Superoxide Generation from eNOS: The Role of Pterins

描述（由申请人提供）：四氢生物蝶呤（BH4）是内皮型一氧化氮合酶（eNOS）必不可少的辅助因子。一氧化氮形成减少和随后的松弛受损被认为是由于内皮BH4水平降低，从而增强了eNOS的超氧化物形成。这一观点得到了电子顺磁共振自旋俘获研究的支持，研究表明完全还原的BH4抑制eNOS的超氧化物释放，而氧化的BH4则没有影响。此外，这些研究表明BH4结合位点的饱和对于抑制超氧化物至关重要，并且被氧化的BH4类似物（如sepapterin和7,8-二氢生物蝶呤）从eNOS中取代辅因子可提高超氧化物的产生。基于这些发现，我们假设完全还原和氧化BH4之间的比例调节内皮型一氧化氮合酶产生的超氧化物。先前的研究使用l -精氨酸和l -精氨酸类似物来暗示eNOS是超氧化物的来源。然而，自旋俘获研究表明，这些化合物对纯化的eNOS的超氧化物释放几乎没有影响。这一悖论表明，eNOS作为超氧化物生产者的鉴定仍然难以捉摸。在这里，eNOS在BH4缺失的人冠状动脉内皮细胞形成超氧化物中的作用将通过使用1,14-双异硫脲、7-硝基吲哚唑或caveolin-1支架肽的药理学方法进行研究。这些药物通过阻碍酶的激活和/或阻断血红素群特异性地抑制氧的激活。此外，将检测转染野生型eNOS和与BH4亲和力较低的C99A突变体的COS-7中超氧化物的形成。这些实验将揭示eNOS/BH4在超氧化物形成中的相互作用。此外，还将研究BH4在内皮细胞中耗尽和再生的机制。为此，我们将研究氧化应激对GTP环水解酶- i活性和GTP反馈调节肽的影响。平行实验将检验GFRP在BH4调控中的作用。最后，我们将研究亚硝化/氧化应激是否会改变bh4与eNOS的结合，从而产生一种通过bh4不敏感机制产生超氧化物的酶。这些信息可能有助于识别危险因素和设计新的治疗干预措施，以改善高血压、动脉粥样硬化和糖尿病等疾病的血管功能。

DESCRIPTION (provided by applicant): Tetrahydrobiopterin (BH4) is an essential cofactor of endothelial nitric oxide synthase (eNOS). Diminished NO formation and subsequent impaired relaxation have been proposed to be due to reduced endothelial BH4 levels, which enhances superoxide formation from eNOS. This idea gained support from electron paramagnetic resonance spin trapping studies showing that fully reduced BH4 inhibits superoxide release form eNOS while oxidized BH4 has no effect. In addition, these studies demonstrated that saturation of BH4- binding site is critical for inhibiting superoxide and that displacement of the cofactor from eNOS by oxidized BH4 analogs such as sepiapterin and 7,8-dihydrobiopterin elevates superoxide production. Based on these findings we hypothesized that the ratio between fully reduced and oxidized BH4 regulates superoxide generation from endothelial nitric oxide synthase. Previous studies used L-arginine and L-arginine analogs to implicate eNOS as a source of superoxide. However, spin trapping studies showed that these compounds have little effect on superoxide release from purified eNOS. This paradox indicates that identification of eNOS as a superoxide producer remains elusive. Here, the role of eNOS in superoxide formation from BH4- depleted human coronary artery endothelial cells will be examined by pharmacological approaches using 1,14-bis-isothiourea, 7-nitroindazole or caveolin-1 scaffolding peptide. These agents specifically inhibit oxygen activation by either impeding enzyme activation and/or blocking heme group. In addition, superoxide formation in COS-7 transfected with wild type eNOS and C99A mutant that has a low affinity off BH4 will be examined. These experiments will reveal the interplay between eNOS/BH4 in superoxide formation. In addition, it will be examined the mechanisms by which BH4 is depleted and regenerated in endothelial cells. To this end, the effect of oxidative stress on GTP cyclohydrolase-I activity and GTP feedback regulatory peptide will be examined. Parallel experiments will examine the role of GFRP in the regulation of BH4. Finally, it will be examined whether nitrosative/oxidative stress changes BH4-binding to eNOS to generate an enzyme that produces superoxide by a BH4-insensitive mechanism. This information is likely to contribute in the identification of risk factors and in the design of new therapeutic interventions to ameliorate vascular function in conditions such as hypertension, atherosclerosis and diabetes.