Immunohistochemical localization, biochemical characterization, and biological activity of neurotensin in the frog adrenal gland

The primary structure of neurotensin has been recently determined for the f rog Rana ridibunda (Endocrinology 189: 4140-4146, 1998). In the present stu dy, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an an tiserum directed against the conserved C-terminal region of the peptide. Ne urotensin-like immunoreactivity was detected in two populations of nerve fi bers: numerous varicose fibers coursing between adrenal cells, and a few pr ocesses located in the walls of blood vessels irrigating the gland. Reverse d-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the sa me retention time as synthetic frog neurotensin. The possible involvement o f neurotensin in the regulation of steroid secretion was studied in vitro u sing perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldos terone 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 reproducible stimulation of steroid output without any tachyphylaxis. Prol onged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of cortic osteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion From dispersed frog adrenal cells. This study demo nstrates that neurotensin is located in nerve processes innervating the adr enal gland of amphibians. The results also show that synthetic frog neurote nsin exerts a direct stimulatory effect on corticosteroid output. Taken tog ether, these data support the view that neurotensin, released by nerve fibe rs, may act as a local regulator of corticosteroid secretion.