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. lonomycin 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. lonomycin-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. lonomycin 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. lonomycin-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. 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.