Characterization of an Early Inhibitory Effect of Glucocorticoids on Stimulated Adrenocorticotropin and Endorphin Release from a Clonal Strain of Mouse Pituitary Cells*

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Abstract
The ability of glucocorticoid steroid hormones to inhibit release of ACTH and endorphin has been studied in the mouse pituitary tumor cell strain, AtT-20/ D l6v. Inhibition of release of both peptides was first detectable at 30 min (to 80–90% of control release) and was maximal (30–50% of control) by 3–4 h of pretreatment with dexamethasone (1 × 10-6 M). Glucocorticoids did not consistently affect basal hormone release, but they inhibited release stimulated 4-to 8-fold by hypothalamic extract, phorbol esters, or 8-Br-cAMP but not high K+-stimulated release. The inhibitory effect was glucocorticoid specific and dose dependent with ED50S for dexamethasone, corticosterone, and cortisol close to their reported KdS for binding to the cytoplasmic glucocorticoid receptor. Inhibition of release was blocked by cycloheximide (1 × 10-5 M), suggesting that protein synthesis was required. Experiments were performed to determine the site of action of glucocorticoids. Steroid pretreatment for up to 6 h had no effect on total intracellular content of ACTH or β-endorphin, hormone biosynthesis, or intracellular and extracellular hormone degradation. These results show that the inhibitory action of steroids was not due to decreased hormone synthesis, as has been reported after longer periods (24–72 h) of glucocorticoid treatment. Experiments with the tumorpromoting phorbol esters (used as model secretagogues) showed that pretreatment of D x 16 cells with dexamethasone (1 × 10-6 M) did not alter the binding of [3H]phorbol dibutyrate to its specific receptor on these cells, indicating that glucocorticoids do not act by interfering with binding of secretagogues to the cells. Furthermore, glucocorticoids did not affect the ED50 for stimulation of ACTH release by phorbol esters, but they reduced the magnitude of maximum response. We conclude that an early action of glucocorticoids on D 16 cells is to inhibit the release of ACTH and β-endorphin by a mechanism that is independent of the synthesis and degradation of the secretory peptides but which involves the synthesis of new protein.