Differential sensitivity of basal and acetylcholine‐stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta

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Abstract
In this study we compared the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)‐stimulated activity of NO in isolated rings of rat aorta. Superoxide dismutase (SOD, 1–300 u ml−1) induced a concentration‐dependent relaxation of phenylephrine (PE)‐induced tone in endothelium‐containing rings which was blocked by NG–nitro‐L‐arginine (l‐NOARG, 30 μm), but had no effect on endothelium‐denuded rings. It was likely therefore that the relaxant action of SOD resulted from protection of basally produced NO from destruction by superoxide anion, generated either within the tissue or in the oxygenated Krebs solution. In contrast, a concentration of SOD (50 u ml−1) which produced almost maximal enhancement of basal NO activity, had no effect on ACh (10 nM−3 μm)‐induced relaxation. In the presence of catalase (3000 u ml−1) to prevent the actions of hydrogen peroxide, superoxide anion generation using hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 16 mu ml−1) produced an augmentation of PE‐induced tone in endothelium‐containing but not endothelium‐denuded rings. This was likely to have resulted from removal of the tonic vasodilator action of basally‐produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml−1), NG–monomethyl‐L‐arginine (L‐NMMA, 30 um) or L‐NOARG (30 μm). Pyrogallol (0.1 mM) had a similar action toHX/XO, but produced an additional augmentation of tone by an endothelium‐independent mechanism. In contrast to their ability to destroy almost completely the 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 at any concentration. An increase in the concentration of HX to 1 mM or pyrogallol to 0.3 mM did, however, lead to a profound decrease in the magnitude and time course of ACh‐induced relaxation at all concentrations. Treatment with diethyldithiocarbamate (DETCA, 0.1 mM, 1 h) to inhibit endogenous Cu‐Zn SOD, augmented PE‐induced tone in endothelium‐containing rings and abolished the ability of HX (0.1 mM)/ XO (16 mu ml−1) and L‐NMMA (30 um) to augment tone. It was likely that DETCA had led to the destruction of basal NO activity by increasing superoxide anion levels since its actions were reversed by exogenous SOD (10–300 u ml−1). In contrast to its ability to destroy basal activity of NO completely, DETCA (0.1 mM) produced only a slight blockade of ACh‐induced relaxation. However, if these tissues were subsequently treated with concentrations of HX (0.1 mM)/XO (16 mu ml−1) or pyrogallol (0.1 mM), which had no effect by themselves on ACh‐induced relaxation, a profound blockade was seen and this was reversed completely with SOD (250 u ml−1). The data suggest that basal activity of NO is more sensitive to inactivation by superoxide anion than ACh‐stimulated activity and this probably results from differential protection by endogenous Cu‐Zn SOD. It is possible therefore that endogenous SOD lowers superoxide anion levels to such an extent that only small amounts of NO, such as those produced under basal conditions, are destroyed. Following generation of superoxide anion withHX/XO or pyrogallol, or inhibition of Cu‐Zn SOD with DETCA, levels of the free radical will increase such that greater amounts of NO, such as those produced following stimulation with ACh, will then be destroyed.