Immunohistochemical distribution, biochemical characterization, and biological action of tachykinins in the frog adrenal gland.

The distribution of tachykinin-like immunoreactivity (LI) was studied in the adrenal gland of the frog Rana ridibunda using the immunofluorescence technique. A dense network of varicose fibers immunoreactive to both substance-P (SP) and neurokinin-A (NKA) was found in the adrenal tissue. In contrast, no positive fibers could be detected using antineurokinin-B (NKB) antibodies. At the electron microscope level, the immunogold technique revealed that tachykinin-LI was sequestered in dense core vesicles of 50-70 nm. Bilateral transection of either splanchnic or vagus nerves or total lesion of celiac sympathetic ganglion did not suppress tachykinin-LI. A combination of HPLC analysis and RIA detection was used to characterize tachykinin-LI in frog adrenal extracts. Two major peaks were resolved, which coeluted, respectively, with synthetic ranakinin, a novel tachykinin previously isolated from the frog brain, and [Leu3,Ile7]NKA previously isolated from the frog gut. No NKB could be detected in the extracts. The effects of various synthetic tachykinins on corticosteroid secretion were studied using perifused frog adrenal slices. For concentrations ranging from 10(-8)-10(-4) M, SP induced a dose-dependent stimulation of corticosterone and aldosterone release. A desensitization phenomenon was observed when iterative or prolonged infusions of SP were administered to the tissue. All mammalian or amphibian tachykinin-related peptides tested in our model also enhanced corticosteroid production. The effectiveness of the tachykinins tested was: [Pro7] NKB > NKA > ranakinin > [Pro9]SP > SP > kassinin > physalaemin > NKB > [Leu3,Ile7]NKA. SP also enhanced prostaglandin E2 and prostacyclin release in the effluent perifusate and the response preceded by 10-15 min the increase in corticosteroid output. Indomethacin (5 x 10(-6) M), a specific blocker of cyclooxygenase activity, totally suppressed SP-evoked steroid secretion. These data indicate that tachykinin-induced stimulation of steroidogenesis was mediated through activation of the arachidonic acid cascade. Taken together, our results show that the frog adrenal gland is innervated by a dense network of peptidergic fibers containing both ranakinin and [Leu3,Ile7]NKA, which, in vitro, stimulates corticosteroid secretion by adrenocortical cells through a prostaglandin-dependent mechanism. The present results support the view that tachykinins released by nerve fibers exert a neuroendocrine control on corticosteroid release in amphibians.

In vitro study of catecholamine release from perifused frog adrenal slices.

The adrenal gland of amphibians is composed of a mixed population of adrenochromaffin and corticosteroid-secreting cells. It has previously been shown that chromaffin cells synthesize several bioactive substances (including biogenic amines and neurotransmitters) which may act locally to regulate corticosteroid secretion. In the present report, we have studied the secretory activity of adrenochromaffin cells in Rana ridibunda. Frozen sections of adrenal gland were immunolabeled with antisera against tyrosine hydroxylase or phenylethanolamine-N-methyltransferase. Comparison of homologous fields on consecutive sections indicated that 77% of catecholaminergic cells produce adrenaline. The concentrations of catecholamines were measured by means of high performance liquid chromatography analysis coupled to electrochemical detection. The concentrations of dopamine, noradrenaline, and adrenaline in fresh adrenal tissue were 24 +/- 4, 763 +/- 68, and 1032 +/- 118 ng/mg wet weight, respectively. After a 12-hr perifusion period, the concentration of adrenaline in the tissue was reduced by 62%, whereas noradrenaline only decreased by 22%. The secretion rates of adrenaline and noradrenaline from perifused adrenal slices significantly diminished during the first 7 hr of the experiment and then remained relatively stable for about 10 hr. Exposure of adrenal tissue to a depolarizing concentration of potassium (55 mM) induced an immediate and substantial rise of adrenaline and noradrenaline release and a delayed increase in corticosterone output. Acetylcholine, which stimulates corticosterone secretion from frog adrenocortical cells, induced a slight but not significant increase of adrenaline and noradrenaline release. Similarly, the selective cholinergic agonists muscarine and nicotine did not significantly affect catecholamine release, while muscarine mimicked the stimulatory action of acetylcholine on corticosterone secretion. This study validates the use of the perifusion model to investigate the mechanism of control of catecholamine release from frog adrenochromaffin tissue. The results presented herein indicate that, in contrast to mammals, the secretion of catecholamines from the amphibian adrenal gland is not regulated by cholinergic inputs.