Regulation by mitochondrial superoxide and nadph oxidase of cellular formation of nitrated cyclic GMP: Potential implications for NO and ROS signaling

Abstract

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of oxidative stress response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of reactive oxygen species (ROS). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of hydrogen peroxide were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and hydrogen peroxide. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that NADPH oxidase 2 (Nox2)-generated hydrogen peroxide regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent hydrogen peroxide production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signaling in the presence of NO.