ANALYSIS OF ENDOGENOUSLY PRODUCED NITRIC OXIDE (NO) EMPLOYING IRON COMPLEXES AS AN NO TRAPPING REAGENT AND PHYSIOLOGICAL STUDY ON NO

铁络合物作为NO捕获剂内源产生一氧化氮(NO)的分析及其生理学研究

• 批准号: 08640781
• 负责人: YOSHIMURA Tetsuhiko
• 金额: $ 1.41万
• 依托单位: INSTITUTE FOR LIFE SUPPORT TECHNOLOGY (ILST), YAMAGATA TECHNOPOLIS FOUNDATION
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08640781/
• 关键词: NITRIC OXIDE; ENDOGENOUSLY PRODUCED NITRIC OXIDE; TRAP; IRON COMPLEX; ELECTRON SPIN RESONANCE; DITHIOCARBAMATE; N- (DITHIOCARBOXY) SARCOSINE

Diatomic molecule, nitric oxide (NO) is an inorganic free radical. It has been known as an atomospheric pollutant and a potential hazard. However, it is now widely known as an endogenous molecule with critical physiological roles in the biological processes of vascular smooth muscle relazation, neuronal transmission and macrophage-induced cytotoxicity. 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.Iron complexes with dithiocarbamate derivatives are noted among the … More 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 measurement. Such dithiocarbamate derivatives include N,N-diethyldithiocarbamate (DETC), N-methyl-D-glucamine dithiocarbamate (MGD), and N-(dithiocarboxy) sarcosine (DTCS). Iron complex with DTCS (Fe-DTCS) and its NO complex (NO-Fe-DTCS) are fairy soluble and stable in aqueous media. Therefore, Fe-DTCS and NO-Fe-DTCS complexes have a potential as a biologically benign, effective NO-trapping reagent and as a water-soluble spin-labeling reagent (or spin probe), respectively. Further, if the EPR signal from endogenously produced or exogenously supplied NO-Fe-DTCS complex is able to measure in the whole body of a live small animal, in vivo EPR imaging of endogenous and exogenous NO would be performed. Such EPR image would permit noninvasive observation of in vivo spatial distribution of NO.To obtain EPR spectra and image, we used a home built 700-MHz-microwave EPR imaging system, which is composed of an electromagnet attached with a pair of field gradient coils, a data-processing computer, and a 700-MHz-microwace EPR unit.In this study, we demonstrated the usefulness of iron dithiocarbamates as an NO trapping reagent. Further, we reported the applications of the NO trapping reagent to the detection and imaging of endogenously produced NO.First, we tried the detection of NO generation from porcine aorta endothelial cells. Second, we demonstrated in vivo EPR imaging of endogenously produced NO,trapped by an Fe-DTCS complex, in the abdomen of a live mouse. This experiment has been cariied out in a septic-shock model by administering lipopolysaccharide to mice via intraperitoneal route. The outline of the image thus obtained corresponds to the liver, suggesting the metabolic pathway of NO-Fe-DTCS complex.We could demonstrate in this study that iron dithiocarbamates as an NO trapping reagent are suitable for in vivo real time detection of NO produced under physiological and pathological conditions. Less

一氧化氮（NO）是双原子分子，是一种无机自由基。它被称为一种大气污染物和潜在危害。然而，它现在被广泛认为是一种内源性分子，在血管平滑肌松弛、神经元传递和巨噬细胞诱导的细胞毒性的生物过程中具有关键的生理作用。为了阐明内源性 NO 的多种作用，有关细胞、组织和器官中 NO 的数量和分布的信息至关重要。然而，由于体内NO形成的浓度非常小，并且NO在生命系统中的半衰期非常短，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，自旋捕获技术与电子顺磁共振（EPR）光谱相结合已被用于体外和体内测定不稳定的自由基。具有二硫代氨基甲酸酯衍生物的铁络合物被认为是NO的自旋捕获试剂之一，因为NO对铁络合物具有高亲和力，并且所得的NO结合铁络合物表现出 体外 EPR 测量中室温下的强烈三线信号。此类二硫代氨基甲酸酯衍生物包括N，N-二乙基二硫代氨基甲酸酯(DETC)、N-甲基-D-葡糖胺二硫代氨基甲酸酯(MGD)和N-(二硫代羧基)肌氨酸(DTCS)。铁与 DTCS 的络合物 (Fe-DTCS) 及其 NO 络合物 (NO-Fe-DTCS) 在水介质中易溶且稳定。因此，Fe-DTCS 和 NO-Fe-DTCS 复合物分别具有作为生物学良性、有效的 NO 捕获试剂和水溶性自旋标记试剂（或自旋探针）的潜力。此外，如果来自内源性产生或外源性提供的NO-Fe-DTCS复合物的EPR信号能够在活体小动物的全身中测量，则将进行内源性和外源性NO的体内EPR成像。这种 EPR 图像将允许无创观察 NO 体内空间分布。为了获得 EPR 光谱和图像，我们使用了自制的 700 MHz 微波 EPR 成像系统，该系统由附有一对场梯度线圈的电磁体、数据处理计算机和 700 MHz 微波 EPR 装置组成。在这项研究中，我们证明了铁的有用性 二硫代氨基甲酸盐作为 NO 捕获剂。此外，我们还报道了NO捕获试剂在内源性NO产生的检测和成像中的应用。首先，我们尝试检测猪主动脉内皮细胞产生的NO。其次，我们在活体小鼠腹部展示了被 Fe-DTCS 复合物捕获的内源性 NO 的体内 EPR 成像。该实验通过腹膜内途径向小鼠施用脂多糖，在败血性休克模型中进行。由此获得的图像轮廓对应于肝脏，表明NO-Fe-DTCS复合物的代谢途径。在本研究中我们可以证明二硫代氨基甲酸铁作为NO捕获剂适合体内实时检测生理和病理条件下产生的NO。较少的

项目成果

吉村 哲彦: "NOのすべて" 医歯薬出版株式会社(平田結喜緒編), 3-7,30-34 (1996)

吉村哲彦：《关于 NO 的一切》石药出版株式会社（平田幸雄编），3-7,30-34 (1996)

鈴木保宏: "In vivo nitric oxide detection in the septic rat brain by electron paramagnetic resonance" Free Radical Research(印刷中). (1998)

Yasuhiro Suzuki：“通过电子顺磁共振检测脓毒症大鼠大脑中的一氧化氮”自由基研究（正在出版）（1998 年）。

藤井敏司: "ニトロシル鉄錯体をスピンプローブとするL-band ESRイメージング" 磁気共鳴と医学. 7. 169-172 (1996)

Satoshi Fujii：“使用亚硝酰基铁络合物作为自旋探针的 L 波段 ESR 成像”《磁共振与医学》7. 169-172 (1996)。

横山秀克: "In vivo ESR-CT imaging of the liver in mice receiving subcutaneous injection of nitric oxide bound iron complex" Magnetic Resonance Imaging. (印刷中). (1997)

Hidekatsu Yokoyama：“接受皮下注射一氧化氮结合铁复合物的小鼠肝脏的体内 ESR-CT 成像”磁共振成像（1997 年出版）。

S.Fujii, Y.Suzuki, T.Yoshimura, H.Kamada: "In vivo three-dimentional EPR imaging of nitric oxide production from isosorbide dinitrate in mouse" American Journal of Physiology. (in press).

S.Fujii、Y.Suzuki、T.Yoshimura、H.Kamada：“小鼠体内硝酸异山梨酯产生一氧化氮的体内三维 EPR 成像”美国生理学杂志。