Abstract
We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Treatment with arachidonic acid or induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co(2+) quenching technique (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725-734). Yet depolarization, as assessed from the changes of mitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapid and extensive with arachidonic acid and slow and partial with. Cyclosporin A-inhibitable release of cytochrome c and cell death correlate with the changes of TMRM fluorescence but not with those of calcein fluorescence. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. Modulation of the open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.
Highlights
We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells
In this study we investigated the occurrence of cell death in rat hepatoma MH1C1 cells treated with AA, a potent PTP inducer that is characterized in the accompanying article [24], or with the ionophore A23187
These results indicate that PTP opening by AA causes mitochondrial depolarization in situ, a finding that was obtained in nominally Ca2ϩ-free media
Summary
We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. In this study we investigated the occurrence of cell death in rat hepatoma MH1C1 cells treated with AA, a potent PTP inducer that is characterized in the accompanying article [24], or with the ionophore A23187 We found that both treatments cause PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co2ϩ quenching technique [25], while depolarization, as assessed from the fluorescence changes of the potentiometric probe TMRM, was rapid and extensive with AA and slow and partial with A23187. Since pore opening must be accompanied by depolarization, these findings suggest that relatively short PTP openings are detected only by trapped calcein and have little impact on cell viability, while detectable changes of TMRM distribution require longer PTP openings, which eventually cause cy-
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