(-)-Epicatechin Activation of Endothelial Cell Endothelial Nitric Oxide Synthase, Nitric Oxide, and Related Signaling Pathways

Recent reports indicate that (-)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)- mediated nitric oxide production in endothelial cells. However, the mechanism by which (-)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (-)-epicatechin- induced effects on eNOS, using human coronary artery endothelial cells in culture. Treatment of cells with (-)-epicatechin led to time- and dose-dependent effects that peaked at 10 minutes at 1 mu mol/L. (-)-Epicatechin treatment activates eNOS via serine 633 and serine 1177 phosphorylation and threonine 495 dephosphorylation. Using specific inhibitors, we have established the participation of the phosphatidylinositol 3-kinase pathway in eNOS activation. (-)-Epicatechin induces eNOS uncoupling from caveolin-1 and its association with calmodulin-1, suggesting the involvement of intracellular calcium. These results allowed us to propose that (-)-epicatechin effects may be dependent on actions exerted at the cell membrane level. To test this hypothesis, cells were treated with the phospholipase C inhibitor U73122, which blocked (-)-epicatechin- induced eNOS activation. We also demonstrated inositol phosphate accumulation in (-)-epicatechin- treated cells. The inhibitory effects of the preincubation of cells with the calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 indicate that (-)-epicatechin-induced eNOS activation is at least partially mediated via the Ca2+/CaMKII pathway. The (-)-epicatechin stereoisomer catechin was only partially able to stimulate nitric oxide production in cells. Together, these results strongly suggest the presence of a cell surface acceptor-effector for the cacao flavanol (-)-epicatechin, which may mediate its cardiovascular effects. (Hypertension. 2010;55:1398-1405.)