Role of Na⁺/Ca²⁺ exchanger in preventing Na⁺ overload and hepatocyte injury: Opposite effects of extracellular and intracellular Ca²⁺ chelation

We have previously shown that an increase of intracellular Na⁺ occurs in isolated rat hepatocytes undergoing ATP depletion and that Na⁺ accumulation is associated with an uncontrolled influx of Ca²⁺ through the activation in reverse mode of the Na⁺/Ca²⁺ exchanger. In the present study we have investigated the relationship between alterations of Na⁺ and Ca²⁺ homeostasis and hepatocyte killing using treatments which differentially chelate extracellular or intracellular Ca²⁺. Chelation of extracellular Ca²⁺ by ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) potentiated Na⁺ overload and cell killing induced in isolated rat hepatocytes by hypoxia or menadione. Similar effects were also observed when Na⁺ accumulation was induced by the combined addition of Na⁺ ionophore monensin and the inhibition of plasma membrane Na⁺/K⁺ ATPase by ouabain. Conversely, the use of the intracellular Ca²⁺ chelator EGTA acetoxymethyl ester (EGTA/AM) reduced Na⁺ overload and hepatocyte death induced by hypoxia or cell treatment with menadione or monensin plus ouabain. The effects of EGTA/AM were reverted in the presence of bepridil, an inhibitor of Na⁺/Ca²⁺ exchanger. Altogether these results indicated that differential chelation of intracellular or extracellular Ca²⁺ influences in opposite ways hepatocyte killing due to ATP depletion by modulating intracellular Na⁺ levels through the reversed activity of the Na⁺/Ca²⁺ exchanger.