Silvia Nelli

Oxidation of nitroxyl anion to nitric oxide by copper ions

This study made use of a nitric oxide‐sensitive electrode to examine possible means of generating nitric oxide from nitroxyl anion (NO−) released upon the decomposition of Angelis salt. Our results show that copper ions (from CuSO4) catalyze the rapid and efficient oxidation of nitroxyl to nitric oxide. Indeed, the concentrations of copper required to do so (0.1–100 μM) are roughly 100‐times lower than those required to generate equivalent amounts of nitric oxide from S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP). Experiments with ascorbate (1 mM), which reduces Cu2+ ions to Cu+, and with the Cu2+ chelators, EDTA and cuprizone, and the Cu+ chelator, neocuproine, each at 1 mM, suggest that the oxidation is catalyzed by copper ions in both valency states. Some compounds containing other transition metals, i.e. methaemoglobin, ferricytochrome c and Mn(III)TMPyP, were much less efficient than CuSO4 in catalyzing the formation of nitric oxide from nitroxyl, while FeSO4, FeCl3, MnCl2, and ZnSO4 were inactive. Of the copper containing enzymes examined, Cu‐Zn superoxide dismutase and ceruloplasmin were weak generators of nitric oxide from nitroxyl, even at concentrations (2500 and 30 u ml−1, respectively) vastly greater than are present endogenously. Two others, ascorbate oxidase (10 u ml−1) and tyrosinase (250 u ml−1) were inactive. Our findings suggest that a copper‐containing enzyme may be responsible for the rapid oxidation of nitroxyl to nitric oxide by cells, but the identity of such an enzyme remains elusive.

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

This study explored the role of the potassium ion in endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilatation in the bovine coronary artery. Bradykinin‐induced, EDHF‐mediated vasodilatation was blocked by the Na+–K+ ATPase inhibitor, ouabain (1 μM), in a time‐dependent manner, with maximal blockade seen after 90 min. In contrast, the KIR channel inhibitor, Ba2+ (30 μM), had no effect. 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. The magnitude of potassium‐induced vasodilatation was similar in endothelium‐containing and endothelium‐denuded rings, and was unaffected by Ba2+ (30 μM), but abolished by ouabain (1 μM). Ouabain (1 μM, 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 KATP channel opener, levcromakalim, was hardly affected. 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 Ba2+‐sensitive, endothelium‐independent vasodilatation, are consistent with this ion contributing to the EDHF response in this tissue.

Role of copper ions and cytochrome P450 in the vasodilator actions of the nitroxyl anion generator, Angeli's salt, on rat aorta

Since copper ions catalyse the oxidation of nitroxyl anion to nitric oxide, we investigated whether this might explain the vasodilator actions of the nitroxyl generator, Angelis salt, in rat aorta. Parallel studies were conducted with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), since Cu ions catalyse the liberation of nitric oxide from this compound. Copper sulphate enhanced relaxation to Angelis salt and SNAP but this resulted from reduced destruction of nitric oxide by superoxide rather than from enhanced generation of nitric oxide, since it was mimicked by superoxide dismutase and by the superoxide dismutase mimetic, MnCl2. Results with the selective Cu2+ chelators, neocuproine and bathocuproine disulfonate, and the Cu2+ chelators, EDTA, cuprizone and diethyldithiocarbamate, confirmed an important role for endogenous copper in mediating relaxation to SNAP but suggested only a minor role for Angelis salt. Relaxation to Angelis salt was, however, powerfully blocked by proadifen, suggesting an important role for cytochrome P450.

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.

Formation of nitric oxide from nitroxyl anion : role of quinones and ferricytochrome c

Our previous finding that copper ions oxidize nitroxyl anion released from Angelis salt to nitric oxide prompted us to examine if copper‐containing enzymes shared this property. The copper‐containing enzyme, tyrosinase, which catalyses the hydroxylation of monophenols to diphenols and the subsequent oxidation of these to the respective unstable quinone, failed to generate nitric oxide from Angelis salt by itself, but did so in the presence of tyrosine. L‐DOPA, the initial product of the reaction of tyrosinase with tyrosine, was not the active species, since it failed to generate nitric oxide from Angelis salt. Nevertheless, L‐DOPA and two other substrates, namely, catechol and tyramine did produce nitric oxide from Angelis salt in the presence of tyrosinase, suggesting involvement of the respective unstable quinones. In support, we found that 1,4‐benzoquinone produced a powerful nitric oxide signal from Angelis salt. Coenzyme Qo, an analogue of ubiquinone, failed to generate nitric oxide from Angelis salt by itself, but produced a powerful signal in the presence of its mitochondrial complex III cofactor, ferricytochrome c. Experiments conducted on rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol, to determine if this pathway was responsible for the vascular conversion of nitroxyl to nitric oxide were equivocal: relaxation to Angelis salt was inhibited but so too was that to unrelated relaxants. Thus, certain quinones oxidize nitroxyl to nitric oxide. Further work is required to determine if endogenous quinones contribute to the relaxant actions of nitroxyl donors such as Angelis salt.

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

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. Acetylcholine‐induced, EDHF‐mediated vasodilatation of the ciliary circulation was blocked following inclusion of ascorbate (50 μM, 120 min) in the perfusion fluid. The blockade was highly selective since ascorbate had no effect on the vasodilator actions of the KATP channel opener, levcromakalim, nor on the tonic vasodepressor action of basally released nitric oxide. 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. Ascorbate at 50 μM 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. Thus, ascorbate (50 μM) 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.

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 pro-oxidant 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 μM)-induced constriction and blockade of EDHF. The blockade of EDHF produced by ascorbate in the presence of CuSO4 (0.5 μM) 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.

Flow‐induced enhancement of vasoconstriction and blockade of endothelium‐derived hyperpolarizing factor (EDHF) by ascorbate in the rat mesentery

We previously reported that ascorbate inhibits flow‐ and agonist‐induced, EDHF‐mediated vasodilatation in the bovine ciliary circulation. This study examined whether ascorbate had similar actions in the rat mesenteric vasculature.

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 μM) 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 μM) 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 μM) 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.