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Contribution of increased mitochondrial free Ca 2+ to the mitochondrial permeability transition induced by tert ‐butylhydroperoxide in rat hepatocytes
Author(s) -
Byrne Aaron M.,
Lemasters John J.,
Nieminen AnnaLiisa
Publication year - 1999
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.510290521
Subject(s) - mitochondrial permeability transition pore , mitochondrion , chemistry , permeability (electromagnetism) , microbiology and biotechnology , biophysics , biology , biochemistry , apoptosis , programmed cell death , membrane
Previously, we showed that the oxidant chemical, tert ‐butylhydroperoxide ( t ‐BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca 2+ in t ‐BuOOH cytotoxicity to 1‐day‐cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter‐indicating fluorophores. t ‐BuOOH (100 μmol/L) caused an early increase of mitochondrial free Ca 2+ , as assessed by confocal microscopy of Rhod‐2 fluorescence. Increased mitochondrial Ca 2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential–indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca 2+ chelator (BAPTA‐AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t ‐BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca 2+ chelation also prevented the increase of mitochondrial Ca 2+ , generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca 2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca 2+ after t ‐BuOOH and prevented cell death. We conclude that oxidative stress induced by t ‐BuOOH enhances mitochondrial Ca 2+ uptake, leading to increased matrix Ca 2+ , increased ROS formation, onset of the MPT, and cell death.