This project is directed towards understanding the processes which regulate cytosolic [Ca] in salivary gland cells. We are studying (i) receptor regulation of phosphatidylinositol bisphosphate-specific phospholipase C, (ii) regulation of Ca entry in salivary cells, and (iii) regulation of Ca flux in rat parotid gland basolateral membrane vesicles. Previously we have characterized a phosphatidyl-inositol- 4,5,bisphosphate specific phospholipase C enzyme in rat parotid gland membranes. In this reporting period we have shown that it is independently regulated by both muscarinic and alpha1-adrenergic receptors via a mechanism mediated by alpha subunits of the Gq/11family of G-proteins. Sustained Ca entry into parotid acini maintains a sustained elevation of Ca in the cytosol and thus facilitates prolonged fluid secretion. While we have earlier shown that Ca entry is correlated with the depletion of Ca from intracellular Ca stores, the exact mechanism which regulates this process is yet unclear. Using Mn as a substitute divalent cation, we have now shown that Ca entry into internal Ca pool depleted cells, via agonist stimulation, is very sensitive to temperature. Additionally, the pattern of its response to temperature distinguishes it from Mn entry into unstimulated acini. In a human salivary gland cell line, HSG, we have identified a Ca entry mechanism which is not regulated by the emptying of intracellular Ca stores, but depends on muscarinic receptor activation of G-protein(s). Consistent with our date with intact acini we have observed that Ca influx into isolated basolateral membrane vesicles is decreased at low temperature. We have observed that low temperature also protects Ca influx in vesicles against inactivation by trypsin and carbodiimides, which suggests that temperature induces a modification of the Ca influx pathway.
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