Fig. 8.
(a) The production of the superoxide anion radical (O2•−) and H2O2 reflect the staining with dihydroethidium (DHE) and dichlorodihydro-fluorescein fluorescence (DCF) in the vessel, respectively. We then examined reactive oxygen species (ROS) generation in the coronary arteries of the SMP30 KO mice based on the hypothesis that a SMP30 deficiency exacerbates ROS generation. The signals of the DHE and DCF staining were enhanced in the coronary arteries with a SMP30 deficiency. Apocynin, an NADPH oxidase inhibitor, decreased these signals in the coronary arteries of the SMP30 KO mice. These results suggest that superoxide generation in SMP30 deficiency depends on NADPH oxidase in the coronary arteries. (b) To assess thiol oxidation in the coronary arteries, the location of thiols was determined by the administration of fluorochromes monochlorobimane (MCB), which compounds covalently to react with reduced thiols. However, if the thiols are oxidized, the compounds do not bind. Acetylcholine (ACh) treatment decreased the fluorescence levels because of thiol oxidation in the coronary arteries of the wild type mice. The fluorescence levels because of MCB in the coronary arteries decreased in the SMP30 KO mice, and the level was restored with dithiothreitol (DTT), a reducing agent treatment. These results suggest that the total thiols were oxidized in the SMP30 KO mice. Elevation of the fluorescence of MCB by DTT indicates thiol reduction. ACh oxidized the intracellular thiols to a sufficient SMP30 level. A deficiency of SMP30 extinguishes additional thiol oxidation with ACh. Thus, reducing thiols might be essential for nitric oxide generation.