Radical Intermediates of Nitric Oxide Synthase & Myocardial Ischemia Reperfusion

一氧化氮合酶自由基中间体

• 批准号: 8220816
• 负责人: AH-LIM TSAI
• 金额: $ 45.03万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220816
• 关键词: Address; Anabolism; Arginine; Binding; Calmodulin; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Catalysis; Cell model; Cells; Coupled; Cysteine; Dependence; Disease; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Enzymes; Equilibrium; Etiology; Event; Flavin Mononucleotide; Flavins; Fluorescence Microscopy; Freezing; Functional disorder; Heart Diseases; Heme; Hydrogen Peroxide; Hydroxyl Radical; Hypoxia; In Vitro; Ischemia; Isoenzymes; Isotopes; Kinetics; Knowledge; Length; Ligands; Measurement; Methods; Modeling; Monitor; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidation-Reduction; Oxidoreductase; Oxygen; Oxygenases; Peroxonitrite; Physiologic pulse; Physiology; Process; Protein Isoforms; Proteins; Reaction; Reactive Nitrogen Species; Reactive Oxygen Species; Regimen; Regulation; Reperfusion Injury; Reperfusion Therapy; Role; Signal Pathway; Site; Site-Directed Mutagenesis; Source; Spin Trapping; Staging; Structure; Sulfhydryl Compounds; Sulfhydryl Reagents; Superoxides; Testing; Therapeutic; Tissues; catalyst; chemical reaction; cofactor; conditioning; dimer; human NOS2A protein; human NOS3 protein; improved; in vivo; inhibitor/antagonist; innovation; insight; macrophage; monomer; mutant; oxidation; prevent; public health relevance; tetrahydrobiopterin

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.

描述（由申请人提供）：一氧化氮（NO）都参与正常心脏生理学和各种心脏病理事件，包括心肌缺血和再灌注损伤。特定NOS同工酶的动态表达和激活发生在疾病过程的不同阶段。由于非NOS催化的化学反应的复杂性，无论是心脏保护性还是心脏抑制剂是否存在争议。底物供应和辅因子结合的编排的改变可以使无氧氧（ROS）或反应性氮（RNS）的催化剂变化，这是心脏病生理学的重要中间体。我们最近的研究揭示了eNOS和NNOS催化中的根本性中间轮廓和调节机制。该提案的中心假设是，在耦合和未偶联的NOS催化过程中，了解各种调节分子的相互作用以及ROS，RN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性梗死和不足再染色损伤的病因至关重要。此外，在稳态条件下使用整个组织，细胞或纯化酶进行自旋陷阱的先前研究不足以获得直接的结构和动力学信息，因此需要其他创新的方法。我们计划阐明三个NOS同工酶中自由基中间体的机制：在AIM 1中，我们希望检验以下假设：Nnosox，Enosox和Inosox中形成了不同的自由基中间体。创新的快速冻结（RFQ）EPR动力学测量和其他脉冲EPR方法将用于表征新的激进中间体及其动力学。在AIM 2中，我们将检验以下假设：防止所有NOS同工型中的BH4氧化需要硫醇，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RFQ 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AIM 3中，我们计划测试还原酶域是否是iNOS中的自由基的主要来源，而不是eNOS或NNOS。将使用CAM/CA+2评估纯化的全长NOS和三种同工型的Nosred，以评估氧诱导的自由基中间体，或破坏血红素协调以从Nosox中解剖自由基的贡献并nosred。心肌细胞和巨噬细胞样细胞都将成为我们的缺血/再灌注模型，以评估最后一个目标中自由基中间谱的硫醇，氧，底物，辅助因子和抑制剂的调节作用。这些方法将在每个NOS同工型的耦合和未耦合条件下提供有关机制的最基本知识，并且对于开发用于治疗再灌注损伤的治疗方案可能是有用的。 公共卫生相关性：该项目的重点是表征激进中间体的结构和时间依赖性，包括ROS和RN，在所有三种一氧化氮合酶同工酶中均由氧诱导。还研究了通过底物，辅助因子和硫醇对这些自由基中间体的调节，无论是在体外还是在体外，以阐明心肌缺血的潜在疾病机制和再灌注损伤。