Under hypoxic conditions, NO plays an important role in regulating O 2 delivery by controlling local blood vessel relaxation. NO is primarily produced by endothelial NO synthase (eNOS), which is reportedly phosphorylated at Ser 635 and thereby activated under conditions of fluid shear stress and O 2 concentration. The aim of this work was to investigate the effects of fluid shear stress and O 2 concentration on the phosphorylation of eNOS at Ser 635 . Bovine aortic endothelial cells were exposed to hypoxia only, a combination of shear stress and hyperoxia, and a combination of shear stress and hypoxia at various time points. Hypoxia did not increase phosphorylation significantly at 15 min but induced a gradual increase to 1.94-fold over 180 min. Under simultaneous exposures to shear stress and hyperoxia, eNOS phosphorylation was detected after 15-min and was 2.75-fold higher than the initial condition at the 60-min time point. In contrast, under a combination of shear stress and hypoxia, eNOS phosphorylation was increased to 2.44-fold at 60 min. However, although phosphorylation levels under these conditions were higher than those after hypoxia only at all time points, they were lower than those after a combination of shear stress and hyperoxia. Our results indicate that hyperoxia and shear stress additively stimulate phosphorylation of eNOS at Ser 635 and suggest a moderating role of hypoxia.
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