Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax.
Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating this event remain elusive. In the present study, using isolated liver mitochondria, we demonstrate that cytochrome c release occurs via distinct mechanisms that are either Ca(2+)-dependent or Ca(2+)-independent. An increase in mitochondrial matrix Ca(2+) promotes the opening of the permeability transition (PT) pore and the release of cytochrome c, an effect that is significantly enhanced when these organelles are incubated in a reaction buffer that is based on a physiologically relevant concentration of K(+) (150 mm KCl) versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca(2+) are sufficient to induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT varies among mitochondria within a single population of these organelles. In contrast, Ca(2+)-independent cytochrome c release is induced by oligomeric Bax protein and occurs without mitochondrial swelling or the release of matrix proteins, although our data also indicate that Bax enhances permeability transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca(2+) concentration, as well as the reaction buffer composition, are key factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both Ca(2+)-dependent and Ca(2+)-independent mechanisms.
Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.
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Gogvadze
J D
JD
Robertson
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B
Zhivotovsky
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S
Orrenius
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006531:HEPES / Q000494:pharmacology; D008297:Male; D008353:Mannitol / Q000494:pharmacology; D008564:Membrane Potentials; D008930:Mitochondria, Liver / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D013395:Sucrose / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D008353:Mannitol; D013395:Sucrose; D011189:Potassium Chloride; D011188:Potassium; D006531:HEPES; D002118:Calcium