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

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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