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首页> 外文期刊>American Journal of Physiology >Thiol-metabolizing proteins and endothelial redox state: differential modulation of eNOS and biopterin pathways.
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Thiol-metabolizing proteins and endothelial redox state: differential modulation of eNOS and biopterin pathways.

机译：硫醇代谢蛋白和内皮氧化还原状态：eNOS和生物蝶呤途径的差异调节。

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The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and pathophysiological roles of reactive oxygen species. The relative contributions of the thioredoxin (Trx) and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on endothelial nitric oxide (NO) synthase (eNOS) and NO-dependent pathways in the endothelium. We designed duplex small interfering RNA (siRNA) constructs to specifically "knock down" the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione-to-oxidized glutathione ratio. siRNA-mediated knockdown of either GR, TrxR1, or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor) and also blocked eNOS enzyme activity (using the [(3)H]arginine/[(3)H]citrulline assay). Pretreatment of endothelial cells with N,N'-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production. siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide (H(2)O(2)) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased H(2)O(2) production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in H(2)O(2) generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall.
机译：基于硫醇的抗氧化剂严格维持细胞内的氧化还原状态，以建立活性氧的生理和病理生理作用的平衡。硫氧还蛋白（Trx）和谷胱甘肽/戊二醛毒素系统对细胞内氧化还原平衡的相对贡献尚不完全清楚，硫醇代谢改变对内皮中一氧化氮（NO）合酶（eNOS）和NO依赖性途径的影响也尚未完全了解。我们设计了双链体小干扰RNA（siRNA）构建体，以专门“敲低”培养的主动脉内皮细胞中三种关键的巯基代谢酶的表达。靶向谷胱甘肽还原酶（GR），胞质Trx还原酶（TrxR1）或线粒体Trx还原酶（TrxR2）的siRNA构建体的转染显着降低了细胞内还原型谷胱甘肽与氧化型谷胱甘肽的比率。 siRNA介导的GR，TrxR1或TrxR2的敲低显着抑制了VEGF诱导的NO生成（通过电化学NO传感器测量），并且还阻断了eNOS酶的活性（使用[（3）H]精氨酸/ [（3）H]瓜氨酸测定）。用GR和TrxR的抑制剂N，N'-双（2-氯乙基）-N-亚硝基脲预处理内皮细胞，可显着降低VEGF诱导的NO产生。 siRNA介导的TrxR2敲低导致内皮细胞中过氧化氢（H（2）O（2））生产的显着增加。相反，GR或TrxR1的敲低只会稍微增加H（2）O（2）的产量。用四氢生物蝶呤补充内皮细胞可防止H（2）O（2）生成的增加，这与siRNA介导的GR击倒有关。这些研究表明，巯基代谢蛋白的差异调节导致氧化和亚硝化应激途径的关键变化。对硫醇代谢蛋白的差异调节的更多了解可能会导致开发与血管壁氧化应激相关疾病的新药理靶标。

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