Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis.
We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.
Takeyama
N
N
Department of Emergency and Critical Care Medicine, Kansai Medical University, Fumizono-cho 10-15, Moriguchi, Osaka, 570-8507, Japan. takeyama@takii.kmu.ac.jp
Miki
S
S
Hirakawa
A
A
Tanaka
T
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003574:Cytochrome c Group / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010539:Permeability / Q000187:drug effects; D021381:Protein Transport; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D006861:Hydrogen Peroxide