Measurement of mitochondrial and non-mitochondrial Ca²⁺ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca²⁺ in a Ca²⁺-free extracellular medium as revealed by changes in the absorbance of the Ca²⁺ indicator arsenazo III. The process is completed in 2 min and the amount of Ca²⁺ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca²⁺ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent ⁴⁵Ca²⁺ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of ⁴⁵Ca²⁺ from ⁴⁵Ca²⁺-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca²⁺ from non-mitochondrial stores (vasopressin and 2,5-di-tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca²⁺ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca²⁺ from mitochondrial stores without significantly affecting active Ca²⁺ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca²⁺ in liver cells.