Self-Regulation of Adrenocorticotropin Secretion by Mouse Pituitary Tumor Cells in Culture*

Abstract

A clonal strain of mouse pituitary tumor cells, AtT20/D16, synthesized and secreted ACTH during 15 yr in continuous culture. When cultures were studied over a period of about 1 mo., ACTH accumulation per ml culture medium per 24 h reached a plateau during the late exponential phase of cell growth so that ACTH production per cell declined as the cultures approached the stationary phase of growth. The time-course of ACTH accumulation in medium was rapid (50% of maximum ACTH concentration was attained within 10 min) and reached a hormone concentration within 30 min which changed little during the ensuing 24 h of incubation. Three sequential medium changes at 15 min intervals resulted in a 3-fold enhancement of ACTH accumulation over that seen in cultures in which the medium was removed and returned to the same dishes 3 times. ACTH accumulation per cell increased as the volume of incubation medium was increased. Porcine ACTH added to cultures inhibited endogenous ACTH secretion at 30 and 120 min of incubation. This inhibition could not be ascribed to the rapid degradation of exogenous ACTH in AtT20/D16 cultures. ACTH added to conditioned medium was stable and the addition of low concentrations of ACTH (1 mU/ml) which did not inhibit endogenous hormone secretion resulted in total ACTH in culture medium equivalent to the sum of exogenous and endogenous ACTH. These results indicate that ACTH secretion by AtT20/D16 cells is sensitive to the concentration of ACTH in the surrounding medium and may be controlled by an ultra short loop negative feedback mechanism.