The long range goal of the proposed research is to elucidate the mechanism(s) by which calcium ions regulate the secretory process. It is becoming increasingly clear that the measurement of ionized or free cytosolic calcium (Ca-i) is essential for obtaining definitive evidence for the regulatory role of intracellular calcium in secretion. It is also clear that strong interrelationships exist between calcium ions and other defined intracellular messengers. Of particular interest in this proposal is to examine the regulation of cyclic AMP metabolism by Ca-i and the interrelationship of calcium, cyclic GMP and sodium ions in such regulation. Mouse parotid acini will be isolated by procedures involving enzymatic digestion, divalent cation depletion and mechanical shearing. Free cytosolic calcium levels in intact cells will be measured by using a highly fluorescent calcium indicator (Quin-2) and the effects of secretogogues on Ca-i and amylase release determined. Cells """"""""permeabilized"""""""" by high voltage will also be used to assess the effects of a wide range of Ca-i on amylase release; (Ca-i) concentrations will be determined by use of Ca-EGTA buffers. Regulation of cyclic nucleotide (cAMP) metabolism by Ca-i will be determined by examining adenylate and phosphodiesterase activities. For studies involving adenylate cyclase isolated parotid membranes will be prepared and calcium-regulated adenylate cyclase activity measured in the absence and presence of sodium ions. The site of interaction between sodium and calcium ions will be examined. The presence of an inhibitory guanine nucleotide binding site and its modulation by sodium will also be determined. For studies involving phosphodiesterases, the forms of phosphodiesterase present in parotid tissue will be identified by DEAE-column chromatography and antibody studies. Regulation of enzyme activity by cholinergic agonists will be determined and the role of calcium and cGMP assessed. Knowledge of the intracellular biochemical events associated with the secretory process may provide valuable insight into disease states where defects in such intracellular events exist.
