The broad, long-term objectives of this proposal are to understand on a molecular level the mechanisms by which neurotransmitters promote receptor- mediated alterations of ion transport events involved in saliva formation (fluid secretion) by the parotid acinar cell. The acinar cell secretes an isotonic primary fluid that is modified by athe salivary ductal system, which adds and removes electrolytes. Secretory events are controlled by the release of neurotransmitters, and athe parotid gland receives parasympathetic and sympathetic innervation. The former is believed to regulate fluid secretion, while the latter is involved in the stimulation of exocytosis and amylase release. ATP is costored and coreleased with neurotransmitters. We have demonstrated that extracellular ATP produces effects on ion transport systems similar to those produced by activation of phospholipase C (:LC)-linked receptors, but by a different mechanism--the activation of a ligand (ATP)-gated Ca2+ permeable ion channel that also appears to be the ATP-binding P2Z purinoceptor. Recently we found that the activation of PLC-linked receptors or P2Z receptors stimulates the tyrosine phosphorylation of PKCdelta, a Ca2+-independent isoform of PKC. In addition, activation of the P2Z receptor is blocked by tyrosine kinase inhibitors and is enhanced by activation of protein kinase C (PKC) by phorbol ester, suggesting that the P2Z channel/receptor is modulated by PKC (perhaps PKCdelta) via the stimulation of protein tyrosine kinases. These results and others in the literature suggest that the activation of fluid secretion by neurotransmitters acting on parotid cells involves the phosphorylation of multiple protei ns on tyrosine and other amino acid residues. This is the overall hypothesis that will be examined. We will determine the involvement of protein kinases and phosphatases ina the activation of ion transport systems (the P2Z receptor, the capacitative Ca2+ entry pathway, and tahe Na-K-2CI cotransporter) involved in fluid secretion. In addition, the activation of phospholipase D, which may contribute to the P2Z receptor activation, will be investigated. We also will examine the subcellular localization and redistribution of multiple PKC isoforms when pLC-linked and P2Z receptors are activated in parotid acinar cells. Together, these studies will provide a more complete understanding of biochemical events involved in fluid secretion and saliva formation, and may suggest potential regulatory sites at which therapeutic agents may alter saliva formation in dysfunctional conditions.
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