Hypoxia causes increased monocyte nitric oxide synthesis which is mediated by changes in dimethylarginine dimethylaminohydrolase 2 expression in animal and human models of normobaric hypoxia.
Tissue hypoxia is a cardinal feature of inflammatory diseases 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.



Peritoneal 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%. Healthy 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.



Intracellular nitrate plus nitrite(NOx) concentration was higher in wild-type murine monocytes after hypoxia and reoxygenation than in normoxia-treated cells (mean(SD) 13·2(2·4) vs 8·1(1·7) pmols/mg protein, p = 0·009). 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, 0·33(0.04) pmols/mg 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 NOx concentration was elevated after hypoxic exposure (3·6(1.8)μM vs 6·4(3·2), p = 0·01), which was associated with a reduction in intracellular ADMA in paired samples from 3·6(0.27) pmols/mg protein to 3·15(0·3) (p < 0·01). This finding was associated with a 1·9-fold(0·6) increase in DDAH2 expression over baseline(p = 0·03).



This 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.
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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S
Lambden
Centre for Altitude, Space and Extreme Environment Medicine, UCL, London, UK.
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D
Martin
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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K
Vanezis
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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B
Lee
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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J
Tomlinson
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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S
Piper
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK.
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O
Boruc
Centre for Altitude, Space and Extreme Environment Medicine, UCL, London, UK.
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M
Mythen
Nitric Oxide Signalling Group, Clinical Sciences Centre, MRC, Hammersmith Hospital, London, UK. Electronic address: james.leiper@csc.mrc.ac.uk.
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J
Leiper
D000293:Adolescent; D000328:Adult; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood; D015687:Cell Hypoxia; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008297:Male; D018345:Mice, Knockout; D008875:Middle Aged; D009000:Monocytes / Q000502:physiology*; D009566:Nitrates / Q000097:blood; D009569:Nitric Oxide / Q000096:biosynthesis*; D009573:Nitrites / Q000097:blood; D055815:Young Adult
D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; C018524:N,N-dimethylarginine; D001120:Arginine; D000581:Amidohydrolases; C059999:dimethylargininase
Asymmetric dimethylarginine; Dimethylarginine dimethylaminohydrolase; Hypoxia; Nitric oxide