Mammalian bombesin-like peptides are widely distributed in the central nervous system as well as in the gastrointestinal tract, where they modulate smooth-muscle contraction, exocrine and endocrine processes, metabolism and behaviour. They bind to G-protein-coupled receptors on the cell surface to elicit their effects. Bombesin-like peptide receptors cloned so far include, gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype-3 (BRS-3). However, despite the molecular characterization of BRS-3, determination of its function has been difficult as a result of its low affinity for bombesin and its lack of an identified natural ligand. We have generated BRS-3-deficient mice in an attempt to determine the in vivo function of the receptor. Mice lacking functional BRS-3 developed a mild obesity, associated with hypertension and impairment of glucose metabolism. They also exhibited reduced metabolic rate, increased feeding efficiency and subsequent hyperphagia. Our data suggest that BRS-3 is required for the regulation of endocrine processes and metabolism responsible for energy balance and adiposity. BRS-3-deficient mice provide a useful new model for the investigation of human obesity and associated diseases.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.

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We have identified the amphibian ghrelin from the stomach of the bullfrog. We also examined growth hormone (GH)-releasing activity of this novel peptide in both the rat and bullfrog. The three forms of ghrelin identified, each comprised of 27 or 28 amino acids, possessed 29% sequence identity to the mammalian ghrelins. A unique threonine at amino acid position 3 (Thr 3 ) in bullfrog ghrelin differs from the serine present in the mammalian ghrelins; this Thr 3 is acylated by either n-octanoic or n-decanoic acid. The frog ghrelin-28 has a complete structure of GLT (O-n-octanoyl)FLSPAD-MQKIAERQSQNKLRHGNM; the structure of frog ghrelin-27 was determined to be GLT(O-n-octanoyl)FLSPAD-MQKIAERQSQNKLRHGN; frog ghelin-27-C10 possessed a structure of GLT(O-n-decanoyl)FLSPADMQKIAER-QSQNKLRHGN. Northern blot analysis demonstrated that ghrelin mRNA is predominantly expressed in the stomach. Low levels of gene expression were observed in the heart, lung, small intestine, gall bladder, pancreas, and testes, as revealed by reverse transcription polymerase chain reaction analysis. Bullfrog ghrelin stimulated the secretion of both GH and prolactin in dispersed bullfrog pituitary cells with potency 2-3 orders of magnitude greater than that of rat ghrelin. Bullfrog ghrelin, however, was only minimally effective in elevating plasma GH levels following intravenous injection into rats. These results indicate that although the regulatory mechanism of ghrelin to induce GH secretion is evolutionary conserved, the structural changes in the different ghrelins result in species-specific receptor binding. Growth hormone (GH)1 secretion from the pituitary gland is regulated by hypothalamic hormones; growth hormone-releasing hormone (GHRH) stimulates GH secretion, whereas somatostatin is inhibitory (1). Derivatives of Met-enkephalin stimulate GH release (2), the first demonstration that small synthetic peptides and nonpeptide molecules, dubbed growth hormone secretagogues (GHSs), can mediate GH release through a receptor distinct from that of GHRH (3-5). The G-protein-coupled GHS receptor (GHS-R) was subsequently identified in swine, rat, and human (6, 7), suggesting that one or more unknown ligands for this receptor are endogenously present.We recently discovered an endogenous ligand for GHS-R from the rat stomach, using an intracellular calcium influx assay in stable cell lines expressing rat GHS-R (8). This novel molecule, a 28-amino acid peptide named ghrelin (from "ghre," the Proto-Indo-European root of "grow"), possesses a unique serine residue at the third N-terminal position (Ser 3 ) that is n-octanoylated (8, 9). Acylation of Ser 3 is essential for ghrelin bioactivity. cDNA analysis revealed that the rat ghrelin sequence follows the 23-residue signal sequence within the 117-residue prepro-ghrelin. Ghrelin stimulates GH secretion both in vivo and in vitro. Accumulating evidence in mammals suggests that, in addition to regulating GH release, ghrelin also influences feeding behavior (10, 11), gastrointestinal function (12, 13), and ener...

Gonadotropin-inhibitory hormone (GnIH), a neuropeptide that inhibits gonadotropin synthesis and release, was first identified in quail hypothalamus. GnIH acts on the pituitary and GnRH neurons in the hypothalamus via GnIH receptor to inhibit gonadal development and maintenance. In addition, GnIH neurons express melatonin receptor and melatonin induces GnIH expression in the quail brain. Thus, it seems that melatonin is a key factor controlling GnIH neural function. In the present study, we investigated the role of melatonin in the regulation of GnIH release and the correlation of GnIH release with LH release in quail. Melatonin administration dose-dependently increased GnIH release from hypothalamic explants in vitro. GnIH release was photoperiodically controlled. A clear diurnal change in GnIH release was observed in quail, and this change was negatively correlated with changes in plasma LH concentrations. GnIH release during the dark period was greater than that during the light period in explants from quail exposed to long-day photoperiods. Conversely, plasma LH concentrations decreased during the dark period. In contrast to LD, GnIH release increased under short-day photoperiods, when the duration of nocturnal secretion of melatonin increases. These results indicate that melatonin may play a role in stimulating not only GnIH expression but also GnIH release, thus inhibiting plasma LH concentrations in quail. This is the first report describing the effect of melatonin on neuropeptide release.