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

1 Pulmonary and Critical Care Medicine and Cardiovascular Center, 2 Department of Biophysics and Free Radical Research Center, and 3 Department of Physiology and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin; and 4 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas Submitted 18 July 2007 ; accepted in final form 13 February 2008 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 p47 phox 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 p47 phox and tyrosine phosphorylation of p47 phox 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. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species Address for reprint requests and other correspondence: E. R. Jacobs, 8701 Watertown Plank Rd., Pulmonary and Critical Care Medicine and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 (e-mail: ejacobs{at}mcw.edu )