Norfuraneol dephosphorylates eNOS at threonine 495 and enhances eNOS activity in human endothelial cells.
OBJECTIVE
Pentoses are widely abundant in organic food. Thermal treatment of pentoses leads to the formation of norfuraneol (NF). The aim of this study was to show whether NF, which is taken up regularly, for example with cooked food, affects the human endothelial nitric oxide synthase (eNOS) system.


RESULTS
The study was performed using cultured human umbilical vein endothelial cells (HUVEC), HUVEC-derived EA.hy926 cells, and bovine aortic endothelial cells. Nitric oxide (NO) release and eNOS activity were measured using diaminofluorescein-2 and [14C]L-arginine/[14C]L-citrulline conversion. Levels of (phospho-)eNOS were detected by western blotting. Reactive oxygen species (ROS) production was assessed using 2',7'-dichlorodihydrofluorescein diacetate. Pharmacokinetic parameters of NF were calculated by VolSurf software. NF dose dependently increased eNOS activity and NO release (30-300 microM), but did not affect total eNOS protein or cellular ROS levels. The increase in eNOS activity coincided with specific dephosphorylation of eNOS-Thr495, known to enhance eNOS activity. Inhibition of protein phosphatase 1 (PP1) by calyculin A, tautomycetin, or siRNA against PP1 reversed NF-induced eNOS-Thr495 dephosphorylation. Phosphorylation at eNOS-Ser1177 was not significantly altered by NF. Inhibition of protein kinase C with bisindolylmaleimide I (GFX) or calphostin C mimicked the effect of NF. In contrast to GFX, however, NF had no effect on phorbol-12-myristate-13-acetate-induced endothelial ROS formation. In silico, NF is stable towards CYP3A4 metabolism, shows low protein binding, and high tissue distribution.


CONCLUSIONS
NF enhances endothelial NO release most likely by promoting specific dephosphorylation of eNOS-Thr495 via PP1 in vitro and may be a promising compound to enhance endothelial function in vivo.
Schmitt
Christoph A
CA
Department of Pharmacognosy, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
Heiss
Elke H
EH
Aristei
Yasmin
Y
Severin
Theodor
T
Dirsch
Verena M
VM
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003198:Computer Simulation; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005663:Furans / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D008954:Models, Biological; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D013912:Threonine; D013997:Time Factors; D014018:Tissue Distribution
D004791:Enzyme Inhibitors; D005663:Furans; D017382:Reactive Oxygen Species; D013912:Threonine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D011493:Protein Kinase C; D054645:Protein Phosphatase 1; C113254:4-hydroxy-5-methyl-3(2H)-furanone