Switching superoxide & NO production by endothelial NOS

NO由血管内皮细胞（EC）组成性产生，在血压和血管张力的即时调节中起关键生理作用。被描述为内皮功能障碍的NO生物活性缺陷导致许多血管疾病和病症的发病机制;这些疾病和病症包括冠状动脉疾病、动脉粥样硬化、高血压和糖尿病性血管病变。最近的研究表明，内皮型一氧化氮合酶（eNOS）辅因子四氢生物蝶呤的管理许多最近的研究已经恢复血管疾病的动物模型和患者的内皮功能。尽管BH 4在这些情况下的效率，BH 4组织含量的测量没有显示明显的生化缺陷。这项研究将评估改变生物蝶呤氧化还原平衡的可能性，而不是数量，为表现为内皮功能障碍的慢性血管疾病提供了共同的分子基础。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，BH 4容易氧化为二氢生物蝶呤（BH 2）。在初步研究中，我们已经发现eNOS以相等的亲和力结合BH 4和BH 2。重要的是，而BH 4结合eNOS专门产生超氧阴离子。超氧化物与失活NO在近扩散限制反应中反应;产物过氧亚硝酸盐（OONO-）是比任何母体分子更有效的氧化剂，并且可以进一步氧化BH 4。因此，在EC中引发BH 4氧化的任何刺激（例如，氧化的LDL）可以潜在地引发前馈级联，其中eNOS衍生的超氧化物使BH 4不足、减弱的NO生物活性和慢性内皮功能障碍持续存在。我们的总体假设是结合蝶呤辅因子的氧化还原状态构成了决定eNOS功能的表型开关。eNOS的进一步调节是由两个自抑制控制元件施加的，我们已经鉴定并证实了它们控制催化速率和活性对细胞内游离钙浓度的依赖性。总体研究计划是阐明蝶呤氧化在eNOS表型转换中的作用，从NO产生到超氧化物产生（AIM 1），定义调节NOS催化NO或超氧化物产生速率的基本控制机制（AIM 2），并阐明具有补救内皮功能障碍和恢复慢性超氧化物产生内皮细胞NO合成的潜力的新治疗策略（AIM 3）。PPG资源和与其他PPG研究者的有效合作（D。哈贾尔河Upmacis和R. Silverstein）对于这项研究的成功至关重要。

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.