ESR ANALYSIS AND IN VIVO ACTION OF ENDOGENOUS NITRIC OXIDE

关于一氧化氮的生理和病理作用的各种信息已被发现对开发药物如伟哥非常有用。为了阐明内源性NO的各种作用，有关NO在细胞、组织和器官中的数量和分布的信息是必不可少的。然而，由于NO在体内形成的浓度极低，且在生命系统中的半衰期极短，因此确定其数量和分布相当困难。作为克服这些困难的分析方法之一，自旋俘获技术结合电子顺磁共振（EPR）光谱已被用于体外和体内不稳定自由基的测定。含二硫代氨基甲酸酯衍生物的铁配合物是一氧化氮的自旋捕获试剂之一，因为一氧化氮对铁配合物有很高的亲和力，由此产生的一氧化氮结合的铁配合物在室温下在体外EPR仪上表现出强烈的三线信号。N-（二硫代羧基）肌氨酸（DTCS）是二硫代氨基甲酸酯的衍生物。我们最近报道了铁与DTCS配合物（Fe-DTCS）及其NO配合物（NO-Fe-DTCS）在水介质中具有相当的可溶性和稳定性，并且Fe-DTCS配合物具有潜在的生物良性，有效的NO捕获试剂。在本研究中，我们采用EPR自旋捕获技术和铁配合物与二硫代氨基甲酸盐作为NO捕获剂，对内源性一氧化氮进行了测量。首先，我们尝试在实验性败血症和脑膜炎期间活体大鼠脑内检测一氧化氮。结果表明，在脓毒症和脑膜炎期间，NO是通过脑内诱导NO合成酶在体内产生的。其次，我们通过体内EPR光谱测量了硝基血管扩张剂硝酸异山梨酯在小鼠体内产生的一氧化氮，并获得了上腹部的三维EPR图像。该项目于1998年作为国际科学研究计划的联合研究项目启动。我们与美国俄克拉何马医学研究基金会的Yashige Kotate博士合作。在这项联合研究中，我们研究了自旋诱捕剂苯基- n-叔丁基硝基酮（PBN）在各种生物系统中的药理活性。结果提示PBN具有非甾体类抗炎药的作用，能降低脑膜炎模型大鼠脑内一氧化氮的生成。少

A variety of information about physiological and pathological actions of nitric oxide has been found very useful for the development of drugs as Viagra. To elucidate a variety of actions of endogenous NO, information concerning the quantities and distributions of NO in cells, tissues, and organs is essential. However, it is rather difficult to determine quantities and distribution due to the very small concentration of NO formed in vivo and the very short half-life of NO in living system. As one of the analytical methods to overcome these difficulties, spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy has been used for the determination of unstable free radicals in vitro and in vivo.1. Iron complexes with dithiocarbamate derivatives are noted among the spin trapping reagents for NO because NO has a high affinity for the iron complexes and resultant NO-bound iron complexes exhibit an intense three-line signal at room temperature on in vitro EPR mea … More surement. N-(dithiocarboxy) sarcosine (DTCS) is a derivative of dithiocarbamate. We recently reported that iron complex with DTCS (Fe-DTCS) and its NO complex (NO-Fe-DTCS) are fairly soluble and stable in aqueous media and Fe-DTCS complex have a potential as a biologically benign, effective NO-trapping reagent. In this study, we measured endogenously produced nitric oxide by employing EPR spin-trapping technique and iron complex with dithiocarbamate as an NO trapping agent. First, we tried in vivo detection of nitric oxide in the brain of living rat during experimental sepsis and meningitis. It was demonstrated that NO is generated in vivo by inducible NO synthase in the brain during sepsis and meningitis. Second, we measured NO production from a nitrovasodilator, isosorbide dinitrate, in mice by in vivo EPR spectroscopy and obtained three-dimensional EPR images of the upper abdominal region.2. This project was started as a joint research of International Scientific Research Program in 1998. We collaborated with Dr. Yashige Kotate of Oklahoma Medical Research Foundation in USA. In this joint research, we investigated the pharmacological activities of a spin-trapping agent, phenyl-N-tert-butyl nitrone (PBN) in various biological systems. The results obtained suggested that PBN has the function like a non-steroidal anti-inflammatory drug and it reduces the nitric oxide production in the rat brain of meningitis model. Less