α‐Tocopherol and Endothelial Nitric Oxide Synthesis

Nitric oxide (NO), a central regulator of vascular tone and homeostasis, is generated upon activation of endothelial NO synthase (eNOS), which is mediated by an increase of intracellular calcium and/or by eNOS phosphorylation. A reduction of NO bioavailability leads to endothelial dysfunction that has been shown to be improved by alpha-tocopherol in certain conditions. The underlying mechanisms, however, are not completely clarified. The present study was performed to investigate whether alpha-tocopherol is able to affect endothelial NO synthesis. The formation of NO was measured in human umbilical vein endothelial cells using citrulline (coproduct) and cGMP (product of the NO-activated soluble guanylate cyclase) as indicator molecules. alpha-Tocopherol (10-200 microM, 24 hr) increased ionomycin-induced citrulline and cGMP formation in intact cells in a concentration-dependent manner. In parallel, ionomycin-stimulated phosphorylation of eNOS at serine 1177, known to support enzyme activation, was increased by alpha-tocopherol, suggesting that this was the mechanism responsible for enhanced NO formation. The effect of alpha-tocopherol was dependent on its hydrophobic structure because it was mimicked by gamma-tocopherol but not by trolox, a hydrophilic derivative of alpha-tocopherol. Coincubation with ascorbic acid (100 microM, 24 hr) amplified the effects of alpha-tocopherol on eNOS phosphorylation and NO formation, which is possibly related to the regeneration of oxidized alpha-tocopherol by ascorbate. Our data suggest that vasoprotective effects of alpha-tocopherol in vivo may be related to an increase of NO formation. The effect of alpha-tocopherol seems to be dependent on tissue saturation with ascorbic acid, and both vitamins may act synergistically to provide optimal conditions for endothelial NO formation.