Identification and characterization of two G protein-coupled receptors for neuropeptide FF

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.     

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster

Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC(50) = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC(50) = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.     

Regulation of endogenous human NPFF2 receptor by neuropeptide FF in SK-N-MC neuroblastoma cell line

Neuropeptide FF has many functions both in the CNS and periphery. Two G protein-coupled receptors (NPFF1 and NPFF2 receptors) have been identified for neuropeptide FF. The expression analysis of the peptide and receptors, together with pharmacological and physiological data, imply that NPFF2 receptor would be the primary receptor for neuropeptide FF. Here, we report for the first time a cell line endogenously expressing hNPFF2 receptor. These SK-N-MC neuroblastoma cells also express neuropeptide FF. We used the cells to investigate the hNPFF2 receptor function. The pertussis toxin-sensitive inhibition of adenylate cyclase activity upon receptor activation indicated coupling to Gi/o proteins. Upon agonist exposure, the receptors were internalized and the mitogen-activated protein kinase cascade was activated. Upon neuropeptide FF treatment, the actin cytoskeleton was reorganized in the cells. The expression of hNPFF2 receptor mRNA was up-regulated by neuropeptide FF. Concomitant with the receptor mRNA, the receptor protein expression was increased. The homologous regulation of hNPFF2 receptor correlates with our previous results in vivo showing that during inflammation, the up-regulation of neuropeptide FF mRNA precedes that of NPFF2 receptor. The regulation of hNPFF2 receptor by NPFF could also be important in the periphery where neuropeptide FF has been suggested to function as a hormone.     

Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

The novel neuropeptide Y Y(1) receptor antagonist J-104870: a potent feeding suppressant with oral bioavailability

Neuropeptide Y (NPY) is known to induce robust feeding through the action of NPY receptors in the hypothalamus. Among the subtypes of NPY receptors, Y(1) receptors may play a key role in feeding regulation. In the present study, we demonstrated that a novel Y(1) antagonist, J-104870, shows high selectivity and potency for the Y(1) receptor with an anorexigenic effect on NPY-mediated feeding. J-104870 displaced [(125)I]peptide YY (PYY) binding to cloned human and rat Y(1) receptors with K(i) values of 0.29 and 0.54 nM, respectively, and inhibited the NPY (10 nM)-induced increase in intracellular calcium levels (IC(50) = 3.2 nM) in cells expressing human Y(1) receptors. In contrast, J-104870 showed low affinities for human Y(2) (K(i) > 10 microM), Y(4) (K(i) > 10 microM), and Y(5) receptors (K(i) = 6 microM). In rat hypothalamic membranes, J-104870 also completely displaced the binding of [(125)I]1229U91, which is known to bind to the typical Y(1) receptor, with a high affinity (K(i) = 2.0 nM). Intracerebroventricular (ICV) injection of J-104870 (200 microg) significantly suppressed NPY (5 microg)-induced feeding in satiated Sprague-Dawley rats by 74%. Furthermore, ICV and oral administration of J-104870 (200 microg and 100 mg/kg, respectively) significantly suppressed spontaneous food intake in Zucker fatty rats. These findings suggested that J-104870 is a selective and potent nonpeptide Y(1) antagonist with oral bioavailability and brain penetrability. In addition, the anorexigenic effect of J-104870 clearly revealed the participation of the Y(1) receptor in NPY-mediated feeding regulation. The potent and orally active Y(1) antagonist J-104970 is a useful tool for elucidating the physiological roles of NPY in obesity.     

Evidence that NPY Y1 receptors are involved in stimulation of feeding by orexins (hypocretins) in sated rats

Neuropeptide Y (NPY) produced in the arcuate nucleus (ARC) of the hypothalamus stimulates feeding both directly by activating NPY receptors and indirectly through release of the orexigenic peptides, galanin and beta-endorphin (beta-END), in the paraventricular nucleus (PVN) and surrounding neural sites. Orexin A and orexin B, produced outside the ARC in the lateral hypothalamic area (LH), have recently been shown to stimulate feeding. In the present studies we tested the hypothesis that NPYergic signaling may mediate feeding stimulated by orexins. In adult male rats injected intracerebroventricularly (i.c.v.) with orexin A (3, 10, 15 nmol) or orexin B (3, 10, 30 nmol) feeding was stimulated in a dose-dependent manner; maximal feeding was seen after 15 nmol orexin A and 30 nmol orexin B. To determine whether NPY may mediate this orexin stimulated feeding, we used 1229U91, a selective NPY Y1 receptor antagonist (NPY-A). Whereas NPY-A on its own was ineffective, it suppressed NPY-induced feeding. Furthermore, NPY-A completely blocked the feeding evoked by either orexin A (15 nmol) or orexin B (30 nmol). These results show that orexin A and B stimulate feeding and further suggest that these excitatory effects may be mediated by NPYergic signaling through Y1 receptors. These findings are in accord with the view that the orexin-NPY pathway may comprise a functional link upstream from NPY within the hypothalamic appetite regulating network.     

Evidence of NPY Y5 receptor involvement in food intake elicited by orexin A in sated rats

Intracerebroventricular (icv) injections of orexin A stimulate feeding in sated rats. Since neuropeptide Y is a potent orexigenic peptide and orexin-containing neurons are morphologically linked with NPY-producing neurons in the hypothalamus, we evaluated the functional relationship between the two orexigenic peptides. The results show that whereas it was ineffective on its own, a selective NPY Y5 receptor antagonist, injected icv 15 min. before orexin A significantly suppressed orexin A-induced feeding. Since previous investigations demonstrated that an NPY Y1 receptor antagonist also inhibits feeding induced by orexin A, the current results further underscore the existence of a functional link between orexin and NPY producing neurons as the orexin network appears to be capable of influencing NPYergic signaling through Y1 and Y5 receptors to stimulate feeding.     

Elevated hypothalamic orexin signaling, sensitivity to orexin A, and spontaneous physical activity in obesity-resistant rats

Selectively-bred obesity-resistant [diet resistant (DR)] rats weigh less than obesity-prone [diet-induced obese (DIO)] rats, despite comparable daily caloric intake, suggesting phenotypic energy expenditure differences. Human data suggest that obesity is maintained by reduced ambulatory or spontaneous physical activity (SPA). The neuropeptide orexin A robustly stimulates SPA. We hypothesized that DR rats have greater: 1) basal SPA, 2) orexin A-induced SPA, and 3) preproorexin, orexin 1 and 2 receptor (OX1R and OX2R) mRNA, compared with DIO rats. A group of age-matched out-bred Sprague-Dawley rats were used as additional controls for the behavioral studies. DIO, DR, and Sprague-Dawley rats with dorsal-rostral lateral hypothalamic (rLHa) cannulas were injected with orexin A (0, 31.25, 62.5, 125, 250, and 500 pmol/0.5 microl). SPA and food intake were measured for 2 h after injection. Preproorexin, OX1R and OX2R mRNA in the rLHa, and whole hypothalamus were measured by real-time RT-PCR. Orexin A significantly stimulated feeding in all rats. Orexin A-induced SPA was significantly greater in DR and Sprague-Dawley rats than in DIO rats. Two-mo-old DR rats had significantly greater rLHa OX1R and OX2R mRNA than DIO rats but comparable preproorexin levels. Eight-mo-old DR rats had elevated OX1R and OX2R mRNA compared with DIO rats, although this increase was significant for OX2R only at this age. Thus DR rats show elevated basal and orexin A-induced SPA associated with increased OX1R and OX2R gene expression, suggesting that differences in orexin A signaling through OX1R and OX2R may mediate DIO and DR phenotypes.     

Identification of an NPY-Y1 receptor subtype in bovine chromaffin cells

Bovine chromaffin cells have been used in a variety of studies designed to reveal different aspects of neuropeptide Y (NPY) action. Pharmacological data have defined five NPY receptor subtypes, only one of which (Y3) has not been cloned. Some studies with bovine chromaffin cells have concluded that the effects of NPY on this cell type are mediated by the Y3 subtype. Previous work from our laboratory demonstrates that a Y1 subtype mediates the effect of NPY in this tissue. In the current studies we provide further evidence for the existence of the Y1 subtype in bovine chromaffin cells. BIBP3226, the selective Y1 antagonist, potently displaces [125I]NPY from its binding site IC50 = 1.91 x 10(-9) M. Moreover, [125I]BIBP3226 binds to bovine chromaffin cell membranes with high affinity (IC50 = 5.9 x 10(-8) M). Examination of BIBP3226 antagonism of NPY inhibition of forskolin stimulated cyclic AMP accumulation reveals that it is a competitive antagonist with a K(B) similar to the IC50 for [125I]BIBP3226 binding. Northern blot analysis using a porcine cDNA clone for the Y1 subtype demonstrates a 3.5-kb mRNA species in chromaffin cells. These data identify the bovine chromaffin cell NPY receptor as a Y1 subtype.     

Expression of mammalian RF-amide peptides neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the PrRP receptor in the peripheral tissues of the rat

The mRNA expression of neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and the UHR-1/GPR10 receptor were examined using in situ hybridization in rat peripheral tissues. In the hypophysis, modest expression of PrRP and receptor mRNA were seen in the anterior lobe. The trigeminal ganglion was devoid of expression signals. PrRP and UHR-1/GPR10 receptor mRNA:s were found in the adrenal medulla and PrRP mRNA was found in the pancreas. NPFF mRNA was detected in the spleen. In the testis and epididymis, PrRP and UHR-1/GPR10 receptor mRNA:s were detected. The results suggest a limited expression of mammalian RF-amide peptides in the peripheral organs.     

The SK-N-MC cell line expresses an orexin binding site different from recombinant orexin 1-type receptor.

Orexin A and B (also known as hypocretins), two recently discovered neuropeptides, play an important role in food intake, sleep/wake cycle and neuroendocrine functions. Orexins are endogenous ligands of two G-protein-coupled receptors, termed OX1 and OX2. This work presents the first short orexin A and B analogues, orexin A 23-33 and orexin B 18-28, with high affinity (119 +/- 49 and 49 +/- 23 nm) for OX1 receptors expressed on SK-N-MC cells and indicates the importance of the C-terminal part of the orexin peptides for this ligand-receptor interaction. However, these C-terminal fragments of orexin did not displace the 125I-labelled orexin B from the recombinant orexin 1 receptor stably expressed in Chinese hamster ovary cells. To examine the role of the shortened orexin A 23-33 in feeding, its effects in mimicking or antagonizing the effects of orexin A were studied in rats after administration via the lateral hypothalamus. In contrast with orexin A, which potently induced feeding up to 4 h after administration, orexin A 23-33 neither induced feeding nor inhibited orexin A-induced feeding. Modafinil (Vigil), which was shown earlier to activate orexin neurons, displayed binding neither to the orexin receptor expressed on SK-N-MC cells nor to the recombinant orexin 1 receptor, which indicates that modafinil displays its antinarcoleptic action via another yet unknown mechanism. PCR and subsequent sequencing revealed expression of the full-length orexin 1 receptor mRNA in SK-N-MC and NT-2 cells. Interestingly, sequencing of several cDNA clones derived from RNA of both SK-N-MC and NT-2 cells differed from the published nucleotide sequence at position 1375. Amino acid prediction of this A -->G change results in an isoleucine --> valine substitution at the protein level, which may provide evidence for an editing process.     

Interactions between orexin A, NPY and galanin in the control of food intake of the goldfish, Carassius auratus

The neuropeptides orexin A (OXA), neuropeptide Y (NPY) and galanin (GAL) have been shown to play a role in the regulation of food intake in mammals. They also significantly stimulate feeding in goldfish. In order to assess the interactions between these peptides in the control of feeding in goldfish, we investigated the effects of central injection of specific receptor antagonists for NPY (BIBP 3226) and GAL (M40) on OXA-induced feeding and the effects of desensitization of orexin receptors on NPY- and GAL-induced feeding. We investigated the effects of BIBP 3226 on GAL-induced feeding and the effects of M40 on NPY-induced feeding. We also examined the effects of coinjection of each pair of neuropeptides on feeding behavior. Injections of 10 ng/g OXA, 5 ng/g NPY and 10 ng/g GAL each induced an increase in feeding. Fish treated with 5 ng/g BIBP or 20 ng/g M40 had food consumption similar to saline controls. BIBP at 5 ng/g significantly reduced NPY- and OXA-induced feeding. Injections of 20 ng/g M40 significantly decreased GAL-induced feeding, but had no effect on OXA-induced feeding. Blocking of orexin receptors by treatment with high doses of OXA (100 ng/g) resulted in a decrease in both NPY- and GAL-induced feeding. Coinjection with 0.5 ng/g OXA and either 0.5 ng/g NPY or 0.5 ng/g GAL resulted in a food intake higher than that observed in saline control fish and in fish treated with NPY or GAL alone at 0.5 ng/g. NPY mRNA expression was increased in the telencephalon and in the hypothalamus compared to saline-treated fish, following injection of OXA. These results indicate that both NPY and GAL are at least, in part, dependent on coaction with OXA for the stimulation of food intake and feeding behavior in goldfish. In addition, the effects of OXA are mediated, in part, by the NPY pathway. This suggests a functional interdependence between these three peptidergic systems in the control of energy balance in goldfish.     

Neuropeptide Y receptor in cultured vascular smooth muscle cells: ligand binding and increase in cytosolic free Ca2+

We have previously shown that neuropeptide Y (NPY) increases cytosolic free Ca2+ concentration [( Ca2+]i) in porcine aortic smooth muscle cells. In this study, specific NPY receptor binding sites were identified in the cells by use of [125I]Bolton-Hunter NPY [( 125I]BH-NPY). Binding was to a single population of the sites with a Kd of 1.1 +/- 0.2 nM and a Bmax of 0.68 +/- 0.10 pmol/mg protein. [125I]BH-NPY binding was displaced by NPY-related peptides including members of the pancreatic polypeptide (PP) family. The potency of these peptides other than human PP for displacing [125I]BH-NPY binding was substantially consistent with their potency for increasing [Ca2+]i. Human PP had no effect on [Ca2+]i even at 10(-5) M, but it inhibited the NPY-induced increase in [Ca2+]i with a potency comparable to that for displacing [125I]BH-NPY binding. NPY(13-36) was about 500 and 300 times less effective than porcine NPY in increasing [Ca2+]i and in displacing [125I]BH-NPY binding, respectively, showing that the NPY receptor in cultured vascular smooth muscle cells is of the Y1-type.     

Neuropeptide Y (NPY) and peptide YY (PYY) receptors in rat brain

1. Specific binding sites for neuropeptide Y (NPY) and peptide YY (PYY) were investigated in rat brain areas using quantitative receptor autoradiography with 125I-Bolton-Hunter NPY (125I-BH-NPY) and 125I-PYY, radioligands for PP-fold family peptides receptors. 2. There were no differences between localization of 125I-BH-NPY and 125I-PYY binding sites in the rat brain. High densities of the binding sites were present in the anterior olfactory nucleus, lateral septal nucleus, stratum radiatum of the hippocampus, posteromedial cortical amygdaloid nucleus, and area postrema. 3. In cold ligand-saturation experiments done in the presence of increasing concentrations of unlabeled NPY and PYY, 125I-BH-NPY and 125I-PYY binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, molecular layer of the cerebellum, and area postrema was single and of a high affinity. There was a significant difference between the affinities of 125I-BH-NPY (Kd = 0.96 nM) and 125I-PYY binding (Kd = 0.05 nM) to the molecular layer of the cerebellum. The binding of the two radioligands to the other areas examined had the same affinities. 4. When comparing the potency of unlabeled rat pancreatic polypeptide (rPP), a family peptide of NPY and PYY, to inhibit the binding to the areas examined, rPP displaced 125I-BH-NPY and 125I-PYY binding to the area postrema more potently than it did the binding to the stratum radiatum of the hippocampus, layer I of the somatosensory frontoparietal cortex, and molecular layer of the cerebellum. 5. Thus, the quantitative receptor autoradiographic method with 125I-BH-NPY and 125I-PYY revealed differences in binding characteristics of specific NPY and PYY binding sites in different areas of the rat brain. The results provide further evidence for the existence of multiple NPY-PYY receptors in the central nervous system.     

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.     

Neuropeptide FF exerts pro- and anti-opioid actions in the parabrachial nucleus to modulate food intake

Neurons that synthesize the morphine modulatory peptide neuropeptide FF (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) densely innervate the parabrachial nucleus (PBN), an area implicated in regulating food intake. We analyzed opioid-related actions of NPFF in feeding in adult male Sprague-Dawley rats. Unilateral infusion of 2 nmol/0.5 microl of the mu-opioid receptor agonist [d-Ala2,NMe-Phe4,glycinol5]enkephalin (DAMGO) into the lateral PBN increased 4-h food intake from 0.7 +/- 0.1 to 3.3 +/- 0.3 g. NPFF (1.25-5.0 nmol) prevented this hyperphagic mu-opioidergic action. In rats fed after 4-h deprivation (baseline = 12.3 +/- 0.3 g/2 h), 5 nmol of NPFF did not alter and larger doses (10 and 20 nmol) actually increased food intake (+36, 54%). Twenty nanomoles also elevated intake of freely feeding rats (from 0.7 +/- 0.1 to 5.1 +/- 1.0 g/4 h). The opioid receptor blocker naloxone (10 nmol) antagonized this increase. These data reveal both pro- and anti-opioid actions of NPFF in the PBN to modulate feeding. The mechanisms for the opposite actions of low and high concentrations of this neuropeptide in parabrachial regulation of food intake remain to be determined.     

The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors.

We have cloned the guinea pig neuropeptide Y (NPY) Y1 receptor and found it to be 92-93% identical to other cloned mammalian Y1 receptors. Porcine NPY and peptide YY (PYY) displayed affinities of 43 pM and 48 pM, respectively. NPY2-36 and NPY3-36 had 6- and 46-fold lower affinity, respectively, than intact NPY. Functional coupling was measured by using a microphysiometer. Human NPY and PYY were equipotent in causing extracellular acidification with EC50 values of 0.59 nM and 0.69 nM, respectively, whereas NPY2-36 and NPY3-36 were about 15-fold and 500-fold less potent, respectively, than NPY. The present study shows that the cloned guinea pig Y1 receptor is very similar to its orthologues in other mammals, both with respect to sequence and pharmacology. Thus, results from previous studies on guinea pig NPY receptors might imply the existence of an additional Y1-like receptor sensitive to B1BP3226.