Radical Intermediates of Nitric Oxide Synthase & Myocardial Ischemia Reperfusion

描述（由申请人提供）：一氧化氮（NO）既参与正常的心脏生理，也参与各种心脏病理事件，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。由于一氧化氮催化的化学反应的复杂性，一氧化氮对心脏是保护还是破坏仍存在争议。改变底物供应和辅因子结合的choreography可以将一氧化氮合酶转化为活性氧（ROS）或活性氮（RNS）的催化剂，而活性氧（ROS）或活性氮（RNS）是心脏病理生理的重要中间体。我们最近的研究揭示了eNOS和nNOS催化过程中不同的自由基中间体结构和调控机制。本研究的中心假设是，了解各种调控分子的相互作用以及ROS、RNS等自由基中间体在偶联和非偶联NOS催化过程中的动态变化，对阐明心肌梗死和缺血再灌注损伤的病因至关重要。此外，以往的研究使用整个组织、细胞或纯化酶在稳态条件下进行自旋捕获，不足以获得直接的结构和动力学信息，需要其他创新方法。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Aim 1中，我们希望验证nNOSox， eNOSox和iNOSox中形成不同自由基中间体的假设。创新的速冻淬火（RFQ） 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及其动力学。在目标2中，我们将测试假设硫醇是防止所有NOS亚型中BH4氧化所必需的，但也是保持nNOS和iNOS结构完整性所必需的。类似的RFQ EPR动力学测量将在存在和不存在硫醇的情况下进行。位点特异性突变体将用于评估关键半胱氨酸的作用。在Aim 3中，我们计划测试还原酶结构域是否是iNOS自由基的主要来源，而不是eNOS或nNOS自由基的主要来源。我们将利用CaM/Ca+2或破坏血红素配位来评估纯化的全长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作为缺血/再灌注的模型，以评估硫醇、氧、底物、辅助因子和抑制剂对自由基中间体谱的调节作用。这些方法将为了解NOS各亚型在耦合和非耦合条件下的机制提供最基本的知识，并有助于制定治疗再灌注损伤的治疗方案。

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.