Reaction between imidazolineoxil N-oxide (carboxy-ptio) and nitric oxide released from cultured endothelial cells: Quantitative measurement of nitric oxide by ESR spectrometry

The performance of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide for the monitoring agent of nitric oxide was investigated. The agent (125–500 μM) was mixed with equal volume of nitric oxide solution, and aliquots of the mixture were applied to ESR spectroscopy. ESR spectra of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl, a product of the agent reacted with nitric oxide, were observed. A linear relationship was observed between the amplitude of the signal and concentrations of nitric oxide up to 80 μM. Endothelial cells cultured on microcarries were packed in a column, perfused with Krebs solutions and the effluent was mixed to the agent. The same ESR spectra were obtained and amplitude of the signal was increased by bradykinin (3–300nM), decreased by preincubation of N^G-monomethyl-L-arginine (3–100 μM) and reversed by following incubation of L-arginine (100 μM).