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 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)
Anwer
M S
MS
Department of Medicine, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536.
D000468:Alkalies / Q000378:metabolism*; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism
D000468:Alkalies; D015759:Ionomycin; D000584:Amiloride; D012964:Sodium; D011188:Potassium; D002118:Calcium