The AtT-20/D16-16 mouse pituitary tumor cell secretes corticotropin (ACTH) in response to corticotropin-releasing factor (CRF), (-)-isoproterenol, and vasoactive intestinal peptide (VIP). These responses are associated with a rapid increase in cyclic AMP formation. Somatostatin (SRIF) markedly decreases the stimulatory effect of CRF, (-)-isoproterenol, and VIP on both cyclic AMP formation and immunoreactive ACTH secretion. Forskolin and cholera toxin, adenylate cyclase activators, also stimulate cyclic AMP formation and ACTH secretion in AtT-20 cells and these responses are all inhibited by SRIF. The ACTH secretory responses to melittin and to the calcium ionophore A23187, neither of which increases cyclic AMP in AtT-20 cells, were not inhibited by SRIF. SRIF did not affect the binding of a tritiated ,B-adrenergic receptor antagonist to AtT-20 membranes nor did it decrease basal cyclic AMP formation even in the presence ofexcess phosphodiesterase inhibitor, indicating that the reduction of cyclic AMP levels by SRIF did not involve either an interference with (-adrenergic agonist binding to receptors or stimulation of cyclic AMP degradation. These results indicate that the inhibition of CRF-, (-)-isoproterenol-, and VIP-stimulated ACTH secretion by SRIF may be regulated by its inhibitory action on adenylate cyclase.The AtT-20 mouse pituitary cell line has been extensively used to investigate the regulation ofsynthesis, storage, and secretion of the corticotropin (ACTH)/P-endorphin family of peptides (1-8). Recent studies in our laboratory have demonstrated the responsiveness ofthis clonal cell line to synthetic corticotropinreleasing factor (CRF) (9), to f32-adrenergic agonists, and to vasoactive-intestinal peptide (VIP) (unpublished data). All three substances increase cyclic AMP formation in the tumor cells and the combination of any two of the agents on cyclic AMP synthesis is additive (unpublished MN). Goat anti-rabbit immunoglobulin was from Cappel Laboratories (Cochranville, PA). All other chemicals were of reagent grade.Cell Culture Methods. AtT-20/D16-16 cells were grown in DME medium containing 10% fetal calf serum at 37°C in a humidified atmosphere of 10% C02/90% air as described (9).Incubation Procedure. Cells were plated in 35-mm diameter culture dishes (Costar) at an initial density of 1.5-2 x 105 cells per well and were used 5-7 days after subculturing (60-80% confluency). Before use, cells were equilibrated for 60 min at 37°C in 1 ml of DME medium/25 mM Hepes, pH 7.4, containing 2% fetal calf serum and bacitracin at 3 ,ug/ml. For ACTH secretion studies, the medium was aspirated and replaced with 1 ml of the same medium containing added test agent(s), and the cells were incubated for an additional 60 min. An aliquot of the medium was removed and centrifuged to remove detached cells and debris. The supernatant was frozen at -20°C until used for ACTH measurement. For cyclic AMP studies, cells were preincubated for 30 min with 0.5 mM IBMX in fresh medium. This medium then was aspirated and r...

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The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/ D16 -16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nucleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.

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