PO(2)-dependent NO Production Determines OPPC Activity in Macrophages

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O(2), L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO(2) on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon gamma (IFNgamma) at pO(2) ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO(2)>0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O(2), without decreasing NOx production. These results are the first to investigate the effect of pO(2) on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.