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330 mT

Fig. 2. The shape of the EPR spectra from 57Fe-(or 56Fe)-DNIC neocuproine (A,B). Doublet spectra above spectra A is low-field part of the latter recorded at higher amplification. The spectra were recorded at 77 K. (From Ref. [25].)

as {3d}7 and the iron center acquires the character of Fe+. In the absence of NO, the Fe2+ ions are not chelated by neocuproine because of rapid formation of water insoluble iron hydroxide complexes [25].

Bathocuproine disulfonate (BCS) is a highly soluble cuproine derivative. Vasodilatory studies of Cys-NO have shown that exogenous copper and iron inhibit the relaxation of precontracted endothelium-denuded rat aorta rings by Cys-NO, and that the inhibitory action of both metal ions is cancelled by BCS [17,18]. The addition of this chelator enhanced the duration of the vessel relaxation induced by Cys-NO in the presence of ascorbate, copper or iron (Fig. 3, curves f,g) [17].

Bathophenanthroline disulfonate (BPDS) is a potent chelator of transition metal ions and optical absorption experiments confirmed that it binds both iron and copper. Nevertheless, BPDS imparted significant protection of Cys-NO against iron only. It showed that chelation by BPDS inhibits the pathway for iron-catalyzed decomposition of Cys-NO thereby enhancing the duration of vessel relaxation (Fig. 3, curve d). In contrast, the copper-catalyzed pathway proceeded practically unimpaired (Fig. 3, curve e) [17].

The stability of Cys-NO was also studied in vitro in solutions containing ascorbate, copper or iron. In vitro, BCS protected Cys-NO effectively against catalytic decomposition by both metals but strong iron chelator bathophenanthroline disulfonate (BPDS) protected against ferrous iron only (Fig. 4) [17]. The observations were attributed to full saturation of the coordination sphere in the Fe2+-BPDS complexes (i.e. the binding of three BPDS ligands to each iron) and preventing the iron from participating in redox reactions with Cys-NO [17,18]. In contrast, the monovalent copper ions bind only two BPDS ligands, and leaving the copper atom accessible to small molecules in the solution [26]. The experimental observations suggest that Cys-NO can penetrate into Cu(BPDS)2 complexes and be decomposed via reduction by Cu+ (Fig. 4) [17].

The catalytic decomposition of Cys-NO by copper-BPDS in the presence of BPDS and ascorbate was noticeable down to very low copper concentrations of about 2 ^M (Fig. 4) [17,18]. This makes it questionable to attribute the protection of Cys-NO by cuproine derivatives to their sequestration of intrinsic Cu+. For example, Fig. 5 (curve b) [17] shows that BPDS gives significant protection of Cys-NO in vasodilation experiments in

BHDS + Asc -t-Cu

2 min

Fig. 3. Representative traces of the relaxant effect of Cys-NO (30 nM) in endothelium-denuded rat aortic rings precontracted with noradrenaline (0.1 ^M). (Curve a) with 0.5 mM ascorbate only, (curve b) With 0.1 mM bathophenanthroline disulfonate (BPDS) and 0.5 mM ascorbate, (curve c) With 0.1 mM bathocuproine sulfonate (BCS) and 0.5 mM ascorbate, (curve d) BPDS + ascorbate + 250 nM Fe2+, (curve e) BPDS + ascorbate + 250 nM Cu2+, (curve f) BCS + ascorbate + 250 nM Fe2+ and, (curve g) BCS + ascorbate + 250 nM Cu2+. (From Ref. [17].)

Krebs buffers. It means that intrinsic copper did not form the main pathway for the decomposition of Cys-NO in these experiments. We estimate the intrinsic copper content to be lower than 2 ^M. Instead, the protection of Cys-NO was attributed to the sequestration and inactivation of intrinsic iron in the solutions. Phrased otherwise, intrinsic ferrous iron rather than intrinsic copper was seen to dominate the decomposition of Cys-NO.

In experiments with nitrosothiols, the presence of spurious quantities of reduced iron and copper is often found to be significant. Sheu et al. [27] investigated the iron and copper levels of sample solutions containing cysteine, glutathione (1-2 mM) or phosphate buffer (100 mM) and reported values of [Fe] ~ 1.8 ^M and [Cu] ~ 0.05 ^M. Therefore, spurious

Fig. 4. Influence of subsequent additions (shown by arrows) of ascorbate, copper and iron on the stability of Cys-NO in the presence of 1 mM bathophenanthroline disulfonate (top) or bathocuproine sulfonate (bottom). Results are expressed as mean ± SE of three experiments [17].

Fig. 4. Influence of subsequent additions (shown by arrows) of ascorbate, copper and iron on the stability of Cys-NO in the presence of 1 mM bathophenanthroline disulfonate (top) or bathocuproine sulfonate (bottom). Results are expressed as mean ± SE of three experiments [17].

iron comfortably exceeded copper (Fig. 4). Under these conditions, only the addition of BPDS would bind the intrinsic iron, extend the lifetime of Cys-NO and thereby make the vessel relaxation last longer.

The addition of BCS also prolongs the vasodilatory action of Cys-NO significantly (Fig. 5, curve c) [17], but the mechanism is very different. As remarked before, the combination of iron, Cys-NO and BCS leads to the formation of BCS-DNIC. This BCS-DNIC is quite stable and long-lived and was shown to induce a long-lasting vasodilation in aortic rings from which the endothelium had been removed. The vasorelaxation observed with BCS-DNIC was sustained significantly longer than that induced by DNIC with phosphate ligands (Fig. 6, curves a,b) [17].

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