The effects of fluid shear stress and O2 concentration on the phosphorylation of eNOS at Ser635 in endothelial cells

Abstract

Under hypoxic conditions, NO plays an important role in regulating O₂ delivery by controlling local blood vessel relaxation. NO is primarily produced by endothelial NO synthase (eNOS), which is reportedly phosphorylated at Ser⁶³⁵ and thereby activated under conditions of fluid shear stress and O₂ concentration. The aim of this work was to investigate the effects of fluid shear stress and O₂ concentration on the phosphorylation of eNOS at Ser⁶³⁵. 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 Ser⁶³⁵ and suggest a moderating role of hypoxia.