Flow cytometric analysis of Ca²⁺-induced membrane permeability transition of isolated rat liver mitochondria

The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca²⁺ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca²⁺ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 μl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca²⁺ sequentially induced ROS generation, depolarization, swelling and Ca²⁺ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca²⁺-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca²⁺-induced phospholipase A₂ suppressed all of the events associated with Ca²⁺-induced MPT. These results suggested that ROS generation and phospholipase A₂ activation by Ca²⁺ underlie the mechanism of the initiation of MPT.