Mechanism of ionomycin‐induced intracellular alkalinization of rat hepatocytes

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 I mmol/L amiloride, 100 μmol/L diisothiocyanostilbene disulfonate or removal of extracellular Na + , indicating that the effect is not mediated by Na + /H + exchange, Cl − /HCO 3 − exchange or Na + /HCO 3 − 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 ionomycininduced increases in intracellular pH by 25% without affecting increases in intracellular Ca + + concentration. Taken together, these results indicate that the increase in basal intracellular pH may primarily be due to ionomycin‐mediated electroneutral Ca + + /2H + exchange across the hepatocyte plasma membrane. Because the effect of ionomycin is not mediated by Na + /H + exchange, the activity of this exchanger under basal conditions is not regulated by intracellular Ca + + . These results also suggest that the pharmacological effects of ionomycin in hepatocytes are mediated by changes in intracellular pH in addition to or independent of changes in intracellular Ca + + concentration. (H EPATOLOGY 1993;18:433–439).