NPY concentration increased threefold after 48 h of infection. Concomitantly, there was an increase in NPY immunoreactive fibers surrounding small vessels in the dorsolateral medulla, especially in the nucleus tractus solitarius. These results suggest that NPY may play a role in inducing some of the hemodynamic changes seen during pneumococcal meningitis.

Characterization of a selective antagonist of neuropeptide Y at the Y2 receptor. Synthesis and pharmacological evaluation of a Y2 antagonist.

Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta-turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 microM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 microM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.

Voluntary alcohol consumption is controlled via the neuropeptide Y Y1 receptor.

We have shown previously that voluntary ethanol consumption and resistance to ethanol-induced sedation are inversely related to neuropeptide Y (NPY) levels in NPY-knock-out (NPY(-/-)) and NPY-overexpressing mice. In the present report, we studied knock-out mice completely lacking the NPY Y1 receptor (Y1(-/-)) to further characterize the role of the NPY system in ethanol consumption and neurobiological responses to this drug. Here we report that male Y1(-/-) mice showed increased consumption of solutions containing 3, 6, and 10% (v/v) ethanol when compared with wild-type (Y1(+/+)) control mice. Female Y1(-/-) mice showed increased consumption of a 10% ethanol solution. In contrast, Y1(-/-) mice showed normal consumption of solutions containing either sucrose or quinine. Relative to Y1(+/+) mice, male Y1(-/-) mice were found to be less sensitive to the sedative effects of 3.5 and 4.0 gm/kg ethanol as measured by more rapid recovery from ethanol-induced sleep, although plasma ethanol levels did not differ significantly between the genotypes. Finally, male Y1(-/-) mice showed normal ethanol-induced ataxia on the rotarod test after administration of a 2.5 gm/kg dose. These data suggest that the NPY Y1 receptor regulates voluntary ethanol consumption and some of the intoxicating effects caused by administration of ethanol.

Quetiapine affects neuropeptide Y and corticotropin-releasing hormone in cerebrospinal fluid from schizophrenia patients: relationship to depression and anxiety symptoms and to treatment response.

Cumulative evidence indicates that neuropeptides play a role in the pathophysiology of schizophrenia. Early data showed increased neuropeptide Y (NPY) in cerebrospinal fluid (CSF) from schizophrenia patients and data from rodents show that antipsychotic drugs modulate NPY levels in and release from selected rat brain regions. In view of these findings we investigated whether the atypical antipsychotic quetiapine, originally used as an antipsychotic but subsequently shown to be efficient also in major depressive disorder and in both poles of bipolar disorder, would affect NPY-like immunoreactivity (-LI), and corticotropin-releasing hormone (CRH)-LI levels in CSF of schizophrenia patients. NPY-LI and CRH-LI in CSF were determined in 22 patients with schizophrenia. Lumbar puncture was performed at baseline and again after 4 wk of quetiapine treatment (600 mg/d). Patients were assessed with the Positive and Negative Syndrome Scale (PANSS) at baseline and at weekly intervals. Quetiapine treatment was associated with a significant increase in NPY-LI (p<0.001) and decrease in CRH-LI (p<0.01). Stepwise multiple regression analysis revealed that ΔNPY-LI and ΔCRH-LI levels predicted 63% (p<0.001) of the variability of the ΔPANSS total score, ΔNPY-LI 42% (p<0.05) of the ΔPANSS anxiety items (G2) and ΔCRH-LI 40% (p=0.05) of the ΔPANSS depression items (G6). These results suggest that while quetiapine's effects on monoamines are probably related to its antipsychotic properties, the modulation of NPY and CRH accounts for its antidepressant and anxiolytic effects and can be markers of response.

Neuropeptide Y secretion from a human insulinoma.

Neuropeptide Y (NPY) is a 36 aminoacid peptide known to inhibit glucose-stimulated insulin secretion. NPY has been shown to be synthesized and secreted by rat islets of Langerhans. More recently, we described the presence on NPY within human islets of Langerhans and in several pancreatic endocrine tumors. In this report, we describe the case of a patient presenting with an insulinoma who underwent the surgical resection of the tumor and was studied in vivo and in vitro for NPY production. Using a highly specific and sensitive two-site amplified enzyme-linked immunosorbent assay, we detected high plasma NPY levels in the patient prior to the surgical resection of the tumor which returned to normal after surgery. NPY was secreted from the tumor when kept in primary cell culture. Furthermore, immunohistochemistry of the insulinoma revealed the presence of NPY and its C-flanking peptide together with insulin, chromogranin and neuron specific enolase. It is concluded that elevated circulating NPY levels observed in this patient with an insulinoma reflected in vivo secretion by the tumor and it is hypothesized that NPY could potentially be used as an endocrine marker in patients with suspected insulinoma.

Beta-adrenoceptor stimulation increases neuropeptide Y release from sympathetic nerves in intact rats.

This study was undertaken to assess in conscious normotensive rats the effects of beta-adrenoceptor stimulation on plasma neuropeptide Y (NPY) levels. Wistar rats were subjected to adrenal demedullation on the right side and were either adrenalectomized or sham-operated on the left side. Eleven days later, the conscious rats were infused i.v. for 30 min with either isoproterenol (10 ng/min) or its vehicle. Plasma NPY levels were significantly lower (23.8 +/- 2.6 pM, means +/- S.E.M., n = 12, P < 0.01) in vehicle-treated medullectomized rats than in corresponding sham-operated controls (36.7 +/- 4.1 pM, n = 12). The medullectomized rats infused with isoproterenol showed plasma NPY levels (36.7 +/- 3.3 pM, n = 11) comparable to those of sham-operated rats having received the vehicle. These data therefore demonstrate that plasma NPY levels are lower in rats without adrenal medulla and that in these animals isoproterenol increases NPY release, most likely by activating pre-synaptic beta-adrenoceptors.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

Neuropeptide Y expression and regulation in a differentiated rat insulin-secreting cell line.

Neuropeptide-Y (NPY) is a 36-amino acid peptide known to inhibit glucose-stimulated insulin secretion in various animal models in vitro and in vivo. NPY is thought to be one of the mediators of sympathetic action in the pancreas through nerve endings surrounding the islets, and it has recently been shown to be synthesized within the islets of Langerhans. To elucidate the potential role of NPY in the endocrine pancreas, we studied the expression and regulation of NPY secretion in a rat insulinoma cell line (INS-1). NPY mRNA and peptide are highly expressed and secreted by INS-1 cells. NPY levels were determined by a sensitive and specific two-site amplified enzyme-linked immunosorbent assay. Incubation of INS-1 cells with various glucose concentrations did not modify NPY secretion; however, stimulation of adenylate cyclase by forskolin induced a dose- and time-dependent increase in NPY release in the medium. The glucagon-like peptide-I-(7-36) amide (GLP-1), a known gluco-incretin in humans, induced at low concentration (10(-9) M) a similar expression of NPY mRNA and peptide secretion in INS-1 cells. On the other hand, the inhibition of cAMP accumulation by the alpha 2-adrenergic agonist clonidine decreased NPY secretion. In conclusion, 1) high levels of gene expression and secretion of NPY are found in a rat insulinoma cell line (INS-1). 2) Accumulation of cAMP induced by forskolin or a gluco-incretin (GLP-1) induces a further increase in NPY gene expression and release. 3) NPY secretion is not modulated by low or high glucose concentrations in the medium. 4) Induction of NPY, a known inhibitor of insulin secretion, may represent a novel counterregulatory mechanism of insulin secretion, limiting the stimulatory effect of GLP-1 on insulin secretion.

Transgenic mice overexpressing neuropeptide Y: An experimental model of metabolic and cardiovascular diseases

Neuropeptide Y (NPY) is an abundant neurotransmitter in the brain and sympathetic nervous system (SNS). Hypothalamic NPY is known to be a key player in food intake and energy expenditure. NPY’s role in cardiovascular regulation has also been shown. In humans, a Leucine 7 to Proline 7 single nucleotide polymorphism (p.L7P) in the signal peptide of the NPY gene has been associated with traits of metabolic syndrome. The p.L7P subjects also show increased stress-related release of NPY, which suggests that more NPY is produced and released from SNS. The main objective of this study was to create a novel mouse model with noradrenergic cell-targeted overexpression of NPY, and to characterize the metabolic and vascular phenotype of this model. The mouse model was named OE-NPY^DBH mouse. Overexpression of NPY in SNS and brain noradrenergic neurons led to increased adiposity without significant weight gain or increased food intake. The mice showed lipid accumulation in the liver at young age, which together with adiposity led to impaired glucose tolerance and hyperinsulinemia with age. The mice displayed stress-related increased mean arterial blood pressure, increased plasma levels of catecholamines and enhanced SNS activity measured by GDP binding activity to brown adipose tissue mitochondria. Sexual dimorphism in NPY secretion pattern in response to stress was also seen. In an experimental model of vascular injury, the OE-NPY^DBH mice developed more pronounced neointima formation compared with wildtype controls. These results together with the clinical data indicate that NPY in noradrenergic cells plays an important role in the pathogenesis of metabolic syndrome and related diseases. Furthermore, new insights on the role of the extrahypothalamic NPY in the process have been obtained. The OE-NPY^DBH model provides an important tool for further stress and metabolic syndrome-related studies.

Neuropeptide Y at the cellular level – Studies on PreproNPY L7P Polymorphism and the Mitochondrial Form of NPY

Neuropeptide Y (NPY) is a widely expressed neurotransmitter in the central and peripheral nervous systems. Thymidine 1128 to cytocine substitution in the signal sequence of the preproNPY results in a single amino acid change where leucine is changed to proline. This L7P change leads to a conformational change of the signal sequence which can have an effect on the intracellular processing of NPY. The L7P polymorphism was originally associated with higher total and LDL cholesterol levels in obese subjects. It has also been associated with several other physiological and pathophysiological responses such as atherosclerosis and T2 diabetes. However, the changes on the cellular level due to the preproNPY signal sequence L7P polymorphism were not known. The aims of the current thesis were to study the effects of the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes in primary cultured and genotyped human umbilical vein endothelial cells (HUVEC), in neuroblastoma (SK-N-BE(2)) cells and in fibroblast (CHO-K1) cells. Also, the putative effects of the L7P polymorphism on proliferation, apoptosis and LDL and nitric oxide metabolism were investigated. In the course of the studies a fragment of NPY targeted to mitochondria was found. With the putative mitochondrial NPY fragment the aim was to study the translational preferences and the mobility of the protein. The intracellular distribution of NPY between the [p.L7]+[p.L7] and the [p.L7]+[p.P7] genotypes was found to be different. NPY immunoreactivity was prominent in the [p.L7]+[p.P7] cells while the proNPY immunoreactivity was prominent in the [p.L7]+[p.L7] genotype cells. In the proliferation experiments there was a difference in the [p.L7]+[p.L7] genotype cells between early and late passage (aged) cells; the proliferation was raised in the aged cells. NPY increased the growth of the cells with the [p.L7]+[p.P7] genotype. Apoptosis did not seem to differ between the genotypes, but in the aged cells with the [p.L7]+[p.L7] genotype, LDL uptake was found to be elevated. Furthermore, the genotype seemed to have a strong effect on the nitric oxide metabolism. The results indicated that the mobility of NPY protein inside the cells was increased within the P7 containing constructs. The existence of the mitochondria targeted NPY fragment was verified, and translational preferences were proved to be due to the origin of the cells. Cell of neuronal origin preferred the translation of mature NPY (NPY1-36) when compared to the non neuronal cells that translated both, NPY and the mitochondrial fragment of NPY. The mobility of the mitochondrial fragment was found to be minimal. The functionality of the mitochondrial NPY fragment remains to be investigated. L7P polymorphism in the preproNPY causes a series of intracellular changes. These changes may contribute to the state of cellular senescence, vascular tone and lead to endothelial dysfunction and even to increased susceptibility to diseases, like atherosclerosis and T2 diabetes.

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.