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A Novel Amphibian Hypothalamic Neuropeptide: Isolation, Localization, and Biological Activity

Laboratory of Brain Science (K.U., H.T., K.T.), Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan; Core Research for Evolutional Science and Technology (K.U., H.T., K.T.), Japan Science and Technology Corporation, Tokyo 150-0002, Japan; Instrument Center for Chemical Analysis (S.O.), Hiroshima University, Higashi-Hiroshima 739-8526, Japan; and Department of Biology (A.K., K.Y., S.K.), School of Education, Waseda University, Nishiwaseda, Tokyo 169-8050, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp

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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