Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes.
Previously, we showed that the oxidant chemical, tert-butylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 micromol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.
Byrne
A M
AM
Department of Anatomy, Case Western Reserve University, Cleveland, OH 44106-4930, USA.
Lemasters
J J
JJ
Nieminen
A L
AL
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
D000634:Aminoquinolines; D002614:Chelating Agents; D017382:Reactive Oxygen Species; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D009249:NADP; C035738:Quin2-acetoxymethyl ester; D020122:tert-Butylhydroperoxide; D003676:Deferoxamine; D002118:Calcium