4-Hydroxy-2-nonenal increases superoxide anion radical in endothelial cells via stimulated GTP cyclohydrolase proteasomal degradation.
OBJECTIVE
4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs).


RESULTS
Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH4) is a limiting factor in non supplemented cells. This was verified by quantification of BH4 by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH4 by 4-HNE is attributable to specific mechanisms involving protein degradation.


CONCLUSIONS
4-HNE by altering BH4 homeostasis mediates eNOS-uncoupling and superoxide generation in BAECs. By also decreasing phosphorylation of eNOS-serine 1179 4-HNE may specifically regulate NO/reactive oxygen species fluxes in the endothelium with important consequences to redox signaling.
Whitsett
Jennifer
J
Department of Biophysics, Medical College of Wisconsin, Milwaukee, USA.
Picklo
Matthew J
MJ
Vasquez-Vivar
Jeannette
J
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000502:physiology; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D006136:GTP Cyclohydrolase / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000502:physiology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D000447:Aldehydes; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D046988:Proteasome Endopeptidase Complex; D006136:GTP Cyclohydrolase; C003402:sapropterin; C027576:4-hydroxy-2-nonenal