Redox-sensitive mechanism of no scavenging by nitronyl nitroxides

Nitronyl nitroxides, NN•, have been increasingly used in the field of NO-related studies as specific antagonists of NO•. We employed a combination of EPR and NMR spin trapping to study the mechanisms of the reaction of NN• with NO• in reducing environments. EPR allowed observation of NO-induced transformation of the paramagnetic trap, NN•, to the corresponding iminonitroxide, IN•. In a complementary way, corresponding EPR-invisible diamagnetic products (the hydroxylamines NN–H and IN–H) were detected by 19F-NMR using newly synthesized fluorinated traps. Addition of reducing agents to a solution of NN• resulted in fast disappearance of its EPR spectra and appearance of a 19F-NMR peak of the corresponding hydroxylamine, NN–H. Addition of NO• as a bolus, or NO• generated on sodium nitroprusside photolysis, resulted in 19F-NMR-detectable accumulation of the hydroxylamine, IN–H. Upon high rates of NO• generation in ascorbate-containing solutions, partial recovery of NN• was observed, which undergoes further reactions with NO• and ascorbate in a competitive manner. Using 19F-NMR and a fluorinated trap, NO-induced conversion of NN–H into IN–H was also observed in vivo in hypertensive ISIAH rats compared with normotensive WAG rats. The results provide insight into a new potential redox-sensitive mechanism of the antagonistic action of NN• against NO•, which may provide insight into previously unexplained behavior of this category of NO-reacting compounds.