Chemical hypoxia increases cytosolic Ca2+ and oxygen free radical formation

'Chemical hypoxia' was produced in isolated rat hepatocytes. The cells were immobilized in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer equilibrated with 95% O2 + 5% CO2 or 95% air + 5% CO2. During 'chemical hypoxia', 2 mM KCN + 0.5 mM iodoacetate (CN-IAA) were added to the perfusate for 30 min. Cytosolic ionized Ca2+ (Cai2+) was measured with aequorin, the formation of oxygen free radicals by lucigenin-enhanced chemiluminescence and cell injury by the rate of LDH released by the cells in the effluent perfusate. As soon as the cells were exposed to CN-IAA in the presence of 95% O2 + 5% CO2, Cai2+ increased rapidly to reach 1.5 microM within 10 min, and oxygen free radical formation increased 5-fold. The increase in LDH release was temporally delayed and occurred only during the recovery phase. The results were not significantly different when the cells were perfused with KHB equilibrated with 95% air + 5% CO2, except that oxygen free radical formation increased 13-fold. These results suggest that both a rise in Cai2+ and a formation of reactive oxygen species could be responsible for the cell injury and the cell death induced by CN-IAA poisoning.