Oxidation by trace Cu2+ ions underlies the ability of ascorbate to induce vascular dysfunction in the rat perfused mesentery.

Ascorbate has both antioxidant and pro-oxidant activities. We have previously shown that plasma levels of ascorbate induce constriction and blockade of dilatation mediated by endothelium-derived hyperpolarizing factor (EDHF). In this study we sought to determine if these detrimental actions were mediated by a prooxidant action of ascorbate. Since trace levels of transition metal ions including, Cu2+ and Fe3+, promote oxidation of ascorbate, we examined the effects of the chelating agents, cuprizone and deferoxamine, and of CuSO4 and FeCl3 on ascorbate-induced constriction and blockade of EDHF in the perfused rat mesentery. Cuprizone abolished and Cu2+ but not Fe3+ ions enhanced both ascorbate (50 microM)-induced constriction and blockade of EDHF. The blockade of EDHF produced by ascorbate in the presence of CuSO4 (0.5 microM) was abolished by the hydrogen peroxide scavenger, catalase, but unaffected by the scavengers of hydroxyl radical or superoxide anion, mannitol and superoxide dismutase (SOD), respectively. Consistent with these observations, the oxidation of ascorbate by CuSO4 led to the rapid production of hydrogen peroxide. Catalase, mannitol and SOD had no effect on ascorbate-induced constriction. 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 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 investigating dietary supplementation with the vitamin on cardiovascular health.

Ascorbate elevates perfusion pressure in the bovine extraocular long posterior ciliary artery: role of endothelium-derived hyperpolarizing factor (EDHF).

Ascorbate blocks agonist-induced, endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine perfused ciliary artery and this is associated with a rise in perfusion pressure. We now report the origins of this ascorbate-induced rise in perfusion pressure. In segments of ciliary artery perfused at 2.5 ml/min, the addition of ascorbate (10-150 microM) enhanced U46619-induced perfusion pressure. Ascorbate produced no enhancement in the absence of U46619, suggesting that its effects resulted not from a constrictor action but through removal of a tonic vasodilator influence. Experiments revealed the endothelial source of this vasodilator influence, and EDHF, but not nitric oxide or prostanoids, appeared to be involved. The ascorbate-induced enhancement of vasoconstrictor tone was not seen in a static myograph or in segments perfused at low rates of flow, but was seen at flow rates of 2.5 ml(-1) and above. We conclude that ascorbate augments vasoconstrictor tone through inhibition of flow-induced EDHF activity.

Requirement for flow in the blockade of endothelium-derived hyperpolarizing factor (EDHF) by ascorbate in the bovine ciliary artery.

We previously reported that ascorbate inhibits endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine perfused ciliary circulation and rat perfused mesentery, but not in rings of bovine or porcine coronary artery. In this study, we have compared the ability of ascorbate to inhibit EDHF-mediated vasodilatation in a single vessel, the bovine long posterior ciliary artery, when perfused and when mounted as rings in a myograph. Both in segments perfused at a flow rate of 2.5 ml min(-1) and in rings mounted in a myograph, bradykinin and acetylcholine each induced vasodilator responses that were mediated jointly by EDHF and nitric oxide, as revealed by their respective blocking agents, apamin/charybdotoxin, and L-NAME. Ascorbate (50 and 150 microm) induced a time (max at 2-3 h)-dependent inhibition of the EDHF-mediated component of vasodilatation to bradykinin or acetylcholine in perfused segments, but not in rings. Ascorbate (50 microm) failed to inhibit bradykinin-induced vasodilatation at a flow rate of 1.25 ml min(-1) or below, but produced graded blockade at the higher flow rates of 2.5 and 5 ml min(-1). Furthermore, using a pressure myograph where pressure and flow were independently controlled, it was confirmed that the inhibitory action of ascorbate (150 microm) was directly related to flow per se and not any associated changes in pressure. Thus, we have shown in the bovine ciliary artery that ascorbate inhibits EDHF-mediated vasodilatation under conditions of flow but not in a static myograph. The mechanism by which flow renders EDHF susceptible to inhibition by ascorbate remains to be determined.

Evaluation of potassium ion as the endothelium-derived hyperpolarizing factor (EDHF) in the bovine coronary artery.

1. This study explored the role of the potassium ion in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine coronary artery. 2. Bradykinin-induced, EDHF-mediated vasodilatation was blocked by the Na(+)-K(+) ATPase inhibitor, ouabain (1 micro M), in a time-dependent manner, with maximal blockade seen after 90 min. In contrast, the K(IR) channel inhibitor, Ba(2+) (30 micro M), had no effect. 3. When the potassium content of the bathing solution was increased in a single step from 5.9 to 7-19 mM, powerful vasodilatation (max. 75.9+/-3.6%) was observed. Vasodilatation was transient and, consequently, cumulative addition of potassium produced little vasodilatation, with vasoconstriction predominating at the higher concentrations. 4. The magnitude of potassium-induced vasodilatation was similar in endothelium-containing and endothelium-denuded rings, and was unaffected by Ba(2+) (30 micro M), but abolished by ouabain (1 micro M). 5. Ouabain (1 micro M, 90 min) powerfully blocked bradykinin-induced, nitric oxide-mediated vasodilatation as well as that induced by the nitrovasodilator, glyceryl trinitrate, but that induced by the K(ATP) channel opener, levcromakalim, was hardly affected. 6. Thus, activation of Na(+)-K(+) ATPase is likely to be involved in the vasodilator responses of the bovine coronary artery to both nitric oxide and EDHF. These findings, together with the ability of potassium to induce powerful, ouabain- but not Ba(2+)-sensitive, endothelium-independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.

Differential effects of ascorbate on endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation in the bovine ciliary vascular bed and coronary artery.

1. The ability of ascorbate to inhibit endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation was compared in the bovine perfused ciliary vascular bed and isolated rings of coronary artery. 2. Acetylcholine-induced, EDHF-mediated vasodilatation of the ciliary circulation was blocked following inclusion of ascorbate (50 micro M, 120 min) in the perfusion fluid. The blockade was highly selective since ascorbate had no effect on the vasodilator actions of the K(ATP) channel opener, levcromakalim, nor on the tonic vasodepressor action of basally released nitric oxide. 3. The possibility that concentration of ascorbate by the ciliary body was a prerequisite for blockade to occur was ruled out, since EDHF was still blocked when the anterior and posterior chambers were continuously flushed with Krebs solution or when both the aqueous and vitreous humour were drained. 4. Ascorbate at 50 micro M failed to affect bradykinin- or acetylcholine-induced, EDHF-mediated vasodilatation in rings of bovine coronary artery. Raising the concentration to 3 mM did produce blockade of EDHF, but this was nonselective, since vasodilator responses to endothelium-derived nitric oxide were also inhibited. 5. Thus, ascorbate (50 micro M) is not a universal blocker of EDHF. Whether its ability to block in the bovine ciliary circulation, but not in the coronary artery, is due to differences in the nature of EDHF at the two sites, differences in vessel size (resistance arterioles versus conduit artery), the presence or absence of flow, or to some other factor remains to be determined.

Role of catalase in the smooth muscle relaxant actions of sodium azide and cyanamide.

The aim of this study was to determine the role of catalase in the smooth muscle relaxant actions of sodium azide and cyanamide. The effects of 3-amino-1,2,4-triazole suggested a role for this enzyme in the relaxant actions of sodium azide on rat aorta and bovine retractor penis muscle and cyanamide on rat aorta. Moreover, results obtained using a difference spectrophotometric assay based upon the oxidation of haemoglobin were consistent with the catalase-dependent oxidation of sodium azide to nitric oxide (NO) and of cyanamide to nitroxyl anion. Surprisingly, however, no free nitric oxide or nitroxyl was detected in solution using a sensitive electrode. This anomaly might be explained if the stable complexes of catalase with nitric oxide or nitroxyl do not release their respective ligand except to sites of high affinity, such as the haemoglobin employed in the difference spectrophotometric assay, or indeed, the soluble guanylate cyclase within the smooth muscle.