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Generation of reactive oxygen species and intracellular signal transduction mediated by nitric oxide synthases

一氧化氮合酶介导的活性氧的产生和细胞内信号转导

基本信息

批准号：
13670745
负责人：
ICHIMORI Kohji
金额：
$ 1.47万
依托单位：
National Institute of Advanced Industrial Science and Technology (2002)Tokai University (2001)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

There are three types of nitric oxide synthases (NOSs) asnd their subunits are comprised of a N-terminal oxygenase domain that is linked to a C-termmal flavoprotein domain by a central calmodulin binding sequence. NOS utilizes L-arginine and N-hydroxy-L-arginine to generate NO. Under a certein condition such as the absence of substrate, electrons from NADPH are wasted to produce superoxide, H_2O_2 or H_2O, which is called as an uncoupling reaction. These reduced oxygen species (ROS) are generated at different branch points in the process, and their production ratio depends on the characteristics of the individual enzyme. It is important to elucidate the mechanisms of uncoupling in each NOS. Our present study addresses this issue by comparing uncoupled electron flux in the three NOSs and their product ratios for superoxide, H_2O_2, and water, and by examining how these are affected by certain substrate analogs. We studied ROS generation in three NOS isoforms by measuring NO, superoxide, … More and H_2O_2 production at 25℃ using oxyhemoglobin oxidation, reduction of succinylated cytochrome c, and the generation of Fe(SCN)_4^- from Fe^<2+>, respectively. NADPH consumption was also measured to determine total electron flux. In the presence of excess L-arginine and tetrahydrobiopterin, NOS1 and NOS2 did not show significant uncoupling but for NOS3 almost half of the NADPH-derived electrons went toward ROS generation during NO synthesis. In the absence of L-arginine the major ROS generated was superoxide for NOS1 and NOS3 but was H_2O_2 for NOS2. Adding pseudo-substrates that increased or decreased electron flux through the NOS heme did not greatly alter ROS product ratios. Their pattern of ROS formation distinguish NOSs from cytochrome P450's which generate mainly H_2O_2 and water during uncoupled NADPH consumption. The different ROS product ratio of NOS1 and NOS2 is surprising, and suggests how variation at discreet steps in the oxygen activation pathway can impact NOS-related inflammation processes. Less

一氧化氮合酶(NOSS)有三种类型，它们的亚基由一个N-末端加氧酶结构域组成，该加氧酶结构域通过一个中央钙调素结合序列与一个C-终端黄素蛋白结构域相连。一氧化氮合酶利用L-精氨酸和N-羟基-L-精氨酸生成NO。在某些特定的条件下，如没有底物，NADPH中的电子被浪费来产生超氧化物、H_2O_2或H_2O，这被称为解偶联反应。这些还原的氧物种(ROS)是在过程的不同分支点产生的，它们的生成比率取决于单个酶的特性。阐明每个一氧化氮合酶的解偶联机制是非常重要的。我们目前的研究通过比较三种NOS中未耦合的电子通量和它们对超氧化物、H_2O_2和水的产物比率，以及通过考察这些比率如何受某些底物类似物的影响来解决这个问题。通过测定NO、超氧化物歧化产物、…的含量，研究了三种一氧化氮合酶的ROS生成在25℃时，分别采用氧化血红蛋白氧化、琥珀酸化细胞色素c还原以及Fe~(2+)~(2+)和Gt~(2+)生成Fe(SCN)_4^-产生H_2O_2和More。NADPH消耗也被测量以确定总的电子通量。在过量的L精氨酸和四氢生物蝶呤存在下，NOS1和NOS2没有表现出明显的解偶联，但对于NOS3，几乎一半的NADPH衍生电子在NO合成过程中流向了ROS的生成。在没有L精氨酸的情况下，NOS1和NOS3的主要ROS是超氧化物歧化，NOS2的主要ROS是H_2O_2。添加伪底物来增加或减少通过NOS血红素的电子通量，并没有显著改变ROS产物的比率。它们的ROS生成模式区别于细胞色素P450‘S，后者在非偶联的NADPH消耗过程中主要产生H_2O_2和水。NOS1和NOS2的ROS产物比例不同令人惊讶，这表明氧激活途径中谨慎步骤的变化如何影响NOS相关的炎症过程。较少

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Superoxide generation mediated by 8-nitroguanosine, a highly redox-active nucleic acid derivative.

由 8-硝基鸟苷介导的超氧化物生成，8-硝基鸟苷是一种高度氧化还原活性的核酸衍生物。

DOI：
发表时间：
2003
期刊：
Biochem.Biophys.Res.Commun. 311
影响因子：
0
作者：
Sawa;T. et al.
通讯作者：
T. et al.

8-Nitroguanosine formation in viral pneumonia and its implication for pathogenesis

DOI：
10.1073/pnas.0235623100
发表时间：
2003-01-21
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Akaike, T;Okamoto, S;Maeda, H
通讯作者：
Maeda, H

Angiotensm II receptor antagonists and angiotensin-converting enzymes inhibitors lower in vitro the formation of advanced glycation end products Biochemical Mechanism

血管紧张素II受体拮抗剂和血管紧张素转换酶抑制剂可降低体外晚期糖基化终末产物的形成生化机制

DOI：
发表时间：
2002
期刊：
J Am Soc Nephrol 13
影响因子：
0
作者：
Miyata;T; van Ypersele de Strihou;C; Ueda;Y; Ichimon;K;Inagi;R; Onogi;H;Ishikawa;N; Nangaku;M; Kurokawa;K;
通讯作者：
K;

Myeloperoxidase has directly-opposed effects on nitration reaction - Study on myeloperoxidase-deficient patient and myeloperoxidase-knockout mice