ESR ANALYSIS AND IN VIVO ACTION OF ENDOGENOUS NITRIC OXIDE

有关一氧化氮的生理和病理行为的各种信息已被发现对伟哥等药物的开发非常有用。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在体内形成的浓度很小，而且在生物系统中的半衰期很短，因此很难确定NO的数量和分布。作为克服这些困难的分析方法之一，自旋捕捉技术与电子顺磁共振(EPR)谱相结合已被用于体外和体内不稳定自由基的测定。二硫代氨基甲酸铁络合物是NO的自旋捕捉剂之一，因为NO与铁络合物有很高的亲和力，所生成的铁络合物在室温下在体外EPR mea…上显示出强烈的三线信号。更多的慰藉。N-二硫代甲酸肌氨酸(DTCS)是二硫代氨基甲酸酯的一种衍生物。我们最近报道了铁与DTCS的络合物(Fe-DTCS)及其NO络合物(NO-Fe-DTCS)在水介质中具有很好的溶解性和稳定性，Fe-DTCS络合物有可能成为一种生物上无害的、有效的无捕捉剂。在这项研究中，我们利用EPR自旋捕获技术和以二硫代氨基甲酸盐为NO捕捉剂的铁络合物来测量内源性产生的一氧化氮。首先，我们尝试在体内检测实验性脓毒症和脑膜炎期间活体大鼠脑内一氧化氮的含量。已有研究表明，在脓毒症和脑膜炎过程中，体内NO是通过脑内诱导型NO合成酶产生的。其次，我们通过体内EPR波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NO，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，我们研究了自旋捕捉剂苯基-N-叔丁基硝酮(PBN)在不同生物体系中的药理活性。提示PBN具有非甾体类抗炎药的作用，并能降低脑膜炎模型大鼠脑内一氧化氮的生成。较少

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