The mitochondrial permeability transition, release of cytochrome c and cell death - Correlation with the duration of pore openings in situ

We investigated the relationship between opening of the permeability transi tion pore (PTP), mitochondrial depolarization, cytochrome c release, and oc currence of cell death in rat hepatoma MH1C1 cells. Treatment with arachido nic acid or A23187 induces PTP opening in situ with similar kinetics, as as sessed by the calcein loading-Co2+ quenching technique (Petronilli, V., Mio tto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biop hys, J. 76, 725-734), Yet depolarization, as assessed from the changes of m itochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapi d and extensive with arachidonic acid and slow and partial with A23187. Cyc losporin A-inhibitable release of cytochrome c and cell death correlate wit h the changes of TMRM fluorescence but not with those of calcein fluorescen ce. Since pore opening must be accompanied by depolarization, we conclude t hat short PTP openings are detected only by trapped calcein and may have li ttle impact on cell viability, while changes of TMRM distribution require l onger PTP openings, which cause release of cytochrome c and may result in c ell death. Modulation of the open time appears to be the key element in det ermining the outcome of stimuli that converge on the PTP.