The overall objective of the proposed research is to elucidate the mechanisms by which calcium ions regulate the secretory process. Evidence has accumulated which suggests that the calcium and cyclic AMP signal transduction pathways act in a coordinated manner to regulate secretion. In mouse parotid gland muscarinic receptor interaction leads to phosphoinositide (PI) turnover, and to both stimulation and inhibition of beta-adrenergic-induced cAMP accumulation. The hypothesis to be tested in this proposal is that muscarinic subtypes couple to the different functional responses and the mechanisms underlying regulation of these functional responses are different.
Specific aims are 1) to characterize and identify muscarinic subtypes in isolated mouse parotid acini by radioligand binding; 2) to correlate muscarinic receptor subtypes with functional responses by comparing selective antagonist binding to receptors with the effects of antagonists on PI turnover and cAMP metabolism and 3) to determine the mechanisms underlying the regulation of functional responses by a) examining the interaction of muscarinic and beta-adrenergic agonists on receptor binding and PI turnover; and by b) examining the role of intracellular mediators on cAMP metabolism. Cellular levels of Cyclic GMP, calcium and protein kinase C (PKC) activity will be correlated with effects on adenylate cyclase and phosphodiesterase activities in intact cells and/or in cell-free preparations. The free intracellular calcium concentration (Cai) will be measured using fura-2; cyclic nucleotides will be determined by radioimmunoassay and PKC activity will be measured by enzyme assay. The proposed studies should provide a significant understanding of the molecular mechanisms involved in the interaction of the two signal transduction pathways in the regulation of salivary gland secretion, and provide a framework for examining the cellular biochemical defects in diseases such as Cystic Fibrosis.
