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Biphasic Action of Forskolin on Growth Hormone and Prolactin Secretion by Rat Anterior Pituitary Cells in Vitro^*

Department of Medicine, Michael Reese Hospital and Medical Center, University of Illinois College of Medicine (M.S., N.E.S.), and the Department of Pediatrics, Wyler Children's Hospital, University of Chicago Pritzker School of Medicine (B.J.C., L.C.), Chicago Illinois 60616
The Department of Pediatrics, Case Western Reserve University (L.C.) Cleveland, Ohio 44106

Address all correspondence and requests for reprints to: Marta Szabo, Ph.D., Division of Endocrinology and Metabolism, Department of Medicine, Michael Reese Hospital and Medical Center, Lake Shore Drive at 31st Street, Chicago, Illinois 60616.

Abstract

To assess the role of cAMP-mediated signal transduction processes in mediation of secretagogue-stimulated GH release, we examined the dose-related effects of the diterpene adenylate cyclase activator forskolin (FSK) in primary monolayer cultures of rat adenohypophyseal cells. In cell cultures prepared from both immature (12 days old) and adult (6 weeks to 4 months old) male or female rats, the dose-related stimulation of GH release by FSK was biphasic. With increasing FSK concentrations from 0.03–3.16 µM, GH release increased progressively to maximal values of 442 ± 19% and 303 ± 10% of basal release in cells from immature and adult rats, respectively. FSK concentrations above 3.16 µM induced progressively diminished GH responses, with net inhibition to below basal release evident at 100 µM FSK. FSK stimulated PRL release to a lesser degree than it did GH release; the PRL response to FSK was also biphasic.

When maximal stimulatory concentrations (E_max) of FSK and GH-releasing factor (GRF; 10 nM) were added in combination, the GH response was significantly less than the individual response to either secretagogue alone. In response to FSK alone, GRF alone, and FSK plus GRF, GH release was 478 ± 7%, 583 ± 11%, and 244 ± 5%; 278 ± 4%, 283 ± 3%, and 175 ± 2%; and 299 ± 12%, 351 ± 5%, and 191 ± 17% of basal release in cells from 12-day-old, adult male, and adult female rats, respectively (P < 0.01 for all responses to combined addition vs. the individual responses). Submaximal stimulatory concentrations of GRF added in combination with submaximal FSK elicited partially additive GH responses; the GH response to Emax GRF, on the other hand, was inhibited in a dose-related manner by all concentrations of FSK that by themselves were stimulatory.

The GH responses were also suppressed when E_mox FSK was added to cultured cells of 12-day-old rats in combination with E_max cholera toxin (2.5 ng/ml) or prostaglandin E2 (10 µM), agents whose actions, like that of GRF, involve adenylate cyclase activation. In contrast, FSK did not suppress but in most cases augmented the maximal GH responses to secretagogues whose action is independent of adenylate cyclase activation: (Bu)2cAMP (0.5 mM), TRH (100 nM), phorbol myristate acetate (50 nM), the Ca²⁺ ionophore A23187 (250 µM), and the dihydropyridine Ca²⁺ channel agonist BAY K8644 (10 µM). Indeed, combined addition of FSK with the latter two agents resulted in synergistic stimulation.

Pretreatment of the cultured cells with pertussis toxin (1–100 ng/ml) for 24 h attenuated the suppressive effect of FSK on GRF-induced GH release, implicating participation of a Gi protein in this phenomenon. Participation of a Gi protein was also suggested by the finding that in cell cultures from adult male rats, GH release induced by combined GRF and FSK was significantly less susceptible to suppression by somatostatin than either GRF-or FSK-induced GH release. The suppressed GH responses obtained upon combined addition of GRF and FSK could not, however, be explained by direct inhibition of GRF-stimulated adenylate cyclase by FSK, as the intracellular cAMP responses were additive at all secretagogue concentrations tested; this finding suggests that FSK exerts its inhibitory action at a site distal to cAMP generation. (Endocrinology 127: 1811–1817,1990)

Footnotes

^* This work was supported in part by NIH Grant P-60-DK-20595 and a grant from the Michael Reese Medical Research Institute Council (to M.S.) and by NIH Grant DK-40221 and the Basil O'Connor Research Starter Award (no. 5–609) from the March of Dimes Birth Defects Foundation (to L.C.). Presented in part at the 70th Annual Meeting of The Endocrine Society, June 1988, New Orleans, LA (Abstract 960).

Received April 20, 1990.

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