Oxidation by trace Cu2+ ions promotes ascorbate-induced constriction and blockade of endothelium-derived hyperpolarizing factor (EDHF) in the rat perfused mesentery

William Martin, John Craig, Silvia Nelli

University of Glasgow, Glasgow, UK

Although acute treatment with ascorbate improves NO-mediated dilatation in patients with a variety of cardiovascular pathologies, little benefit to cardiovascular health has emerged from clinical trials involving dietary supplementation with the vitamin (Duarte & Lunec, 2005). Transition metal ions, including Cu2+, are know to promote oxidation of ascorbate, raising the possibility that a detrimental pro-oxidant action may override any acute vascular benefit. We have previously shown that plasma levels of ascorbate induce two potentially detrimental effects, namely constriction and blockade of endothelium-derived hyperpolarizing factor (EDHF; McNeish et al, 2002; Stirrat et al, 2008). In this study we sought to determine if these detrimental effects were mediated by a pro-oxidant action of ascorbate.

Experiments were conducted on the rat mesentery, perfused with Krebs solution at 15 ml min-1 in the presence of the NO synthase inhibitor, L-NAME (100 μM), and the cyclooxygenase inhibitor, indomethacin (3 μM). Perfusion pressure was raised to ∼120 mmHg using U46619 (70–100 nM). In all experiments, n>5 and differences were determined by ANOVA followed by the Bonferroni post hoc test.

As previously reported (Stirrat et al, 2008), treatment with ascorbate (50 μM) induced a rapid constriction (53.8 ± 11.8 mmHg) and a slowly developing blockade of acetylcholine (10 nmol)-induced, EDHF-mediated dilatation (reduced from 49.8 ± 5.4 to 25.2 ± 5.6% at 3 h, P<0.01). The Cu2+ chelator, cuprizone (30 μM), abolished the ability of ascorbate to induce constriction (-2.1 ± 0.9 mmHg, P<0.001) and block EDHF-mediated dilatation (43.3 ± 6.0%). Moreover, treatment with CuSO4 (0.5 μM) enhanced the ability of ascorbate to induce constriction (93.5 ± 19.2 mmHg, P<0.01) and block EDHF-mediated dilatation (10.5 ± 2.6%, P<0.001). The blockade of EDHF produced by ascorbate in the presence of CuSO4 (0.5 μM) was prevented by the hydrogen peroxide scavenger, catalase (1,200 u ml-1; 49.9 ± 6.5%), but not by the scavengers of hydroxyl radical or superoxide anion, mannitol (10 mM, 25.0 ± 6.3%, P<0.01) and superoxide dismutase (100 u ml-1; 2.5 ± 2.1%, P<0.001), respectively. Ascorbate-induced constriction in the presence of CuSO4 (0.5 μM) was unaffected by catalase (66.1 ± 17.1 mmHg), mannitol (95.9 ± 8.1 mmHg) or superoxide dismutase (104.1 ± 9.0 mmHg).

Thus, in the rat perfused mesentery, both the constrictor and EDHF-blocking actions of ascorbate arise from its oxidation by trace Cu2+ ions. The blockade of EDHF appears to results from the consequent generation of hydrogen peroxide, but the factor producing constriction remains unidentified. These detrimental actions of ascorbate may help explain the disappointing outcome of clinical trials studying the effects of dietary supplementation with the vitamin on cardiovascular health.

Duarte & Lunec, 2005. Free Radic Res 39: 671-686.

McNeish et al, 2002. Br J Pharmacol 135: 1801-1809.

Stirrat et al, 2008. Br J Pharmacol 153: 1162-1168.

Supported by the Wellcome Trust