20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells

Meetha Medhora, Yuenmu Chen, Stephanie Gruenloh, Daniel Harland, Sreedhar Bodiga, Jacek Zielonka, Debebe Gebremedhin, Ying Gao, J R Falck, Siddam Anjaiah, Elizabeth R. Jacobs

Research output: Contribution to journalArticle

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Abstract

Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20- hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47phox and tyrosine phosphorylation of p47phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume294
Issue number5
DOIs
StatePublished - May 2008

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Superoxides
Pulmonary Artery
Oxidoreductases
Endothelial Cells
Reactive Oxygen Species
NADPH Oxidase
Cell Growth Processes
Fluorescence
20-hydroxy-5,8,11,14-eicosatetraenoic acid
Cell Physiological Phenomena
Nitric Oxide Synthase Type III
Polyethylene
Vascular Smooth Muscle
Cytochrome P-450 Enzyme System
Blood Vessels
Tyrosine
Aorta
Phosphorylation
Lipids
Lung

Keywords

  • CYP4A
  • Hydrogen peroxide
  • Rac1/2
  • Reactive oxygen species
  • Superoxide

ASJC Scopus subject areas

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

Cite this

20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells. / Medhora, Meetha; Chen, Yuenmu; Gruenloh, Stephanie; Harland, Daniel; Bodiga, Sreedhar; Zielonka, Jacek; Gebremedhin, Debebe; Gao, Ying; Falck, J R; Anjaiah, Siddam; Jacobs, Elizabeth R.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 294, No. 5, 05.2008.

Research output: Contribution to journalArticle

Medhora, Meetha ; Chen, Yuenmu ; Gruenloh, Stephanie ; Harland, Daniel ; Bodiga, Sreedhar ; Zielonka, Jacek ; Gebremedhin, Debebe ; Gao, Ying ; Falck, J R ; Anjaiah, Siddam ; Jacobs, Elizabeth R. / 20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells. In: American Journal of Physiology - Lung Cellular and Molecular Physiology. 2008 ; Vol. 294, No. 5.
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AU - Medhora, Meetha

AU - Chen, Yuenmu

AU - Gruenloh, Stephanie

AU - Harland, Daniel

AU - Bodiga, Sreedhar

AU - Zielonka, Jacek

AU - Gebremedhin, Debebe

AU - Gao, Ying

AU - Falck, J R

AU - Anjaiah, Siddam

AU - Jacobs, Elizabeth R.

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AB - Reactive oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20- hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time- and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47phox and tyrosine phosphorylation of p47phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid.

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