Radical Intermediates of Nitric Oxide Synthase & Myocardial Ischemia Reperfusion

描述（由申请人提供）：一氧化氮（NO）参与正常心脏生理和各种心脏病理事件，包括心肌缺血和再灌注损伤。特异性NOS同工酶的动态表达和激活发生在疾病过程的不同阶段。由于NOS催化的化学反应的复杂性，NO是心脏保护性的还是心脏破坏性的仍然存在争议。改变底物供应和辅因子结合的编排可以将NO合酶转化为活性氧（ROS）或活性氮（RNS）的催化剂，活性氧或活性氮是心脏病理生理学的重要中间体。我们最近的研究揭示了eNOS和nNOS在催化过程中非常不同的自由基中间体特征和调控机制。该提议的中心假设是，理解各种调节分子的相互作用以及在偶联和非偶联NOS催化期间ROS、RNS和其他自由基中间体的动态变化对于阐明心肌梗死和缺血-再灌注损伤的病因至关重要。此外，以前的研究使用整个组织，细胞，或纯化的酶在稳态条件下与自旋捕获是不够的，以获得直接的结构和动力学信息，并需要其他创新的方法。我们计划阐明三种NOS同工酶中自由基中间体动力学的机制：在目的1中，我们希望测试不同的自由基中间体在nNOSox，eNOSox和iNOSox中形成的假设。创新的快速冷冻淬灭（EPR）动力学测量和其他脉冲EPR方法将用于表征新的自由基中间体以及它们的动力学。在目标2中，我们将测试硫醇在所有NOS同种型中防止BH 4氧化所需的假设，但对于保持nNOS和iNOS的结构完整性也是必要的。在存在和不存在硫醇的情况下，将进行类似的EPR动力学测量。位点特异性突变体将用于评估关键半胱氨酸的作用。在目标3中，我们计划测试还原酶结构域是否是iNOS中自由基的主要来源，而不是eNOS或nNOS。将使用CaM/Ca+2或血红素配位的破坏来评估三种同种型的纯化全长NOS和NOSred的氧诱导自由基中间体，以剖析NOSox和NOSred的自由基贡献。心肌细胞和巨噬细胞样细胞将是我们的缺血/再灌注模型，以评估巯基，氧，底物，辅因子和抑制剂对自由基中间体的调节作用，在最后的目标。这些方法将提供最基本的知识，在耦合和非耦合条件下的每一个NOS亚型的机制，并可用于开发治疗再灌注损伤的治疗方案。 公共卫生相关性：本项目的重点是表征的结构和时间依赖性的自由基中间体，包括活性氧和RNS，在所有三个一氧化氮合酶同工酶的氧诱导。这些自由基中间体的底物，辅因子和硫醇的调节也进行了研究，在体外和离体，以阐明心肌缺血和再灌注损伤的潜在疾病机制。

DESCRIPTION (provided by applicant): Nitric oxide (NO) participates both in the normal cardiac physiology and various cardiac pathological events including myocardial ischemia and reperfusion injury. Dynamic expression and activation of specific NOS isozyme occurs at different stages of the disease processes. Whether NO is cardioprotective or cardiodestructive remains controversial due to the complexity of the chemical reactions catalyzed by NOS. Changing of the choreography of the substrate supply and cofactor binding could transform NO synthase to catalyst for the reactive oxygen species (ROS) or reactive nitrogen species (RNS) that are important intermediates for cardiac pathophysiology. Our recent studies disclosed very different radical intermediate profile and regulation mechanism in eNOS and nNOS catalysis. The central hypothesis of this proposal is that understanding the interplay of the various regulatory molecules and the dynamic changes of the ROS, RNS and other radical intermediates during coupled and uncoupled NOS catalysis are crucial to elucidation of the etiology of myocardial infarction and ischemia- reperfusion injury. Furthermore, previous studies using whole tissue, cells, or purified enzyme under steady-state condition with spin-trapping are insufficient to obtain direct structural and kinetic information and require other innovative approach. We plan to elucidate the mechanism of radical intermediates dynamics in three NOS isozymes: In Aim 1, we wish to test the hypothesis that different radical intermediates are formed in the nNOSox, eNOSox and iNOSox. Innovative rapid-freeze quench (RFQ) EPR kinetic measurements and other pulsed EPR methods will be used to characterize new radical intermediates as well as their kinetics. In Aim 2, we will test the hypothesis that thiol is required in preventing BH4 oxidation in all NOS isoforms but is also necessary for keeping structural integrity of the nNOS and iNOS. Similar RFQ EPR kinetic measurements will be conducted in the presence and absence of thiol. Site-specific mutants will be used to assess the role of the key cysteines. In Aim 3, we plan to test whether the reductase domain is the main source of radicals in iNOS but not eNOS or nNOS. Purified full length NOS and NOSred of three isoforms will be evaluated for oxygen-induced radical intermediates using CaM/Ca+2 or disruption of heme coordination to dissect the radical contribution from the NOSox and NOSred. Both cardiomyocytes and macrophage-like cells will be our models for ischemia/reperfusion to assess the regulatory roles of thiol, oxygen, substrate, cofactor and inhibitors on the radical intermediate profile in the last aim. These approaches will provide the most basic knowledge on the mechanism under coupled and uncoupled conditions of each NOS isoforms and can be useful in developing therapeutic regimens for treating reperfusion injury. PUBLIC HEALTH RELEVANCE: This project focuses on characterizing the structure and temporal dependence of the radical intermediates, including ROS and RNS, induced by oxygen in all three nitric oxide synthase isozymes. The regulation of these radical intermediates by substrate, cofactors and thiol also are studied, both in vitro and ex vivo, in order to elucidate the underlying disease mechanism of myocardial ischemia and reperfusion injury.