Alpha-tocopherol amplifies phosphorylation of endothelial nitric oxide synthase at serine 1177 and its short-chain derivative trolox stabilizes tetrahydrobiopterin.
Alpha-tocopherol has been shown to increase nitric oxide (NO)-dependent relaxation but the underlying mechanisms have not been fully characterized. The present study investigates the effect of alpha-tocopherol and its derivative trolox on the synthesis of NO in human umbilical vein endothelial cells. NO was assayed as citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) on ionomycin stimulation of cells. Ionomycin induced citrulline and cGMP formation partially through phosphorylation of endothelial NO synthase (eNOS) at its serine residue 1177, which was mediated mainly by calmodulin-dependent kinase II. Preincubation of cells with alpha-tocopherol or trolox increased eNOS activity in a concentration-dependent manner without changing eNOS expression. The effect of the water-soluble trolox was due to chemical stabilization of the eNOS cofactor tetrahydrobiopterin. On the contrary, alpha-tocopherol, located mainly in cellular membranes, did not affect tetrahydrobiopterin but increased ionomycin-induced eNOS phosphorylation at serine 1177. The effects of alpha-tocopherol on citrulline and cGMP formation and eNOS phosphorylation were amplified by co-incubation with ascorbate, which is suggested to regenerate oxidized alpha-tocopherol and to act synergistically with alpha-tocopherol. Our data describe a new vasoprotective function of alpha-tocopherol that may contribute to the prevention of endothelial dysfunction in vivo.
Institute of Molecular Cell Biology, Friedrich-Schiller-University of Jena, 99089 Erfurt, Germany. heller@zmkh.ef.uni-jena.de
Regine
R
Heller
Martin
M
Hecker
Nadine
N
Stahmann
Jens J
JJ
Thiele
Gabriele
G
Werner-Felmayer
Ernst R
ER
Werner
D000975:Antioxidants / Q000494:pharmacology; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism*; D002478:Cells, Cultured; D002839:Chromans / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine; D014471:Umbilical Veins; D024502:alpha-Tocopherol / Q000494:pharmacology*
D000975:Antioxidants; D002839:Chromans; D001708:Biopterins; D012694:Serine; D015759:Ionomycin; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D024502:alpha-Tocopherol; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid