Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase.

Background— Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality. Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression. The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase–induced actin cytoskeletal changes in vascular endothelial cells. Methods and Results— To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O2) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 μmol/L), for various durations (0 to 48 hours). Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66±3% and 57±5%, and inhibited eNOS activity by 48±9%. All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase...