Protein tyrosine nitration in cytokine-activated murine macrophages - Involvement of a peroxidase/nitrite pathway rather than peroxynitrite

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O . (-)(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O . (-)(2), H2O2, peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O . (-)(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at similar to6 and similar to9 h after stimulation with interferon-gamma /lipopolysaccharide and interferon-gamma /zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 mum 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O . (-)(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.