COPPER CHELATION-INDUCED REDUCTION OF THE BIOLOGICAL-ACTIVITY OF S-NITROSOTHIOLS

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 mu M) 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 10(9) 1(-1). 4 In BCS-treated platelet suspensions, the addition of Cu2+ ions (0.37-2.37 mu M) 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 mu M) broke down at a rate of 3.3 mu M min(-1). BCS (10 mu M) reduced this to 0.5 mu M min(-1). 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.