on September 13, 2007
Published online before print September 13, 2007, doi: 10.1161/ATVBAHA.107.153742
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Submitted on November 20, 2006
Accepted on August 24, 2007
4-Hydroxy-2-Nonenal Increases Superoxide Anion Radicalin Endothelial Cells via Stimulated GTP Cyclohydrolase Proteasomal Degradation
Jennifer Whitsett ;
From the Department of Biophysics (J.W.), and Free Radical Research Center (J.V.V.), Medical College of Wisconsin, Milwaukee; and the Department of Pharmacology, Physiology, and Therapeutics (M.J.P.), University of North Dakota, Grand Forks.
* To whom correspondence should be addressed. E-mail: jvvivar{at}mcw.edu.
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).
Methods and 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.
Key words: tetrahydrobiopterin • eNOS phosphorylation • 2-hydroxyethidium • glutathione • ascorbate
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