125I-NPY Binding Research Articles

Human syncytiotrophoblast NPY receptors are located on BBM and activate PLC-to-PKC axis.

Neuropeptide Y (NPY) is abundant in plasma and amniotic fluid of women throughout pregnancy, during which its involvement in placental hormonogenesis has been proposed. In accordance with its putative role, the aim of this study was to characterize the human placental syncytiotrophoblast receptivity to NPY. Thus we performed this study on brush-border membranes (BBM) and basal plasma membranes (BPM). Specific 125I-labeled NPY (125I-NPY) binding to BBM was rapid (20 min), saturable, with a maximum binding capacity of 604 +/- 100 fmol/mg protein, and of high affinity, with a dissociation constant of 11 +/- 3 nM. No saturable binding could be shown in BPM. The rank order of affinity of NPY and related peptides to compete for 125I-NPY binding sites was peptides YY (PYY) > NPY = [Leu31,Pro34]NPY > 13-36NPY >> pancreatic polypeptide (PP). It is noteworthy that PYY displaced only 45% of the binding sites. In BBM, both NPY and PYY were potent phospholipase C (PLC) stimulators, leading to a four- to fivefold increase of control phosphodiesterase activity. The latter effect could be prevented by preincubation of membranes with 5 microM U-73122, a known inhibitor of G protein-linked receptor activation of PLC-beta. Furthermore, 5 microM BIBP-3226, a Y1-receptor antagonist, shifted both dose-response curves to the right in a similar fashion for both peptides. In accordance with the PLC stimulation, both peptides also induced stimulation of protein kinase C (PKC) activity, which could be partially but additively prevented by U-73122 and LY-294002, a selective inhibitor of phosphatidyl-inositol-3 kinase (PI3K). Taken together, these data suggest that placental and blood-derived NPY binds to a mixed population of receptors composed of Y1 and Y3 subtypes on the maternal side of the syncytiotrophoblast, where it can mediate its physiological purposes via PLC-beta and PI3K activation, both of which lead to PKC activation. However, because BIBP-3226 antagonized both effects, the physiological relevance of the apparent Y3 fraction is still unsolved.

Molecular Characterization of a Second Mouse Pancreatic Polypeptide Receptor and Its Inactivated Human Homologue

The family of mammalian neuropeptide Y (NPY)/peptide YY (PYY)/pancreatic polypeptide (PP) receptors comprises several G protein-coupled receptors, i.e. Y1, Y2, and Y4/PP1. We now report cloning of a novel member of this family named PP2. The coding region of the mouse PP2 gene reveals no introns and predicts a seven transmembrane domain (TM) receptor of 371 amino acids. Percent identities of the mouse PP2 to mouse Y1, mouse Y4/PP1 and human Y2 receptors are 53, 42, and 31, respectively. The mouse PP2 receptor expressed in COS cells binds rat 125I-PP with high affinity, i.e. IC50 = 65 pM. Pharmacological characterization of 125I-PP binding shows a rank order of potency of PP >> PYY >/= NPY, which is similar to that of the mouse Y4/PP1 receptor. Mouse PP2 transcripts were not detectable by Northern analysis in adult tissues and in 11-, 15-, and 17-day-old embryos. However, a 9.8-kb PP2 transcript was detectable in 7-day-old mouse embryo, i.e. prior to the organogenesis of pancreas and the onset of PP production. We have also cloned the human homologue of PP2, which is a single copy gene and maps to human chromosome 5q31. Surprisingly, the human PP2 cDNAs and gene sequences display a single base deletion in the coding region. This frameshifting mutation predicts a truncated receptor of 290 amino acids without TM7. Transfection of COS-7 cells with several different human PP2 expression constructs failed to confirm any specific binding of 125I-PP, 125I-PYY, or 125I-NPY to cell membranes. These data suggest that in mouse there are at least two PP receptors, Y4/PP1 and PP2, whereas in humans, PP2 is either functionally inactive or it has acquired a PP-independent function.

Purification and Biochemical Characterization of Neuropeptide Y2 Receptor

Neuropeptide Y (NPY) receptors consist of three subtypes, designated NPY1, NPY2, and NPY3. The Y1 receptor has been cloned. The present study reports the purification of the NPY-Y2 receptor from porcine brain and its biochemical characterization. NPY receptors were solubilized and purified by sequential hydrophobic interaction, ion exchange, and NPY-affinity chromatography. By use of SDS-polyacrylamide gel electrophoresis, high performance liquid chromatography gel permeation chromatography, and chemical cross-linking studies, the affinity-purified brain NPY-Y2 receptor was identified as a monomeric glycoprotein with a molecular mass of 60 kDa. Following deglycosylation, the molecular mass of the Y2 receptor was decreased to 45 kDa. Although the 125I-NPY binding to the purified NPY receptor was considerably decreased by N-ethylmaleimide, guanine nucleotides had no effect. Therefore, the purified NPY-Y2 receptor is probably not associated with G-proteins, but may have intramolecular-free sulfhydryl groups. The specific activity of the isolated NPY-Y2 receptor is 15.8 nmol/mg of protein. The isolated receptor retained its capacity to bind to 125I-NPY, specific to NPY and peptide YY, and showed no cross-reactivity with any other peptides. Highly purified (10(9)-fold purification) NPY receptor from the brain was identified as the Y2 subtype as demonstrated by its affinity to C-terminal fragments of NPY, including NPY-(13-36).

Neuropeptide Y and the Development of Cancer Anorexia

The authors determined whether radioligand binding of neuropeptide Y (NPY) to hypothalamus taken from nonanorectic and anorectic tumor-bearing rats was altered as compared with similar tissue taken from freely-feeding and food-restricted control rats. Previous results indicate that tumor-bearing rats exhibit a refractory feeding response to NPY, the most potent feeding stimulus known. Additional studies indicate that the concentration of NPY in the hypothalamus of anorectic tumor-bearing rats is decreased as compared with freely-feeding or food-restricted control rats. Because these observations of decreased response to exogenous peptide in the presence of decreased endogenous levels suggest an alteration in hypothalamic NPY receptors, this study investigated binding of 125I-NPY to hypothalamic membranes of tumor-bearing and control rats. Determinations of receptor affinity for NPY (half maximal concentration for displacement) indicated a 20-fold decrease in affinity with the development of anorexia, which changed to an 80-fold decrease during severe anorexia. Receptor density, as indicated by specific binding, exhibited only a 30% decrease, even during severe anorexia. These results suggest major alterations in NPY receptor mechanisms in experimental cancer anorexia, with receptor affinity being decreased progressively as the rats become more anorectic. The absence of a compensatory up-regulation in receptor density in the presence of decreased endogenous NPY concentrations indicate dysfunction in receptor regulatory mechanisms. This receptor aberration may be the central nervous system basis for the etiology of cancer anorexia.

A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations. Similarly, cells transfected with the corresponding bovine clone (LCR1) did not show specific NPY/PYY binding exceeding that resulting from endogenous binding sites; mock-transfected COS7 cells, used frequently for heterologous expression of receptors, were found to have endogenous specific 125I-NPY binding sites ( B max = 112 fmol/mg protein; K d = 0.25 nM ). Moreover, the hFB22 transfected cells, when compared to control transfected cells, did not display de novo NPY- or PYY-induced second messenger responses, i.e., (1) inhibition of forskolin-stimulated cAMP accumulation or (2) 45Ca 2+ influx. The presence of hFB22 mRNA was detected in several human neuroblastoma cell lines, none of which was found to express Y3-like NPY binding sites. hFB22 displays 39% amino acid sequence identity (in the transmembrane regions) to the human interleukin-8 receptor, and 32–36% amino acid identity to the human receptors of angiotensin II, bradykinin, and n-formylpeptide, but only 23% amino acid identity to the previously described human NPY/PYY receptor of the Y1 receptor subtype. Our results show that hFB22 and LCR1 do not encode NPY receptors, and their true ligand(s) remains to be identified.

Autoradiographic localization of binding sites for neuropeptide Y and bradykinin on astrocytes.

The cellular localization of binding sites for the vasoactive peptides 125I-neuropeptide Y (NPY) and 3H-bradykinin (BK) was studied in explant cultures of rat cerebellum, brain stem and spinal cord by means of autoradiography. The majority of astrocytes in these cultures expressed binding of 125I-NPY and 3H-BK over their cell bodies and processes. Simultaneous staining of the cultures with anti-glial fibrillary acidic protein (GFAP) has shown that the labelled cells were GFAP-positive and could therefore be identified as astrocytes. In addition to glial cells, a great number of neurones also revealed binding sites for the neuropeptides. Our autoradiographic findings together with recent electrophysiological studies provide good evidence for the existence of NPY- and BK-receptors on astrocytes.

Y2 receptors for peptide YY and neuropeptide Y on dispersed chief cells from guinea pig stomach.

Peptide YY (PYY) and neuropeptide Y (NPY) inhibit agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production and pepsinogen secretion from chief cells. We used radiolabeled PYY and NPY to characterize receptors on chief cells from guinea pig stomach. Binding of 125I-labeled PYY was rapid (70% maximal within 10 min) and specific (not inhibited by secretin, vasoactive intestinal peptide, cholecystokinin, carbachol, prostaglandin E2, forskolin, or cholera toxin). Measurement of the ability of PYY to inhibit binding of 125I-PYY indicated the presence of 1.8 x 10(3) high-affinity [dissociation constant (Kd) = 1.7 nM] and 5.1 x 10(4) low-affinity (Kd = 83.3 nM) sites/cell. Internalization of bound 125I-PYY was suggested by slow and incomplete dissociation in the presence of unlabeled PYY (50% after 2 h) and was examined further by measuring residual binding after washing with acetic acid (pH 2.5), glycine (pH 10.5), or trypsin. After 30 min at 37 degrees C, internalization of radioligand was evidenced by the failure of washing with these solutions to remove 50-65% of bound radioactivity. At 4 degrees C, internalization of 125I-PYY was nearly abolished. Binding of 125I-PYY and 125I-NPY was inhibited by NPY-(13-36) but not by [Leu31,Pro34]NPY indicating that these are Y2 receptors. In guinea pig chief cells, PYY and NPY modulate cAMP-mediated pepsinogen secretion by interacting with specific high-affinity Y2 receptors.

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.

Effects of intracerebroventricular D-myo-inositol-1,2,6-trisphosphate (PP56), a proposed neuropeptide Y (NPY) antagonist, on locomotor activity, food intake, central effects of NPY and NPY-receptor binding

D-myo-Inositol-1,2,6-trisphosphate (PP56) is a novel experimental drug which is structurally related to the intracellular second messenger IP 3. Among other pharmacological effects, PP56 has been shown to antagonize neuropeptide Y (NPY) induced vasoconstriction with a high degree of specificity. We examined the effects of i.c.v. PP56 on locomotor activity and food intake in rats, and on the hypoactivity and hyperphagia induced by NPY. In the open field, PP56 give alone increased locomotor activity by up to 85%. It did not prevent NPY induced hypoactivity to any extent. PP56 given alone did not affect food intake except for a small increase after the highest dose tested (200 nmol). When NPY was given after pretreatment with PP56, NPY induced hyperphagia was significantly reduced. A similar effect, however, was seen with regard to the hyperphagia produced by another orexigenic peptide, galanin. PP56 did not affect the binding of 125I-NPY to brain membranes in vitro, or to cells of two different neuroblastoma cell lines which selectively express NPY Y 1 or Y 2 receptors. In summary, PP56 acted as a locomotor stimulant per se. Only one of the two tested central effects of NPY could be antagonized by PP56, and then only partially and in a non-specific manner. The central effects of PP56 do not seem to be produced at the level of NPY receptors.

Neuropeptide Y: an endogenous inhibitor of norepinephrine-stimulated melatonin secretion in the rat pineal gland.

The biosynthesis of the hormone melatonin (MEL) by the mammalian pineal gland has been thought to be regulated strictly by stimulatory factors, most predominantly norepinephrine (NE), released from the sympathetic nerve fibers which heavily innervate the gland. Evidence from many investigators suggests that sympathetic fibers may colocalize other neuroactive factors in addition to NE. One of these factors is neuropeptide Y (NPY), which has been found in the nerve fibers of the pineal gland. The present study sought to explore potential interactions between NE and NPY in the regulation of pineal MEL secretion. Specific, saturable, and reversible binding of 125I-NPY to intact cultured pinealocytes was measured with an affinity constant of 1 nM and an NPY binding site density of 0.04 pmol/mg of protein. In addition, cell culture studies revealed that NPY represents a potent (IC50 of 0.4 nM) endogenous inhibitor of NE-stimulated MEL secretion. However, this inhibition is accompanied by only a modest reduction (35%) of cyclic AMP accumulation. These findings reinforce the view that the mammalian pineal gland, which appears to integrate both inhibitory as well as stimulatory signals, is an important model of autonomic function, particularly in the context of biological rhythmicity.

Specific neuropeptide Y binding sites in chicken brain.

In the present study, 125I-labeled neuropeptide Y (NPY) binding to chicken brain regions was evaluated. Cerebellum and cerebral cortex membranes bound significantly more 125I-NPY specifically than did membranes from other brain regions. Scatchard plots of NPY binding to cerebellar membranes were curvilinear; the high-affinity component had an affinity (Kd) of 1.1 nM, with a receptor concentration (Ro) of 182 fmol/mg membrane protein. Scatchard plots of NPY binding to chicken cerebral cortex membranes were linear, with a Kd of 0.63 nM and Ro of 90 fmol/mg. Unlabeled avian pancreatic polypeptide (APP) inhibited 125I-NPY binding to cerebellar membranes with a constant at which 50% inhibition occurs of 0.5 nM but showed essentially no affinity for cerebral cortex NPY binding sites. As previously reported, 125I-APP bound to cerebellar membranes with a Kd of 0.365 nM and an Ro of 323 fmol/mg, and unlabeled NPY showed about one order of magnitude lower affinity than did unlabeled APP for 125I-APP binding sites. Pseudo-Hill coefficients for APP binding to cerebellar APP receptors and NPY binding to cerebellar NPY receptors were 0.9. In contrast, pseudo-Hill plots for APP competition for 125I-NPY binding were curvilinear. It is concluded that the chicken cerebellum contains distinct APP and NPY receptors, whereas cerebral cortex contains only NPY receptors. APP is capable of binding with high affinity to the cerebellar, but not the cortical, NPY receptor.

Solubilization of the Neuropeptide Y Receptor from Rat Brain Membranes

The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free α-NH 2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-α-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-α-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-α-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.

Neuropeptide Y (18-36) is a competitive antagonist of neuropeptide Y in rat cardiac ventricular membranes.

Neuropeptide Y (NPY), a hexatriacontapeptide amide, is present in high concentrations in the mammalian heart. Specific receptors of NPY in rat cardiac ventricular membranes have been characterized recently in our laboratory. Structure-activity studies with selected partial sequences of NPY revealed that NPY(18-36) inhibited the binding of 125I-NPY to rat cardiac ventricular membranes but had no effect on the cardiac adenylate cyclase activity. NPY, as previously reported, inhibited the cardiac adenylate cyclase activity. These observations suggested that NPY (18-36) may be an antagonist of NPY in cardiac membranes. Consistent with this observation, the presence of NPY (18-36) (1 microM) shifted the inhibitory adenylate cyclase activity dose-response curve of NPY to the right in a parallel fashion. Furthermore, NPY(18-36) (1 microM) completely abolished the effect of NPY (10 nM) that alone caused 80% of the maximum inhibition of adenylate cyclase activity. These findings confirm that NPY(18-36) is a competitive antagonist of NPY in rat cardiac ventricular membranes. NPY cardiac receptor antagonist, NPY(18-36), or analogs based on this sequence may have potential clinical application, since NPY has been implicated in the pathophysiology of congestive heart failure.

Characterization of neuropeptide Y binding sites in rat cardiac ventricular membranes

Neuropeptide Y (NPY) binding sites in rat cardiac ventricular membranes have been characterized in detail. 125I-NPY bound to the membranes with high affinity. Binding was saturable, reversible and specific, and depended on time, pH and temperature. Analysis of the binding data obtained under optimal conditions, 2 hr, 18°C and at pH 7.5, revealed the presence of low and high affinity binding sites. The high affinity binding sites had an apparent dissociation constant (K d) of 0.38 nM and a binding capacity (B max) of 7.13 fmol/mg protein. The apparent K d and B max for low affinity binding sites were 22.34 nM and 261.25 fmol/mg protein, respectively. Peptides unrelated to NPY did not compete with 125I-NPY for the binding sites even at 1 μM concentrations, whereas homologous peptides, peptide YY (PYY) and pancreatic polypeptide (PP), and NPY(13–36) inhibited 125I-NPY binding but with lower potency compared to NPY. 125I-NPY binding was sensitive to the nonhydrolyzable GTP analog, Gpp(NH)p, suggesting that the NPY receptor is coupled to the adenylate cyclase system. The ventricular membrane receptor characterized in this study may play an important role in mediating the physiological effects of NPY in the heart.

Neuropeptide Y binding and inhibition of cAMP accumulation in human neuroepithelioma cells

The specific binding of 125I-labeled neuropeptide Y (NPY) and the biological response to NPY receptor activation were measured in cultured human neuroepithelioma (SK-N-MC) cells. A single class of high-affinity binding sites [dissociation constant (KD) = 0.2 nM] was characterized both by equilibrium binding of 125I-NPY concentrations less than 1 nM and kinetically by the initial rates of 125I-NPY association and dissociation. Specific 125I-NPY binding was decreased in a concentration-dependent manner by inclusion of guanine nucleotides in the incubation medium. The existence of multiple affinity states or NPY receptor subtypes was suggested by 1) a Hill coefficient of less than 1.0 obtained when analyzing equilibrium binding with 125I-NPY concentrations greater than 1 nM, 2) biphasic dissociation of 125I-NPY, 3) an increase in the component of rapid dissociation and decrease in the component of slow dissociation when guanine nucleotides were present during dissociation of 125I-NPY, and 4) displacement of 125I-NPY by unlabeled peptide with a slope factor of 0.6. Exposure of intact cells to NPY caused a concentration-dependent pertussis toxin-sensitive inhibition of forskolin-stimulated cellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation [50% effective concentration (EC50) = 0.4 nM]. In contrast, NPY had no effect on cellular inositol phosphate content or protein kinase C activation. These results demonstrate that NPY binds specifically to a G protein-linked receptor that inhibits adenylate cyclase in SK-N-MC cells.

Interaction of 125I-Neuropeptide Y with rat cardiac membranes

125I-Neuropeptide Y (NPY) bound specifically with high affinity to rat atrial and ventricular membranes. Scatchard analysis revealed the presence of single class of binding sites in both atrial and ventricular membranes. The apparent K d and B max for atrial membranes were 0.63 nM and 70 fmol/mg protein, respectively; ventricular membranes had an apparent k d of 0.39 nM and a B max of 283 fmol/mg protein. NPY structural homologues peptide YY (PYY) and pancreatic polypeptide (PP) bound to the ventricular membranes NPY receptor, but with several fold lower potency compared to NPY. Binding of 125I-NPY to ventricular membranes was sensitive to guanosine triphosphate (GTP) suggesting that the NPY receptor is linked to adenylate cyclase system. The receptor characterized in this system may play a crucial role in mediating the cardiac effects of NPY.

Electroconvulsive shocks increase the concentration of neocortical and hippocampal neuropeptide Y (NPY)-like immunoreactivity in the rat

Studies on humans and rats have suggested that neuropeptide Y (NPY) is involved in major depression and anxiety. Therefore, we conducted the present study in order to elucidate the effect of repeated (13 or 14 days) treatment of rats with electroconvulsive shocks (ECS) on the concentration of NPY-like immunoreactivity (-LI) in various brain regions, adrenals and plasma. In addition, the effect of ECS on 125I-NPY binding was studied in 3 brain regions. The effects of ECS were compared to effects of 3 control treatments: one group not being handled at all during the time period, one group handled like the ECS-group but not receiving shocks, and one group receiving shocks below the threshold for induction of convulsions. The latter group developed behavioural signs reminiscent of the inescapable shock-induced ‘learned helplessness’ syndrome (a proposed animal model of depression). We found that the concentration of NPY-LI in the frontal and parietal cortex and in the hippocampus were approximately doubled in the ECS-group as compared to the 3 control groups. No changes in NPY-LI were detected in the striatum, hypothalamus, pons, olfactory bulbs or cerebellum, nor in plasma or adrenals. In spite of the marked changes NPY-LI concentration, the binding characteristics of 125I-NPY in the frontal and parietal cortex and in the hippocampus were similar in all 4 groups of rats. Finally, we confirmed the previous observationnthat ECS increase [ 3H]prazosin binding in cortex. In conclusion, ECS treatment increases neocortical and hippocampal NPY-LI concentrations, while leaving 125I-NPY binding unaffected. Subconvulsive shocks were without effect.

125I-neuropeptide Y binding activity of pig spleen cell membranes: Effect of solubilisation

Crude preparations of pig spleen cell membranes were obtained by differential centrifugation. 125I-NPY bound specifically to these membranes with a K D of 56±13 pM and B max of 44±4.0 fmols/mg protein. After treatment with 1% CHAPS and 10mM 2-mercaptoethanol in the presence of 2μM leupeptin, 2μM pepstatin A, 10μM phosphoramidon, 200μM PMSF and 0.1% bacitracin, followed by centrifugation at 100,000g a soluble preparation was obtained that contained a single population of specific 125I-NPY binding sites. The K D of the soluble receptor was significantly higher at 1.38±0.2 nM (P<0.01) but the B max of 59.6±6.6 fmols/mg protein was similar (N.S.). This is the first description of a method for obtaining NPY receptors in soluble form and should enable their purification and characterisation, though the low affinity of the soluble receptor may reflect disruption of the ligand binding site upon removal of the receptor from the membrane.

Binding sites for 125I-neuropeptide Y (NPY) on membranes from bovine adrenal medulla

Bovine adrenal medulla membranes were examined for the presence of specific 125I-neuropeptide Y ( 125I-NPY) binding sites using rapid centrifugation to measure the amount of bound ligand. Specific binding was determined from the difference between 125I-NPY bound in the presence and absence of 10 −7 M unlabeled NPY. The binding was saturable and reached equilibrium within 5 min at 0°C. Analysis of specific 125I-NPY binding using the LIGAND computer program indicated a best fit for a two site model with a K d of 0.26 nM and a B max of 12 fmol/mg protein for the high affinity site and a K d of 170 nM and a B max of 6 pmol/mg protein for the low affinity site. The rate of dissociation (k −1) was 0.071/min with a t 1 2 of 9 min. Displacement curves for avian or human pancreatic polypeptide revealed that these peptides displaced 125I-NPY from both sites with IC 50 values greater than 10 nM.