Methodological proof of immunochemistry for specific identification of 8-nitroguanosine 3′,5′-cyclic monophosphate formed in glia cells

The biological significance of nitrated guanine derivatives, especially 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), has become evident. Therefore it is important to determine the presence and relative abundance of 8-nitro-cGMP formed in cells and tissues. In the present study, we performed immunocytochemistry with monoclonal antibodies specific for 8-nitroguanine (clone NO2-52) and 8-nitro-cGMP (clone 1G6) in rat C6 glioma cells and rat primary cultured astrocytes. Immunocytochemistry utilizing the anti-8-nitro-cGMP monoclonal antibody (1G6) indicated that immunostaining increased markedly in C6 cells expressing increased amounts of inducible nitric oxide synthase (iNOS) after treatment with lipopolysaccharide (LPS) plus cytokines. Treatment of C6 cells with inhibitors for NOS and soluble guanylate cyclase (sGC) completely nullified the elevated 1G6 immunoreactivity. These results were consistent with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Immunocytochemistry performed using NO2-52 also showed that treatment of cells with inhibitors for NOS and sGC completely nullified the elevated immunoreactivity; this indicated that 8-nitro-cGMP is a major component of 8-nitroguanine derivatives produced in cells. Similar results were obtained in the primary astrocytes stimulated with LPS plus cytokines. Because immunocytochemistry is a conventional, powerful, and fairly straightforward method for determining the presence, localization, and relative abundance of an antigen of interest in cultured cells, anti-8-nitroguanine (NO2-52) and anti-8-nitro-cGMP (1G6) antibodies could be useful tools for analyzing nitrated guanine nucleotides, especially 8-nitro-cGMP, by means of immunocytochemistry.