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⁻/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 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. (HEPATOLOGY 1993;18:433–439).