Potent and selective tools to investigate neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5)

We have evaluated 3 newly developed neuropeptide Y receptor antagonists in various in vitro binding and bioassays: BIBO3304 (Y1), T4[NPY33-36]4 (Y2), and CGP71683A (Y5). In rat brain homogenates, BIBO3304 competes for the same population of [125I][Leu31,Pro34] peptide YY (PYY) binding sites (75%) as BIBP3226, but with a 10 fold greater affinity (IC50 of 0.2 +/- 0.04 nM for BIBO3304 vs. 2.4 +/- 0.07 nM for BIBP3226),while CGP71683A has high affinity for 25% of specific [125I][Leu31,Pro34]PYY binding sites. Both BIBO3304 and CGP71683A (at 1.0 microM) were unable to compete for a significant proportion of specific [125I]PYY3-36/Y2 sites. The purported Y2 antagonist T4[NPY33-36]4 competed against [125I]PYY3-36 binding sites with an affinity of 750 nM. These results were confirmed in HEK 293 cells transfected with either the rat Y1, Y2, Y4, or Y5 receptor cDNA. BIBO3304, but not CGP71683A, competed with high affinity for [125I][Leu31,Pro34]PYY binding sites in HEK 293 cells transfected with the rat Y1 receptor cDNA, whereas the reverse profile was observed upon transfection with the rat Y5 receptor cDNA. Additionally, both molecules were inactive at Y2 and Y4 receptor subtypes expressed in HEK 293 cells. Receptor autoradiographic studies revealed the presence of [125I][Leu31,Pro34]PYY/BIBO3304-insensitive sites in the rat brain as reported previously for BIBP3226. Finally, the selective antagonistic properties of BIBO3304 were demonstrated in a Y1 bioassay (rabbit saphenous vein; pA2 value of 9.04) while being inactive in Y2 (rat vas deferens) and Y4 (rat colon) bioassays. These results confirm the high affinity and selectivity of BIBO3304 and CGP71683A for the Y1 and Y5 receptor subtypes, respectively, while the purported Y2 antagonist, T4[NPY33-36]4 possesses rather low affinity for this receptor.     

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.     

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.     

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.     

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.     

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-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.     

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.     

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.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

Molecular dynamics of NPY Y1 receptor activation

A three-dimensional model of the human neuropeptide Y(NPY)Y₁ receptor (hY₁) was constructed, energy refined and used to simulate molecular receptor interactions of the peptide ligands NPY, [L31, P34]NPY, peptide YY (PYY) and pancreatic polypeptide (PP), and of the nonpeptide antagonist R-N²-(diphenylacetyl)-N-(4-hydroxyphenyl)methyl-argininamide (BIBP3226) and its S-enantiomer BIBP3435. The best complementarity in charges between the receptor and the peptides, and the best structural accordance with experimental studies, was obtained with amino acid 1–4 of the peptides interacting with Asp194, Asp200, Gln201, Phe202 and Trp288 in the receptor. Arg33 and Arg35 of the peptides formed salt bridges with Asp104 and Asp287, respectively, while Tyr36 interacted in a binding pocket formed by Phe41, Thr42, Tyr100, Asn297, His298 and Phe302. Calculated electrostatic potentials around NPY and hY₁ molecules indicated that ligand binding is initiated by electrostatic interactions between a highly positive region in the N- and C-terminal parts of the peptides, and a negative region in the extracellular receptor domains. Molecular dynamics simulations of NPY and BIBP3226 interactions with the receptor indicated rigid body motions of TMH5 and TMH6 upon NPY binding as mechanisms of receptor activation, and that BIBP3226 may act as an antagonist by constraining these motions.     

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.     

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 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.     

[3H]-BIBP3226 and [3H]-NPY Binding to Intact SK-N-MC Cells and CHO Cells Expressing the Human Y1 Receptor

Abstract

We have studied the binding of [³H]-NPY and the newly developed non-peptide Y₁ receptor antagonist [³H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y₁ receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [³H]-NPY binding was slow, the binding kinetics of [³H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y₁ agonists NPY, PYY and [Leu³¹-Pro³⁴]-NPY completely and potently displaced [³H]-NPY binding, they could only displace 70 to 80 % of the [³H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [³H]-NPY binding sites in CHO-Y1 cells whereas [³H]-BIBP3226 binding parameters remained unchanged.     

Physiological role of neuropeptide Y in the regulation of the ascending phase of the peristaltic reflex

The physiological role of neuropeptide Y (NPY) and of specific NPY receptors in regulating the intestinal peristaltic reflex was examined in three-compartment flat-sheet preparations of rat colon. Graded muscle stretch or mucosal stimulation applied to the central compartment inhibited NPY release in the orad compartment where ascending contraction was measured. NPY and the Y1-receptor agonist [Leu31, Pro34]NPY inhibited, whereas the selective Y1-receptor antagonist BIBP 3226 augmented ascending contraction and substance P (SP) release in the orad compartment induced by muscle stretch or mucosal stimulation. Neither agonist nor antagonist had any effect on descending relaxation or VIP release in the caudad compartment. The Y2-receptor agonist NPY13-36 and antagonist BIIE 0246 had no effect on peptide release or mechanical response. The results indicate that suppression of a tonic inhibitory influence of NPY neurons on excitatory neurotransmitter release contributes substantially to the orad contractile phase of the peristaltic reflex. The effect of NPY on neurotransmitter release is mediated by Y1 receptors.     

Presence of neuropeptide Y and the Y1 receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium

The aims of the present study were to investigate the presence and distribution of NPY and the Y1 receptor in endocardial endothelial cells (EECs), to verify if EECs can release NPY, and to determine if the effect of NPY on intracellular calcium is mediated via the Y1 receptor. Immunofluorescence, 3-D confocal microscopy and radioimmunoassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1 receptor are present in human EECs (hEECs) and that their distributions are similar, the fluorescence labelling being higher in the nucleus and more particularly at the level of the nuclear envelope when compared with the cytosol. Using radioimmunoassay, we demonstrated that EECs are a source of NPY and can secrete this peptide upon a sustained increase of intracellular calcium ([Ca]i). Using fluo-3 and 3-D confocal microscopy technique, superfusion of hEECs as well as EECs isolated from rat adult hearts with increasing concentrations of NPY induced a dose-dependent, sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of NPY on EEC [Ca]i was completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1 receptor antagonist. The results suggest that NPY and Y1 receptors are present in the EECs of 20-week-old human fetal heart and they share the same distribution and localization inside the cell. In addition, EECs are able to secrete NPY in response to an increase in [Ca]i, and the Y1 receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]i in these cells. Thus, NPY released by EECs may modulate excitation-secretion coupling of these cells.     

Role of serotonin (5-HT) in the antidepressant-like properties of neuropeptide Y (NPY) in the mouse forced swim test

Neuropeptide Y (NPY) is thought to be implicated in depressive disorders. The mouse forced swim test (FST) is an animal model widely used as a predictor of the efficacy of antidepressant drugs. The present study was undertaken to explore the possible contribution of endogenous serotonin (5-HT) systems in the behavioral effects elicited by NPY in this model. The selective serotonin re-uptake inhibitor (SSRI), fluoxetine, was also tested for comparison. 5-HT was depleted prior to testing by the administration of the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg, i.p., each day for 3 days; control mice received saline-vehicle over the same period). On the fourth day, mice received NPY (3 nmol, I.C.V.), fluoxetine (16 mg/kg, i.p.) or saline injections before testing in the FST. Both NPY and fluoxetine significantly reduced immobility time in saline-treated control animals. Pre-treatment with PCPA significantly blocked the effects of fluoxetine in the FST, confirming the role of endogenous 5-HT. Similarly, pre-treatment with PCPA also significantly attenuated the anti-immobility effects of NPY, thus suggesting a role for 5-HT in the effects of NPY in the FST. Quantitative receptor autoradiography revealed increases in specific [125I][Leu31, Pro34]PYY sites that were sensitive to BIBP3226 (Y1-like sites) in various brain regions. Specific [125I]GR231118 and [125I]PYY(3-36) binding levels were not changed following PCPA treatment, suggesting that depletion of endogenous 5-HT resulted in an apparent increase in the level of Y1 sites in their high-affinity state. Taken together, these results suggest a role for 5-HT-related systems in the antidepressant-like properties of NPY.     

Neuropeptide Y receptor alterations in the hypothalamus of lactating rats.

Hypothalamic neuropeptide Y (NPY) neurons are influenced by circulating levels of insulin and leptin and are thought to be involved in mediating hunger following underfeeding. We have investigated hypothalamic NPY receptor subtypes in lactating rats, which are markedly hyperphagic throughout the day and night. NPY receptors were measured by using [125I] peptide YY, a high-affinity ligand, and Y1 receptors were masked by using the highly specific antagonist BIBP 3226. Freely fed lactating rats showed no changes in the densities of Y1, or non-Y1, NPY binding sites in whole hypothalamic homogenates or in individual hypothalamic regions (measured by quantitative autoradiography) examined during the day or night (P > 0.05; n = 10/group, and n = 6/group, respectively). However, reducing food intake by 35% had a more profound effect on NPY receptor density in lactating than in control rats, producing down-regulation of non-Y1 receptors in the ventromedial, dorsomedial, and perifornical lateral areas (all P < 0.05; n = 7/group) and reduction of plasma insulin and leptin levels (both P < 0.01). Thus, although the NPY system may not have a major role in the hyperphagia of freely fed lactating rats, it appears to have an important function in the response to undernutrition in such animals.