MIFEPRISTONE MODULATION OF ACTH AND CRH REGULATION OF BOVINE ADRENOCORTICOSTEROIDOGENESIS IN-VITRO

Mifepristone (RU486), bovine corticotropin-releasing hormone (CRH), ar ginine vasopressin (VP), adrenocorticotropin (ACTH(1-24)), and protein kinase activators (forskolin, [FSK]; phorbol 12-myristate 13-acetate, [PMA]) were used in vitro to investigate their direct effect on adren ocorticosteroidogenesis. Bovine adrenocortical fasciculata/reticularis cells (2 x 10(5) viable cells/well) were cultured for 3 d in medium s upplemented with 10% fetal calf serum. After incubation for an additio nal 24 hr in serum-free medium, cells were treated with serum-free med ium alone (Control) or various concentrations of ACTH, CRH, VP, FSK, P MA, RU486, and/or various combinations for 1, 2, 4, or 24 hr. Medium c ontent of cortisol and progesterone were determined by radioimmunoassa ys. ACTH, CRH, FSK, and PMA each stimulated (P < 0.05) secretion of co rtisol in time- and dose-related manners. Although these agents stimul ated (P < 0.05) secretion of progesterone in a dose-related manner, me dium content of progesterone declined (P < 0.05) over time. The minima l effective doses of ACTH and CRH required to stimulate (P < 0.05) sec retion of cortisol relative to the Control over a 4-hr culture period were 0.01 nM and 3 nM, respectively. Relative to observations at 1 hr posttreatment, 24-hr treatment with ACTH or CRH increased the medium c ontent of cortisol by an additional 19.8- acid 48-fold, respectively ( whereas content of progesterone declined over that time period). VP-st imulated secretion of cortisol was time- (P < 0.05) but not dose-relat ed. Specifically, by 24-hr posttreatment, the medium content of cortis ol was increased (P < 0.05) 4.6-fold relative to the quantity of corti sol secreted by 1-hr postaddition of VP (0.01 to 1 mu M). Co-treatment with RU486 (1 mu M) decreased (P < 0.05) FSK-, ACTH- and CRH-stimulat ed secretion of cortisol by 77, 27, and 56%, respectively. Similarly, the stimulatory effects of ACTH and CRH on progesterone secretion were reduced (P < 0.05) by 40 and 22%, respectively, by co-addition of RU4 86. The inhibitory action of RU486 on production of cortisol was no lo nger apparent by 24 hr after treatment. These observations indicate th at RU486 can act as a steroid agonist and as well as an antagonist. Th ese data characterize time- and dose-related direct actions of ACTH, C RH, and RU486 on adrenocorticosteroidogenesis. This information will a ssist efforts to clarify complex intra-adrenal interactions of neuroho rmones, growth factors, and endogenous steroids.