Hepatocyte growth factor (HGF) causes endothelium-dependent vasodilation, but its relation to endothelial nitric oxide synthase (eNOS) activity remains to be elucidated. Treatment of bovine aortic endothelial cells with HGF increased eNOS activity within minutes, accompanied by an increase of activity-related site-specific phosphorylation of eNOS. The phosphorylation was completely abolished by pretreatment of the cells with a phosphoinositide 3-kinase (PI3K) inhibitor (wortmannin) and by transfection of dominant-negative Akt, and the enzyme activity was inhibited by wortmannin. In addition, eNOS activity and phosphorylation were abolished by pretreatment of the cells with an intracellular Ca2+-chelator, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM), with a suppression of Akt phosphorylation. These results suggest that HGF stimulates eNOS activity by a PI3K/Akt-dependent phosphorylation in a Ca2+-sensitive manner in vascular endothelial cells.
Abbreviations used: BAEC, bovine aortic endothelial cells; BAPTA/AM, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester); CaM, calmodulin; HGF, hepatocyte growth factor; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; l-NAME, NG-nitro-l-arginine methyl ester; NO, nitric oxide; eNOS, endothelial NO synthase; iNOS, inducible NO synthase; PI3K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor.