title: Role of oxidative stress in the ammonia-induced mitochondrial permeability transition in cultured astrocytes.
abstract: Ammonia is a neurotoxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE) and other neurological disorders, and astrocytes are thought to be the principal target of ammonia toxicity. While the precise mechanisms of ammonia neurotoxicity remain to be more clearly defined, altered bioenergetics and oxidative stress appear to be critical factors in its pathogenesis. It has recently been demonstrated that pathophysiological concentrations of ammonia induce the mitochondrial permeability transition (MPT) in cultured astrocytes, a process associated with mitochondrial dysfunction, and frequently caused by oxidative stress. This study investigated the potential role of oxidative stress in the induction of the MPT by ammonia. Accordingly, the effect of various antioxidants on the induction of the MPT by ammonia in cultured astrocytes was examined. Astrocytes were subjected to NH4Cl (5 mM) treatment for 2 days with or without various antioxidants. The MPT was assessed by quantitative fluorescence imaging for the mitochondrial membrane potential (DeltaPsim), employing the potentiometric dye TMRE; by changes in mitochondrial calcein fluorescence and by 2-deoxyglucose-6-phosphate (2-DG-6-P) changes in mitochondrial permeability. Astrocytes treated with ammonia significantly dissipated the DeltaPsim, which was blocked by the MPT inhibitor, cyclosporin A, caused a decrease in mitochondrial calcein fluorescence and increased 2-DG-6-P permeability into mitochondria. All of these findings are consistent with induction of the MPT. Pretreatment with SOD, catalase, desferroxamine, Vitamin E, PBN and the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), completely blocked the ammonia-induced MPT. These data provide strong evidence that oxidative stress is involved in the induction of the MPT by ammonia, and suggest that oxidative stress and the subsequent induction of the MPT contribute to the pathogenesis of HE and other hyperammonemic disorders.
authors.affiliation: Department of Pathology (D-33), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101, USA.
authors.forename: K V
authors.initials: KV
authors.lastname: Rama Rao
authors.forename: A R
authors.initials: AR
authors.lastname: Jayakumar
authors.forename: M D
authors.initials: MD
authors.lastname: Norenberg
mesh_terms: D000641:Ammonia / Q000378:metabolism / Q000494:pharmacology*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000378:metabolism* / Q000503:physiopathology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D005456:Fluorescent Dyes; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006501:Hepatic Encephalopathy / Q000378:metabolism / Q000503:physiopathology; D022124:Hyperammonemia / Q000378:metabolism* / Q000503:physiopathology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats
chemical_list: D000975:Antioxidants; D005456:Fluorescent Dyes; D000643:Ammonium Chloride; D009569:Nitric Oxide; C015785:2-deoxyglucose-6-phosphate; D019298:Glucose-6-Phosphate; D000641:Ammonia; D016572:Cyclosporine