CA2+ ANTAGONISTS DO NOT PROTECT ISOLATED-PERFUSED RAT HEPATOCYTES FROM ANOXIC INJURY

Ca2+ antagonists were studied during anoxia in perfused isolated rat hepatocytes. Cytosolic free calcium (Ca(i)2+) was measured with aequorin. Anoxia was induced for 2 h by saturating the perfusate with 95% N2/5% CO2. Anoxia increased Ca(i)2+ in two distinct phases reaching a maximum of 1.5 muM. The increase in Ca(i)2+ was caused by Ca2+ influx from the extracellular fluids because the main Ca(i)2+ surge was totally abolished in Ca2+-free media. LDH release increased 6-fold during the second hour of anoxia, but when Ca2+ was removed from the perfusate during the anoxic period, LDH rose only 2.7-fold. Ca2+ antagonists (10(-7) to 10(-5) M) did not prevent the increase in Ca(i)2+ and the rise in LDH release. On the contrary, high concentrations (10(-6) and 10(-5) M) of the blockers nifedipine and diltiazem significantly increased anoxic cell injury. The observation that the increase in LDH and the rise in Ca(i)2+ were not suppressed by Ca2+ antagonists suggests that (i) Ca2+ antagonists protect the whole liver from anoxic injury by acting on cells other than parenchymal cells; (ii) the influx of Ca2+ responsible for the massive increase in hepatocyte Ca(i)2+ evoked by anoxia did not take place through voltage-sensitive Ca2+ channels but must have occurred via the Na+-Ca2+ antiporter operating in the reverse mode (Ca2+ influx vs. Na+ efflux), and (iii) high concentrations of Ca2+ antagonists may be deleterious to the parenchymal cells of the liver.