The overall objective is to understand the mechanisms of the multihormonal control of ACTH production. To avoid the complex interactions that occur in an intact animal, primary cultures of rat anterior pituitary cells will be used. Corticotropin-releasing factor (CRF) has been shown to stimulate ACTH secretion, increase cAMP content, activate cAMP-dependent protein kinase, increase pro-opiomelanocortin (POMC) mRNA content and stimulate POMC gene transcription. CRF-induced ACTH secretion appears to be mediated via a cAMP mechanism and is calcium-dependent. Other agents, such as vasopressin and angiotensin II, stimulate ACTH release from anterior pituitary cells without changing cAMP levels. This alternate mechanism may involve activation of phospholipid/calcium-dependent protein kinase C, because activators of this enzyme, such as phorbol esters, also cause ACTH release without increasing cAMP. This research will determine whether the protein kinase C pathway regulates POMC gene expression in cultured rat anterior pituitary cells by measuring total ACTH production, POMC mRNA content and POMC gene transcription rates before and after stimulation by phorbol myristate acetate. The possibility that these two mechanisms of ACTH release may independently regulate POMC gene expression will be explored by studying the interaction of combined stimulation and by determining whether stimulation of POMC gene expression by each mechanism is similarly inhibited by dexamethasone. In addition, because calcium is required for ACTH release, its role in POMC gene expression will be determined by assessing whether calcium will increase total ACTH production and increase POMC mRNA levels and whether calcium is required for stimulation of POMC gene expression by the cAMP-dependent and cAMP-dependent pathways.
