The objectives of this proposal are to study the mechanisms by which Ca++, catecholamines and glucagon interact with a unique cell culture model of the C-cell (the rMTC 6-23 and 44-2 cell lines), which I and a colleague established, to stimulate secretion of calcitonin (CT), a calcium regulating hormone, and neurotensin (NT), a hypothalamic peptide whose biologic function is not clearly established. I propose to study (1) the role of cyclic nucleotides in the mediation of glucagon- and isoproterenol-stimulated secretion of CT and NT. I will evaluate the effects of cAMP analogues, phosphodiesterase inhibitors, prostaglandins, cholera toxin, Ca++, Mg++ and control peptides which do not stimulate CT or NT secretion on the activation of adenylate cyclase (AC) and phosphodiesterase (PDE), cAMP generation and peptide secretion. (2) I will determine the role of calmodulin (CDR) in the control of CT and Nt secretions by studying the effects of trifluoperazine (TFP) and W-compounds (both CDR antagonists) on cellular events though to be important in the secretory process including 45Ca++ movement into the cell, activation of AC and PDE, cAMP and protein phosphorylation. (3) I will determine whether glucagon- and isoproterenol-(cAMP-mediated) or Ca++ and K++ stimulated secretion is associated with phosphorylation of specific membrane or cytosolic proteins and study the dependence of such phosphorylation on cAMP or CDR. Protein phosphorylation will be assessed by incorporation of [Gamma -32)-ATP followed by cold ATP] or by inhibiting protein kinase activity by chelating Mg++. These studies are significant because they will provide insight into the mechanisms by which Ca++, a physiologic regulator of CT secretion, and glucagon and catecholamines control cyclic nucleotide metabolism and CT and NT secretion in a model system which has many secretory characteristics of the C-cell.
