Role of the permeability transition pore in cytochrome C release from mitochondria during ischemia-reperfusion in rat liver

Ischemia and reperfusion cause mitochondrial dysfunctions that initiate the mitochondrial apoptosis pathway. They involve the release of cytochrome C and the activation of the caspase cascade but the mechanism(s) leading to cytochrome C release is(are) poorly understood. The aim of this study was to analyse the relation between cytochrome C release and the opening of the permeability transition pore (PTP) during in situ liver ischemia and reperfusion. Liver ischemia was induced for 30, 60 and 120 min and blood re-flow was subsequently restored for 30 and 180 min. Ischemia hugely altered mitochondrial functions, i.e., oxidative phosphorylation and membrane potential, and was accompanied by a time-dependent mitochondrial release of cytochrome C into the cytosol and by activations of caspases-3 and -9. PTP opening was not observed during ischemia, as demonstrated by the absence of effect of an in vivo pre-treatment of rats with cyclosporin A (CsA), a potent PTP inhibitor. Cytochrome C release was due neither to a direct effect of caspases onto mitochondria nor to an interaction of Bax or Bid with the mitochondrial membrane but could be related to a direct effect of oxygen deprivation. In contrast, during reperfusion, CsA pre-treatment inhibits cytochrome C release, PTP opening and caspase activation. At this step, cytochrome C release is likely to occur as a consequence of PTP opening. In conclusion, our study reveals that cytochrome C release, and thus the induction of the mitochondrial cell death pathway, occur successively independently and dependent on PTP opening during liver ischemia and reperfusion, respectively.