Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations

Abstract—The bioactivity of nitric oxide (YNO) depends, in part, on its interaction with superoxide. Usually, superoxide dismutase (SOD) preserves YNO bioactivity by limiting the availability of superoxide. Ascorbic acid also effectively scavenges superoxide, but the extent to which this interaction is necessary for intact YNO bioactivity is not known. Therefore, the present study examined the effect of ascorbic acid on YNO bioactivity with isolated rabbit arterial segments. A steady flux of superoxide (1.15 to 2.3 mmol z L21 z min21) produced either by pyrogallol autoxidation or a hypoxanthine/xanthine oxidase system inhibited endothelium-derived YNO-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 mmol/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 (3.260.3-fold) by inhibiting vascular copper-zinc SOD with diethyldithiocarbamate. This increased endogenous flux of superoxide produced an impairment of YNO-mediated arterial relaxation that was reversed by EUK-8 (300 mmol/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 YNO interaction with superoxide and found that SOD and EUK-8 compete more effectively with YNO for superoxide than does ascorbic acid. These data indicate that preservation of YNO bioactivity by superoxide scavengers depends not only on superoxide scavenging activity, but also on the rate of superoxid