Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress.
Glutathione (GSH), the major cellular protectant against reactive oxygen and nitrogen species, is compartmentalized in a cytosolic (c) and a mitochondrial (mt) pool. We investigated how c-GSH and mt-GSH are differentially affected by endogenously produced nitric oxide (NO). Microglial cell line (N9) cultures were immunostimulated with lipopolysaccharide/interferon-gamma to elicit the inducible isoform of NO synthase (iNOS). Despite a significant reduction in total GSH, the mt-GSH remained nearly unaffected by iNOS-mediated NO production. To investigate possible consequences of GSH depletion on the mitochondrial membrane potential, we used buthionine sulfoximine (BSO) to reduce separately the c-GSH, whereas ethacrynic acid (EA) was applied to deplete both mt-GSH and c-GSH. The mitochondrial membrane potential was more vulnerable to NO exposure in EA-pretreated cultures than in BSO-pretreated cultures, indicated by a potentiated release of tetramethylrhodamine from mitochondria into the cytosol. To relate the EA-mediated decrease in mitochondrial membrane potential to the oxidant buildup after GSH depletion, we loaded the cells with the oxidant-sensitive fluorochrome 2',7'-dihydrodichlorofluorescein (DCF) diacetate. EA treatment caused an increase in DCF fluorescence over time that was potentiated when the iNOS expression was stimulated. Inhibition of NO production abolished this effect. We conclude that endogenous NO production in microglial cells does not compromise the mt-GSH pool which, in turn, might explain the ability of these cells to combat high-output NO production.
Roychowdhury
Sanjoy
S
Institute for Medical Neurobiology, Otto-von-Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany.
Wolf
Gerald
G
Keilhoff
Gerburg
G
Horn
Thomas F W
TF
D019328:Buthionine Sulfoximine / Q000494:pharmacology; D018929:Cell Culture Techniques; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D004976:Ethacrynic Acid / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D005978:Glutathione; D004976:Ethacrynic Acid