The long range goal of the proposed research is to gain an understanding of the mechanisms by which calcium ions regulate the secretory process. In order to accomplish this goal, the role of other mediators of secretion including calmodulin (the calcium-binding protein), sodium ions and cyclic AMP (c-AMP), and the relationship between these mediators and calcium must be clearly defined. In addition the effects of calcium on specific intracellular processes must be determined. Mouse parotid acini will be isolated by procedures involving enzymatic digestion, divalent cation depletion and mechanical shearing. Calmodulin activity, content and distribution will be determined, as well as calcium-calmodulin dependent membrane protein phosphorylation. The functional role of calmodulin in amylase release will be assessed by utilizing a variety of calmodulin inhibitors. The mechanism(s) by which sodium ions regulate amylase release will be evaluated by examining the effects of sodium ions on Beta-adrenergic and forskolin stimulation of adenylate cyclase activity in isolated membranes versus intact cells. The interaction between sodium and calcium ions on adenylate cyclase activity will also be assessed. Reserpine will be utilized as a pharmacological tool to further evaluate the role of calcium in secretion under conditions of impaired stimulus-secretion coupling. Amylase release and changes in calcium content will be examined and related to changes in c-AMP dependent protein kinase activity and membrane phosphorylation. 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.
