Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols - Implications for uncoupling endothelial nitric-oxide synthase
Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols - Implications for uncoupling endothelial nitric-oxide synthase
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DOI：
10.1074/jbc.m302227200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Dikalov, S
中科院分区：
文献类型：
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作者：
Kuzkaya, N;Weissmann, N;Dikalov, S
四氢生物蝶呤（BH 4）是内皮型一氧化氮合酶（eNOS）的重要辅助因子. BH 4的缺乏导致eNOS解偶联，这与超氧化物的增加和NO的减少有关。生产BH 4已被认为是过氧亚硝酸盐（ONOO（-））氧化的目标，抗坏血酸已被证明可以保持BH 4水平并提高内皮NO（.）然而，这些过程背后的机制仍然没有得到很好的界定。为了进一步了解这些相互作用，ONOO（-）与BH 4的反应进行了研究，使用电子自旋共振和自旋探针1-羟基-3-羧基-2，2，5-四甲基吡咯烷。ONOO-与BH 4反应的速度比与抗坏血酸或硫醇反应的速度快6 - 10倍。ONOO-与BH_4反应的直接产物是三氢生物蝶呤自由基（BH_3），其被抗坏血酸还原回BH 4，而硫醇在BH 4的再循环中不是有效的。用自旋探针1-羟基-3-甲氧羰基-2，2，5，5-四甲基吡咯烷（1-hydroxy- 3- methoxycarbonyl-2，2，5，5- tetramethylpyrrolidine）和NO（.）自旋捕获二乙基二硫代氨基甲酸铁。大剂量ONOO（-）、ONOO（-）供体3- morpholinosydnonimine和BH 4合成抑制剂（2，4- diamino- 6- hydroxypyrimidine）解偶联eNOS，增加超氧化物和减少NO（.）生产补充外源性BH_4可使内皮细胞NO（.）生产在ONOO（-）之前用BH 4和抗坏血酸盐处理BAEC可防止eNOS被ONOO（-）解偶联。这项研究表明，内皮BH 4是一个重要的目标氧化ONOO（-）和BH 4的反应速率常数超过硫醇或抗坏血酸。我们证实ONOO（-）通过氧化四氢生物蝶呤使eNOS解偶联，抗坏血酸不能完全保护BH 4免受氧化，但能抑制BH 3。回到BH 4。
Tetrahydrobiopterin ( BH4) serves as a critical co- factor for the endothelial nitric- oxide synthase ( eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO (.) production. BH4 has been suggested to be a target for oxidation by peroxynitrite ( ONOO (-)), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO (.) production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO (-) with BH4 was studied using electron spin resonance and the spin probe 1- hydroxy- 3- carboxy- 2,2,5- tetramethylpyrrolidine. ONOO- reacted with BH4 6 - 10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells ( BAECs) by measuring superoxide and NO. using spin probe 1- hydroxy- 3- methoxycarbonyl- 2,2,5,5- tetramethylpyrrolidine and the NO (.)- spin trap iron- diethyldithiocarbamate. Bolus ONOO (-), the ONOO (-) donor 3- morpholinosydnonimine, and an inhibitor of BH4 synthesis ( 2,4- diamino- 6- hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO (.) production. Exogenous BH4 supplementation restored endothelial NO (.) production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO (-) prevented uncoupling of eNOS by ONOO (-). This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO (-) and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO (-) uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.