Production of Steroids by<i>in Vitro</i>Superfusion of Endocrine Tissue. III. Corticosterone Output from Rat Adrenals Stimulated by Adrenocorticotropin or Cyclic 3′,5′-Adenosine Monophosphate and the Inhibitory Effect of Cycloheximide

Abstract

Decapsulated rat adrenals were continuously superfused and corticosterone outputs assayed by competitive protein binding or soda fluorescence. During 5 hr superfusions, corticosterone output rate of adrenals (H) from acutely hypophysectomized rats remained almost constant (0.3–0.5 μg/rat/hr), whereas that of adrenals (I) from intact rats declined rapidly from high initial values (7 μgxylrat/hr) and approached that of adrenals (H). Release of preformed steroid could not explain this decline, which is ascribed to the decaying in vivo ACTH stimulus. Continuous ACTH infusion (64 mU/ ml) to adrenals (H) increased corticosterone output rate 13-fold within 1 hr almost to that of adrenals (I) similarly infused. After 2 hr the output rates of adrenals (H and I) each continuously infused with ACTH showed similar substantial declines, which were not attributable to irreversible changes in the tissue during superfusion. Dose response curves for adrenals (H) showed that higher concentrations of continuously infused ACTH or cyclic AMP increased both maximum corticosterone output rates and total outputs at any time during superfusion, making it difficult to attribute the late decline in output rate to depletion of precursors. Cycloheximide (1 mil) rapidly inhibited corticosterone output rate of adrenals (H) when continuously infused 30 or 120 min after stimulation by continuously infused ACTH or cyclic AMP. Cycloheximide also immediately and totally suppressed stimulation of corticosterone output rate by simultaneously infused ACTH or cyclic AMP. However, this inhibition was partially reversed by ceasing cycloheximide infusion after 3 hr. Similar infusions of cycloheximide alone halved the basal output rate of adrenals (H) to a virtually constant value, implying that nonspecific effects can maximally account for only half the cycloheximide inhibition of ACTH stimulated steroidogenesis. Moreover, continuous infusion of progesterone to adrenals (H) substantially increased corticosterone output rate, while cycloheximide (1 HIM) did not affect this stimulation. These dynamic in vitro studies provide data consistent with a mechanism for ACTH stimulated steroidogenesis, mediated by cyclic AMP and rapidly turning-over protein (s). (Endocrinology86: 487, 1970)