Identification of an NPY-Y1 receptor subtype in bovine chromaffin cells

Bovine chromaffin cells have been used in a variety of studies designed to reveal different aspects of neuropeptide Y (NPY) action. Pharmacological data have defined five NPY receptor subtypes, only one of which (Y3) has not been cloned. Some studies with bovine chromaffin cells have concluded that the effects of NPY on this cell type are mediated by the Y3 subtype. Previous work from our laboratory demonstrates that a Y1 subtype mediates the effect of NPY in this tissue. In the current studies we provide further evidence for the existence of the Y1 subtype in bovine chromaffin cells. BIBP3226, the selective Y1 antagonist, potently displaces [125I]NPY from its binding site IC50 = 1.91 x 10(-9) M. Moreover, [125I]BIBP3226 binds to bovine chromaffin cell membranes with high affinity (IC50 = 5.9 x 10(-8) M). Examination of BIBP3226 antagonism of NPY inhibition of forskolin stimulated cyclic AMP accumulation reveals that it is a competitive antagonist with a K(B) similar to the IC50 for [125I]BIBP3226 binding. Northern blot analysis using a porcine cDNA clone for the Y1 subtype demonstrates a 3.5-kb mRNA species in chromaffin cells. These data identify the bovine chromaffin cell NPY receptor as a Y1 subtype.     

Effects of BIBP3226 and BIBP3435 on cytosolic calcium in neuropeptide Y Y1 receptor-transfected Chinese hamster ovary cells and wild type CHO-K1 cells.

The NPY Y1-receptor selective antagonist BIBP3226 exerts a dual control on the cytosolic free calcium concentration ([Ca2+]i) in NPY Y1 receptor-transfected Chinese Hamster Ovary Cells (CHO-Y1 cells). It is a potent inhibitor of the NPY-evoked increase in [Ca2+]i. This can be ascribed to its antagonistic properties for the NPY Y, receptor since its less active stereoisomer, BIBP3435, is much less potent. However, when its concentration exceeds 1 microM, BIBP3226 produces a large increase in [Ca2+]i on its own. This effect is mimicked by BIBP3435 and it also occurs in wild type CHO-K1 cells. These latter cells do not contain high affinity binding sites for [3H]NPY and [3H]BIBP3226 and, hence, no endogenous NPY Y1 receptors. It is concluded that, at moderately high concentrations, the NPY Y1 receptor antagonist BIBP3226 and its entantiomer BIBP3435 are able to increase the [Ca2+ ]i in CHO cells either by stimulating another receptor or by directly affecting cellular mechanisms that are involved in calcium homeostasis.     

Neuronal Regulation of Interleukin 6 Secretion in Murine Spleen: Adrenergic and Opioidergic Control

Abstract: The PNS was anticipated to be involved in the modulation of immune responses. To study aspects of this neuronal-immune communication, a recently developed tissue slice method was used to study the effects of adrenergic and opioidergic transmitters on interleukin 6 (IL-6) secretion in the spleen. The α₂-adrenergic agonist p -aminoclonidine (10⁻⁷ M ) inhibited IL-6 secretion (control vs. p -aminoclonidine, 100.0 ± 4.76 vs. 59.3 ± 6.6% of control values; p < 0.001). The α₁-adrenergic agonist methoxamine (10⁻⁸ M ) also inhibited IL-6 secretion (100.0 ± 4.8 vs. 71.5 ± 3.8%; p < 0.001). The endogenous opioids β-endorphin (10⁻¹⁰ M ), methionine-enkephalin (10⁻⁹ M ), and leucine-enkephalin (10⁻⁹ M ) inhibited IL-6 secretion as well ( p = 0.0051, p = 0.0337, and p = 0.0226, respectively). Electrical stimulation of spleen slices inhibited IL-6 secretion (100.0 ± 4.3 vs. 56.7 ± 4.6% of control values; p < 0.001). The involvement of α-adrenergic and opioidergic molecules in this electrically induced inhibition was shown by the use of antagonists. Electrical inhibition of IL-6 secretion was attenuated by phentolamine (10⁻⁷ M ; p = 0.0345), by naloxone (10⁻⁶ M ; p = 0.0046), by cyprodime (10⁻⁸ M ; p = 0.0014), and by the combination of cyprodime (10⁻⁷ M ) plus phentolamine (10⁻⁸ M ; p < 0.0001). We conclude from the complementary studies that the inhibition of IL-6 secretion induced by electrical pulses was mostly mediated by α-adrenergic and μ-opioidergic endogenous transmitters.     

PYY preference is a common characteristic of neuropeptide Y receptors expressed in human, rat, and mouse gastrointestinal epithelia

This investigation describes the relative potencies of four peptide agonists, namely, peptide YY (PYY), [Leu3l,Pro34]PYY (Pro34pYY), neuropeptide Y (NPY), and [Leu31,Pro34]NPY (Pro34NPY), as antisecretory agents in human, rat, and mouse gastrointestinal preparations. The inhibition of agonist responses by the Y1-receptor antagonist BIBP 3226 was also tested in each preparation. An unexpectedly pronounced preference for PYY and Pro34PYY was observed in functional studies of two human epithelial lines stably transfected with the rat Y1 receptor (Y1-7 and C1Y1-6). NPY and Pro34NPY were at least an order of magnitude less effective than PYY in these functional studies but were only marginally less potent in displacement binding studies using membrane preparations of the same clonal lines. The orders of agonist potency obtained in Y1-7 and C1Y1-6 epithelia were compared with those obtained from a single human colonic adenocarcinoma cell line (Colony-6, which constitutively expresses Y1 receptors) and also from mucosal preparations of rat and mouse descending colon. Similar peptide orders of potency were obtained in rat and mouse colonic mucosae and Colony-6 epithelia, all of which exhibited PYY preference (although less pronounced than with Y1-7 and C1Y1-6 epithelia) and significant sensitivity to the Y1 receptor antagonist, BIBP 3226. We have compared the pharmacology of these five mammalian epithelial preparations and provide cautionary evidence against the reliance upon agonist concentration-response relationships alone, in the characterization of NPY receptor types.     

Neuropeptide Y stimulates DNA synthesis in human vascular smooth muscle cells through neuropeptide Y Y1 receptors

We investigated the mitogenic effect, measured as [3H]thymidine incorporation, of neuropeptide Y (NPY) on smooth muscle cells (SMCs) from human subcutaneous arteries (diameter: 0.4 mm). NPY stimulated DNA synthesis in a concentration-dependent manner, Emax 32 +/- 5% relative to control. The effect was potently antagonised by the NPY Y1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine-a mide), indicating the effect to be mediated via the NPY Y1 receptor. Noradrenaline (NA) also induced mitogenesis, Emax 35 +/- 10% relative to control. When added together, NPY and NA potentiated the [3H]thymidine incorporation, Emax 109 +/- 38% relative to control. Also, this effect seems to be mediated by the NPY Y1 receptor, since BIBP3226 blocked the effect (44 +/- 9% relative to control). The mitogenic effect of NPY and NA, two important transmitters of the sympathetic nervous system, might have clinical consequences on conditions with elevated sympathetic nerve activity.     

EFFECTS OF BIBP3226 AND BIBP3435 ON CYTOSOLIC CALCIUM IN NEUROPEPTIDE Y Y1 RECEPTOR-TRANSFECTED CHINESE HAMSTER OVARY CELLS AND WILD TYPE CHO-K1 CELLS

The NPY Y₁-receptor selective antagonist BIBP3226 exerts a dual control on the cytosolic free calcium concentration ([Ca²⁺]_i) in NPY Y₁ receptor- transfected Chinese Hamster Ovary Cells (CHO-Y₁ cells). It is a potent inhibitor of the NPY-evoked increase in [Ca²⁺]_i. This can be ascribed to its antagonistic properties for the NPY Y₁ receptor since its less active stereoisomer, BIBP3435, is much less potent. However, when its concentration exceeds 1 μM, BIBP3226 produces a large increase in [Ca²⁺]_i on its own. This effect is mimicked by BIBP3435 and it also occurs in wild type CHO-K1 cells. These latter cells do not contain high affinity binding sites for [³H]NPY and [³H]BIBP3226 and, hence, no endogenous NPY Y₁ receptors. It is concluded that, at moderately high concentrations, the NPY Y₁ receptor antagonist BIBP3226 and its entantiomer BIBP3435 are able to increase the [Ca²⁺]_i in CHO cells either by stimulating another receptor or by directly affecting cellular mechanisms that are involved in calcium homeostasis.     

Orexigenic effect of the melanocortin MC4 receptor antagonist HS014 is inhibited only partially by neuropeptide Y Y1 receptor selective antagonists

Neuropeptide Y (NPY) and melanocortin (MC) peptides have opposite effects on food intake: NPY-like peptides and MC receptor antagonists stimulate feeding and increase body weight, whereas melanocortins and NPY antagonists inhibit food intake. In this study we tested whether the orexigenic effect of the selective MC4 receptor antagonist HS014 (1 nmol) could be inhibited by three different NPY antagonists, (R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]D-argininam ide (BIBP3226), (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2(diphenyl acetyl)-argininamidetrifluoroacetate (BIBO3304), and decapeptide [D-Tyr(27,36)D-Thr32]NPY(27-36), after icv administration in freely feeding male rats. All three NPY receptor antagonists inhibited the orexigenic effects of HS014 partially and with markedly different potency. [D-Tyr(27,36)D-Thr32]NPY(27-36) was active only in subconvulsive dose. The NPY Y1 selective antagonist BIBP3226 was more effective in inhibiting the effect of HS014 than BIBO3304 despite in vitro data indicating that BIBP3226 is about 10 times less potent than BIBO3304 at NPY Y1 receptor. An enantiomer of BIBO3304, BIBO3457, failed to inhibit HS014-induced feeding, indicating that the effects of BIBO3304 were stereoselective. These results suggest that stimulation of food intake caused by weakening of melanocortinergic tone at the MC4 receptor is partially but not exclusively related to NPY Y1 receptor activation.     

Nicotine evoked improvement in learning and memory is mediated through NPY Y1 receptors in rat model of Alzheimer's disease

We investigated the role of endogenous neuropeptide Y (NPY) system in nicotine-mediated improvement of learning and memory in rat model of Alzheimer's disease (AD). Intracerebroventricular (icv) colchicine treatment induced AD-like condition in rats and showed increased escape latency (decreased learning), and amnesic condition in probe test in Morris water maze. In these rats, nicotine (0.5mg/kg, intraperitoneal), NPY (100 ng/rat, icv) or NPY Y1 receptor agonist [Leu(31), Pro(34)]-NPY (0.04 ng/rat, icv) decreased escape latency by 54.76%, 55.81% and 44.18%, respectively, on day 4 of the acquisition. On the other hand, selective NPY Y1 receptor antagonist, BIBP3226 (icv) produced opposite effect (44.18%). In the probe test conducted at 24h time point, nicotine, NPY or [Leu(31), Pro(34)]-NPY increased the time spent by 72.72%, 44.11% and 26.47%, respectively; while BIBP3226 caused reduction (8.82%). It seems that while NPY or [Leu(31), Pro(34)]-NPY potentiated, BIBP3226 attenuated the learning and memory enhancing effects of nicotine. Brains of colchicine treated rats showed significant reduction in NPY-immunoreactivity in the nucleus accumbens shell (cells 62.23% and fibers 50%), bed nucleus of stria terminalis (fibers 71.58%), central nucleus of amygdala (cells 74.33%), arcuate nucleus (cells 70.97% and fibers 69.65%) and dentate gyrus (cells 58.54%). However, in these rats nicotine treatment for 4 days restored NPY-immunoreactivity to the control level. We suggest that NPY, perhaps acting via NPY Y1 receptors, might interact with the endogenous cholinergic system and play a role in improving the learning and memory processes in the rats with AD-like condition.     

Neuropeptide Y-Y1 receptor agonist worsens while antagonist improves survival of cultured Y1-expressing neuronal cells following oxygen and glucose deprivation

In this in vitro study, we investigated the influence of neuropeptide Y (NPY) Y1 receptor activation or inhibition on the viability of cultured neuronal or glial cells following oxygen glucose deprivation (OGD). Viability of cultured cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. When compared to the vehicle-treated control group, treatment with NPY or [Leu³¹,Pro³⁴]-NPY (Y1 agonist) reduced viability of cultured SK-N-MC (Y1-expressing) human neuronal cells at 24 h after 1 h of OGD, while BIBP3226 (Y1 antagonist) improved viability. Except at the highest concentration of NPY used in the study, treatment with NPY or NPY3-36 (Y2 agonist) did not influence viability of cultured SH-SY5Y (Y2-expressing) human neuronal cells at 24 h after 1 h of OGD. In addition, treatment with NPY, [Leu³¹,Pro³⁴]-NPY, NPY3-36, or BIBP3226 did not affect viability of cultured primary astrocytes at 24 h after 4 h of OGD. The present results agree with those of a recent in vivo study. Activation of NPY-Y1 receptors may mediate ischemic pathophysiological processes, and inhibiting the Y1 receptors may be protective. The combination of OGD and cultured neuronal cells may be useful in future studies on the neuroprotective and harmful mechanisms of NPY-Y1 receptor inhibition and activation during ischemia, respectively.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

The effect of a neuropeptide Y antagonist, BIBP 3226, on short-term arterial pressure control in conscious unrestrained rats with congestive heart failure

The effects of a neuropeptide Y (NPY) Y1-receptor antagonist (BIBP 3226) on mean arterial pressure (MAP) and heart rate were investigated in conscious unrestrained rats with chronic congestive heart failure. The rats were randomly assigned to 2 groups, and received either BIBP 3226 or its inactive enantiomer (BIBP 3435) as an intravenous infusion (6 mg/kg/h for 1.5 h, respectively). Before, during and after the infusion, rats were stressed with a jet of air and received a bolus injection of NPY (2 nmol/kg iv.). There was no difference between the 2 groups in resting MAP and heart rate before, during or after infusion (BIBP 3226 vs. BIBP 3435). The effects of exogenous NPY on MAP were significantly attenuated in BIBP 3226 group during and 1 h after the infusion (p<0.05). The tissue NPY levels in heart, adrenal gland and kidney in heart failure rats were not different from those in sham-operated rats. The results suggest that Y1-receptor mechanisms are of minor importance in the short-term control of basal MAP and heart rate in conscious unrestrained rats with congestive heart failure.     

Neuropeptide Y induced increase of cytosolic and nuclear Ca2+ in heart and vascular smooth muscle cells

It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L-type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide.     

Neuropeptide Y-Y1 receptor modulates nitric oxide level during stroke in the rat

In a rat endovascular middle cerebral artery occlusion (MCAO) stroke model, we previously showed that intracerebroventricular (ICV) injection of neuropeptide Y (NPY) or an Y1 receptor agonist, [Leu(31),Pro(34)]-NPY, increased the infarct volume, that an Y1 receptor antagonist, BIBP3226, reduced the infarct volume, and that an Y2 receptor agonist, NPY3-36, had no effect. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (NO) and examined how ICV administration of NPY or its receptor analogs would modulate the brain NO level between the bregma levels +2 and -4 mm during MCAO, since excessive NO mediates ischemic damage. The relative brain NO concentration was increased to 131.94 +/- 7.99% (mean +/- SEM; n = 8) at 15 min of MCAO. NPY treatment further increased the relative brain NO concentration to 250.94 +/- 50.48% (n = 8), whereas BIBP3226 significantly reduced the brain NO concentration to 69.63 +/- 8.84% (n = 8). [Leu(31),Pro(34)]-NPY (137.61 +/- 14.54%; n = 7) or NPY3-36 (129.23 +/- 21.77%; n = 8) did not affect the brain NO concentration at 15 min of MCAO. Our results suggest that the NPY-Y1 receptor activation mediates ischemic injury via NO overproduction and that inhibition of the Y1 receptor may confer protection via suppression of excessive NO production during ischemia.     

Neuropeptide Y regulates catecholamine release evoked by interleukin-1beta in mouse chromaffin cells

Activation of the hypothalamic-pituitary-adrenal gland (HPA) axis can modulate the immune system. Cytokines and neuropeptide Y (NPY) are potent regulators of the HPA axis and are both produced by the adrenal medulla. The cytokine interleukin-1beta (IL-1beta) belongs to the interleukin-1 family along with interleukin-1alpha and the interleukin receptor antagonist (IL-1ra). The aim of the present study was to determine the interaction between NPY and IL-1beta in catecholamine (norepinephrine, NE and epinephrine, EP) release from mouse chromaffin cells in culture. We found that IL-1beta increased the constitutive release of NPY, NE and EP from mouse chromaffin cells. This IL-1beta stimulatory effect was blocked by IL-1ra. The immunoneutralization of NPY and the use of the NPY Y(1) receptor antagonist (BIBP 3226) inhibited the stimulatory effect of IL-1beta on catecholamine release from these cells. The present work shows that IL-1beta induces catecholamine release, and in turn this peptide will induce an additional increase in catecholamine release acting through the Y(1) receptor. This work suggests that NPY is involved in the regulatory loop between the immune and the adrenal system in some pathophysiological conditions where plasmatic IL-1beta increases, like in sepsis, rheumatoid arthritis, stress or hypertension.     

Interactions between orexin A, NPY and galanin in the control of food intake of the goldfish, Carassius auratus

The neuropeptides orexin A (OXA), neuropeptide Y (NPY) and galanin (GAL) have been shown to play a role in the regulation of food intake in mammals. They also significantly stimulate feeding in goldfish. In order to assess the interactions between these peptides in the control of feeding in goldfish, we investigated the effects of central injection of specific receptor antagonists for NPY (BIBP 3226) and GAL (M40) on OXA-induced feeding and the effects of desensitization of orexin receptors on NPY- and GAL-induced feeding. We investigated the effects of BIBP 3226 on GAL-induced feeding and the effects of M40 on NPY-induced feeding. We also examined the effects of coinjection of each pair of neuropeptides on feeding behavior. Injections of 10 ng/g OXA, 5 ng/g NPY and 10 ng/g GAL each induced an increase in feeding. Fish treated with 5 ng/g BIBP or 20 ng/g M40 had food consumption similar to saline controls. BIBP at 5 ng/g significantly reduced NPY- and OXA-induced feeding. Injections of 20 ng/g M40 significantly decreased GAL-induced feeding, but had no effect on OXA-induced feeding. Blocking of orexin receptors by treatment with high doses of OXA (100 ng/g) resulted in a decrease in both NPY- and GAL-induced feeding. Coinjection with 0.5 ng/g OXA and either 0.5 ng/g NPY or 0.5 ng/g GAL resulted in a food intake higher than that observed in saline control fish and in fish treated with NPY or GAL alone at 0.5 ng/g. NPY mRNA expression was increased in the telencephalon and in the hypothalamus compared to saline-treated fish, following injection of OXA. These results indicate that both NPY and GAL are at least, in part, dependent on coaction with OXA for the stimulation of food intake and feeding behavior in goldfish. In addition, the effects of OXA are mediated, in part, by the NPY pathway. This suggests a functional interdependence between these three peptidergic systems in the control of energy balance in goldfish.     

NPY signaling through Y1 receptors modulates thalamic oscillations

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

Effects of neuropeptide Y on alpha 1-and beta-adrenoceptor-stimulated second messenger systems in rat frontal cortex

Neuropeptide Y (NPY) (1 microM) significantly reduced the basal cAMP concentration in slices of rat frontal cortex. However, NPY (10(-9)-10(-6)M) did not alter the isoproterenol-stimulated (10(-9)-10(-5) M) accumulation of cAMP in the frontal cortical slices, showing that Y2 NPY receptors do not modulate the beta-adrenoceptor-stimulated adenylase cyclase activity. NPY (10(-8)-2.5 x 10(-5) M) was also demonstrated to stimulate inositol phosphate accumulation in rat frontal cortex slices in a dose-dependent manner. However, NPY (1 microM) did not potentiate the ability of phenylephrine (5 X 10(-8)-10(-4) M), an alpha 1-adrenoceptor agonist, to stimulate inositol phosphate hydrolysis. The combined effects of phenylephrine and NPY (1 microM) on inositol phosphate hydrolysis were additive, suggesting that the alpha 1-adrenoceptor and NPY Y1 receptor sites are located on different postsynaptic sites in rat frontal cortex. This study demonstrates the existence of both Y2 and Y1 NPY receptors in the rat frontal cortex based on second messenger systems, but there does not appear to be an interaction of NPY with either alpha 1- or beta-adrenoceptors.     

Neuropeptide Y and its receptor analogs differentially modulate the immunoreactivity for neuronal or endothelial nitric oxide synthase in the rat brain following focal ischemia with reperfusion

Summary An intracerebroventricular (icv) injection of neuropeptide Y (NPY) or [Leu³¹, Pro³⁴]-NPY (non-Y2 receptor agonist) given during middle cerebral artery occlusion (MCAO) increases the infarct volume and nitric oxide (NO) overproduction in the rat brain. An icv injection of NPY3-36 (non-Y1 receptor agonist) has no effects, while BIBP3226 (selective Y1 receptor antagonist) reduces the infarct volume and NO overproduction. This study examined the effects of NPY or its receptor analog on the immunoreactivity (ir) for three isoforms of NO synthase (NOS) following 1h of MCAO and 3h of reperfusion. Focal ischemia/reperfusion led to increased ir for neuronal NOS (nNOS) within the ipsilateral caudate putamen and insular cortex. NPY or [Leu³¹, Pro³⁴]-NPY enhanced but BIBP3226 suppressed such increase in the nNOS-ir. Focal ischemia/reperfusion also led to an ipsilateral increase in extent and/or intensity of the ir for endothelial NOS (eNOS) in the caudate putamen and/or parietal cortex. NPY or [Leu³¹, Pro³⁴]-NPY suppressed but BIBP3226 enhanced such change in the eNOS-ir. NPY3-36 did not consistently influence the nNOS-ir or eNOS-ir following MCAO. Specific ir for inducible NOS was undetectable. These opposing effects of NPY-Y1 receptor activation or inhibition on nNOS and eNOS may lead to harmful or beneficial consequences following ischemia/reperfusion.     

In vivo inhibition of neuropeptide FF agonism by BIBP3226, an NPY Y1 receptor antagonist

BIBP3226 {(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide} was recently shown to display relatively high affinities for neuropeptide FF (NPFF) receptors and exhibit antagonist activities towards NPFF receptors in vitro. The present study was undertaken to investigate the antagonistic effects of BIBP3226 on several in vivo pharmacologic profiles induced by exogenous NPFF and NPVF. (1) BIBP3226 (5 nmol) injected into the third ventricle completely antagonized the hypothermic effects of NPFF (30 nmol) and NPVF (30 nmol) after cerebral administration in mice; (2) BIBP3226 (5 nmol, i.c.v.) prevented the anti-morphine actions of NPFF (10 nmol, i.c.v.) in the mouse tail-flick assay; (3) in urethane-anaesthetized rats, both NPFF (200 nmol/kg, i.v.) and NPVF (200 nmol/kg, i.v.) increased the mean arterial blood pressure, which were significantly reduced by pretreatment with BIBP3226 (500 nmol/kg, i.v.). Collectively, these data suggest that BIBP3226, a mixed antagonist of NPY Y1 and NPFF receptors, shows in vivo antagonistic effects on NPFF receptors. In addition, it seems to be clear that the in vivo pharmacological profiles of NPFF are mediated directly by NPFF receptors.