Fluid secretion in salivary glands is modulated by changes in the cytosolic [Ca2+], which involves Ca2+ release from intracellular pools and Ca2+ entry from the external medium. We have directed our efforts towards understanding the processes which regulate cytosolic [Ca2+]. Three main areas are being investigated: (i)regulation of signal generation, (ii) regulation of intracellular Ca2+ mobilization, and (iii) regulation of Ca2+ flux in cell membranes. In the present reporting period a major effort was directed towards characterizing the mechanism of Ca2+ entry in rat parotid acini. We report that agonists stimulate Mn2+ entry into parotid acini in a manner similar to Ca2+. Using Mn2+ as a tool we show that divalent cation entry likely occurs via a cytosolic route and is regulated by the [Ca2+] in the intracellular pool. We also show that hyperpolarizing conditions stimulate while depolarizing conditions inhibit cation entry, independent of intracellular [Ca2+]. We have observed that in isolated basolateral membrane vesicles the permeability to Ca2+ is increased by the presence of permeant ions and inhibited by La3+ and Mg2+. Additionally continuing our studies on the regulation of IP3 generation, using an in vitro assay for the PIP2-specific phospholipase C, we show that GTPYS and carbachol, but not carbachol alone, can stimulate this activity in an isolated, plasma membrane-enriched, membrane preparation.
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