L-Arginine-dependent formation of N-nitrosamines by the cytosol of macrophages activated with lipopolysaccharide and interferon-γ

Abstract

The cytosol fraction of J774-1 murine macrophages activated with lipopolysaccharide (LPS) + interferon-_γ(IFN-_γ) was found to nitrosate a wide range of secondary and tertiary amines. The reaction was dependent on L-argmlne and NADPH. The optimal pH for nitrosation was 7.2–7.3. Nitrosation was inhibited by arginine derivatives such as monomethyl-L-arginine and N^G-nitro-L-arginine, well-known inhibitors of nitric oxide (NO) synthase. These results indicate that nitrosation is mediated by NO synthase, which catalyzes formation of NO and L-citrulline from L-arginine. Nitrosamine formation also required oxygen and was inversely correlated with the basicity of nitrosatable amines. The nitrosation was inhibited by oxyhemoglobin, an NO trapping agent, and enhanced by superoxide dismutase, which stabilizes NO. LPS + IFN-^γ induced ˜500˜600 times greater nitrosatlon activity than that of non-activated macrophages. Macrophages treated with LPS alone exhibited 3–4 times greater nitrosation activity than untreated macrophages, whereas macrophages treated with IFN-^γ alone did not show enhanced nitrosatlon activity. A combination of the cytosols from macrophages treated with LPS alone and IFN-^γ alone did not nitrosate morpholine as rapidly as the cytosol of macrophages treated with both compounds together. The activity for forming L-citrulline and nitrite/nitrate from L-arglnlne was markedly induced by treat ment with either LPS alone or LPS + IFN-^γ but not with IFN-^γ. Those results suggest that some other factor(s) in addition to NO synthase is involved for efficient nitrosation by the macrophage cytosol. This factor(s) was not induced in macrophages by either LPS- or IFN-^γ alone, but was induced only in the presence of the two compounds.