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26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.

The biosynthesis of various hypothalamic neuropeptides has been previously reported in anterior pituitary cells but not in intermediate lobe cells. We have recently demonstrated the occurrence of two somatostatin isoforms in the frog brain, namely somatostatin-14 (SS1) and [Pro(2),Met(13)]somatostatin-14 (SS2). In the present study, we demonstrate that the gene encoding the SS2 precursor (PSS2) is actively expressed in the intermediate lobe of the frog pituitary. High concentrations of PSS2 mRNA have been detected by Northern blot analysis and in situ hybridization in the frog pars intermedia but not in the pars distalis or pars nervosa. The distribution of PSS1- and PSS2-derived peptides has been investigated by immunohistochemistry using two antisera directed against SS1 and the sequence 54-66 of PSS2 (PSS2(54-66)), respectively. The SS1 antiserum stained only a network of fibers in the neural lobe and a few nerve processes in the intermediate lobe. In contrast, the PSS2(54-66) antiserum produced intense labeling of melanotrope cells in the pars intermedia. Biochemical characterization of the immunoreactive materials present in pituitary extracts was performed by combining high-performance liquid chromatography analysis and RIA detection. The SS1 RIA revealed the existence of two major immunoreactive peaks that exhibited the same retention times as synthetic SS1 and SS2. The PSS2(54-66) RIA detected a single peak that likely corresponds to the N-flanking peptide of SS2 (PSS2(1-66)). The present study reveals that melanotrope cells of the frog pituitary selectively express the PSS2 gene and fully process PSS2 to generate the mature somatostatin variant SS2. Taken together, these data provide the first evidence that the gene encoding a hypophysiotropic neuropeptide is intensely expressed in the intermediate lobe of the pituitary.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda (Endocrinology 139: 4140-4146, 1998). In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10(-9) M and 5.8 x 10(-10) M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily, was isolated in pure form from the brain of the European green frog, Rana ridibunda. The primary structure of the peptide indicates that evolutionary pressure to conserve the complete amino acid sequence has been very strong. Frog PACAP comprises 38 amino acid residues and contains only 1 substitution (isoleucine for valine at position 35) compared with human/ovine/rat PACAP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, synthetic ovine PACAP-(1-38) produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 2.1 +/- 0.6 x 10(-7) M; mean +/- SE; n = 6). Maximum stimulation (an approximately 8-fold increase in concentration over basal values) was produced by 10(-6) M peptide. The truncated form of PACAP [PACAP-(1-27)] also produced a dose-dependent increase in cAMP in frog anterior pituitary fragments, and the potency of the peptide (ED50 = 5.9 +/- 0.6 x 10(-8) M) was comparable to that of PACAP-(1-38). The data suggest, therefore, that the function as well as the structure of PACAP have been conserved during the evolution of amphibia to mammals.