The principal object of this project is to extend our studies on the effect of Ca2+ mobilizing hormones on the Ca2+ permeability and Ca2+ pump of the intracellular stores of pancreatic acinar cells. This constitutes as essential stage towards understanding the mechanisms of release and reloading of the intracellular stores with Ca2+upton cell stimulation and removal of stimuli. To date, we have developed techniques; to measure free cytosolic Ca2+ in intact pancreatic acinar cells using the Ca2+ sensitive dye FURA 2; to specifically label the hormone mobilizable intracellular pool with 45Ca in intact cells; to measure Ca2+ channel mediated - 45Ca fluxes in intact and permeabilized cells; and to measure Ca2+ pump mediated -45Ca fluxes in permeabilized cells. Using these techniques, the immediate aims are (a) demonstration that Ca2+ release from the intracellular pool is sufficient to activate the plasma membrane Ca2+ channel. Activation of this channel is not directly mediated and does not require the presence of the hormone. (b) Show that the hormone, like IP3, activates a Ca2+ channel in the endoplasmic reticulum membrane. (c) Compare the effects of IP3, GTP and arachidonic acid with the hormone activated Ca2+ release from the intracellular pool. (d) Study the contribution of IP3, GTP and arachidonic acid to the hormone dependent Ca2+ release (e) define the requirements for the newly discovered, activation of the endoplasmic reticulum Ca2+ pump by the Ca2+ mobilizing hormone (f) attempt to characterize the mechanism of activation of the endoplasmic reticulum Ca2+ pump by the Ca2+ pump by the Ca2+ mobilizing hormones. Our basic studies are providing techniques to test hypotheses on hormonal regulation of free cytosolic Ca2+ and Ca2+ content in the intracellular stores of pancreatic acinar cells both during and at the termination of cell stimulation.
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