This application seeks to retrain and improve knowledge of adrenocortical glomerulosa cell physiology, aldosterone synthesis and secretion, divalent cation control mechanisms for aldosterone synthesis, molecular biology, protein characterization, and cholesterol metabolism during steady-state and stimulated steroidogenesis. Research will be conducted with a laboratory having state-of-the-art molecular and protein characterization techniques that is also investigating cholesterol metabolism during steady-state and stimulated steroidogenesis. Over an 18-month period, facility will be developed with techniques, data collected, and affiliations developed which will further the missions of Meharry Medical College. Following completed retraining, the principal investigator proposes investigating the physiological and morphological effects of divalent cations on steady-state and stimulated aldosterone secretion by a cultured human aldosterone-secreting clonal adrenocortical tumor cell, H295R. Approaches used will be similar to those developed for studying glucocorticoid synthesis and secretion by a cultured mouse adrenocortical cell line, Y-1. Non-lethal CdCl2 and CdAc2 inhibitory effects on unstimulated and stimulated H295R cell models will specifically be examined and characterized because the applicants previously showed that CdAc2 had profound steroid inhibitory effects on mouse adrenal cells at low molar concentrations. Study aims include characterizing: 1) CdCl2 and CdAc2 inhibition of (a) mitochondrial cholesterol and 25-hydroxy-cholesterol utilization, (b) stimulation by Angiotensin II, Ca++, and K+; 2) competition between Cd++ and Ca++ ions for plasma membrane transport channel sites; 3) characterization of Steroidogenic Acute Regulatory protein involvement in stimulated aldosterone synthesis; and 4) Examination of steady-state mitochondrial cholesterol metabolism. To investigate mitochondrial 25-hydroxy and endogenous cholesterol metabolism, adrenal cells will be "loaded" with 3H labeled compounds, incubated with or without stimulating factors in the presence or absence of Cd++, mitochondria will be isolated, inner and outer membranes obtained, and label quantitated. Agents stimulating steroid secretion act through second messengers; both CdAc2 inhibition of stimulated IP3 and steroid secretion will be examined. In their other studies, plasma membrane adenyl cyclase activity was inhibited through a CdCl2-Ca++ competition, CdCl2- and CdAc2-Ca++ competition will be examined in this study, but lesser CdAc2 and Ca++ concentrations will be used.