investigator's application): Anterior pituitary corticotropes secrete adrenocorticotropic hormone (ACTH) during periods of physical and emotional stress. ACTH release is stimulated by corticotropin releasing hormone (CRH) and inhibited by glucocorticoids. The goal of this proposal is to understand the molecular mechanisms by which CRH and glucocorticoids regulate cytosolic Ca2+ and granule fusion. The first specific aim will determine whether CRH and gluocorticoids regulate exocytosis by modulating Ca influx and by additional actions on the exocytotic machinery that are independent of changes in cytosolic Ca.
The second aim will determine whether chronic exposure to CRH and glucocorticoids changes Ca channel mRNA expression. Experiments will be performed on single corticotropes or the ACTH secreting AtT-20 cell line. Simultaneous measurements of cytosolic Ca, Ca influx, and exocytosis will be performed by quantitative fluorescence microscopy, voltage clamp analysis of whole cell currents, and lock-in phase measurements of membrane capacitance. Physiological regulation of Ca channel expression will be monitored by RNase protection assays and in situ hybridization measurements of Ca channel mRNA and by measurements of dihydropyridine binding sites and Ca current density. The ultimate goal is to understand the immediate and long term mechanisms involved in regulation of Ca channel activity and ACTH secretion in corticotropes.
