Mitochondrial Permeability Transition in Rat

42 Onset of the mitochondrial permeability transition (MPT) is the penultimate event 43 leading to lethal cellular ischemia/reperfusion injury, but the mechanisms precipitating 44 the MPT after reperfusion remain unclear. Here, we investigated the role of 45 mitochondrial free Ca and reactive oxygen species (ROS) in pHand MPT-dependent 46 reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic 47 Krebs-Ringer-HEPES buffer (KRH) at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to 48 simulate ischemia/reperfusion. Some cells were loaded with the Ca chelators, 49 BAPTA/AM and Quin 2/AM, either by a cold loading protocol for intramitochondrial 50 loading or by warm incubation for cytosolic loading. Cell death was assessed by 51 propidium iodide fluorometry and immunoblotting. Mitochondrial Ca, inner membrane 52 permeability, membrane potential and ROS formation were monitored with Rhod-2, 53 calcein, tetramethylrhodamine methylester and dihydrodichlorofluorescein, respectively. 54 Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM 55 cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of 56 Rhod-2, calcein and dichlorofluorescein revealed that an increase of mitochondrial Ca 57 and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca 58 chelation, but not cytosolic Ca chelation, prevented ROS formation and subsequent 59 necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or 60 diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, 61 inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to 62 prevent reoxygenation-induced cell death. In conclusion, Ca-dependent mitochondrial 63