Mechanism of ionomycin-induced intracellular alkalinization of rat hepatocytes

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Calcium ionophores such as ionomycin and A23187 are often used to determine the role of intracellular Ca++ in cellular processes. Ionomycin but not Ca+(+)-mobilizing agonists increases basal intracellular pH in hepatocytes. To explain this difference in effects of agents that increase intracellular Ca++ concentration, the mechanism of ionomycin-induced increases in basal intracellular pH in isolated rat hepatocytes was studied. Changes in intracellular pH and intracellular Ca++ concentration were measured with the fluorescent probes BCECF (2',7'-bis-2-[carboxyethyl ester]-5[6]carboxyfluorescein) and quin-2, respectively. Ionomycin produced dose-dependent increases in intracellular pH and intracellular Ca++ concentration, with the increase in intracellular Ca++ concentration preceded by the increase in intracellular pH. Ionomycin-induced increases in intracellular pH were not affected by 1 mmol/L amiloride, 100 mumol/L diisothiocyanostilbene disulfonate or removal of extracellular Na+, indicating that the effect is not mediated by Na+/H+ exchange, Cl-/HCO3- exchange or Na+/HCO3- cotransport. Ionomycin failed to increase intracellular pH or intracellular Ca++ concentration in the absence of extracellular Ca++, and both intracellular pH and intracellular Ca++ concentration increased promptly when extracellular Ca++ was reintroduced. Ionomycin-induced increases in intracellular Ca++ concentration but not intracellular pH were smaller in hepatocytes loaded with the Ca++ buffering agent MAPTA. Thapsigargin increased intracellular Ca++ concentration but failed to increase intracellular pH. Thus the effect of ionomycin is independent of the effect of ionomycin on intracellular Ca++ concentration and dependent on extracellular intracellular Ca++ concentration. Experimental conditions that produce cell depolarization did not increase basal intracellular pH but lowered ionomycin-induced increases in intracellular pH by 25% without affecting increases in intracellular Ca++ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)