Cellular respiration during hypoxia: Role of cytochrome oxidase as the oxygen sensor in hepatocytes

We previously reported that hepatocytes exhibit a reversible suppression of respiration during prolonged hypoxia (PO₂ = 20 torr for 3-5 h). Also, isolated bovine heart cytochrome c oxidase undergoes a reversible decrease in apparent V(max) when incubated under similar conditions. This study sought to link the hypoxia-induced changes in cytochrome oxidase to the inhibition of respiration seen in intact cells. Hepatocytes incubated at PO₂ = 20 torr exhibited decreases in respiration and increases in [NAD(P)H] after 2-3 h that were reversed upon reoxygenation (PO₂ = 100 torr). Respiration during hypoxia was also inhibited when N,N,N',N'-tetramethyl-p-phenylenediamine (0.5 mM) and ascorbate (5 mM) were used to reduce cytochrome c, suggesting that cytochrome oxidase was partially inhibited. Similarly, liver submitochondrial particles revealed a 44% decrease in the apparent V(max) of cytochrome oxidase after hypoxic incubation. In hepatocytes loaded with tetramethylrhodamine ethyl ester (10 nM) to quantify mitochondrial membrane potential, acute hypoxia (<30 min) produced no change in fluorescence, consistent with the absence of an acute change in respiration. However, fluorescence increased during acute reoxygenation after prolonged hypoxia, suggesting an increase in potential. The control exhibited by NADH over mitochondrial respiration was not altered during hypoxia. Thus, changes in the V(max) of cytochrome oxidase during prolonged hypoxia correlate with the changes in respiration and mitochondrial potential. This suggests that the oxidase functions as an oxygen sensor in the intact hepatocyte.