Roychowdhury Sanjoy, Wolf Gerald, Keilhoff Gerburg, Horn Thomas F W
Institute for Medical Neurobiology, Otto-von-Guericke University, Leipziger Strasse 44, D-39120 Magdeburg, Germany.
Nitric Oxide. 2003 Feb;8(1):39-47. doi: 10.1016/s1089-8603(02)00146-5.
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.
谷胱甘肽（GSH）是细胞对抗活性氧和氮物种的主要保护剂，分为胞质（c）和线粒体（mt）池。我们研究了内源性产生的一氧化氮（NO）如何对c-GSH和mt-GSH产生不同影响。用脂多糖/干扰素-γ对小胶质细胞系（N9）培养物进行免疫刺激，以诱导一氧化氮合酶（iNOS）的诱导型同工型。尽管总GSH显著减少，但mt-GSH几乎不受iNOS介导的NO产生的影响。为了研究GSH耗竭对线粒体膜电位的可能影响，我们使用丁硫氨酸亚砜胺（BSO）分别降低c-GSH，而应用依他尼酸（EA）来耗尽mt-GSH和c-GSH。与BSO预处理的培养物相比，EA预处理的培养物中的线粒体膜电位对NO暴露更敏感，这通过四甲基罗丹明从线粒体向细胞质的增强释放来表明。为了将EA介导的线粒体膜电位降低与GSH耗竭后的氧化剂积累联系起来，我们用对氧化剂敏感的荧光染料2',7'-二氢二氯荧光素（DCF）二乙酸酯加载细胞。EA处理导致DCF荧光随时间增加，当iNOS表达受到刺激时这种增加会增强。抑制NO产生可消除这种效应。我们得出结论，小胶质细胞内源性NO产生不会损害mt-GSH池，这反过来可能解释了这些细胞对抗高产量NO产生的能力。
