title: Mechanism of oxidative stress-induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells.
abstract: 1. Following ischaemic reperfusion, large amounts of superoxide anion (.O2-), hydroxyl radical (.OH) and H2O2 are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H2O2 (100 microM) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 +/- 0.04 pH units) and C6 glioma cells (0.33 +/- 0.07 pH units). 2. Two membrane-crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H2O2-induced intracellular acidosis, while the non-membrane-crossing iron chelator apo-transferrin had no effect. Furthermore, the acidosis was completely inhibited by two potent membrane-crossing .OH scavengers, N-(2-mercaptopropionyl)-glycine (N-MPG) and dimethyl thiourea (DMTU). Since .OH can be produced during iron-catalysed H2O2 breakdown (Fenton reaction), we have shown that a large reduction in pH1 in glial cells can result from the production of intracellular .OH via H2O2 oxidation. 3. We have ruled out the possible involvement of: (i) an increase in intracellular Ca2+ levels; and (ii) inhibition of oxidative phosphorylation. 4. Our results suggest that .OH inhibits glycolysis, leading to ATP hydrolysis and intracellular acidosis. This conclusion is based on the following observations: (i) in glucose-free medium, or in the presence of iodoacetate or 2-deoxy-D-glucose, H2O2-induced acidosis is completely suppressed; (ii) H2O2 and iodoacetate both produce an increase in levels of intracellular free Mg2+, an indicator of ATP breakdown; and (iii) direct measurement of intracellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatment with 100 microM H2O2. 5. Inhibition of the pH1 regulators (i.e. the Na(+)-H+ exchange and possibly the Na(+)-HCO3(-)-dependent pH1 transporters) resulting from H2O2-induced intracellular ATP reduction may also be involved in the H2O2-evoked intracellular acidosis in glial cells.
authors.affiliation: Institute of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
authors.forename: K L
authors.initials: KL
authors.lastname: Tsai
authors.forename: S M
authors.initials: SM
authors.lastname: Wang
authors.forename: C C
authors.initials: CC
authors.lastname: Chen
authors.forename: T H
authors.initials: TH
authors.lastname: Fong
authors.forename: M L
authors.initials: ML
authors.lastname: Wu
mesh_terms: D000138:Acidosis / Q000139:chemically induced / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000502:physiology; D002952:Citric Acid Cycle / Q000502:physiology; D003676:Deferoxamine / Q000494:pharmacology; D004579:Electron Transport / Q000502:physiology; D005260:Female; D005910:Glioma; D006019:Glycolysis / Q000502:physiology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019271:Hypoxanthine / Q000494:pharmacology; D007773:Lactates / Q000378:metabolism; D008297:Male; D018384:Oxidative Stress / Q000502:physiology*; D010618:Phenanthrolines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017262:Siderophores / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D014168:Transferrin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D014969:Xanthine Oxidase / Q000494:pharmacology
chemical_list: D007773:Lactates; D010618:Phenanthrolines; D017262:Siderophores; D017923:Sodium-Hydrogen Exchangers; D014168:Transferrin; D019271:Hypoxanthine; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D003676:Deferoxamine