Quantitation of peroxynitrite production by macrophages

Reaction of peroxynitrite with coumarin boronic acid [1] was used for quantitation of peroxynitrite produced by activated murine macrophages (RAW 264.7) in cell culture. Highly fluorescent 7-hydroxycoumarin produced in reaction leads to fluorescence increase, affording real-time detection. Quiescent cells exhibit low rate of fluorescence increase which is further decreased in the presence of catalase, while catalase does not significantly affect the rate for activated cells.  Specificity of the assay is supported by the dependence of the rate on the presence of arginine in the medium, inhibition by N 6 -(1-iminoethyl) L-lysine (inhibitor of iNOS) and by ebselen (peroxynitrite scavenger). The rate of peroxynitrite generation by stimulated RAW 264.7 was estimated as ~0.5 amol/(cell·s) that can be compared with NO generation rate of ~ 1.7 amol/(cell·s) determined earlier for the same cells [2]. Under the activation scheme used in this work (gamma-interferon + LPS), human THP-1 derived macrophages do not produce any peroxynitrite detectable by coumarin boronic acid. The level of iNOS mRNA in activated THP-1 derived macrophages does not exceed the level in control cells. 1 Zielonka, J.; Sikora, A.; Hardy, et al. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chemical Research in Toxicology. (2012), 25 (9), 1793–1799. 2 Woldman, Y. Y., Eubank, T. D., Mock, A. J., et al. Detection of nitric oxide production in cell cultures by luciferine-luciferase chemiluminescence. Biochemical and Biophysical Research Communications (2015), 465 232-238