LA3-CELLS( AND PH SENSITIVITY OF CA2+ ENTRY AND INTRACELLULAR STORE FILLING IN GASTRIC PARIETAL)

The fluorescent Ca2+ indicator fura 2 was used to measure cytosolic fr ee [Ca2+] ([Ca2+](i)) in order to obtain information about relative ra tes of Ca2+ influx into parietal cells during treatment with carbachol (a cholinergic agonist) or thapsigargin (TG, a Ca2+-mobilizing agent) or during reloading of the internal Ca2+ stores. In Ca2+-containing s olutions, carbachol-, TG-, and reloading-stimulated Ca2+ entry exhibit ed nearly identical sensitivity to La3+ [inhibition constant (K-i) sim ilar to 10 mu M] or low pH (pK(i) similar to 7.0). In experiments in w hich carbachol and TG were used, there was no additional increase in [ Ca2+](i) when TG was added to carbachol-treated cells or when carbacho l was added to cells previously treated with TG. Thus it is likely tha t a single Ca2+ entry pathway serves a signaling function as well as a role in refilling the Ca2+ store during reloading. Because the Ca2+ p athway is exquisitely sensitive to pH and serosal pH increases during stimulant-induced H+ secretion (which is activated by increases in [Ca 2+](i)), this mechanism will exert positive feedback on parietal cells in the intact stomach. When parietal cells were pretreated with carba chol in Ca2+-free solutions, reloading was independent of pH and La3+, suggesting that Ca2+-containing solutions should be used to determine the properties of the influx pathway.