Role of dimethylarginine dimethylaminohydrolase 2 in the regulation of nitric oxide synthesis in animal and observational human models of normobaric hypoxia

Abstract

BackgroundTissue hypoxia is a cardinal feature of inflammation at the site of infection or systemically and modulates monocyte function. Nitric oxide is a crucial component of the immune cell response. This study explored the metabolism of the endogenous inhibitor of nitric oxide production asymmetric dimethylarginine (ADMA) by monocyte dimethylarginine dimethylaminohydrolase 2 (DDAH2), and the role of this pathway in the regulation of the cellular response and the local environment during hypoxia. MethodsPeritoneal macrophages were isolated from a macrophage-specific DDAH2 knockout mouse that we developed and compared with appropriate controls. Cells were exposed to 3% oxygen followed by reoxygenation at 21%. 15 healthy male volunteers underwent an 8 h exposure to normobaric hypoxia with an inspired oxygen percentage of 12%. Peripheral blood mononuclear cells were isolated from blood samples taken before and at the end of this exposure. Nitric oxide, DDAH2, and ADMA concentrations in monocytes were compared before and after hypoxic challenge in both murine and human models. FindingsIntracellular nitrate plus nitrite concentration was higher in wild-type murine monocytes after hypoxia and reoxygenation than in normoxia-treated cells (mean 13·2 pmols/mg protein [SD 2·4] vs 8·1 [1·7], p=0·009), and accumulation of extracellular nitric oxide increased after hypoxic challenge (13·7 μM [3·1] vs 1·9 [0·18], p=0·002). DDAH2 protein was 4·5-fold (SD 1·3) higher than in control cells (p=0·03). This increase led to a 24% reduction in ADMA concentration (mean 0·33 pmols/mg [SD 0·04] to 0·24 [0·03], p=0·002). DDAH2-deficient murine monocytes demonstrated no increase in nitric oxide production after hypoxic challenge. These findings were recapitulated in a human observational study. Mean plasma nitric oxide concentration was elevated after hypoxic exposure (3·6 μM [SD 1·8] vs 6·4 [3·2], p=0·01), which was associated with a 30% reduction in plasma ADMA (0·43 μM [0·13] to 0·29 [0·06], p=0·001) and a reduction in intracellular ADMA in paired samples (3·6 pmols/mg protein [0·27] to 3·15 [0·3], p=0·0009). This finding was mediated by a 1·9-fold (0·6) increase in DDAH2 expression over baseline (p=0·03). InterpretationThis study shows that in both human and murine models of acute hypoxia, increased DDAH2 expression mediates a reduction in intracellular ADMA concentration which in turn leads to elevated nitric oxide concentrations both within the cell and in the local environment. Cells deficient in DDAH2 were unable to mount this response. We suggest that this mechanism forms part of the early adaptive response to hypoxia and regulates changes in both cell function and the local microenvironment. FundingMedical Research Council, Royal College of Anaesthetists.