Tannin 1-α-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatildylinositol 3-kinase/Akt pathway in endothelial cells

Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures , punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca2+ concentration preceded NO production. The Ca2+ ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pre-treatment with Ca2+ chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloyipunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK) 1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.