EPR Detection of Glutathionyl and Protein-tyrosyl Radicals during the Interaction of Peroxynitrite with Macrophages (J774)

Abstract

Peroxynitrite is one of the biological oxidants whose addition to cells has been shown to either activate signaling pathways or lead to cell injury, depending on cell type and oxidant concentration. The intermediacy of free radicals in these processes has been directly demonstrated only during the interaction of peroxynitrite with erythrocytes, a particular cell type, due to its high hemoglobin content. Here, we demonstrate that the addition of peroxynitrite to a macrophage cell line (J774) led to the production of glutathionyl and protein-tyrosyl radicals. The glutathionyl radical was characterized by EPR spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide. Protein-tyrosyl radical formation was suggested by direct EPR spectroscopy and confirmed by EPR spin-trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid and Western blot analysis of nitrated proteins in treated macrophages. Time dependence studies of free radical formation indicate that intracellular glutathione and unidentified proteins are the initial peroxynitrite targets in macrophages and that their derived radicals trigger radical chain reactions. The results are likely to be relevant to the understanding of the bioregulatory and biodamaging effects of peroxynitrite.