Contribution of Increased Mitochondrial Free Ca2+ to the... : Hepatology (original) (raw)
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Contribution of Increased Mitochondrial Free Ca2+ to the Mitochondrial Permeability Transition Induced By Tert –Butylhydroperoxide in Rat Hepatocytes
1_Department of Anatomy, Case Western Reserve University, Cleveland, OH_
2_Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC_
* Department of Anatomy, Case Western Reserve University, School of Medicine W520, 10900 Euclid Avenue, Cleveland, OH 44106–4930. fax: (216) 368–1144; E-mail: [email protected].
Received: 24 July 1998; Accepted: 28 January 1999
Abstract
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 μmol/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.
Copyright © 1999 American Association for the Study of Liver Diseases.