Copper chelation‐induced reduction of the biological activity of S‐nitrosothiols

Abstract

1 The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells.2 Chelation of trace copper with BCS (10 gLM) in washed platelet suspensions reduced the inhibition of thrombin-induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti-aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3 The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 x 109 1 -'. 4 In BCS-treated platelet suspensions, the addition of Cu2" ions (0.37-2.37 fiM) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5 The anti-aggregatory activity of GSNO was reduced in a concentration-dependent manner by the copper (I)-specific chelators BCS and neocuproine, and to a smaller extent by desferal.No effect was observed with the copper (II) specific chelator, cuprizone, the iron-specific chelator, bathophenanthroline sulphonate, or the broader-specificity copper chelator, D-penicillamine. 6 In both BCS-treated and -untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS-treated platelet suspensions there was no significant difference between the anti-aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7 The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absorbance at 334 nm. In Tyrode buffer, cysNO (10 !LM) broke down at a rate of 3.3 tAM min-'. BCS (10 tLM) reduced this to 0.5 ZlM min-'. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS. 8 We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti-aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S-nitrosothiol breakdown. The enhancement by copper of th anti-aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.

Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses

“…Decomposed DEA͞NO (10 M), on its own or in the presence of 1 M DEA͞NO, did not inf luence cGMP accumulation. Washed human platelets were prepared and incubated as described (15). When used, phosphodiesterase (PDE) inhibitors were added 10 min before DEA͞NO.…”

A New Pathway of Nitric Oxide/Cyclic GMP Signaling InvolvingS-Nitrosoglutathione

“…As shown in Fig. 7A, the endotheliumdependent vasodilator bradykinin stimulated a N G -nitro-L-arginine (L-NNA)-sensitive release of cGMP into the coronary effluent, which was strongly inhibited by the Cu ϩ -selective chelators neocuproine and BCS but not by the Cu 2ϩ chelator cuprizone (38). To exclude that these effects were due to an interference of the drugs with sGC stimulation or cGMP outward transport, similar experiments were performed with the NONOate SPER/NO, which is expected to activate cardiac sGC directly.…”

“…It is well established that GSNO decomposition is catalyzed by Cu ϩ ions (29,38,67,68). Although a marked mobilization of redoxactive copper occurs in myocardial ischemia/reperfusion injury (69), the cellular availability of Cu ϩ is probably limited under physiological conditions, suggesting that enzymatic pathways of GSNO decomposition may be more relevant than the nonenzymatic Cu ϩ mechanism.…”

S‐nitrosothiols as selective antithrombotic agents – possible mechanisms

“…The susceptibility of GSNO's antiplatelet action to NO scavenging by haemoglobin suggests instead that it undergoes platelet-mediated metabolism, either by a (so far uncharacterized) copper-dependent surface enzyme (Gordge et al, 1995;, or by csPDI, which is known to be present on platelets (Essex et al, 1995) and capable of releasing NO (Root et al, 2004). If differences exist in csPDI expression between platelets and vascular cells, then it might be possible to exploit this to provide selective antithrombotic action.…”