Inhibition of nitric oxide by reactive oxygen species

1. The aim of this study was to determine the role of the antioxidant enzymes, superoxide dismutase and catalase, in the regulation of vascular tone in isolated rings of rat aorta. 2. In the first part of the study a comparison was made of the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)-stimulated NO. 3. Superoxide dismutase (SOD, 1-300 u ml-1) had no effect on endothelium- denuded rings but it produced a concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings which was blocked by the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 30 muM). In contrast, SOD (50 u mh-1) had no effect on ACh-induced relaxation. These results demonstrated a selective potentiation of basal but not ACh-stimulated activity of NO by SOD. 4. Superoxide anion generation using hypoxanthine (HX, 0,1 mM)/xanthine oxidase (XO, 16 mu ml-1) augmented PE-induced tone in endothelium- containing but not endothelium-denuded rings. This was likely to have resulted from removal of the tonic vasodilator actions of basally-produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml-1), NG-mono-methyl-L-arginine (L-NMMA, 30 muM) or L-NOARG (30 muM). Pyrogallol (0.1 mM) had a similar action to HX/XO, but produced an additional augmentation of tone by an endothelium-independent mechanism which was unaffected by SOD (250 u ml-1). In contrast to their ability to almost completely destroy basal activity of NO, HX (0.1 mM)/XO (16 mu ml-1) and pyrogallol (0.1 mM) had no effect on ACh-induced relaxation. Increasing the concentration of HX to 1 mM (keeping XO at 16 mu ml-1) or pyrogallol to 0.3 mM, however, profoundly blocked ACh-induced relaxation and this was prevented by treatment with SOD (250 u ml-1). 5. Treatment with diethyldithiocarbamate (DETCA, 0.1 mM, 1h, followed by washout) to irreversibly inhibit endogenous Cu-Zn SOD, augmented PE- induced tone in endothelium-containing, but not endothelium-denuded rings, and abolished the ability of HX (0.1 mM)/XO (16 mu ml-1) and L-NMMA (30 muM) to augment tone. It was likely that DETCA had led to destruction of basal NO by increasing superoxide anion levels since its actions were reversed by SOD (10-300 u ml-1). 6. In contrast to its ability to completely destroy basal activity of NO, DETCA (0.1 mM) produced only a slight blockade of ACh-induced relaxation. Furthermore, DETCA potentiated the ability of HX (0.1 mM)/XO (16 mu ml-1) or pyrogallol (0.1 mM) to block ACh-induced relaxation and this was prevented by pretreatment with SOD (250 u ml-1). 7. The data suggest that basal activity of NO is more sensitive to inactivation by superoxide anion than ACh-stimulated activity and that endogenous Cu-Zn SOD is vital for the protection of endothelial NO. 8. In the second part of the study the role of catalase in the relaxation induced by sodium azide, hydroxylamine, glyceryl trinitrate and hydrogen peroxide was investigated in isolated rings of rat aorta. (Abstract shortened by ProQuest.)