Nitric oxide promotes recycling of 8-nitro-cGMP, a cytoprotective mediator, into intact cGMP in cells.

8-Nitro-cGMP is an endogenous nucleotide discovered under inflammation conditions as an important mediator of nitric oxide (NO) signaling. Besides cGMP-like behaviour, 8-nitro-cGMP exerts unique cytoprotective effects against oxidative stress. Although the formation of 8-nitro-cGMP from 8-nitro-GTP has previously been proposed, the mechanism by which excess or unused 8-nitro-cGMP is removed from cells remains unknown. In this study, we report a nitric oxide-dependent cellular conversion of 8-nitro-cGMP to intact cGMP in RAW 264.7 macrophage cells. In our experiments, we synthesized isotopically labeled 8-nitro-cGMP as a tool for metabolites analysis and identified 8-amino-cGMP as an initial metabolite of 8-nitro-cGMP using a LC-MS/MS technique. We also proved that endogenous 8-nitro-cGMP can be converted into 8-amino-cGMP by immunocytochemical staining with an antibody that specifically recognizes 8-amino-cGMP. Moreover, we showed that isotopically labeled 8-amino-cGMP is metabolized into cGMP under inflammation conditions. We propose that nitrosylation of 8-amino-cGMP occurs by NO formation under stress conditions and gives putative 8-diazonium-cGMP, which subsequently decomposes into cGMP. To the best of our knowledge, this study is the first to report reductive deamination of aminoguanine nucleotide at the C-8 position. The findings of this study collectively indicate that NO plays a crucial role not only in the production of 8-nitro-cGMP but also in its elimination under oxidative stress or inflammation.