Kuzkaya Nermin, Weissmann Norbert, Harrison David G, Dikalov Sergey
Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2003 Jun 20;278(25):22546-54. doi: 10.1074/jbc.M302227200. Epub 2003 Apr 10.
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-tetramethyl-pyrrolidine. 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-tetramethyl-pyrrolidine 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.
四氢生物蝶呤（BH4）是内皮型一氧化氮合酶（eNOS）的关键辅因子。BH4缺乏会导致eNOS解偶联，这与超氧化物生成增加和一氧化氮（NO*）生成减少有关。有人提出BH4是过氧亚硝酸根（ONOO-）氧化的靶点，并且已证明抗坏血酸可维持BH4水平并增强内皮细胞中NO的生成；然而，这些过程背后的机制仍不清楚。为了进一步深入了解这些相互作用，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研究了ONOO-与BH4的反应。ONOO-与BH4的反应速度比与抗坏血酸或硫醇的反应速度快6至10倍。ONOO-与BH4反应的直接产物是三氢生物蝶呤自由基（BH3.），它可被抗坏血酸还原回BH4，而硫醇在BH4的循环利用方面效率不高。通过使用自旋探针1-羟基-3-甲氧基羰基-2,2,5,5-四甲基吡咯烷和NO自旋捕获剂铁二乙氨基二硫代甲酸盐测量超氧化物和NO*，研究了培养的牛主动脉内皮细胞（BAECs）中过氧亚硝酸根引起的eNOS解偶联。单次注射ONOO-、ONOO-供体3-吗啉代 sydnonimine和BH4合成抑制剂（2,4-二氨基-6-羟基嘧啶）会使eNOS解偶联，增加超氧化物生成并减少NO生成。外源性补充BH4可恢复内皮细胞中NO的生成。在加入ONOO-之前用BH4和抗坏血酸处理BAECs可防止ONOO-导致的eNOS解偶联。这项研究表明，内皮细胞中的BH4是ONOO-氧化的关键靶点，并且BH4的反应速率常数超过硫醇或抗坏血酸的反应速率常数。我们证实ONOO-通过氧化四氢生物蝶呤使eNOS解偶联，并且抗坏血酸不能完全保护BH4免受氧化，但能将BH3.自由基循环回BH4。
