Electron spin resonance and chemiluminescence analyses to elucidate the vasodilating mechanism of sodium nitroprusside

The objective of this study was to elucidate, both in in vitro and in ex vivo models, the mechanism of action of the well known vasodilating agent sodium nitroprusside (SNP). An in vivo experimental model based on the i.v. infusion of SNP solution (2 mM, 0.83 ml/min for 24 min, corresponding to a dose of 1.67 mmoli/Kg) in the anesthetized pig has been developed, and the following parameters monitored: 1) blood levels of HbFe(II)NO by Electron Spin Resonance (ESR) analysis as an index of the bioconservative pathway; 2) methemoglobin (ESR analysis) and plasma levels of nitrates (chemiluminescence analysis) as markers of NO dependent oxidative pathway. The results indicate that: a) only »17 % of the administered dose is converted to HbFe(II)NO (Cmax 4.15 ± 0.69 vs. 0.80 ± 0.52 μM) at the end of infusion; b) no formation of methemoglobin is observed in SNP-treated animals, to indicate that NO administered as SNP does not undergo bioinactivation (oxidative metabolism); d) SNP induces a significant reduction in systemic arterial pressure and the vasodilating effect starts from the beginning of the infusion. Since the administration of an equimolar dose of genuine NO is efficiently converted to HbFe(II)NO without inducing any vasodilating response, the hemodynamic effect induced by SNP is not mediated by HbFe(II)NO, at least under physiological conditions; hence a direct release of NO form SNP in the vascular target should be considered. To demonstrate this mechanism, we developed an in vitro model based on endothelial cells (EA.hy926 cell lines or isolated from umbilical cord), incubated with different amounts of SNP. The release of NO from the bioactivation of SNP by endothelial cells has been determined by stripping NO from the culture medium through an helium flow, able to transport the released NO directly in the chemiluminescence analyser. The results indicate that the endothelium is able to metabolize SNP, with the formation of stoichiometric amounts of NO, the agent responsible for vasodilation. Hence the amount of the administered drug that escapes the bioconservative pathway (more than 80% of the dose), reaches the endothelium: this means that the pharmacological response of SNP is mediated by a direct mechanism of NO release/metabolization of the parent compound at the cellular target