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 microM) 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 microM. Endothelial cells cultured on microcarriers 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 (3-300 nM), decreased by preincubation of NG-monomethyl-L-arginine (3-100 microM) and reversed by following incubation of L-arginine (100 microM).