Novel cell line selectively expressing neuropeptide Y-Y2 receptors

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH-SY5Y, CHP-234, and MHH-NB-11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP-234 and MHH-NB-11 binding of [3H]propionyl-NPY was observed with Kd-values of 0.64 +/- 0.07 nM and 0.53 +/- 0.12 nM, respectively, determined by saturation analysis with non-linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu31, Pro34]-NPY (NPY-Y1, NPY-Y5), [Ahx(5-24)]-NPY (NPY-Y2), [Ala31, Aib32]-NPY (NPY-Y5), NPY [3-36] (NPY-Y2, NPY-Y5), and NPY [13-36] (NPY-Y2). Both cell lines, CHP-234 and MHH-NB-11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY-Y2 receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin-induced cAMP production in an ELISA.     

Dependence of peptide self-association on intermolecular interaction by PFGNMR in TFE aqueous solution: C-terminal analogues of NPY as model peptides

We have investigated the dependence of peptide oligomerization on intermolecular interaction in terms of both energetic and structural effect by PFGNMR. Three peptides, NPY[20-36], Pro34-NPY[20-36] and NPY[21-31], which are related to human NPY, were synthesized as models in this work. In contrast to NPY[20-36], both Pro34-NPY[20-36] and NPY[21-31] were found with descendent affinity with TFE cluster and continuous dissociating with increased temperature. The observed results can be accounted by the entropic change with temperature and the varied hydrophobic interactions between species due to the differed structures of peptides from each other. The removal of helical secondary structure or residues from C-terminal region may increase the energetic difference between peptide-peptide self-associating and peptidesolvent binding. This increased energetic difference leads to larger dependence of association-dissociation equilibrium on temperature and entropic increase while dissociating.     

Novel analogues of neuropeptide Y with a preference for the Y1-receptor.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and acts in humans via at least three receptor subtypes: Y1, Y2, and Y5. Whereas selective agonists and antagonists are known for the Y2- and Y5-receptors, the Y1-receptor still lacks a highly selective agonist. This work presents the first NPY-based analogues with Y1-receptor preference and agonistic properties. Furthermore, the importance of specific amino acids of NPY for binding to the Y-receptor subtypes is presented. Amongst the analogues tested, [Phe7,Pro34]pNPY (where pNPY is porcine neuropeptide Y) showed the most significant Y1-receptor preference (> 1 : 3000-fold), with subnanomolar affinity to the Y1-receptor, and Ki values of approximately 30 nM for the Y2- and Y5-subtype, respectively. Variations of position 6, especially [Arg6,Pro34]pNPY and variations within positions 20-23 of NPY were found to result in further analogues with significant Y1-receptor preference (1 : 400-1 : 2000). In contrast, cyclo S-S [Cys20,Cys24]pNPY was found to be a highly selective ligand at the Y2-receptor, binding only threefold less efficiently than NPY. Analogues containing variations of positions 31 and 32 showed highly reduced affinity to the Y1-receptor, while binding to the Y5-receptor was affected less. Inhibition of cAMP-accumulation of selected peptides with replacements within position 20-23 of NPY showed preserved agonistic properties. The NPY analogues tested give insights into ligand-receptor interaction of NPY at the Y1-, Y2- and Y5-receptor and contribute to our understanding of subtype selectivity. Furthermore, the Y1-receptor-preferring peptides are novel tools that will provide insight into the physiological role of the Y1-receptor.     

Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY

The structure of [Ala(31), Pro(32)]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y(5)-receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y(5)-receptor-selective agonist [Ala(31), Aib(32)]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala(31), Aib(32)]-NPY, the N-terminal residues Tyr(1)-Asp(16) are disordered in solution. Starting from residue Leu(17), an alpha helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala(31)-Tyr(36) indicates that the alpha helix has become interrupted through the [Ala(31), Pro(32)] mutation. This finding is further supported by (15)N-relaxation data through which we can demonstrate that the well-defined alpha helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased (3)J(HN)(alpha) scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y(5)-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala(31), Pro(32)]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn(29) and Leu(30) were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala(31), Pro(32)]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala(31), Pro(32)]-NPY.     

Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide's effect.

Neuropeptide Y (NPY) elicits eating when injected directly into the paraventricular nucleus (PVN) or perifornical hypothalamus (PFH). To identify the essential regions of the NPY molecule and the relative contributions of Y1 and Y2 receptors, the eating stimulatory potency of NPY was compared to that of its fragments, analogues, and agonists when injected into the PVN or PFH of satiated rats. Additionally, antisera to NPY was injected into the cerebral ventricles (ICV) to determine whether passive immunization suppresses the eating produced by mild food deprivation. Tests with NPY fragments revealed that NPY(2-36) was surprisingly potent, nearly three times more so than intact NPY. In contrast, fragments with further N-terminal deletions were progressively less effective or ineffective, as was the free acid form of NPY. Collectively, this suggests that both N- and C-terminal regions of NPY participate in the stimulation of eating. Tests with agonists revealed that the putative Y1 agonist [Pro34]NPY elicited a strong dose-dependent feeding response, while the putative Y2 agonist, C2-NPY, had only a small effect at the highest doses. Although this suggests mediation by Y1 receptors, the uncharacteristically high potency of NPY(2-36) may additionally suggest that the receptor subtype underlying feeding is distinct from that mediating other responses. Additional results revealed that ICV injection of antisera to NPY, which should inactivate endogenous NPY, produced a concentration-dependent suppression of eating induced by mild food deprivation. This finding, along with published work demonstrating enhanced levels of hypothalamic NPY in food-deprived rats, suggests that endogenous NPY mediates the eating produced by deprivation.     

Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock out mice does not result in increased receptor function

The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [(35)S]GTPgammaS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu(31),Pro(34)-NPY increased [(35)S]GTPgammaS binding with a regional distribution consistent with that produced when labeling adjacent sections with [(125)I]-Leu(31),Pro(34)-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [(35)S]GTPgammaS binding in numerous brain areas with a regional distribution similar to the binding observed with [(125)I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors.     

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.     

NPY receptors and opioidergic system are involved in NPY-induced feeding in goldfish

The present study evaluated the effects of both intraperitoneal (i.p. ) and intracerebroventricular administration of selective Y(1) [(Leu(31), Pro(34))-NPY] and Y(2) [(Pro(13), Tyr(36))-NPY (13-36)] receptor agonists on food intake in satiated goldfish. Food intake (FI) was significantly increased by central administration of the Y(1) agonist (1 microg), but not by the Y(2) agonist, at 2 h postinjection. The feeding increase induced by (Leu(31), Pro(34))-NPY was in a similar magnitude to that obtained after ICV injection of the neuropeptide Y, and both feeding stimulations were reversed by the NPY (27-36), a general NPY antagonist. The i.p. administration of the agonists either did not significantly modify (Y(2) agonist) or decreased (Y(1) agonist) food intake in goldfish. These data indicate that it is the Y(1)-like (similar to Y(1) and/or Y(5)) receptor, and not Y(2), that is involved in the central modulation of the feeding behavior in goldfish. We also investigated the possible involvement of opioid peptides as mediators of the NPY stimulatory action on food intake in goldfish. The ICV administration of naloxone (10 microg), a general opioid antagonist, blocked the NPY-induced feeding in goldfish, suggesting that the opioidergic system is involved in feeding regulation by NPY.     

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 (NPY) functional group mimetics: design, synthesis, and characterization as NPY receptor antagonists

N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethylguanyl]cystamine 3 and N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethyl]-1,6-hexanediguanidine 4 have been designed as neuropeptide Y (NPY) functional group mimetics. Both 3 and 4 displace N-[propionyl-³H]-NPY from rat brain binding sites, and are NPY receptor antagonists in rat femoral artery ring segments.     

Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded that Y1 receptor stimulation is associated with both mobilization of intracellular calcium and inhibition of adenylate cyclase 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.     

Conformation of two somatostatin analogues in aqueous solution. Study by NMR methods and circular dichroism

Cyclic-disulfide-containing analogues of somatostatin, Xaa1-Cys2-Xaa3-DTrp4-Lys6-Thr5-Xaa7- Xaa8 [Xaa1 = H or DPhe; Xaa3 = Phe or Tyr; Xaa7 = Cys, Me2Cys or Me2DCys; Xaa8 = OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H-NMR spectroscopy and circular dichroism. The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.     

Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors.

153N-6 (H-[Met5,Pro6,D-Phe7,D-Trp9,Phe10]-MSH(5-13)) has emerged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCI receptor expressed by murine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-MSH and the potent synthetic agonist, [Nle4,D-Phe7]alpha-MSH, at the murine MC1-R. However, the Ki of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle4,D-Phe7]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (Ki of 153N-6 = 9.0 X 10(-6) M). Because Met4 is an important component of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivative of 153N-6 with Ala4 did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met4 is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MCI-R. Pro6 and Phe10 (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6.     

Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.     

Redox-dependent structural reorganization in putidaredoxin, a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida

Putidaredoxin (Pdx), a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida, transfers electrons from NADH-putidaredoxin reductase to cytochrome P450cam. Pdx exhibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the monooxygenase reaction catalyzed by this hemoprotein. To understand how the reduced form of Pdx is stabilized and how reduction of the [2Fe-2S] cluster affects molecular properties of the iron-sulfur protein, crystal structures of reduced C73S and C73S/C85S Pdx were solved to 1.45 angstroms and 1.84 angstroms resolution, respectively, and compared to the corresponding 2.0 angstroms and 2.03 angstroms X-ray models of the oxidized mutants. To prevent photoreduction, the latter models were determined using in-house radiation source and the X-ray dose received by Pdx crystals was significantly decreased. Structural analysis showed that in reduced Pdx the Cys45-Ala46 peptide bond flip initiates readjustment of hydrogen bonding interactions between the [2Fe-2S] cluster, the Sgamma atoms of the cysteinyl ligands, and the backbone amide nitrogen atoms that results in tightening of the Cys39-Cys48 metal cluster binding loop around the prosthetic group and shifting of the metal center toward the Cys45-Thr47 peptide. From the metal center binding loop, the redox changes are transmitted to the linked Ile32-Asp38 peptide triggering structural rearrangement between the Tyr33-Asp34, Ser7-Asp9 and Pro102-Asp103 fragments of Pdx. The newly established hydrogen bonding interactions between Ser7, Asp9, Tyr33, Asp34, and Pro102, in turn, not only stabilize the tightened conformation of the [2Fe-2S] cluster binding loop but also assist in formation of a specific structural patch on the surface of Pdx that can be recognized by P450cam. This redox-linked change in surface properties is likely to be responsible for different binding affinity of oxidized and reduced Pdx to the hemoprotein.     

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3 Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile2-psi[CN4]-Ala3', Pro3-psi[CN4]-Ala4 and Val6-psi[CN4]-Ala7 were synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT).     

Effects of neuropeptide Y on food intake and brain biogenic amines in the rainbow trout (Oncorhynchus mykiss)

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in mammals, but very little is known about NPY actions in fish. The present study investigated the role of NPY in food intake in the rainbow trout (Oncorhynchus mykiss). Food intake was monitored at different times after intracerebroventricular administration of porcine NPY (4 or 8 microg). Both doses significantly increased food intake at 2 and 3 h, and this effect was dose-dependent. However, 50 h after administration of NPY, food intake was significantly lower than in control fish, and cumulative food intake had returned to levels similar to those seen in the control group. The NPY antagonist (D-Tyr27,36, D-Thr32)-NPY (10 microg) inhibited food intake 2 h after icv administration, but did not block the orexigenic effect of NPY when administered jointly with 4 microg NPY. To identify the NPY receptor subtypes involved in the effects of NPY on food intake, we studied the effects of the Y1 receptor agonist (Leu31, Pro34)-NPY (4 microg), the Y2 receptor agonist NPY(3-36) (4 microg), and the highly specific Y5 receptor agonist (cPP(1-7), NPY19-23, Ala31, Aib32, Gln34)-hPP (4 microg). Short-term (2 h) food intake was moderately stimulated by the Y1 agonist, more strongly stimulated by the Y2 agonist, and unaffected by the Y5 agonist. We found that administration of NPY (8 microg icv) had no effect on aminergic systems in several brain regions 2 and 50 h after NPY administration. These results indicate that NPY stimulates feeding in the rainbow trout, and suggest that this effect is cooperatively mediated by Y2- and Y1-like NPY receptors, not by Y5-like receptors.