Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes

To simulateischemia and reperfusion, cultured rat hepatocytes were incubated inanoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. Duringanoxia, intracellular pH (pH_i)decreased to 6.3, mitochondria depolarized, and ATP decreased to <1%of basal values, but the mitochondrial permeability transition (MPT)did not occur as assessed by confocal microscopy from theredistribution of cytosolic calcein into mitochondria. Moreover, cellviability remained >90%. After reperfusion at pH 7.4, pH_i returned to pH 7.2, the MPToccurred, and most hepatocytes lost viability. In contrast, afterreperfusion at pH 6.2 or withNa⁺-free buffer at pH 7.4, pH_i did not rise and cellviability remained >80%. After acidotic reperfusion, the MPT did notoccur. When hepatocytes were reperfused with cyclosporin A (0.5-1µM) at pH 7.4, the MPT was prevented and cell viability remained>80%, although pH_i increased to7.2. Reperfusion with glycine (5 mM) also prevented cell killing butdid not block recovery of pH_i orthe MPT. Retention of cell viability was associated with recovery of30-40% of ATP. In conclusion, preventing the rise ofpH_i after reperfusion blocked theMPT, improved ATP recovery, and prevented cell death. Cyclosporin Aalso prevented cell killing by blocking the MPT without blocking recovery of pH_i. Glycine preventedcell killing but did not inhibit recovery ofpH_i or the MPT.