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Identification and Characterization of Novel Mammalian Neuropeptide FF-like Peptides That Attenuate Morphine-induced Antinociception

2001; Elsevier BV; Volume: 276; Issue: 40 Linguagem: Inglês

10.1074/jbc.m105308200

1083-351X

Qingyun Liu, Xiao Ming Guan, William J. Martin, Terrence P. McDonald, Michelle K. Clements, Qingping Jiang, Zhizhen Zeng, Marlene A. Jacobson, David L. Williams, Hong Yu, Douglas Bomford, David J. Figueroa, John Mallee, Ruiping Wang, Jilly F. Evans, Robert J. Gould, Christopher P. Austin,

Cardiovascular, Neuropeptides, and Oxidative Stress Research

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism. The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism. Phe-Met-Arg-Phe-NH2 Phe-Met-Arg-Phe-NH2-related peptide Leu-Pro-Leu-Arg-Phe-NH2 intracerebral ventricular central nervous system Chinese hamster ovary nuclear factor of activated T cells expressed sequence tag fluorescence imaging plate reader The Phe-Met-Arg-Phe-NH2(FMRFamide)1-related peptides (FaRPs) constitute a large family of neuropeptides that are widely distributed in invertebrates, and function as neurotransmitters and neuromodulators (1Greenberg M.J. Price D.A. Prog. Brain Res. 1992; 92: 25-37Crossref PubMed Scopus (143) Google Scholar, 2Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). Existence of FaRPs in vertebrates was initially demonstrated by the observation of specific immunoreactive staining in rat brain using anti-FMRFamide antisera (3Weber E. Evans C.J. Samuelsson S.J. Barchas J.D. Science. 1981; 214: 1248-1251Crossref PubMed Scopus (145) Google Scholar). The first vertebrate FaRP, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRFa), was isolated from chicken brain using anti-FMRFamide antisera (4Dockray G.J. Reeve Jr., J.R. Shively J. Gayton R.J. Barnard C.S. Nature. 1983; 305: 328-330Crossref PubMed Scopus (225) Google Scholar). Two mammalian FaRP-like peptides, NPAF and NPFF, were then isolated from bovine brain by a similar approach (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar). Isolation of the gene encoding NPAF and NPFF revealed that the two peptides are generated from the processing of a single large precursor (6Perry S.J. Huang E.Y.-K. Cronk D. Bagust J. Sharma R. Walker R.J. Wilson S. Burke J.F. FEBS Lett. 1997; 409: 426-430Crossref PubMed Scopus (197) Google Scholar, 7Vilim F.S. Aarnisalo A.A. Nieminen M.L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar).A great deal of evidence suggests that NPAF and NPFF play important roles in the control of pain and analgesia through interactions with the opioid system (see Refs. 8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar and 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar for review). Intracerebroventricular administration of FMRFamide, NPFF, or NPAF attenuated morphine-induced analgesia whereas injection of antisera against FMRFamide or NPFF had the opposite effect (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 10Kavaliers M. Yang H.Y. Peptides. 1989; 10: 741-745Crossref PubMed Scopus (94) Google Scholar). Administration of NPFF into morphine-tolerant rats induced symptoms of the withdrawal whereas administration of anti-NPFF IgG reversed morphine tolerance (11Malin D.H. Lake J.R. Hammond M.V. Fowler D.E. Rogillio R.B. Brown S.L. Sims J.L. Leecraft B.M. Yang H.Y. Peptides. 1990; 11: 969-972Crossref PubMed Scopus (148) Google Scholar, 12Lake J.R. Hammond M.V. Shaddox R.C. Hunsicker L.M. Yang H.Y. Malin D.H. Neurosci. Lett. 1991; 132: 29-32Crossref PubMed Scopus (110) Google Scholar). Such observations led to the classification of NPFF as one type of anti-opioid peptides, which have been hypothesized to be partially responsible for the rapid development of opioid tolerance and dependence in animal models and clinical use of opioids (13Harrison L.M. Kastin A.J. Zadina J.E. Peptides. 1998; 19: 1603-1630Crossref PubMed Scopus (175) Google Scholar). NPFF, on the other hand, was also shown to have pro-opioid effect following intrathecal administration (14Gouarderes C. Sutak M. Zajac J.M. Jhamandas K. Eur. J. Pharmacol. 1993; 237: 73-81Crossref PubMed Scopus (139) Google Scholar). Furthermore, recent data indicate that ICV-administrated NPFF was able to attenuate neuropathic pain independent of the opioid system (15Altier N. Dray A. Menard D. Henry J.L. Eur. J. Pharmacol. 2000; 407: 245-255Crossref PubMed Scopus (28) Google Scholar). In addition, FMRFamide and NPFF were also shown to affect the cardiovascular system and cause other behavioral changes in mammals (8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar). Recently, molecular identification of two receptors (FF1 and FF2) have been reported for NPFF and NPAF (16Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 18Kotani M. Mollereau C. Detheux M. Le Poul E. Brezillon S. Vakili J. Mazarguil H. Vassart G. Zajac J.M. Parmentier M. Br. J. Pharmacol. 2001; 133: 138-144Crossref PubMed Scopus (112) Google Scholar). A novel mammalian gene encoding NPFF-like peptides (RFRP1 and RFRP3) and their receptor (OT7T022) was also reported (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar). A quail gene encoding neuropeptides highly similar to the chicken peptide LPLRFa was just published (20Satake H. Hisada M. Kawada T. Minakata H. Ukena K. Tsutsui K. Biochem. J. 2001; 354: 379-385Crossref PubMed Scopus (175) Google Scholar). Herein we describe the comparison of the two NPFF receptors in their responses to various NPFF-like peptides and in their expression pattern in the rat central nervous system (CNS). We also describe the independent identification of novel NPFF-related peptides (designated NPVF) and their role in attenuating morphine-induced analgesia. Potential roles of the two receptors/four peptide ligands in nociception modulation are discussed.DISCUSSIONFaRPs constitute a large family of neuropeptides in invertebrates (1Greenberg M.J. Price D.A. Prog. Brain Res. 1992; 92: 25-37Crossref PubMed Scopus (143) Google Scholar, 2Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). The diversity of FaRPs in vertebrates, however, appears to be more limited. In addition to NPFF, NPAF, and LPLRFa, only one other RFamide neuropeptide, the prolactin-releasing peptide, has been isolated from vertebrates (33Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (529) Google Scholar). In this report, we described independent identification and characterization of a mammalian gene designated NPVF and its encoded peptides, which most likely represents the mammalian ortholog of the chicken peptide LPLRFa. We also described independent identification and characterization of two NPFF receptors by comparing their affinity for various FaRPs and detailed analysis of their expression pattern in the rat CNS. Furthermore, we showed that NPVF-derived peptides were more potent than NPFF in attenuating morphine-induced analgesia. The molecular pharmacological properties of FF1 and FF2 toward various NPFF and NPVF peptides and their expression pattern in the rat CNS presented here offer new insights into potential mechanisms of the pain-modulatory functions associated with NPFF.In characterizing affinities of the two receptors to various FaRPs, we noticed FF1 could be activated by many more FaRPs than FF2. Particularly, we were surprised to find that FF1 showed a better response to the chicken peptide LPLRFa than to NPFF, which led to the search for mammalian LPLRFa-like peptides and the eventual identification of NPVF. In the report by Bonini et al. (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), FF1 showed much lower affinity to NPFF (EC50 = 16 nm) than FF2 did (EC50 = 2.0 nm). But no data were presented for LPLRFa. Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) discovered NPVF by looking for mammalian LPLRFa-like peptides directly and then tested them on the orphan receptor FF1/OT7T022. We have now carried out a side-by-side comparison of NPFF- and NPVF-related peptides on FF1 and FF2, and the in vitro data presented here and those from Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) clearly indicated that NPVF-related peptides are more potent ligands for FF1 and NPFF-related peptides are much better ligands for FF2.We mapped the expression pattern of both FF1 and FF2 in the entire rat CNS by in situ hybridization, and the data were nearly completely consistent with the results from radioligand binding analysis. Using 125I-labeled (1DNme)bNPFF, which turned out to have equal affinities to rat FF1 and FF2 (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), Dupouy et al. (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar) mapped NPFF binding sites in the entire rat CNS. Specific binding was detected widely in the CNS with intense signals in the parafascicular, reticular thalamic nuclei, lateral septum, and nucleus of the solitary tract, and in the superficial layers in the dorsal horn and around central canal of the spinal cord, but only a single class of binding was noticed. Examination of FF1 and FF2 expression by in situ hybridization has now allowed us to compare the distribution of the two receptors. FF1 is much more widely expressed, with especially strong signals in brain regions regulating the expression of fear and anxiety, and affective aspects of pain (35Davis M. Biol. Psychiatry. 1998; 44 (1147): 1239Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 36Price D.D. Science. 2000; 288: 1769-1772Crossref PubMed Scopus (1495) Google Scholar). FF2 mRNA levels, on the other hand, are much more limited, with the highest levels found in the spinal cord. It is of interest to note, however, that some discrepancies exist between the patterns of receptor binding and receptor mRNA expression. For example, high density NPFF bindings were observed in the presubiculum and spinal trigeminal tract nucleus (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar, 37Allard M. Zajac J.M. Simonnet G. Neuroscience. 1992; 49: 101-116Crossref PubMed Scopus (112) Google Scholar), but these brain regions express little or no detectable FF1/FF2 mRNA. Although the precise reason for such mismatch is unknown, it could be due to subtle differences between the iodinated NPFF analogues used as the radioligands and native NPFF in binding to the receptors or it could be the consequence of transportation of mature/functional receptor proteins (binding sites) away from their origin of synthesis (mRNA), which were measured by autoradiography and in situ hybridization, respectively. Alternatively, these results may suggest existence of additional receptor(s) for NPFF.The genes encoding NPFF and NPVF are similar in structure but distinct in expression pattern, receptor pharmacology, and presumably physiological functions. NPFF is primarily expressed in the paraventricular and supraoptic nuclei of the hypothalamus, the nucleus of the solitary tract, and the superficial layers in the dorsal horn of spinal cord (7Vilim F.S. Aarnisalo A.A. Nieminen M.L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar), areas with predominant FF2 expression. In contrast, NPVF is only expressed in a population of neurons between the dorsomedial hypothalamic and ventromedial hypothalamic nucleus, which were shown to project to a number of limbic structures including the lateral septal nucleus, bed nucleus of the stria terminalis, amygdala, and hypothalamus as well as to the periaqueductal gray (38Aarnisalo A.A. Panula P. Neuroscience. 1995; 65: 175-192Crossref PubMed Scopus (45) Google Scholar), areas where FF1 is strongly expressed. Furthermore, in vitrobinding and functional analyses also indicate that FF2 can only be activated NPFF-related peptides, whereas FF1 is activated by NPVF-related peptides with a higher affinity than by NPFF-related peptides. Taken together, these data strongly suggest that the physiologically relevant ligands for FF2 are the NPFF-related peptides whereas the NPVF-related peptides are the preferred ligands for FF1.NPFF-related peptides have been shown to be involved in various somatosensory and visceral functions (8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar). Perhaps the best characterized action for NPFF is its role in nociception and analgesia (for recent reviews, see Refs. 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar and 39Panula P. Kalso E. Nieminen M. Kontinen V.K. Brandt A. Pertovaara A. Brain Res. 1999; 848: 191-196Crossref PubMed Scopus (150) Google Scholar). There is a body of evidence that central administration of NPFF-related peptides produces hyperalgesia and reverses morphine-induced analgesia (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar). Intrathecal administration of NPFF, however, produces antinociceptive, pro-opioid effects (14Gouarderes C. Sutak M. Zajac J.M. Jhamandas K. Eur. J. Pharmacol. 1993; 237: 73-81Crossref PubMed Scopus (139) Google Scholar). Since our in situ data showed only FF2 expression in rat spinal cord, FF2 most likely mediated the pro-opioid effect of NPFF following intrathecal injection. On the other hand, the anti-opioid effects of intracerebroventricularly administered NPFF were most likely mediated by FF1 based on the following observations. Our data showed that NPSF-(1–37), which had poor if any agonist activity for FF2 but potent activity for FF1, was more potent than NPFF in suppressing morphine-induced analgesia in two models of nociception (Fig. 7). FMRFamide, which displayed no significant agonist activity for FF2 but potent activity for FF1, was equally as potent as NPFF in anti-opioid activity (40Tang J. Yang H.Y. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5002-5005Crossref PubMed Scopus (214) Google Scholar). Only FF1 is expressed in the periaqueductal gray (Table II), a region critical for the control of nociception. All together, these in vitro and in vivo observations strongly suggest that the anti-opioid effects of NPFF were actually mediated by the FF1 receptor, and the real endogenous anti-opioid peptides are NPVF-related peptides. Furthermore, the strong expression of FF1 in the limic system may suggest an important role of NPVF in manifestation of the affective aspects of pain. The Phe-Met-Arg-Phe-NH2(FMRFamide)1-related peptides (FaRPs) constitute a large family of neuropeptides that are widely distributed in invertebrates, and function as neurotransmitters and neuromodulators (1Greenberg M.J. Price D.A. Prog. Brain Res. 1992; 92: 25-37Crossref PubMed Scopus (143) Google Scholar, 2Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). Existence of FaRPs in vertebrates was initially demonstrated by the observation of specific immunoreactive staining in rat brain using anti-FMRFamide antisera (3Weber E. Evans C.J. Samuelsson S.J. Barchas J.D. Science. 1981; 214: 1248-1251Crossref PubMed Scopus (145) Google Scholar). The first vertebrate FaRP, Leu-Pro-Leu-Arg-Phe-NH2 (LPLRFa), was isolated from chicken brain using anti-FMRFamide antisera (4Dockray G.J. Reeve Jr., J.R. Shively J. Gayton R.J. Barnard C.S. Nature. 1983; 305: 328-330Crossref PubMed Scopus (225) Google Scholar). Two mammalian FaRP-like peptides, NPAF and NPFF, were then isolated from bovine brain by a similar approach (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar). Isolation of the gene encoding NPAF and NPFF revealed that the two peptides are generated from the processing of a single large precursor (6Perry S.J. Huang E.Y.-K. Cronk D. Bagust J. Sharma R. Walker R.J. Wilson S. Burke J.F. FEBS Lett. 1997; 409: 426-430Crossref PubMed Scopus (197) Google Scholar, 7Vilim F.S. Aarnisalo A.A. Nieminen M.L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar). A great deal of evidence suggests that NPAF and NPFF play important roles in the control of pain and analgesia through interactions with the opioid system (see Refs. 8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar and 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar for review). Intracerebroventricular administration of FMRFamide, NPFF, or NPAF attenuated morphine-induced analgesia whereas injection of antisera against FMRFamide or NPFF had the opposite effect (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 10Kavaliers M. Yang H.Y. Peptides. 1989; 10: 741-745Crossref PubMed Scopus (94) Google Scholar). Administration of NPFF into morphine-tolerant rats induced symptoms of the withdrawal whereas administration of anti-NPFF IgG reversed morphine tolerance (11Malin D.H. Lake J.R. Hammond M.V. Fowler D.E. Rogillio R.B. Brown S.L. Sims J.L. Leecraft B.M. Yang H.Y. Peptides. 1990; 11: 969-972Crossref PubMed Scopus (148) Google Scholar, 12Lake J.R. Hammond M.V. Shaddox R.C. Hunsicker L.M. Yang H.Y. Malin D.H. Neurosci. Lett. 1991; 132: 29-32Crossref PubMed Scopus (110) Google Scholar). Such observations led to the classification of NPFF as one type of anti-opioid peptides, which have been hypothesized to be partially responsible for the rapid development of opioid tolerance and dependence in animal models and clinical use of opioids (13Harrison L.M. Kastin A.J. Zadina J.E. Peptides. 1998; 19: 1603-1630Crossref PubMed Scopus (175) Google Scholar). NPFF, on the other hand, was also shown to have pro-opioid effect following intrathecal administration (14Gouarderes C. Sutak M. Zajac J.M. Jhamandas K. Eur. J. Pharmacol. 1993; 237: 73-81Crossref PubMed Scopus (139) Google Scholar). Furthermore, recent data indicate that ICV-administrated NPFF was able to attenuate neuropathic pain independent of the opioid system (15Altier N. Dray A. Menard D. Henry J.L. Eur. J. Pharmacol. 2000; 407: 245-255Crossref PubMed Scopus (28) Google Scholar). In addition, FMRFamide and NPFF were also shown to affect the cardiovascular system and cause other behavioral changes in mammals (8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar). Recently, molecular identification of two receptors (FF1 and FF2) have been reported for NPFF and NPAF (16Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 18Kotani M. Mollereau C. Detheux M. Le Poul E. Brezillon S. Vakili J. Mazarguil H. Vassart G. Zajac J.M. Parmentier M. Br. J. Pharmacol. 2001; 133: 138-144Crossref PubMed Scopus (112) Google Scholar). A novel mammalian gene encoding NPFF-like peptides (RFRP1 and RFRP3) and their receptor (OT7T022) was also reported (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar). A quail gene encoding neuropeptides highly similar to the chicken peptide LPLRFa was just published (20Satake H. Hisada M. Kawada T. Minakata H. Ukena K. Tsutsui K. Biochem. J. 2001; 354: 379-385Crossref PubMed Scopus (175) Google Scholar). Herein we describe the comparison of the two NPFF receptors in their responses to various NPFF-like peptides and in their expression pattern in the rat central nervous system (CNS). We also describe the independent identification of novel NPFF-related peptides (designated NPVF) and their role in attenuating morphine-induced analgesia. Potential roles of the two receptors/four peptide ligands in nociception modulation are discussed. DISCUSSIONFaRPs constitute a large family of neuropeptides in invertebrates (1Greenberg M.J. Price D.A. Prog. Brain Res. 1992; 92: 25-37Crossref PubMed Scopus (143) Google Scholar, 2Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). The diversity of FaRPs in vertebrates, however, appears to be more limited. In addition to NPFF, NPAF, and LPLRFa, only one other RFamide neuropeptide, the prolactin-releasing peptide, has been isolated from vertebrates (33Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (529) Google Scholar). In this report, we described independent identification and characterization of a mammalian gene designated NPVF and its encoded peptides, which most likely represents the mammalian ortholog of the chicken peptide LPLRFa. We also described independent identification and characterization of two NPFF receptors by comparing their affinity for various FaRPs and detailed analysis of their expression pattern in the rat CNS. Furthermore, we showed that NPVF-derived peptides were more potent than NPFF in attenuating morphine-induced analgesia. The molecular pharmacological properties of FF1 and FF2 toward various NPFF and NPVF peptides and their expression pattern in the rat CNS presented here offer new insights into potential mechanisms of the pain-modulatory functions associated with NPFF.In characterizing affinities of the two receptors to various FaRPs, we noticed FF1 could be activated by many more FaRPs than FF2. Particularly, we were surprised to find that FF1 showed a better response to the chicken peptide LPLRFa than to NPFF, which led to the search for mammalian LPLRFa-like peptides and the eventual identification of NPVF. In the report by Bonini et al. (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), FF1 showed much lower affinity to NPFF (EC50 = 16 nm) than FF2 did (EC50 = 2.0 nm). But no data were presented for LPLRFa. Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) discovered NPVF by looking for mammalian LPLRFa-like peptides directly and then tested them on the orphan receptor FF1/OT7T022. We have now carried out a side-by-side comparison of NPFF- and NPVF-related peptides on FF1 and FF2, and the in vitro data presented here and those from Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) clearly indicated that NPVF-related peptides are more potent ligands for FF1 and NPFF-related peptides are much better ligands for FF2.We mapped the expression pattern of both FF1 and FF2 in the entire rat CNS by in situ hybridization, and the data were nearly completely consistent with the results from radioligand binding analysis. Using 125I-labeled (1DNme)bNPFF, which turned out to have equal affinities to rat FF1 and FF2 (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), Dupouy et al. (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar) mapped NPFF binding sites in the entire rat CNS. Specific binding was detected widely in the CNS with intense signals in the parafascicular, reticular thalamic nuclei, lateral septum, and nucleus of the solitary tract, and in the superficial layers in the dorsal horn and around central canal of the spinal cord, but only a single class of binding was noticed. Examination of FF1 and FF2 expression by in situ hybridization has now allowed us to compare the distribution of the two receptors. FF1 is much more widely expressed, with especially strong signals in brain regions regulating the expression of fear and anxiety, and affective aspects of pain (35Davis M. Biol. Psychiatry. 1998; 44 (1147): 1239Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 36Price D.D. Science. 2000; 288: 1769-1772Crossref PubMed Scopus (1495) Google Scholar). FF2 mRNA levels, on the other hand, are much more limited, with the highest levels found in the spinal cord. It is of interest to note, however, that some discrepancies exist between the patterns of receptor binding and receptor mRNA expression. For example, high density NPFF bindings were observed in the presubiculum and spinal trigeminal tract nucleus (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar, 37Allard M. Zajac J.M. Simonnet G. Neuroscience. 1992; 49: 101-116Crossref PubMed Scopus (112) Google Scholar), but these brain regions express little or no detectable FF1/FF2 mRNA. Although the precise reason for such mismatch is unknown, it could be due to subtle differences between the iodinated NPFF analogues used as the radioligands and native NPFF in binding to the receptors or it could be the consequence of transportation of mature/functional receptor proteins (binding sites) away from their origin of synthesis (mRNA), which were measured by autoradiography and in situ hybridization, respectively. Alternatively, these results may suggest existence of additional receptor(s) for NPFF.The genes encoding NPFF and NPVF are similar in structure but distinct in expression pattern, receptor pharmacology, and presumably physiological functions. NPFF is primarily expressed in the paraventricular and supraoptic nuclei of the hypothalamus, the nucleus of the solitary tract, and the superficial layers in the dorsal horn of spinal cord (7Vilim F.S. Aarnisalo A.A. Nieminen M.L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar), areas with predominant FF2 expression. In contrast, NPVF is only expressed in a population of neurons between the dorsomedial hypothalamic and ventromedial hypothalamic nucleus, which were shown to project to a number of limbic structures including the lateral septal nucleus, bed nucleus of the stria terminalis, amygdala, and hypothalamus as well as to the periaqueductal gray (38Aarnisalo A.A. Panula P. Neuroscience. 1995; 65: 175-192Crossref PubMed Scopus (45) Google Scholar), areas where FF1 is strongly expressed. Furthermore, in vitrobinding and functional analyses also indicate that FF2 can only be activated NPFF-related peptides, whereas FF1 is activated by NPVF-related peptides with a higher affinity than by NPFF-related peptides. Taken together, these data strongly suggest that the physiologically relevant ligands for FF2 are the NPFF-related peptides whereas the NPVF-related peptides are the preferred ligands for FF1.NPFF-related peptides have been shown to be involved in various somatosensory and visceral functions (8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar). Perhaps the best characterized action for NPFF is its role in nociception and analgesia (for recent reviews, see Refs. 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar and 39Panula P. Kalso E. Nieminen M. Kontinen V.K. Brandt A. Pertovaara A. Brain Res. 1999; 848: 191-196Crossref PubMed Scopus (150) Google Scholar). There is a body of evidence that central administration of NPFF-related peptides produces hyperalgesia and reverses morphine-induced analgesia (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar). Intrathecal administration of NPFF, however, produces antinociceptive, pro-opioid effects (14Gouarderes C. Sutak M. Zajac J.M. Jhamandas K. Eur. J. Pharmacol. 1993; 237: 73-81Crossref PubMed Scopus (139) Google Scholar). Since our in situ data showed only FF2 expression in rat spinal cord, FF2 most likely mediated the pro-opioid effect of NPFF following intrathecal injection. On the other hand, the anti-opioid effects of intracerebroventricularly administered NPFF were most likely mediated by FF1 based on the following observations. Our data showed that NPSF-(1–37), which had poor if any agonist activity for FF2 but potent activity for FF1, was more potent than NPFF in suppressing morphine-induced analgesia in two models of nociception (Fig. 7). FMRFamide, which displayed no significant agonist activity for FF2 but potent activity for FF1, was equally as potent as NPFF in anti-opioid activity (40Tang J. Yang H.Y. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5002-5005Crossref PubMed Scopus (214) Google Scholar). Only FF1 is expressed in the periaqueductal gray (Table II), a region critical for the control of nociception. All together, these in vitro and in vivo observations strongly suggest that the anti-opioid effects of NPFF were actually mediated by the FF1 receptor, and the real endogenous anti-opioid peptides are NPVF-related peptides. Furthermore, the strong expression of FF1 in the limic system may suggest an important role of NPVF in manifestation of the affective aspects of pain. FaRPs constitute a large family of neuropeptides in invertebrates (1Greenberg M.J. Price D.A. Prog. Brain Res. 1992; 92: 25-37Crossref PubMed Scopus (143) Google Scholar, 2Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (181) Google Scholar). The diversity of FaRPs in vertebrates, however, appears to be more limited. In addition to NPFF, NPAF, and LPLRFa, only one other RFamide neuropeptide, the prolactin-releasing peptide, has been isolated from vertebrates (33Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (529) Google Scholar). In this report, we described independent identification and characterization of a mammalian gene designated NPVF and its encoded peptides, which most likely represents the mammalian ortholog of the chicken peptide LPLRFa. We also described independent identification and characterization of two NPFF receptors by comparing their affinity for various FaRPs and detailed analysis of their expression pattern in the rat CNS. Furthermore, we showed that NPVF-derived peptides were more potent than NPFF in attenuating morphine-induced analgesia. The molecular pharmacological properties of FF1 and FF2 toward various NPFF and NPVF peptides and their expression pattern in the rat CNS presented here offer new insights into potential mechanisms of the pain-modulatory functions associated with NPFF. In characterizing affinities of the two receptors to various FaRPs, we noticed FF1 could be activated by many more FaRPs than FF2. Particularly, we were surprised to find that FF1 showed a better response to the chicken peptide LPLRFa than to NPFF, which led to the search for mammalian LPLRFa-like peptides and the eventual identification of NPVF. In the report by Bonini et al. (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), FF1 showed much lower affinity to NPFF (EC50 = 16 nm) than FF2 did (EC50 = 2.0 nm). But no data were presented for LPLRFa. Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) discovered NPVF by looking for mammalian LPLRFa-like peptides directly and then tested them on the orphan receptor FF1/OT7T022. We have now carried out a side-by-side comparison of NPFF- and NPVF-related peptides on FF1 and FF2, and the in vitro data presented here and those from Hinuma et al. (19Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar) clearly indicated that NPVF-related peptides are more potent ligands for FF1 and NPFF-related peptides are much better ligands for FF2. We mapped the expression pattern of both FF1 and FF2 in the entire rat CNS by in situ hybridization, and the data were nearly completely consistent with the results from radioligand binding analysis. Using 125I-labeled (1DNme)bNPFF, which turned out to have equal affinities to rat FF1 and FF2 (17Bonini J.A. Jones K.A. Adham N. Forray C. Artymyshyn R. Durkin M.M. Smith K.E. Tamm J.A. Boteju L.W. Lakhlani P.P. Raddatz R. Yao W.J. Ogozalek K.L. Boyle N. Kouranova E.V. Quan Y. Vaysse P.J. Wetzel J.M. Branchek T.A. Gerald C. Borowsky B. J. Biol. Chem. 2000; 275: 39324-39331Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), Dupouy et al. (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar) mapped NPFF binding sites in the entire rat CNS. Specific binding was detected widely in the CNS with intense signals in the parafascicular, reticular thalamic nuclei, lateral septum, and nucleus of the solitary tract, and in the superficial layers in the dorsal horn and around central canal of the spinal cord, but only a single class of binding was noticed. Examination of FF1 and FF2 expression by in situ hybridization has now allowed us to compare the distribution of the two receptors. FF1 is much more widely expressed, with especially strong signals in brain regions regulating the expression of fear and anxiety, and affective aspects of pain (35Davis M. Biol. Psychiatry. 1998; 44 (1147): 1239Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 36Price D.D. Science. 2000; 288: 1769-1772Crossref PubMed Scopus (1495) Google Scholar). FF2 mRNA levels, on the other hand, are much more limited, with the highest levels found in the spinal cord. It is of interest to note, however, that some discrepancies exist between the patterns of receptor binding and receptor mRNA expression. For example, high density NPFF bindings were observed in the presubiculum and spinal trigeminal tract nucleus (34Dupouy V. Zajac J.M. Synapse. 1996; 24: 282-296Crossref PubMed Scopus (41) Google Scholar, 37Allard M. Zajac J.M. Simonnet G. Neuroscience. 1992; 49: 101-116Crossref PubMed Scopus (112) Google Scholar), but these brain regions express little or no detectable FF1/FF2 mRNA. Although the precise reason for such mismatch is unknown, it could be due to subtle differences between the iodinated NPFF analogues used as the radioligands and native NPFF in binding to the receptors or it could be the consequence of transportation of mature/functional receptor proteins (binding sites) away from their origin of synthesis (mRNA), which were measured by autoradiography and in situ hybridization, respectively. Alternatively, these results may suggest existence of additional receptor(s) for NPFF. The genes encoding NPFF and NPVF are similar in structure but distinct in expression pattern, receptor pharmacology, and presumably physiological functions. NPFF is primarily expressed in the paraventricular and supraoptic nuclei of the hypothalamus, the nucleus of the solitary tract, and the superficial layers in the dorsal horn of spinal cord (7Vilim F.S. Aarnisalo A.A. Nieminen M.L. Lintunen M. Karlstedt K. Kontinen V.K. Kalso E. States B. Panula P. Ziff E. Mol. Pharmacol. 1999; 55: 804-811PubMed Google Scholar), areas with predominant FF2 expression. In contrast, NPVF is only expressed in a population of neurons between the dorsomedial hypothalamic and ventromedial hypothalamic nucleus, which were shown to project to a number of limbic structures including the lateral septal nucleus, bed nucleus of the stria terminalis, amygdala, and hypothalamus as well as to the periaqueductal gray (38Aarnisalo A.A. Panula P. Neuroscience. 1995; 65: 175-192Crossref PubMed Scopus (45) Google Scholar), areas where FF1 is strongly expressed. Furthermore, in vitrobinding and functional analyses also indicate that FF2 can only be activated NPFF-related peptides, whereas FF1 is activated by NPVF-related peptides with a higher affinity than by NPFF-related peptides. Taken together, these data strongly suggest that the physiologically relevant ligands for FF2 are the NPFF-related peptides whereas the NPVF-related peptides are the preferred ligands for FF1. NPFF-related peptides have been shown to be involved in various somatosensory and visceral functions (8Panula P. Aarnisalo A.A. Wasowicz K. Prog. Neurobiol. 1996; 48: 461-487Crossref PubMed Scopus (200) Google Scholar). Perhaps the best characterized action for NPFF is its role in nociception and analgesia (for recent reviews, see Refs. 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar and 39Panula P. Kalso E. Nieminen M. Kontinen V.K. Brandt A. Pertovaara A. Brain Res. 1999; 848: 191-196Crossref PubMed Scopus (150) Google Scholar). There is a body of evidence that central administration of NPFF-related peptides produces hyperalgesia and reverses morphine-induced analgesia (5Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 9Roumy M. Zajac J.M. Eur. J. Pharmacol. 1998; 345: 1-11Crossref PubMed Scopus (211) Google Scholar). Intrathecal administration of NPFF, however, produces antinociceptive, pro-opioid effects (14Gouarderes C. Sutak M. Zajac J.M. Jhamandas K. Eur. J. Pharmacol. 1993; 237: 73-81Crossref PubMed Scopus (139) Google Scholar). Since our in situ data showed only FF2 expression in rat spinal cord, FF2 most likely mediated the pro-opioid effect of NPFF following intrathecal injection. On the other hand, the anti-opioid effects of intracerebroventricularly administered NPFF were most likely mediated by FF1 based on the following observations. Our data showed that NPSF-(1–37), which had poor if any agonist activity for FF2 but potent activity for FF1, was more potent than NPFF in suppressing morphine-induced analgesia in two models of nociception (Fig. 7). FMRFamide, which displayed no significant agonist activity for FF2 but potent activity for FF1, was equally as potent as NPFF in anti-opioid activity (40Tang J. Yang H.Y. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5002-5005Crossref PubMed Scopus (214) Google Scholar). Only FF1 is expressed in the periaqueductal gray (Table II), a region critical for the control of nociception. All together, these in vitro and in vivo observations strongly suggest that the anti-opioid effects of NPFF were actually mediated by the FF1 receptor, and the real endogenous anti-opioid peptides are NPVF-related peptides. Furthermore, the strong expression of FF1 in the limic system may suggest an important role of NPVF in manifestation of the affective aspects of pain.