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Immunohistochemical Localization, Biochemical Characterization, and Biological Activity of Neurotensin in the Frog Adrenal Gland¹

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, (INSERM U-413), Unité Affiliée au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen, (F.S., H.V., B.B., N.C., J.L., C.D.), 76821 Mont-Saint-Aignan, France; and Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University Medical School (J.M.C.), Omaha, Nebraska 68178

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda . 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.

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