Radical Intermediates of Nitric Oxide Synthase & Myocardial Ischemia Reperfusion

一氧化氮合酶自由基中间体

• 批准号: 7783873
• 负责人: AH-LIM TSAI
• 金额: $ 48.83万
• 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/7783873
• 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; 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; 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.

说明(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。特定一氧化氮合酶同工酶的动态表达和激活发生在疾病过程的不同阶段。由于一氧化氮合酶催化的化学反应的复杂性，一氧化氮对心脏的保护作用或对心脏的破坏作用仍存在争议。改变底物供应的编排和辅因子结合可以将一氧化氮合酶转化为心脏病理生理的重要中间体--活性氧(ROS)或活性氮(RNS)的催化剂。我们最近的研究揭示了eNOS和nNOS催化过程中截然不同的自由基中间产物及其调控机制。该方案的核心假设是，了解偶联和非偶联NOS催化过程中各种调节分子的相互作用以及ROS、RNS和其他自由基中间产物的动态变化，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，以前在稳态条件下使用整个组织、细胞或纯化的酶进行自旋捕获的研究不足以获得直接的结构和动力学信息，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产物的动力学机制：在目标1中，我们希望检验nNOSox、eNOSox和iNOSox中形成不同自由基中间产物的假设。创新的快速冷冻淬火(RFQ)、EPR动力学测量和其他脉冲EPR方法将被用来表征新的自由基中间体及其动力学。在目标2中，我们将测试这一假设，即硫醇是防止所有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。在硫醇存在和不存在的情况下，将进行类似的RFQ EPR动力学测量。定点突变将被用来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域是否是iNOS中自由基的主要来源，而不是eNOS或nNOS。纯化的全长NOS和NOSred的三种亚型将使用CaM/Ca+2或破坏血红素配位来评估氧诱导的自由基中间产物，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，以评估硫醇、氧、底物、辅因子和抑制剂在最终目标中对自由基中间产物的调节作用。这些方法将提供关于每种NOS亚型在偶联和非偶联条件下的机制的最基本的知识，并可用于开发治疗再灌注损伤的治疗方案。 与公共卫生相关：该项目重点描述了氧诱导的三种一氧化氮合酶同工酶中自由基中间体的结构和时间依赖性，包括ROS和RNS。在体外和体外研究了底物、辅因子和硫醇对这些自由基中间体的调节作用，以阐明心肌缺血再灌注损伤的潜在发病机制。