Advertisement

Mechanism of ionomycin-induced intracellular alkalinization of rat hepatocytes

Authors

  • M. Sawkat Anwer

    Corresponding author
    1. Departments of Medicine and Pharmacology, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
    • Tufts University, 200 Westboro Road, North Grafton, MA 01536
    Search for more papers by this author

Abstract

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).

Ancillary