Switching superoxide & NO production by endothelial NOS

NO是由血管内皮细胞(EC)结构性产生的，在血压和血管张力的瞬间调节中起着关键的生理作用。NO生物活性缺乏，称为内皮功能障碍，导致许多血管疾病和疾病的发生；这些疾病包括冠状动脉疾病、动脉粥样硬化、高血压和糖尿病血管病变。最近的研究表明，给予内皮一氧化氮合酶(ENOS)辅因子四氢生物蝶呤，在血管疾病的动物模型和患者中，许多最近的研究已经恢复了内皮功能。尽管BH4在这些病例中有效，但对BH4组织含量的测量并未显示明显的生化缺陷。这项研究将评估改变的生物蝶呤氧化还原平衡，而不是数量，为表现为内皮功能障碍的慢性血管疾病提供共同的分子基础的可能性。事实上，在动脉粥样硬化、高血压和糖尿病血管中占主导地位的氧化条件下，BH4很容易被氧化成二氢生物蝶呤(BH2)。在初步研究中，我们发现eNOS以同样的亲和力结合BH4和BH2。重要的是，而BH4结合的eNOS只产生超氧阴离子。在近乎扩散限制的反应中，超氧化物与失活的NO反应；产物过氧亚硝酸根(Oono-)是一种比母体分子更强的氧化剂，可以进一步氧化BH4。因此，任何在EC中启动BH4氧化的刺激(例如，氧化型低密度脂蛋白)都可能启动一种费前级联反应，在该级联反应中，eNOS产生的超氧化物持续存在BH4不足，减弱NO生物活性和慢性内皮功能障碍。我们的总体假设是，结合蝶呤蛋白辅因子的氧化还原状态构成了决定eNOS功能的表型开关。对eNOS的进一步调节是由两个自抑制调控元件施加的，我们已经发现并证实了这两个元件控制催化速率和活性对细胞内游离钙浓度的依赖。总体研究计划是阐明蝶呤氧化在eNOS表型转换中的作用，从产生NO到产生超氧化物(AIM 1)，确定调节NOS催化-NO或超氧化物产生(AIM 2)的基本控制机制，并阐明可能治疗内皮功能障碍和恢复慢性产生超氧化物内皮细胞(AIM 3)NO合成的新的治疗策略。PPG资源和与其他PPG调查人员(D.Hajjar、R.Upmacis和R.Silverstein)的有效合作是这项研究成功的关键。

NO is constitutively produced by vascular endothelial cells (EC) where it plays a key physiological role in the moment-to-moment regulation of blood pressure and vascular tone. Deficient NO bioactivity, described as endothelial dysfunction, contributes to the pathogenesis of numerous vascular diseases and conditions; these include coronary artery disease, atherosclerosis, hypertension and diabetic vasculopathy. May recent studies have demonstrated that administration of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin Many recent studies have restore endothelial function in animals models of vascular disease and patients. Despite the efficiency of BH4 in these cases, measurements of BH4 tissue content has not revealed overt biochemical deficiency. This research will evaluate the possibility that altered biopterin redox balance, not amount, provides a common molecular basis for chronic vascular diseases that manifest with endothelial dysfunction. Indeed, BH4 is readily oxidized to dihydrobiopterin (BH2) under the oxidative conditions that can predominate in atherosclerotic, hypertensive and diabetic blood vessels. In preliminary studies we have discovered that eNOS binds BH4 and BH2 with equal affinity. Importantly, whereas BH4-bound eNOS exclusively produces superoxide anion. Superoxide reacts with an inactivates NO in a near diffusion-limited reaction; the product peroxynitrite (OONO-) is a more potent oxidant than either parent molecule and can further oxidize BH4. Thus any stimulus which initiates BH4 oxidation in EC (e.g.., oxidized LDL) can potentially initiate a fee-forward cascade wherein eNOS- derived superoxide perpetuates BH4 insufficiency, attenuated NO bioactivity and chronic endothelial dysfunction. Our overall hypothesis is that the redox status of bound pterin cofactor constitutes a phenotypic switch that determines eNOS function. Further regulation of eNOS is imposed by two autoinhibitory control elements that we have identified and confirmed to control catalytic rate and dependence of activity on free intracellular calcium concentration. The overall research plan is to elucidate the role of pterin oxidation in the eNOS phenotype switch, from NO-producing to superoxide-producing (AIM 1) define fundamental control mechanisms that modulate the rate of NOS catalysis-NO or superoxide production (AIM 2), and elucidate novel therapeutic strategies with potential to remedy endothelial dysfunction and restore NO synthesis to chronically superoxide-producing endothelial cells (AIM 3). PPG resources and effective collaborations with other PPG investigators (D. Hajjar, R. Upmacis and R. Silverstein) are essential to the success of this research.