By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

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The distribution of neuropeptide FF receptors (NPFF(1) and NPFF(2)) was analyzed throughout the central nervous system of rodents (rat, mouse, Octodon degus, and guinea pig), rabbit, and marmoset monkey brains, representing three orders of mammals. Quantitative in vitro receptor autoradiography with [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)) and [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) as specific radioligands for NPFF(2) and NPFF(1) receptors, respectively, was used. The NPFF(2) receptor is predominantly expressed in all species, except in the central nervous system of Octodon degus, in which it is undetectable. The density of the NPFF(1) subtype is low in rat and mice, moderate in octodon, rabbit, and monkey, and relatively high in the guinea pig. The present study reveals prominent species differences in the NPFF receptors expression in the brain. The distribution pattern of NPFF(2) receptors in the diencephalon and the superficial layers of the spinal cord is consistent with a hypothesized potential role for NPFF in the modulation of sensory input and opioid analgesia. In contrast, the constant presence of NPFF(1) receptors in the septum, the nucleus of the tractus solitarius, and the hypothalamus suggest its participation in neuroendocrine functions.

1 Neuropeptide FF (NPFF) has been shown to produce antinociceptive effects and enhance morphineinduced antinociception after intrathecal (i.t.) injection. In this study, the spinal effects of two NPFF analogues, [D-Tyr',(NMe) (11 nmol 5 The results of this study show that spinal ,-and 6-opioid receptors play a role in antinociception produced by NPFF analogues. These results also suggest a role for NPFF in modulation of nociceptive signals at the spinal level.