The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study.
The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study.
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DOI：
10.1042/bj3620733
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发表时间：
2002-03
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通讯作者：
J. Vasquez-Vivar;P. Martásek;J. Whitsett;J. Joseph;B. Kalyanaraman
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作者：
J. Vasquez-Vivar;P. Martásek;J. Whitsett;J. Joseph;B. Kalyanaraman
超氧化物水平的增加与高血压、糖尿病和高胆固醇血症中的松弛受损有关。纯化的内皮型一氧化氮合酶（eNOS）在5，6，7，8-四氢生物蝶呤（BH（4））的有限可用性下产生超氧化物。因此，假设内皮BH（4）水平的改变刺激eNOS产生超氧化物。通过测定BH（4）及其类似物对eNOS形成超氧化物的浓度依赖性效应来检验这种可能性。通过EPR自旋捕获定量超氧化物，这是唯一可用的技术来定量eNOS中的超氧化物。使用5-乙氧羰基-5-甲基-吡咯啉N-氧化物，我们表明只有完全还原的BH（4）才能减少eNOS的超氧化物释放，其中BH（4）>6-甲基-BH（4）>5-甲基-BH（4）。相比之下，部分氧化的BH（4）类似物、7，8-二氢生物蝶呤（7，8-BH（2））和sepiapterin没有影响。L-精氨酸和N（G）-硝基-L-精氨酸甲酯（L-NAME）均不能抑制超氧阴离子的形成。BH（4）和L-精氨酸共同刺激NO产生，最大速率为148 nmol/min/mg蛋白质。这些结果表明，BH（4）作为一个“氧化还原开关”，减少超氧化物的释放和促进NO的形成。通过将7，8-BH（2）或sepiapterin添加到完全活性的eNOS中来验证这种作用。7，8-BH（2）和sepiapterin均能促进超氧阴离子的释放，而抑制（.）无编队。总的来说，这些结果表明，氧化和还原BH（4）代谢物之间的比例严格调节超氧化物的形成从eNOS。这种情况下的病理意义进行了讨论。
Augmentation of superoxide levels has been linked to impaired relaxation in hypertension, diabetes and hypercholesterolaemia. Purified endothelial nitric oxide synthase (eNOS) generates superoxide under limited availability of 5,6,7,8-tetrahydrobiopterin (BH(4)). Thus alterations in endothelial BH(4) levels have been postulated to stimulate superoxide production from eNOS. This possibility was examined by determining the concentration-dependent effects of BH(4), and its analogues, on superoxide formation by eNOS. Superoxide was quantified by EPR spin trapping, which is the only available technique to quantify superoxide from eNOS. Using 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, we show that only fully reduced BH(4) diminished superoxide release from eNOS, with efficiency BH(4)>6-methyl-BH(4)>5-methyl-BH(4). In contrast, partially oxidized BH(4) analogues, 7,8-dihydrobiopterin (7,8-BH(2)) and sepiapterin had no effect. Neither l-arginine nor N(G)-nitro-l-arginine methyl ester (l-NAME) abolished superoxide formation. Together, BH(4) and l-arginine stimulated .NO production at maximal rates of 148 nmol/min per mg of protein. These results indicate that BH(4) acts as a "redox switch", decreasing superoxide release and enhancing .NO formation. This role was verified by adding 7,8-BH(2) or sepiapterin to fully active eNOS. Both 7,8-BH(2) and sepiapterin enhanced superoxide release while inhibiting (.)NO formation. Collectively, these results indicate that the ratio between oxidized and reduced BH(4) metabolites tightly regulates superoxide formation from eNOS. The pathological significance of this scenario is discussed.