This project is directed towards understanding the processes which regulate cytosolic [Ca] in salivary gland cells. In these cells, Ca entry which is critical for prolonged fluid secretion in this gland, is regulated by a """"""""Ca release-activated Ca entry""""""""-type of mechanism, found in a number of non-excitable cells. This largely uncharacterized mechanism appears to be stimulated by the depletion of Ca in the intracellular Ca store(s). In this reporting period we have demonstrated that Ca influx in rat parotid and HSG cells (derived from the human submandibular gland) is regulated by (i)Ser/Thr phosphorylation (inhibition) and dephosphorylation (stimulation). We had described previously that internal Ca pool-depleted parotid acinar cells have two Ca influx components with high and low affinities for Ca, Kd=65microM and Kd=3.3mM, respectively. In this reporting period we have shown that the high affinity component is more sensitive to inhibition by depolarization and Zn, while both components are inhibited by micronazole and carbodiimide. Further, we have examined the kinetics of Ca influx into isolated basolateral membrane vesicles (BLMV) at a lower temperature (30 degrees C) and have demonstrated the presence of two Ca influx components with Kd similar to that found in intact cells (108microM and 3l.5mM). In the last reporting period we had described reconstitution of the high affinity Ca2+ influx component from BLMV into artificial lipid vesicles, by using a detergent, octylglucoside, dilution method. By using lectin chromatography and the same reconsititution procedure, we have now partially purified this Ca influx component. Continuing our recent studies on the effects of IFN-gamma- and TNF-alpha on the proliferatin and Ca signalling mechanisms in HSG cells, we have now shown that there is a decrease in the SERCA 2 type of CA pumps in IFN-gamma-treated cells. This is a novel observation and likely accounts for the decreased Ca content of internal Ca store(s) in IFN- gamma-treated HSG cells.
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