Neuropeptide Y receptor-mediated sensitization of ATP-stimulated inositol phosphate formation.

Activation of bovine chromaffin cell neuropeptide Y (NPY) receptors coupled to Gi (Y1) results in the enhancement of ATP-stimulated inositol phosphate formation. NPY alone does not alter inositol phosphate (InsP) formation in these cells, suggesting that some form of receptor cross talk is involved in this process. In some cell types, serial stimulation of Gi-linked and Gs- or Gq-linked receptors results in an increase in intracellular messenger production (cyclic AMP or InsP), a process referred to as heterologous sensitization. NPY preincubation with bovine chromaffin cells followed by the addition of ATP results in a dose-dependent increase in ATP-stimulated InsP formation (EC50 = 2.0 x 10-8 M), which is maximal within 1 min. InsP formation resulting from NPY preincubation persists for more than an hour after NPY removal, declining with time in a linear fashion. [Leu31Pro34]NPY and NPY are equally effective at producing sensitization, whereas NPY13-36 is ineffective, suggesting that NPY acts through the Y1 receptor. Confirmation of the receptor subtype identity was made by including the Y1-selective antagonist HU-404 during the preincubation, which prevented the sensitizing effect of NPY. NPY sensitization was blocked by pertussis toxin pretreatment, demonstrating Gi/Go involvement. ATP-stimulated InsP formation, with and without NPY preincubation, was sensitive to the phospholipase C inhibitor, U73122 [1-(6-([17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino)hexyl)-1H-pyrrole-2,5-dione]. In conclusion, short-term exposure of bovine chromaffin cells to NPY results in a long-lasting increase in the subsequent stimulation of InsP formation by ATP.

ATP stimulated cyclic AMP formation in bovine chromaffin cells is enhanced by neuropeptide Y.

ATP increases cAMP formation in bovine chromaffin cells, EC(50) = 7.1 x 10(-6) M. NPY, EC(50) = 4.1 x 10(-8) M, increases the efficacy of ATP (1.5-2 fold). Inclusion of the selective Y1 receptor antagonist 1229U91 produced a decrease in NPY potency (EC(50) = 2.7 x 10(-7) M). PTX pretreatment did not abolish either the effect of ATP nor the enhancement by NPY. NPY could also enhance the ability of angiotensin and bradykinin to increase cAMP formation. The selective phospholipase C inhibitor, U73122, and the selective protein kinase C inhibitors, bisindolylmaleimide I and RO-31-8425, were effective inhibitors of the enhancing effect of NPY.

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.

Neuropeptide Y secretion from bovine chromaffin cells inhibits cyclic AMP accumulation.

Neuropeptide Y (NPY) is secreted from bovine chromaffin cells in response to nicotinic receptor stimulation and may exhibit autocrine, paracrine or endocrine effects. Stimulation of bovine chromaffin cells with nicotine followed by the addition of forskolin (FSK) to the media results in a decrease in cyclic AMP accumulation compared to that seen in the absence of nicotine. Pertussis toxin (PTX) treatment or the addition of BIBP 3226, a selective NPY Y1 receptor antagonist prevents the inhibitory effect of nicotine. Fractionation of media obtained from cells stimulated with nicotine reveals an NPY-like substance that inhibits FSK-stimulated cAMP accumulation. Thus, an NPY-like substance can be secreted from bovine chromaffin cells in quantities sufficient to inhibit FSK-stimulated cAMP accumulation. These results suggest that NPY can act in an autocrine fashion to regulate chromaffin cell function.

BIBP 3226 inhibition of nicotinic receptor mediated chromaffin cell secretion.

(R)-N 2-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-argininamide (BIBP 3226) is a selective neuropeptide Y Y1 receptor antagonist with structural similarity to the C-terminal tripeptide of neuropeptide Y. Based on this similarity we questioned whether BIBP 3226 could act as an agonist. Incubation of BIBP 3226 with bovine chromaffin cells in culture results in the inhibition of nicotinic receptor-stimulated catecholamine secretion (IC50 = 2.4 microM). The effect of BIBP 3226 is independent of neuropeptide Y action since the presence of neuropeptide Y in the culture medium does not alter the effect of BIBP 3226. BIBP 3226 decreased the efficacy of the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP), but did not change its potency suggesting non-competitive inhibition. BIBP 3226 has a similar effect on nicotinic receptor-stimulated 45Ca2+ influx. BIBP 3226 does not inhibit [3H]norepinephrine release induced by high K+ and its effect is not pertussis toxin-sensitive. We conclude that not only can BIBP 3226 act as a neuropeptide Y receptor antagonist in bovine chromaffin cells but also act as an agonist and inhibit catecholamine secretion.

Neuropeptide Y enhances ATP-induced formation of inositol phosphates in chromaffin cells.

Bovine chromaffin cells contain high affinity NPY binding sites coupled through a pertussis toxin-sensitive G protein to inhibition of cAMP accumulation. NPY alone does not alter [3H]inositol phosphate formation from [3H]phosphoinositides in these cells. Increasing NPY concentrations, in the presence of ATP (300 microM), produced a dose-dependent enhancement in [3H]-inositol phosphate formation, EC50 = 3.2 nM. Inclusion of the selective NPY-Y1 receptor antagonist BW1229 (1 microM) produced a marked decrease in NPY potency (EC50 = 3.3 microM). The Y1 receptor agonist, [Leu31, Pro34]-NPY, was equally effective with NPY, whereas NPY18-36, a Y2 receptor agonist, was much less effective. Inclusion of NPY with ATP also produced an enhancement in the release of intracellular Ca2+. The ability of NPY to enhance both [3H]inositol phosphate formation and the release of intracellular Ca2+ was pertussis toxin-insensitive. NPY action on bovine chromaffin cell receptor(s) appears to facilitated by different G proteins: one which can inhibit cAMP accumulation via a pertussis toxin-sensitive process and another which can enhance ATP activation of the inositol phosphate signaling pathway by a pertussis toxin-insensitive process.

Methylprednisolone and full-dose aprotinin reduce reperfusion injury after cardiopulmonary bypass.

OBJECTIVE: To compare the effects of low- and full-dose aprotinin to methylprednisolone (MPS) in reducing cardiopulmonary bypass (CPB)-induced interleukin-6 (IL-6) release. DESIGN: Prospective, randomized, blinded study. SETTING: Cytokine Laboratory, pharmacology department, in a university teaching hospital. PARTICIPANTS: Forty adult male human patients scheduled for myocardial revascularization were divided into four groups (n = 10): (1) control; (2) MPS, 1 g IV before CPB; (3) aprotinin-low-dose protocol; and (4) aprotinin-full-dose protocol. MEASUREMENTS AND MAIN RESULTS: Plasma levels of IL-6 were measured at baseline and 1 and 24 hours after CPB by enzyme-linked immunosorbent assay technique. Group 1 demonstrated a significant (p < 0.05) increase in IL-6 at 1 and 24 hours post-CPB. Groups 2 and 4 demonstrated significant (p < 0.05) reduction of IL-6 at 1 (group 2 only) and 24 (groups 2 and 4) hours post-CPB when compared with group 1 at the same time periods. CONCLUSIONS: These results demonstrate that MPS, 1 g before CPB, and full-dose aprotinin, but not half-dose aprotinin, achieve significant reduction in IL-6 release after CPB. These results further suggest that MPS and full-dose aprotinin may reduce reperfusion injury after CPB.

Neuropeptide Y inhibits chromaffin cell nicotinic receptor-stimulated tyrosine hydroxylase activity through a receptor-linked G protein-mediated process.

Acetylcholine stimulation of bovine chromaffin cells results in increased norepinephrine and epinephrine secretion accompanied by a corresponding increase in synthesis. The addition of neuropeptide Y (NPY) to the culture medium prevents the increase in catecholamine synthesis but not secretion. Treatment of chromaffin cells with nicotine produces a concentration-dependent increase in tyrosine hydroxylase activity (IC50 = 1.2 microM) that is reduced if NPY is present during stimulation. Tyrosine hydroxylase activity decreases in a concentration-dependent fashion if increasing amounts of NPY are included in the culture medium, IC50 = 0.2 nM. Treatment with pertussis toxin completely prevents the effect of NPY. The rank order of potency for inhibition of tyrosine hydroxylase activity is NPY > or = [Leu31,Pro34]NPY > or = peptide YY > NPY2-36 > NPY13-36 > NPY18-36 > or = NPY26-36 >> NPY1-30, suggesting a NPY-Y1 receptor subtype. Examination of the effect of NPY on nicotine stimulation of chromaffin cell protein phosphorylation showed that NPY produces a concentration-dependent decrease in a 60-kDa protein, IC50 = 6.4 nM. The effect of NPY is pertussis toxin-sensitive. The rank order of potency is [Leu31,Pro34]NPY > or = NPY >> NPY18-36. Immunoprecipitation confirmed the identity of the 60-kDa protein as tyrosine hydroxylase.

Neuropeptide Y (18-36) modulates chromaffin cell catecholamine secretion by blocking the nicotinic receptor ion channel.

Neuropeptide Y (NPY) is a widely distributed peptide with varied activities including inhibition of [3H]NE secretion from chromaffin cells. In the present study, we investigated the mechanism through which NPY and NPY fragments inhibit nicotinic receptor induced influx of 22Na+ and 45Ca++ into bovine chromaffin cells. Fragments of NPY, including NPY13-36, NPY18-36 and NPY26-36, are more potent inhibitors of 45Ca++ and 22Na+ influx than NPY. High [K+]- and BAY K 8644-induced 45Ca++ influx and veratridine-induced 22Na+ influx are not inhibited by either NPY or NPY fragments. Thus, the site of NPY or NPY fragment action is not voltage-gated Ca++ or Na+ channels. A significant amount of acetylcholine-induced 45Ca++ influx still occurs in the presence of the voltage-gated Ca++ channel blockers: nifedipine (L-type), omega-conotoxin-GVIA (N-type) and omega-agatoxin-IVA (P-type). NPY18-36, in the presence of these channel blockers, inhibited the residual nicotinic receptor-induced Ca++ influx. The response to NPY 18-36 is not pertussis toxin sensitive. The rank orders of potency for inhibition of 45Ca++ and 22Na+ are the same: NPY18-36 > or = NPY26-36 > NPY13-36 > NPY13-36 > NPY > or = NPYfree acid. Moreover, the IC50 values for NPY18-36 inhibition of 45Ca++ influx and 22Na+ influx are similar, 0.9 x 10(-6) M and 2.03 x 10(-6) M, respectively. Regression analysis for inhibition of these two phenomena produced a correlation coefficient of .9697 (P < .0003).(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y inhibition of nicotinic receptor-mediated chromaffin cell secretion.

Neuropeptide Y (NPY), a widely distributed peptide with varied activities, inhibits nicotinic receptor-induced [3H]norepinephrine ([3H]NE) secretion from bovine chromaffin cells. The secretion produced by membrane depolarization with high KCl concentrations or veratridine is not inhibited. Fragments of NPY, such as NPY18-36, are potent inhibitors of [3H]NE secretion, whereas [Leu31,Pro34]-NPY and peptide YY have no effect. The response to NPY18-36 is not sensitive to pertussis toxin pretreatment of chromaffin cells. NPY fragments also inhibit nicotinic receptor-induced 45Ca++ influx but not that induced by KCl or veratridine. The rank orders of potency for inhibition of [3H]NE secretion and 45Ca++ influx are the same: NPY18-36 > or = NPY26-36 > NPY13-36. NPY and NPYfree acid are weak inhibitors of secretion but not 45Ca++ influx. Moreover, the IC50s for NPY18-36 inhibition of [3H]NE secretion and 45Ca++ influx are comparable, 1.4 x 10(-6) M and 0.9 x 10(-6) M, respectively. Regression analysis produced a correlation coefficient of 0.9842 (P < .0001). It was concluded that NPY inhibits [3H]NE secretion by a modification of the nicotinic receptor-mediated increase in Ca++ influx. The characterization of the response suggests that the NPY effect is mediated by a previously undefined NPY receptor subtype that was designated Y4.

Neuropeptide Y promotes GTP photo-incorporation into a 55 kDa protein.

Incubation of bovine hippocampal membranes with [alpha-32P]GTP and exposure to ultraviolet light resulted in the labelling of seven species with apparent molecular masses of 200, 74, 55, 53, 50, 43 and 40 kDa. Labelling of the 55 kDa species was greatly enhanced in the presence of carboxyl terminal fragments [neuropeptide Y-(18-36)] of neuropeptide Y. Labelling occurred with [alpha-32P]GTP but not [alpha-32P]ATP. A group of putative direct G protein activating peptides including mastoparan, melittin, substance P and adrenocorticotropic hormone (ACTH)-(1-24), were also able to stimulate the labelling of this protein. Labelling of the 55 kDa protein could be demonstrated in bovine brain but not peripheral tissues. Western blot analysis using an antibody against the common alpha subunit of G proteins recognized a protein co-migrating with the 55 kDa GTP-binding protein. These findings demonstrate the existence of a previously uncharacterized neuronal protein, with an apparent molecular mass of 55 kDa, that binds GTP in response to neuropeptide Y and other peptides.

Neuropeptide Y inhibits pertussis toxin-catalyzed ADP-ribosylation in bovine adrenal chromaffin cell membranes.

Evidence is presented that the neuropeptide Y receptor is directly coupled to an inhibitory G protein existing in cultured bovine adrenal chromaffin cell membranes. Pertussis toxin catalyzes the [32P]ADP-ribosylation of a 41 kDa plasma membrane protein. 5'-Guanylylimidodiphosphate inhibited the [32P]ADP labelling of this protein in a dose-dependent manner whereas GTP had no effect. Preincubation of the plasma membranes with high concentrations of neuropeptide Y followed by a brief exposure to a low concentration of 5'-guanylylimidodiphosphate significantly inhibited ADP-ribosylation beyond that observed with 5'-guanylylimidodiphosphate alone. These results suggest that the neuropeptide Y receptor in bovine adrenal chromaffin cells is directly coupled to a 41 kDa PTX substrate (presumably the alpha subunit of an inhibitory G protein).

Characterization of cocaine-sensitive dopamine uptake in PC12 cells.

Dopamine uptake in rat pheochromocytoma (PC12) cells is a carrier-mediated process which follows Michaelis-Menten kinetics. Uptake was saturable with an apparent Km of 0.71 microM for dopamine and a Vmax of 3.2 pmol/2 x 10(5) cells/min. The rank order of potency for various amines was norepinephrine > or = dopamine > epinephrine. Uptake increased with increasing temperature and showed a sharp break in the Arrhenius plot at 27.5 degrees C. The Q10 was 1.39 above and 2.95 below 27.5 degrees C. Cocaine inhibited uptake in a dose-dependent manner with a Ki of 0.97 microM. The presence of cocaine lowered the apparent Km but did not affect the Vmax, indicating competitive inhibition. Tunicamycin inhibited [3H]dopamine accumulation in a dose- and time-dependent fashion suggesting the dopamine uptake site in PC12 cells is an asparagine-linked glycoprotein. Kinetic analysis showed a decrease in Vmax but not in the apparent Km after tunicamycin treatment, consistent with the notion that tunicamycin treatment results in the loss of a substantial amount of active carrier molecules.

Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase in bovine adrenal chromaffin cells via a pertussis toxin-sensitive process.

The effect of neuropeptide Y (NPY) on adenylate cyclase activity and the role of G-proteins mediating NPY's effect were investigated in cultured bovine adrenal chromaffin cells. The equilibrium binding of [125I]NPY to sucrose gradient purified bovine adrenal medulla plasma membranes revealed high- (GTP gamma S sensitive) and low-affinity binding sites with calculated IC50 values of 0.27 nM and 0.14 microM, respectively. Inhibition of forskolin-stimulated cyclic AMP accumulation was dependent upon the NPY concentration (IC50 = 0.9 nM) and independent of cyclic AMP (cAMP) phosphodiesterase activity. NPY-related peptides, except peptide YY, and NPY fragments exhibited minimal inhibitory activity. The inhibitory effect of NPY on forskolin-stimulated adenylate cyclase activity was completely abolished by pretreatment of the cells with pertussis toxin (PTX). Incubation of membranes with PTX and [32P]nicotinamide adenine dinucleotide revealed a protein band with an apparent molecular mass of 41 kDa. The time course and dose dependence of PTX pretreatment for in vitro ADP-ribosylation were similar to those for PTX to attenuate the NPY effect on forskolin-stimulated adenylate cyclase activity. The direct relation between the NPY receptor and the PTX-sensitive G-protein was further shown by the ability of NPY to inhibit PTX-catalyzed in vitro ADP-ribosylation. ADP-ribosylation of the 41-kDa protein was partially inhibited by 5'-guanylylimidodiphosphate and further inhibited by high concentrations of NPY. An antibody against Gi1/i2 alpha 1 recognized two species of which a 41-kDa protein comigrated with the PTX substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteamine selectively enhances neuropeptide Y2 receptor binding activity.

Affinity labeling of [125I]NPY to the bovine hippocampal NPY receptor has revealed a 50 kDa specific binding protein, the Y2 receptor. Cysteamine (10 microM - 10 mM) specifically enhanced NPY specific labeling of the Y2 receptor without affecting cross-linking efficiency. Several structurally related agents, including reduced glutathione, cysteine, beta-mercaptoethanol and ethanolamine, were without effect on receptor binding. The enhancement of binding by cysteamine could be reversed by washing the membranes. These studies suggest that cysteamine may change the conformation of the NPY Y2 receptor and increase its binding activity.

Role of sulfhydryl groups in Y2 neuropeptide Y receptor binding activity.

Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.

Neuropeptide Y receptor in bovine hippocampus is a Y2 receptor.

[125I]NPY bound to a single class of saturable binding sites on bovine hippocampus membranes with a KD of 0.1 mM and Bmax of 165 fmol/mg of protein. The rank order of potency of NPY fragments and other structurally related peptides to inhibit [125I]NPY binding was: PYY greater than or equal to NPY much greater than BPP greater than or equal to APP and NPY greater than NPY-(13-36) greater than NPY-(18-36) greater than or equal to NPY-(20-36) much greater than NPY-(26-36) greater than NPY-(free acid). The identity of the NPY binding site was investigated by affinity labeling. Gel electrophoresis followed by autoradiography revealed a band with a mol mass of 50 kDa. Unlabeled NPY or PYY, but not BPP, HPP and APP, inhibited labeling of [125I]NPY to the 50 kDa protein band. Moreover, labeling was inhibited by NPY greater than NPY-(18-36) greater than or equal to NPY-(13-36) greater than or equal to NPY-(20-36) greater than NPY-(26-36) greater than NPY-(free acid). The binding of [125I]NPY and the intensity of the cross-linked band were reduced in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.7 nM and 0.6 mM, respectively). These studies demonstrate that bovine hippocampal membranes contain a 50 kDa [125I]NPY binding site that has the ligand specificity characteristic of the Y2 receptor subtype.

Differential secretion of enkephalin-like peptides from bovine adrenal chromaffin cells.

Stimulation of chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) or depolarizing concentrations of K+ resulted in a significant secretion of high and low molecular weight enkephalin-like peptides (ELPs) into the culture medium. BioGel P-10 column chromatography was used to characterize the ELPs in chromaffin cell extracts and in culture media before and after stimulation with either DMPP or K+. DMPP (50 microM) stimulation produced a significant secretion of primarily low molecular weight (less than 3 kDa) ELPs whereas 56 mM K+ caused a secretion of both high and low molecular weights ELPs. The expected decrease in cellular content of low molecular weight peptides was not observed regardless of stimulation type. Our results support the hypothesis that the precursor/product ratio of secreted ELPs is dependent upon the nature of the chromaffin cell stimulus. Moreover the cellular content of low molecular weight ELPs is not depleted with either type of stimulation.

Characterization of neuropeptide Y (NPY) receptors in human hippocampus.

We identified a 50 kDa neuropeptide Y (NPY) receptor from human hippocampus by affinity labeling. NPY specific binding and labeling of the receptor were inhibited in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.27 nM and 0.18 nM, respectively). Peptide YY (PYY), but none of the pancreatic polypeptides, was as effective as NPY in displacing [125I]NPY. NPY fragments inhibited binding with the rank order of potency: NPY greater than NPY13-36 greater than NPY20-36 greater than or equal to NPY18-36 greater than NPY1-36 free acid greater than or equal to NPY26-36. These results demonstrate that the human hippocampal NPY receptor is a 50 kDa protein fitting the classification of a Y2 receptor subtype.

Benextramine irreversibly inhibits [125I]neuropeptide Y affinity labeling of the Y2 binding protein in bovine hippocampus.

Affinity labeling of iodinated neuropeptide Y (NPY) to bovine hippocampal binding proteins revealed that benextramine inhibited specific NPY labeling of the 50 kDa NPY binding protein (Y2 binding protein) in a dose-dependent manner (IC50 = 33 microM). Hippocampal membranes, which were pretreated with benextramine and washed, exhibited decreased [125I]NPY labeling of binding proteins in a similar dose-dependent manner. These findings demonstrate that benextramine irreversibly blocks specific NPY binding to the 50 kDa NPY Y2 binding protein.