The major emphasis of this research is to study mechanisms of exocytosis in the parotid gland. The focus will be on the role of extracellular ATP and ectoATPase in parotid gland function. The presence and location of an ectoATPase will be investigated. EctoATPase will be established by the ability of the ATPase to be expressed on the exterior of intact cells. This ATPase will be compared in its characteristics to that of the plasma membrane ATPase already described. The border(s) of the acinar cell involved in ectoATPase activity win be identified. The effect of kinases on phosphorylation and activity of the ectoATPase will be studied. The ability of the intact cell to metabolize nucleotides and adenosine will be evaluated. Antibodies win be raised against the ectoATPase and specific peptide regions of the ATPase. These antibodies will be used to probe the enzyme. The function of the enzyme will be studied as to hydrolysis of ATP, the effect of calcium on the enzyme, and the interaction of the ectoATPase with kinases. The effect of ATP at the P, receptor of the parotid gland will be assessed both with ATP alone and with ATP plus the secretogogues, isoproterenol carbachol, phenylephrine and substance P. These effects will include, changes in intracellular cyclic AMP and calcium as well as amylase release. Finally we will test the ability of ectokinases to alter the ectoATPase function and to mediate phosphorylation of the membrane from the external surface of the cell. The purpose will be to expand our knowledge of the effect of extracellular AT? both as a secretagogue itself and as a modulator at the neuroeffector sites in the gland. The anti-ectoATPase antibodies will aid in the assessment. The recent evidence pointing to the importance of ectoATPase and the receptors for AT? suggests an important role for extracellular ATP in secretion.
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