2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells

Graphical abstract

Abstract

Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-( E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity.

DPPB (1–10 μM) treatment for 24 h induced a significant and dose-dependent increase in eNOS activity as determined by the [ ¹⁴C] l-arginine/[ ¹⁴C] l-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser ¹¹⁷⁷ and decrease in eNOS-Thr ⁴⁹⁵ phosphorylation, as well as increased AMPK phosphorylation at Thr ¹⁷², whereas Akt phosphorylation at Ser ⁴⁷³ was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca ²⁺ chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser ¹¹⁷⁷ and AMPK-Thr ¹⁷² phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca ²⁺ concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca ²⁺ levels and increases signaling through a CaMKKβ–AMPK dependent pathway.