Superoxide Generation from eNOS: The Role of Pterins

eNOS 产生超氧化物：蝶呤的作用

• 批准号: 6969913
• 负责人: JEANNETTE M. VASQUEZ VIVAR
• 金额: $ 18.31万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-16 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6969913
• 关键词: antimetabolites; clinical research; enzyme activity; guanosine triphosphate; human tissue; nitric oxide; nitric oxide synthase; oxidative stress; pteridine analog; superoxides; tetrahydrobiopterin; tissue /cell culture; vascular endothelium

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.

描述（由申请人提供）：四氢生物蝶呤（BH 4）是内皮型一氧化氮合酶（eNOS）的一种必需辅因子。已经提出减少NO形成和随后的松弛受损是由于内皮BH 4水平降低，这增强了eNOS的超氧化物形成。电子顺磁共振自旋捕获研究表明，完全还原的BH 4抑制eNOS释放超氧化物，而氧化的BH 4没有影响。此外，这些研究表明，BH 4结合位点的饱和对于抑制超氧化物是至关重要的，并且通过氧化的BH 4类似物如sepiapterin和7，8-二氢生物蝶呤从eNOS置换辅因子提高了超氧化物的产生。基于这些发现，我们假设，完全还原和氧化的BH 4之间的比例调节超氧化物生成内皮型一氧化氮合酶。先前的研究使用L-精氨酸和L-精氨酸类似物来暗示eNOS作为超氧化物的来源。然而，自旋捕获研究表明，这些化合物对纯化的eNOS释放超氧化物的影响很小。这种矛盾表明，eNOS作为超氧化物生产者的鉴定仍然是难以捉摸的。在这里，eNOS的超氧化物形成的作用，从BH 4耗尽的人冠状动脉内皮细胞将通过药理学方法使用1，14-双异噻唑，7-硝基吲唑或小窝蛋白-1支架肽检查。这些试剂通过阻碍酶活化和/或阻断血红素基团特异性地抑制氧活化。此外，将检查用野生型eNOS和对BH 4具有低亲和力的C99 A突变体转染的COS-7中的超氧化物形成。这些实验将揭示eNOS/BH 4在超氧化物形成中的相互作用。此外，还将研究BH 4在内皮细胞中耗尽和再生的机制。为此，将检查氧化应激对GTP环水解酶-I活性和GTP反馈调节肽的影响。平行实验将检查GFRP在BH 4调节中的作用。最后，将检查亚硝化/氧化应激是否改变BH 4与eNOS的结合以产生通过BH 4不敏感机制产生超氧化物的酶。这些信息可能有助于识别风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。