title: Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species.
abstract: Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.
authors.affiliation: Univ. of Florida, Dept. of Surgery,Gainesville, FL 32610, USA. Jae.Kim@surgery.ufl.edu
authors.forename: Jae-Sung
authors.initials: JS
authors.lastname: Kim
authors.forename: Jin-Hee
authors.initials: JH
authors.lastname: Wang
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
mesh_terms: D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D022781:Hepatocytes / Q000378:metabolism*; D007425:Intracellular Membranes / Q000502:physiology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D010539:Permeability; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D010100:Oxygen; D002118:Calcium