Abstract
Endothelial dysfunction and activation occur in the vasculature and are believed to contribute to the pathogenesis of cardiovascular diseases. We have shown that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a cytochrome P450 4A-derived eicosanoid that promotes vasoconstriction in the microcirculation, uncouples endothelial nitric-oxide synthase (eNOS) and reduces nitric oxide (NO) levels via the dissociation of the 90-kDa heat shock protein (HSP90) from eNOS. It also causes endothelial activation by stimulating nuclear factor-κB (NF-κB) and increasing levels of pro-inflammatory cytokines. In this study, we examined signaling mechanisms that may link 20-HETE-induced endothelial dysfunction and activation. Under conditions in which 20-HETE inhibited NO production, it also stimulated inhibitor of NF-κB (IκB) phosphorylation. Both effects were prevented by inhibition of tyrosine kinases and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). It is noteworthy that inhibitor of IκB kinase (IKK) activity negated the 20-HETE-mediated inhibition of NO production. Immunoprecipitation experiments revealed that treatment of ionophore-stimulated cells with 20-HETE brings about a decrease in HSP90-eNOS association and an increase in HSP90-IKKβ association, suggesting that the activation by 20-HETE of NF-κB is linked to its action on eNOS. Furthermore, addition of inhibitors of tyrosine kinase MAPK and IKK restored the 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries. The results indicate that 20-HETE mediates eNOS uncoupling and endothelial dysfunction via the activation of tyrosine kinase, MAPK, and IKK, and these effects are linked to 20-HETE-mediated endothelial activation.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL34300] (to M.L.S.); the National Institutes of Health National Institute of General Medical Sciences [Grant GM31278] (to J.R.F.); the Robert A. Welch Foundation (to J.R.F.); and a American Heart Association Predoctoral Fellowship [Grant 0715781T] (to J.C.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159863
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ABBREVIATIONS:
- NO
- nitric oxide
- eNOS
- endothelial nitric-oxide synthase
- SMC
- smooth muscle cell
- 20-HETE
- 20-hydroxyeicosatetraenoic acid
- HSP90
- 90-kDa heat shock protein
- MAPK
- mitogen-associated protein kinase
- ERK
- extracellular signal-regulated kinase
- BAEC
- bovine aortic endothelial cell
- FBS
- fetal bovine serum
- l-NMMA
- NG-monomethyl-l-arginine
- AG82
- cyano-(3,4,5-trihydroxy)cinnamonitrile
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene
- 17-AAG
- 17-(allylamino)-17-demethoxygeldanamycin
- SC514
- 4-amino-[2′,3′-bithiophene]-5-carboxamide
- NBD
- NEMO-binding domain peptide
- NEMO
- NF-κB essential modulating protein
- A23187
- calcium ionophore
- tiron
- 4,5-dihydroxy-1,3-benzene disulfonic acid
- IKK
- inhibitor of IκB kinase
- EET
- epoxyeicosatrienoic acid
- EGFR
- epidermal growth factor receptor
- IKK
- IκB kinase
- IκB
- inhibitor of NF-κB.
- Received August 3, 2009.
- Accepted October 16, 2009.
- © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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