Fluid secretion in salivary glands is modulated by changes in the cytosolic [Ca], which involves the release from intracellular pools and entry from the external medium. We have directed our efforts towards understanding the processes which regulate cytosolic [Ca]. Three main areas were investigated during this reporting period:(i) regulation of signal generation, (ii) regulation of intracellular Ca mobilization, and (iii) regulation of Ca flux in cell membranes. Using an in vitro assay system for phospholipase C, we have observed that rat parotid membranes have an enzyme with a high specificity for phosphatidyl inositol 4,5,bisphosphate, which appears to be immunologically distinct from the PLC~ enzyme found in rat brain membranes. We have also shown that this enzyme can be stimulated by c-AMP in the presence of A-kinase. Agonists such as carbachol, stimulate Mn entry into parotid acini, but the detection of Mn entry (via Ca channels) does not coincide with internal Ca release and an initial lag period is observed. We have established that this lag is most likely due to a retardation of initial Mn entry due to the initial rise in cytosolic [Ca]. We have also shown that Ca entry is inhibited by treatment of cells with DCCD. However, in DCCD treated cells Mn entry can be stimulated by hyperpolarization. Parotid acini are also permeable to Co, but only when hyperpolarized. These data suggest a heterogeneity in the divalent cation influx system in these cells. Using isolated basolateral membrane vesicles, we report that the Ca flux via the putative channel is driven by the electrochemical gradient of Ca and directly regulated by the [Ca] on the cytoplasmic side.
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