Modulation of low shear stress‑induced eNOS multi‑site phosphorylation and nitric oxide production via protein kinase and ERK1/2 signaling.

Physiological shear stress has been demonstrated to serve an atheroprotective function by stimulating endothelial nitric oxide synthase (eNOS) multi‑site phosphorylation. Low shear stress (LSS) serves an atheroprone role by increasing endothelial cell apoptosis and inflammation. The present study assessed whether LSS inhibited nitric oxide (NO) production in human umbilical vein endothelial cells by modulating eNOS phosphorylation and potential signaling pathways. A parallel flow chamber imposed with 2 dyn/cm2 shear stress on endothelial cells was used. Western blotting and 4,5‑diaminofluorescein diacetate were used to analyze the protein expression levels and NO production. LSS activated eNOS‑Ser1177 and eNOS‑Thr495, but inhibited eNOS‑Ser633. NO production was decreased after a transient increase at 5 min. LSS‑stimulated phosphorylation of eNOS‑Ser1177 and ‑Thr495 were suppressed by the Akt inhibitor, perifosine, and extracellular signal regulated kinases1/2 (ERK1/2) inhibitor, PD98059, respectively. Additionally, the phosphorylation of eNOS‑Ser633 inhibited by LSS was restored by the protein kinase A activator, 8‑Bromo‑cAMP. PD98059 completely inhibited the LSS‑induced downregulation of NO production. NO downregulation in response to LSS was intensified by perifosine and was partly inhibited by 8‑Bromo‑cAMP. These results indicated that LSS‑induced activation of ERK1/2/eNOS‑Thr495 serves a major role in inhibiting endothelial NO synthase, which may explain the proinflammatory and proatherosclerotic properties of LSS.