Duval D L, Sieg D J, Billings R E
Cell and Molecular Pharmacology and Physiology Graduate Program, University of Nevada School of Medicine, Reno 89557.
Arch Biochem Biophys. 1995 Feb 1;316(2):699-706. doi: 10.1006/abbi.1995.1093.
Regulation of induced nitric oxide synthase in rat hepatocyte primary cultures was explored. Nitric oxide synthase (NOS) induction by tumor necrosis factor-alpha (TNF alpha) is synergized by interferon-gamma, and both NOS activity and gene expression are maximal by 10 h and maintained through 24 h. Glutathione depletion by diethylmaleate, which conjugates reduced glutathione, 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), a glutathione reductase inhibitor, or buthionine sulfoxamine, a glutathione synthesis inhibitor, abolishes or reduces NOS induction in TNF alpha-treated hepatocytes, whereas N-acetylcysteine has little effect. Thus, reduced glutathione is critical to NOS mRNA induction and activity in TNF alpha-treated hepatocytes. NOS induction in TNF alpha-treated cells is reduced by rotenone, a mitochondrial complex 1 inhibitor. Concurrent treatment with TNF alpha and the antioxidant, Trolox, or the iron-chelating agent, desferrioxamine, also reduces NOS activity. Dithiothreitol, a thiol antioxidant, reduced TNF alpha induction of NOS. Trolox and BCNU, combined, blocked TNF alpha stimulation of NOS greater than either agent alone. These results suggest that TNF alpha increases mitochondrial production of reactive oxygen intermediates (ROI), which contributes to NOS induction. Hepatocytes exposed to extracellular ROI generation through a xanthine/xanthine oxidase superoxide-generating system expressed increased NOS activity and mRNA levels. NOS induction by superoxide also requires reduced glutathione since diethylmaleate blocks induction by xanthine/xanthine oxidase while N-acetylcysteine elevates NOS expression. Thus, the generation of ROI by cytokines or other physiological processes stimulates the induction of NOS and this process is regulated by cellular levels of reduced glutathione.
本研究探讨了大鼠原代肝细胞培养物中诱导型一氧化氮合酶的调控机制。肿瘤坏死因子-α（TNFα）诱导的一氧化氮合酶（NOS）可被干扰素-γ协同增强，且NOS活性和基因表达在10小时时达到最大值，并持续至24小时。二乙基马来酸盐（可与还原型谷胱甘肽结合）、1,3-双（氯乙基）-1-亚硝基脲（BCNU，一种谷胱甘肽还原酶抑制剂）或丁硫氨酸亚砜胺（一种谷胱甘肽合成抑制剂）导致的谷胱甘肽耗竭，会消除或降低TNFα处理的肝细胞中的NOS诱导，而N-乙酰半胱氨酸几乎没有影响。因此，还原型谷胱甘肽对于TNFα处理的肝细胞中NOS mRNA的诱导和活性至关重要。鱼藤酮（一种线粒体复合物1抑制剂）可降低TNFα处理细胞中的NOS诱导。TNFα与抗氧化剂Trolox或铁螯合剂去铁胺同时处理，也会降低NOS活性。二硫苏糖醇（一种硫醇抗氧化剂）可降低TNFα对NOS的诱导。Trolox和BCNU联合使用对TNFα刺激NOS的阻断作用大于单独使用任何一种药物。这些结果表明，TNFα增加了活性氧中间体（ROI）的线粒体产生，这有助于NOS的诱导。通过黄嘌呤/黄嘌呤氧化酶超氧化物生成系统暴露于细胞外ROI产生的肝细胞表现出增加的NOS活性和mRNA水平。超氧化物对NOS的诱导也需要还原型谷胱甘肽，因为二乙基马来酸盐可阻断黄嘌呤/黄嘌呤氧化酶的诱导，而N-乙酰半胱氨酸可提高NOS表达。因此，细胞因子或其他生理过程产生的ROI刺激了NOS的诱导，并且这一过程受细胞内还原型谷胱甘肽水平的调节。
