The purpose of the research proposed in this application is to test the hypothesis that changes in the phosphorylation state of tyrosine groups in proteins of the submandibular gland acinar cells are involved in the biochemical cascade that occurs when muscarinic agonists interact with cholinergic receptors in the membranes of these cells. In order to test this hypothesis, the tyrosine phosphate content of cellular proteins will be determined in resting and activated cells. The phosphotyrosine content will be measured using both immunoblotting with an antibody specific for phosphotyrosine residues, and by immunoprecipitation of 32P-labelled cellular extracts with the same antibody, followed by denaturing polyacrylamide gel electrophoresis and autoradiography. The relationship between agonist dose and phosphotyrosine levels for the various proteins will be examined, as will the time course of the phosphorylation and/or dephosphorylation events. The question of whether these changes are involved in the signal transduction mechanism will be addressed by comparing these dose response and time course data to those of other changes in cell function, including the production of inositol trisphosphate, mobilization of Ca2+ from intracellular stores and through entry, from the medium, and by, the Ca2+- dependent opening of Cl(-) channels. Inhibitors of tyrosine kinase and tyrosine phosphatase activity will also be used to investigate the role of these reactions in the signal transduction process.
