Mechanisms of activation of eNOS by 20-HETE and VEGF in bovine pulmonary artery endothelial cells

Yuenmu Chen, Meetha Medhora, John R. Falck, Kirkwood A. Pritchard, Elizabeth R. Jacobs

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

We have demonstrated that VEGF-induced dilation of bovine pulmonary arteries is associated with activation of cytochrome P-450 family 4 (CYP4) enzymes and eNOS. We hypothesized that VEGF and the CYP4 product 20-HETE would trigger common downstream pathways of intracellular signaling to activate eNOS. We treated bovine pulmonary artery endothelial cells (BPAECs) with 20-HETE (1 μM) or VEGF (8.3 nM) and examined three molecular events known to activate eNOS: 1) phosphorylation at serine 1179, 2) phosphorylation of protein kinase B (Akt), which subsequently phosphorylates eNOS, and 3) association of eNOS with 90-kDa heat shock protein (Hsp90). Both 20-HETE and VEGF increase the phosphorylation of eNOS at serine 1179 and Akt at serine 473. The CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) blocks VEGF-induced phosphorylation of eNOS. VEGF had no effect on the binding of Hsp90 with eNOS, whereas 20-HETE decreased the association of the protein partners. Inhibition of Akt-phosphatidylinositol 3-kinase with wortmannin blocks both 20-HETE and VEGF-induced relaxation of pulmonary arteries, supporting the functional contribution of Akt phosphorylation to the vasoactive actions of both agents. Treatment with radicicol had no effect on 20-HETE-induced relaxation of pulmonary arteries, consistent with an absence of effect on association of Hsp90 to eNOS, whereas radicicol partially blocked VEGF-evoked relaxations, possibly secondary to effects on endpoints other than Hsp90 association with eNOS. In conclusion, VEGF and 20-HETE share eNOS activation pathways, including phosphorylation of serine 1179 and phosphorylation of Akt. Unlike aortic endothelial cells, eNOS activation in BPAECs by either VEGF or 20-HETE does not appear to require increased association of Hsp90.

Original languageEnglish (US)
Pages (from-to)L378-L385
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume291
Issue number3
DOIs
StatePublished - Sep 13 2006

Keywords

  • Intracellular calcium concentration
  • Phosphorylation
  • VEGF-mediated dilation

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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