Neuronal NOS, Nitroarenes, and Neurotoxicity

神经元 NOS、硝基芳烃和神经毒性

• 批准号: 7409586
• 负责人: Richard Timothy Miller
• 金额: $ 19.6万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7409586
• 关键词: Aerobic; Affect; Area; Arginine; Bacillus megaterium; Behavior; Binding; Biological Assay; Brain region; Bypass; Calcium; Calmodulin; Catalysis; Characteristics; Chemicals; Citrulline; Complex; Condition; Data; Dependence; Detection; Diffusion; Dinitrobenzenes; Electron Transport; Electrons; Environmental Pollutants; Enzymes; Exposure to; Fingerprint; Flavins; Flavoproteins; Functional disorder; Future; Goals; Heme; High Pressure Liquid Chromatography; Homeostasis; Hydrogen Peroxide; Imidazole; In Vitro; Laboratories; Lead; Length; Measures; Mediating; Mediator of activation protein; Metabolism; Methodology; Methods; NADP; NOS1 protein, human; Neuraxis; Nitric Oxide; Nitric Oxide Synthase Type I; Nitro Compounds; Numbers; Outcome; Oxidants; Oxidation-Reduction; Oxidoreductase; Oxygen; Oxygen Consumption; POR gene; Pattern; Peptides; Peroxonitrite; Physiological; Play; Process; Production; Property; Protein Isoforms; Proteins; Purpose; Rate; Recombinants; Research Personnel; Role; Running; Sampling; Subcellular Fractions; Superoxides; Techniques; Testing; Time; Tissues; Titrations; Toxic effect; Tyrosine; alpha-resorcylic acid; base; brain tissue; cytochrome c; enzyme activity; experience; gain of function; in vivo; inhibitor/antagonist; iron nitrosyl; neurotoxic; neurotoxicity; nitration; oxidation; prevent; programs; reaction rate; research study; success

DESCRIPTION (provided by applicant): Unraveling the structural secrets of neuronal nitric oxide synthase (nNOS) has become an important goal for the purpose of understanding how nNOS can be differentially regulated and/or modulated by specific chemicals. The interaction of nNOS with environmental pollutants such as nitroarenes, resulting in the production of reactive intermediates and toxicity, is the subject of this proposal. In order to investigate the mechanisms of 1,3-dinitrobenzene (1,3-DNB)-mediated neurotoxicity, we hypothesize that in the presence of nitroarenes, nNOS is converted from a purely nitric oxide (NO*) and L-citrulline synthase to a peroxynitrite (ONOO-) and L-citrulline synthase. ONOO- is a very potent and reactive oxidant formed when nNOS simultaneously produces NO* and superoxide anion radical (O2-). O2- is formed by the nNOS-mediated reduction and subsequent reoxidation of nitroarenes. Concomitantly, nNOS maintains adequate electron flow to the heme to produce its normal products, NO* and L-citrulline. The simultaneous production of both NO* and O2- in close proximity leads immediately to ONOO- formation via the combination of these two radicals at a near diffusion-controlled reaction rate. The ONOO- that is produced, along with partially-reduced intermediates of the nitroarene, are proposed to play a role in the neurotoxicity associated with exposure to 1,3-DNB. The long-term objective of this project is to determine how metabolism of nitroarenes, resulting in the production of reactive intermediates (such as ONOO-, O2-, H2O2, NOx), and active reduced metabolites, mediate toxicity within the central nervous system. Further, we will determine how enzymatic activity of nNOS can be regulated by nitroarenes, O2-, and active reduced metabolites of nitroarenes such as the nitroso- and N-hydroxy-species. Toward this goal, our immediate specific aims are: Aim #1: To dissect electron transfer from nNOS to nitroarenes such as 1,3-DNB by using recombinantly-expressed and purified nNOS and nNOS constructs. Aim #2: To test the hypothesis that interaction of NOS with neurotoxic nitroarenes, including 1,3- DNB, results in modulation of NOS activity, stimulation of O2- production, and a gain of function by becoming a ONOO-generating enzyme.

描述（由申请人提供）：解开神经元一氧化氮合酶（nNOS）的结构秘密已成为了解特定化学物质如何差异调节和/或调制 nNOS 的一个重要目标。 nNOS 与环境污染物（如硝基芳烃）的相互作用，导致反应中间体的产生和毒性，是该提案的主题。为了研究 1,3-二硝基苯 (1,3-DNB) 介导的神经毒性机制，我们假设在硝基芳烃存在的情况下，nNOS 从纯一氧化氮 (NO*) 和 L-瓜氨酸合酶转化为过氧亚硝酸盐 (ONOO-) 和 L-瓜氨酸合酶。 ONOO- 是一种非常有效的活性氧化剂，当 nNOS 同时产生 NO* 和超氧阴离子自由基 (O2-) 时形成。 O2- 是由 nNOS 介导的还原和随后硝基芳烃的再氧化形成的。与此同时，nNOS 保持足够的电子流向血红素，以产生其正常产物 NO* 和 L-瓜氨酸。同时产生的 NO* 和 O2- 非常接近，通过这两个自由基以接近扩散控制的反应速率结合，立即导致 ONOO- 形成。所产生的 ONOO- 与部分还原的硝基芳烃中间体一起，被认为在与接触 1,3-DNB 相关的神经毒性中发挥作用。该项目的长期目标是确定硝基芳烃的代谢如何产生反应性中间体（例如 ONOO-、O2-、H2O2、NOx）和活性还原代谢物，从而介导中枢神经系统内的毒性。此外，我们将确定硝基芳烃、O2-和硝基芳烃的活性还原代谢物（例如亚硝基和 N-羟基物质）如何调节 nNOS 的酶活性。为了实现这一目标，我们当前的具体目标是： 目标#1：通过使用重组表达和纯化的 nNOS 和 nNOS 构建体来剖析从 nNOS 到硝基芳烃（如 1,3-DNB）的电子转移。 目标#2：检验以下假设：NOS 与神经毒性硝基芳烃（包括 1,3-DNB）相互作用，导致 NOS 活性的调节、O2 产生的刺激以及通过成为 ONOO 生成酶而获得功能。