scholarly journals Critical Role of Reactive Oxygen Species and Mitochondrial Permeability Transition in Microcystin-Induced Rapid Apoptosis in Rat Hepatocytes

Hepatology ◽  
2000 ◽  
Vol 32 (3) ◽  
pp. 547-555 ◽  
Author(s):  
Wen-Xing Ding ◽  
Han-Ming Shen ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Mitochondrial Permeability Transition ◽  
Critical Role ◽  
Reactive Oxygen ◽  
Permeability Transition ◽  
Rat Hepatocytes ◽  
Oxygen Species ◽  
Mitochondrial Permeability

scholarly journals Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

2012 ◽  
Vol 302 (7) ◽  
pp. G723-G731 ◽  
Author(s):  
Jae-Sung Kim ◽  
Jin-Hee Wang ◽  
John J. Lemasters
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Permeability Transition ◽  
Rat Hepatocytes ◽  
Oxygen Species ◽  
Mitochondrial Permeability ◽  
Ros Formation

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 Ca2+ 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 Ca2+ 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 Ca2+, 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 Ca2+ and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca2+ chelation, but not cytosolic Ca2+ 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, Ca2+-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.

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