|Budget Amount *help
¥5,100,000 (Direct Cost : ¥5,100,000)
Fiscal Year 1999 : ¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1998 : ¥3,000,000 (Direct Cost : ¥3,000,000)
(1) Reactive nitrogen species (RNS) including peroxynitrite and nitrogen dioxide, which are formed in the reaction of nitrogen oxide (NO) with superoxide anion (OィイD22ィエD2ィイD1-ィエD1) and peroxidase-dependent mechanisms, have a potent inflammatory action. We investigated the role of peroxynitrite in the airway microvascular hyperpermeability during the late allergic response (LAR) in sensitized guinea pigs in vivo. The occurrence of LAR was assessed as a 100% increase in the transpulmonary pressure, which was monitored by the esophageal catheter technique. Airway microvascular permeability was assessed by Monastral blue dye trapping between the endothelium using an image analyzer. In the LAR phase (4 to 6 h after antigen inhalation), microvascular hyperpermeability and eosinophil infiltration within the airway wall were observed. NO production and xanthine oxidase (XO)/xanthine dehydrogenase activity, which are responsible for OィイD22ィエD2ィイD1-ィエD1 production, were enhanced during the LAR.
Peroxynitrite formation assessed by nitrotyrosine immunostaining was also exaggerated at that time. The microvascular hyperpermeability during the LAR was largely reduced by NO synthase inhibitor (L-NAME, 72.7% inhibition ; p<0.05), XO inhibitor (AHPP, 60.8% inhibition ; p<0.05) and peroxynitrite scavenger (ebselen, 81.0% inhibition ; p<0.05). L-NAME had a small but significant inhibitory effect on airway eosinophil accumulation, but AHPP and ebselen had no effect. These results suggest that excessive production of OィイD22ィエD2ィイD1-ィエD1 and NO occurs in the LAR. These two molecules appear to cause airway microvascular hyperpermeability via peroxynitrite formation.
(2) Next, we quantified the RNS using immunostaining of nitrotyrosine and inducible NO synthase (iNOS) in airway inflammatory cells obtained by he induced sputum technique as well as the exhaled NO concentration in chronic obstructive pulmonary disease (COPD), asthma and healthy subjects (HS). iNOS immunoreactivity observed in the airway inflammatory cells was significantly and similarly higher in COPD and asthma compared with HS, although the exhaled NO levels were elevated in asthma but not in COPD and HS. The nitrotyrosine immunoreactivity in the inflammatory cells was obvious in COPD and to a lesser extent in asthma but not in HS. There was a significant negative correlation between the percent predicted values of forced expiratory volume in one second and the amount of nitrotyrosine formation in COPD but not in asthma and HS. These results suggest that 1) RNS may be involved in the pathobiology of the airway inframmatory and obstructive process in COPD, 2) NO produced in the airways, presumably via iNOS, seems to be consumed by its reaction with superoxide anion and/or peroxidase-dependent mechanisms. Less