Background and Aims: The mitochondrial membrane permeability transition (MMPT) has been proposed as a mechanism of cell necrosis. In contrast, it has been suggested that enhanced activity of calpain-like proteases causes cell necrosis. To integrate these concepts, the hypothesis that stimulation of mitochondrial calpain-like protease activity induces the MMPT was developed. Methods: Calpain-like protease activity and the MMPT were measured in rat liver mitochondria. The mitochondrial membrane potential and cell necrosis were measured in rat hepatocytes. Results: The protease inhibitor Cbz-Leu- Leu-Tyr-CHN2 inhibited both calpain-like protease activity and induction of the MMPT by Ca2+ and tert-butyl hydroperoxide. This effect of Cbz-Leu-Leu- Tyr-CHN2 was specific because serine, aspartate, and metalloprotease inhibitors did not inhibit the MMPT. The protease inhibitor Cbz-Leu-Leu-Tyr- CHN2 also delayed the onset of mitochondrial depolarization and cell necrosis during treatment of rat hepatocytes with tert-butyl hydroperoxide, a model of oxidative stress relevant to human disease. Conclusions: These data suggest a unifying hypothesis linking calpain-like protease activity to the MMPT in cell necrosis. We propose for the first time that activation of mitochondrial calpain-like protease activity can function as a cytolytic trigger initiating the MMPT in cell necrosis.
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