The neuropeptide Y (NPY) Y2 receptors are largely dimeric in the kidney, but monomeric in the forebrain

The neuropeptide Y(NPY) Y2 receptors are detected largely as dimers in the clonal expressions in CHO cells and in particulates from rabbit kidney cortex. However, in two areas of the forebrain (rat or rabbit piriform cortex and hypothalamus), these receptors are found mainly as monomers. Evidence is presented that this difference relates to large levels of G proteins containing the Gi alpha -subunit in the forebrain areas. The predominant monomeric status of these Y2 receptors should also be physiologically linked to large synaptic inputs of the agonist NPY. The rabbit kidney and the human CHO cell-expressed Y2 dimers are converted by agonists to monomers in vitro at a similar rate in the presence of divalent cations.     

Dimers of the Neuropeptide Y (NPY) Y2 Receptor Show Asymmetry in Agonist Affinity and Association with G Proteins

In conditions precluding activation of G proteins, the binding of agonists to dimers of the neuropeptide Y (NPY) Y2 receptor shows two components of similar size, but differing in affinity. The dimers of all NPY receptors are solubilized as ～ 180-kDa complexes containing one G protein α β γ trimer. These heteropentamers are stable to excess agonists, chelators, and alkylators. However, dispersion in the weak surfactant cholate releases ～ 300-kDa complexes. These findings indicate that both protomers in the Y2 dimer are associated with G protein heterotrimers, but the extent of interaction depends on affinity for the agonist peptide. The G protein in contact with the first-liganded, higher-affinity protomer should have a stronger interaction with the receptor and a larger probability of activation.     

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.     

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.     

A pool of Y2 neuropeptide Y receptors activated by modifiers of membrane sulfhydryl or cholesterol balance.

The cloned guinea-pig Y2 neuropeptide Y (NPY) receptors expressed in Chinese hamster ovary (CHO) cells, as well as the Y2 receptors natively expressed in rat forebrain, are distributed in two populations. A smaller population that is readily accessed by agonist peptides on the surface of intact cells constitutes less than 30% of Y2 receptors detected in particulates after cell homogenization. A much larger fraction of cell surface Y2 sites can be activated by sulfhydryl modifiers. A fast and large activation of these masked or cryptic sites could be obtained with membrane-permeating, vicinal cysteine-bridging arsenical phenylarsine oxide. A lower activation is effected by N-ethylmaleimide, an alkylator that slowly penetrates lipid bilayers. The restricted-access alkylator, 2-[(trimethylammonium)ethyl]methanethiosulfonate, was not effective in unmasking these sites. Some of the hidden cell surface Y2 sites could be activated by polyene filipin III through complexing of membrane cholesterol. The results are consistent with the presence of a large Y2 reserve in a compartment that can be accessed by alteration of sulfhydryl balance or fluidity of the cell membrane, and by treatments that affect the anchoring and aggregation of membrane proteins.     

Ligand association with the rabbit kidney and brain Y1, Y2 and Y5-like neuropeptide Y (NPY) receptors shows large subtype-related differences in sensitivity to chaotropic and alkylating agents

The binding to rabbit kidney or hypothalamic particulates of the subtype-selective neuropeptide Y (NPY) receptor ligands [125I](Leu31,Pro34)hPYY (as Y1 site label at 2 nM human pancreatic polypeptide (hPP)), [125I]-hPYY(3-36) (Y2 label), and [125I]-hPP (Y5 label) displayed great differences in sensitivity to alkylators and chaotropic agents. Sensitivity to a nonionic chaotrope, urea, was much higher for the Y1 binding than for the Y5-like binding or the Y2 binding. The non-selective alkylator N-ethylmaleimide (NEM) and several alkylators selective for aminergic receptors were much more efficacious against the Y1 relative to the Y2 binding. Similar differences could be confirmed with the attachment of Y1 and Y2-selective tracers to CHO cells expressing the cloned guinea-pig Y1 or Y2 receptors. The Y5-like binding was quite insensitive to NEM, but sensitive to chloroethylclonidine (CEC) and prazobind, which were less potent at the Y1, and especially at the Y2 site. The unrestricted-access alkylator 2-aminoethyl methanethiosulfonate inhibited the binding to all subtypes, while the restricted-access agent 2-(trimethylammonium)ethylmethanethiosulfonate poorly inhibited the Y5-like binding, or the guanine nucleotide-insensitive Y2 binding. These results are compatible with an active conformation of the Y5-like site dependent on maintenance of a shared hydrophobic cavity. The Y2 sites resistant to guanosine polyphosphates and restricted-access alkylators were detected mainly in particulates slowly solubilized by cholate at 0-5 degrees C; these sites could be clustered.     

Self-regulation of agonist activity at the Y receptors

Neuropeptide Y (NPY) is one of the most abundant neuropeptides, and is likely to be present at nanomolar levels over extended periods in the synaptic space of many forebrain areas. This might be linked to an evolved generalized toning activity through a number of other peptide receptors that use C-terminally amidated agonists (with LHRH and orexin receptors and GIR as examples). However, the Y1 and Y2 receptors (which constitute the bulk of Y receptors active in the neural matrix) possess subnanomolar affinities that, at saturating NPY levels, could produce excessive signaling, as well as receptor losses via repeated endocytosis. The related Y4 receptor shows an even higher agonist affinity, and faces the same problem in visceral and neural locations accessible to pancreatic polypeptide (PP). An examination of agonist peptide interaction with Y receptors shows that Y1 and Y4 receptors in particular (as located on either the intact cells, or on particulates derived from various cell types) develop a blockade dependent on ligand concentration, with the blocking ranks of [NPY]>[peptide YY] (PYY) for the Y1, and [human PP]>[PYY-related Y4 agonist] for the Y4 receptor. This blockade is also echoed in a concentration-related reduction in biological activity of primary agonists (NPY and PP), resembling a partial agonism, and is influenced especially by the allosteric interactivity of agonists. With the Y2 receptor, the blocking by agonists is less pronounced, but the signaling by NPY-related peptides is apparently less than with PYY-related agonists. The extended occupancy and self-attenuation of primary agonist activity at Y receptors could represent an evolutionary solution contributing to a balancing of metabolic signaling, agonist clearance and receptor conservation.     

Dimers of the neuropeptide Y (NPY) Y2 receptor show asymmetry in agonist affinity and association with G proteins

In conditions precluding activation of G proteins, the binding of agonists to dimers of the neuropeptide Y (NPY) Y2 receptor shows two components of similar size, but differing in affinity. The dimers of all NPY receptors are solubilized as approximately 180-kDa complexes containing one G protein alpha beta gamma trimer. These heteropentamers are stable to excess agonists, chelators, and alkylators. However, dispersion in the weak surfactant cholate releases approximately 300-kDa complexes. These findings indicate that both protomers in the Y2 dimer are associated with G protein heterotrimers, but the extent of interaction depends on affinity for the agonist peptide. The G protein in contact with the first-liganded, higher-affinity protomer should have a stronger interaction with the receptor and a larger probability of activation.     

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.     

Distribution of neuropeptide Y immunoreactivity in the forebrain of the rat

The distribution of neuropeptide Y (NPY) like immunoreactivity was investigated in the forebrain of the rat with immunohistochemical methods. Specificity of the antisera was established by the absence of all immunoreactive staining in tissue incubated in antisera which had been preabsorbed with the pure NPY antigen. NPY containing cells were distributed widely in the forebrain. These included neocortex, basal forebrain, septum, bed nucleus of the stria terminalis, neostratium, hypothalamic arcuate nucleus, and intergeniculate leaflet. This study also demonstrated an extensive network of NPY fibers in various areas of the forebrain such as the prepotic area, the hypothalamus and the paraventricular nucleus of the thalamus. The distribution of avian pancreatic polypeptide and NPY was compared, and the possible importance of NPY is briefly discussed.     

Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands

The neuropeptide Y-family receptor Y4 differs extensively between human and rat in sequence, receptor binding, and anatomical distribution. We have investigated the differences in binding profile between the cloned human, rat, and guinea pig Y4 receptors using NPY analogues with single amino acid replacements or deletion of the central portion. The most striking result was the increase in affinity for the rat receptor, but not for human or guinea pig, when amino acid 34 was replaced with proline; [Ahx(8-20),Pro(34)]NPY bound to the rat Y4 receptor with 20-fold higher affinity than [Ahx(8-20)]NPY. Also, the rat Y4 tolerates alanine in position 34 since p[Ala(34)]NPY bound with similar affinity as pNPY while the affinity for hY4 and gpY4 decreased about 50-fold. Alanine substitutions in position 33, 35, and 36 as well as the large loop-deletion, [Ahx(5-24)]NPY, reduced the binding affinity to all three receptors more than 100-fold. NPY and PYY competed with (125)I-hPP at Y4 receptors expressed in CHO cells according to a two-site model. This was investigated for gpY4 by saturation with either radiolabeled hPP or pPYY. The number of high-affinity binding-sites for (125)I-pPYY was about 60% of the receptors recognized by (125)I-hPP. Porcine [Ala(34)]NPY and [Ahx(8-20)]NPY bound to rY4 (but not to hY4 or gpY4) according to a two-site model. These results suggest that different full agonists can distinguish between different active conformations of the gpY4 receptor and that Y4 may display functional differences in vivo between human, guinea pig, and rat.     

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.     

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 modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.     

Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors

It is well documented that neuropeptide Y (NPY) exerts a wide range of biological functions through at least five NPY Y receptor subtypes (Y1-Y5), but its immunological effects only recently came into focus. Using NPY family peptides and NPY-related receptor-specific peptides as well as Y1 and Y2 receptor antagonists, we have tested which NPY Y receptors are involved in NPY-induced modulation of rat peritoneal macrophage function in vitro. NPY and PYY increased oxidative burst in phorbol myristate acetate (PMA)-stimulated macrophages involving activation of protein kinase C (PKC), and decreased it in zymosan-stimulated cells resembling inhibition of signaling pathways subsequent to binding of zymosan particles for the iC3b fragment receptor on macrophages. The combined treatment with NPY and NPY Y receptor antagonists revealed that NPY-induced potentiation of oxidative burst in PMA-stimulated cells is mediated through Y1 and Y2 receptors, while NPY-induced suppression in zymosan-stimulated cells is mediated through Y2 receptors only. NPY-related peptides differently modulated macrophage function, confirming involvement of NPY Y2 receptor in both potentiation and suppression of oxidative burst in these cells. Additionally, it was shown that NPY Y5 receptor mediated suppression of oxidative burst in PMA- and zymosan-stimulated macrophages. Taken together, the present data reveal an NPY Y1 and Y2/Y5 receptor interaction in NPY-induced modulation of macrophage functions related to inflammation.     

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

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

Differential regulation of neuropeptide Y receptors in the brains of NPY knock-out mice

To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.     

Blockade of pancreatic polypeptide-sensitive neuropeptide Y (NPY) receptors by agonist peptides is prevented by modulators of sodium transport. Implications for receptor signaling and regulation

Ligand binding to rodent pancreatic polypeptide-responding neuropeptide Y (NPY) receptors (here termed PP/NPY receptors), or to cloned Y4 or Y5 receptors, is selectively inhibited by amiloride, peptide or alkylating modulators of sodium transport. The PP/NPY and Y4 receptors are also selectively blocked by human or rat pancreatic polypeptide (PP) and the blocking peptides are not dissociated by high concentrations of alkali chlorides (which restore most of the binding of subtype-selective agonists to Y1 and Y2 sites). The PP/NPY receptors could also be blocked by NPY and related full-length peptides, including Y1-selective agonists (IC50 300-400 pM). The cloned Y(4) receptors from three species are much less sensitive to NPY or PYY. The sensitivity of both the PP/NPY sites and the Y(4) sites to Y2-selective peptides is quite low. The ligand attachment to PP/NPY sites is also very sensitive to peptidic Y1 antagonist ((Cys31,NVal34NPY27-36))2, which however blocks these sites at much higher molarities. Blockade of PP/NPY and Y4 sites by agonist peptides can be largely prevented by N5-substituted amiloride modulators of Na+ transport, and by RFamide NRNFLRF.NH2, but not by Ca2+ channel blockers, or by inhibitors of K+ transport. Protection of both PP/NPY and Y4 sites against blockade by human or rat pancreatic polypeptide is also afforded by short N-terminally truncated NPY-related peptides. The above results are consistent with a stringent and selective activity regulation for rabbit PP/NPY receptor(s) that may serve to differentiate agonists and constrain signaling, and could involve transporter-like interactants.     

A novel cyclic analog of neuropeptide Y specific for the Y2 receptor.

The low-molecular-mass, cyclic analog of neuropeptide Y, [Ahx5-24, gamma-Glu2-epsilon-Lys30] NPY (YESK-Ahx-RHYINKITRQRY; Ahx, 6-aminohexanoic acid; NPY, neuropeptide Y), was synthesized and investigated for receptor binding, inhibition of forskolin-stimulated cAMP accumulation, inhibition of electrically stimulated rat vas deferens contractions and ability to increase blood pressure. Like the linear peptide [Ahx5-24] NPY (YPSK-Ahx-RHYINLITRQRY), the more rigid, cyclic analog showed good correlation between receptor binding to rabbit kidney membranes and biological activity in the vas deferens assay. Binding of this peptide to a new Y2-receptor-expressing cell line was slightly reduced, compared to the linear peptide [Ahx5-24] NPY, however inhibition of cAMP accumulation was even more efficient. Unlike the linear peptide [Ahx5-24] NPY, the cyclic analog did not induce a blood pressure increase in rats. Reduced binding to Y1 receptor-expressing SK-N-MC cells, as well as the loss of capability of signal transduction, suggest that only Y2-mediated activity is preserved after cyclization. The selectivity of the cyclic compound for Y2 subtypes of NPY receptors with respect to inhibition of cAMP accumulation is more than fortyfold increased, as compared to the linear NPY-(13-36) peptide, which has been used to determine Y2 selectivity so far.     

Sensitivity of orexin-A binding to phospholipase C inhibitors, neuropeptide Y, and secretin

The binding of [(125)I] orexin-A (Ox-A) to particulates from Chinese hamster ovary (CHO) cells expressing the cloned orexin-A receptor, or from rat forebrain areas, was sensitive to blockers of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) U-73122 and ET-18-OCH(3), little affected by phospholipase A(2) inhibitor quinacrine, and not sensitive to D609, a xanthate inhibitor of phosphatidylcholine-selective PLC. Interaction of the receptor with a PtdIns-PLC was further indicated by a large sensitivity of the binding to Ca(2+). Up to 50% of the binding was sensitive to the G-protein nucleotide site agonist GTP-gamma-S. Ligand attachment to the orexin-A receptor thus depends on an association with both PtdIns-PLC and G-protein alpha-subunits. In all paradigms examined, the binding of [(125)I]orexin-A was competed by human/rat neuropeptide Y (hNPY) and porcine secretin with a potency similar to orexin-A (IC(50) range 30-100 nM). The rank order of potency for NPY-related peptides was hNPY > porcine peptide YY (pPYY) > (Leu(31), Pro(34)) human PYY > human PYY(3-36) > hNPY free acid > human pancreatic polypeptide. Among secretin-related peptides, the rank order of potency was porcine secretin > or = orexin-A > human pituitary adenylate cyclase-activating peptide > orexin-B > porcine vasoactive intestinal peptide. Among opioid peptides, rat beta-endorphin and camel delta-endorphin were much less active than NPY and secretin, and two enkephalins were inactive at 1 microM. In view of high abundance of NPY in forebrain, the above cross-reactivity could indicate a significant contribution of NPY to signaling via orexin-A receptors.