La3+ and pH sensitivity of Ca2+ entry and intracellular store filling in gastric parietal cells

The fluorescent Ca2+ indicator fura 2 was used to measure cytosolic free [Ca2+] ([Ca2+]i) in order to obtain information about relative rates of Ca2+ influx into parietal cells during treatment with carbachol (a cholinergic agonist) or thapsigargin (TG, a Ca(2+)-mobilizing agent) or during reloading of the internal Ca2+ stores. In Ca(2+)-containing solutions, carbachol-, TG-, and reloading-stimulated Ca2+ entry exhibited nearly identical sensitivity to La3+ [inhibition constant (Ki) approximately 10 microM] or low pH (pKi approximately 7.0). In experiments in which carbachol and TG were used, there was no additional increase in [Ca2+]i when TG was added to carbachol-treated cells or when carbachol was added to cells previously treated with TG. Thus it is likely that a single Ca2+ entry pathway serves a signaling function as well as a role in refilling the Ca2+ store during reloading. Because the Ca2+ pathway is exquisitely sensitive to pH and serosal pH increases during stimulant-induced H+ secretion (which is activated by increases in [Ca2+]i), this mechanism will exert positive feedback on parietal cells in the intact stomach. When parietal cells were pretreated with carbachol in Ca(2+)-free solutions, reloading was independent of pH and La3+, suggesting that Ca(2+)-containing solutions should be used to determine the properties of the influx pathway.