Ascorbate Prevents the Interaction of Superoxide and Nitric Oxide Only at Very High Physiological Concentrations

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Abstract
—The bioactivity of nitric oxide ( • NO) depends, in part, on its interaction with superoxide. Usually, superoxide dismutase (SOD) preserves • NO bioactivity by limiting the availability of superoxide. Ascorbic acid also effectively scavenges superoxide, but the extent to which this interaction is necessary for intact • NO bioactivity is not known. Therefore, the present study examined the effect of ascorbic acid on • NO bioactivity with isolated rabbit arterial segments. A steady flux of superoxide (1.15 to 2.3 μmol · L −1 · min −1 ) produced either by pyrogallol autoxidation or a hypoxanthine/xanthine oxidase system inhibited endothelium-derived • NO-mediated arterial relaxation elicited by acetylcholine. This effect of superoxide was completely blocked by SOD (300 IU/mL) and the manganese SOD mimic EUK-8 (300 μmol/L) and partially inhibited by ascorbic acid (10 mmol/L). Lower concentrations of ascorbic acid were ineffective despite scavenging >90% of superoxide. We increased the endogenous flux of superoxide (3.2±0.3-fold) by inhibiting vascular copper-zinc SOD with diethyldithiocarbamate. This increased endogenous flux of superoxide produced an impairment of • NO-mediated arterial relaxation that was reversed by EUK-8 (300 μmol/L) but not ascorbic acid (10 mmol/L) despite equivalent scavenging of the endogenous superoxide flux. We used 3-nitrotyrosine formation (from peroxynitrite) as an indicator of • NO interaction with superoxide and found that SOD and EUK-8 compete more effectively with • NO for superoxide than does ascorbic acid. These data indicate that preservation of • NO bioactivity by superoxide scavengers depends not only on superoxide scavenging activity, but also on the rate of superoxide scavenging. Normal extracellular concentrations of ascorbic acid (30 to 150 μmol/L) are not likely to prevent the interaction of • NO with superoxide under physiological conditions.