Antagonistic Action of Imidazolineoxyl N-Oxides against Endothelium-Derived Relaxing Factor/*NO through a Radical Reaction

A labile inorganic free radical, nitric oxide (*NO), is produced by nitric oxide synthase from the substrate L-arginine in various cells and tissues. It acts as an endothelium-derived relaxing factor (EDRF) or as a neurotransmitter in vivo. We investigated the reactivity of stable radical compounds, imidazolineoxyl N-oxides such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), carboxy-PTIO, and carboxymethoxy-PTIO against *NO/EDRF in both chemical and biological systems. By using electron spin resonance (ESR) spectroscopy, imidazolineoxyl N-oxides were found to react with *NO in a stoichiometric manner (PTIO/*NO = 1.0) in a neutral solution (sodium phosphate buffer, pH 7.4) with rate constants of −10⁴ M⁻¹ s⁻¹, resulting in the generation of NO₂⁻/NO₃⁻ and imidazolineoxyls such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI), carboxy-PTI, or carboxymethoxy-PTI. Furthermore, the effects of imidazolineoxyl N-oxides on acetylcholine- or ATP-induced relaxation of the smooth muscle of rabbit aorta were tested. The vasorelaxations were inhibited by all three imidazolineoxyl N-oxides markedly. The inhibitory effects of carboxy-PTIO was almost 2-fold stronger than those of *NO synthesis inhibitors, N^ω-nitro-L-arginine and N^ω-monomethyl-L-arginine. Generation of EDRF/*NO was identified by reacting the PTIO in aortic strips and quantitating the reaction product with ESR spectroscopy. Thus, it was clarified that imidazolineoxyl N-oxide antagonize EDRF/*NO via a unique radical-radical reaction with *NO.