Mitochondrial Ca2+ homeostasis in the regulation of apoptotic and necrotic cell deaths

Using distinct models of apoptosis and necrosis, we have investigated the effect of mitochondrial Ca2+ (Ca-m) homeostasis in the regulation of cell death in neuroblastoma cells as well as cardiac myocytes. The steady state level of Ca-m was determined as the FCCP-releasable Ca2+. Culturing cells with low concentration of extracellular Ca2+ (Ca-o) or with EGTA triggered an early reduction in both the Ca-m store and the membrane potential (Delta Psi(m)). This was followed by the detection of cytochrome c release, caspase activation, and apoptosis. Inhibitors of the mitochondrial permeability transition pore such as cyclosporin A and Bcl-2 blocked the release of Ca-m and inhibited apoptosis. In contrast, mitochondrial Ca2+ overload resulted in necrotic cell death. Culturing cells in the presence of excess Ca-o led to increased Ca-m load together with a decrease of Delta Psi(m) that reached maximum at 1 h, with necrosis occurring at 2 h. While the decline of Ca-m and Delta Psi(m) was a coupled reaction for apoptosis, this relationship was uncoupled during necrosis. Clonazepam, a relatively specific inhibitor of the mitochondrial Na/Ca exchanger, was able to protect the cells from necrosis by reducing Ca-m overload. Importantly, combination of clonazepam and cyclosporin showed a cooperative effect in further reducing the Ca-m overload and abolished cell death. The data imply the participation of Ca-m homeostasis in the regulation of apoptosis and necrosis. (C) 2000 Harcourt Publishers Ltd.