20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H₂O₂, and PI3-kinase/Akt

Abstract

. 20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt. Am J Physiol Lung Cell Mol Physiol 298: L564 -L574, 2010. First published January 8, 2010 doi:10.1152/ajplung.00298.2009We have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide (H2O2) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Ser1179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on H2O2 as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs. pulmonary endothelium; superoxide; hydrogen peroxide; endothelial nitric oxide synthase WE HAVE PREVIOUSLY DEMONSTRATED that 20-HETE, a product of arachidonic acid catalyzed by CYP4, induces activation of nitric oxide synthase (NOS) from pulmonary artery endothelial cells in a [Ca 2ϩ ] i -dependent manner (9,23,36). Furthermore, 20-HETE releases NO, which contributes to endotheliumdependent vasodilation in pulmonary arteries (9, 19). Growing evidence from our own work as well as that from others indicates that 20-HETE activates the vascular NADPH oxidase, leading to increased production of superoxide anion (O 2•Ϫ ) and hydrogen peroxide (2,23,32 /calmodulin-dependent protein kinase II/Janus-kinase 2-dependent pathway in bovine aortic endothelial cells (6, 12). Furthermore, it has been shown that exogenous H 2 O 2 in micromolar concentrations activates eNOS from porcine aortic endothelial cells to cause endothelial NO release (7,28). Superoxide may also react with NO in a diffusion-limited fashion to form peroxynitrite. This species is believed to result in the loss of many of the beneficial effects of NO, including vasodila...

The Cytochrome P450 4A/F-20-Hydroxyeicosatetraenoic Acid System: A Regulator of Endothelial Precursor Cells Derived from Human Umbilical Cord Blood

“…Phosphorylation of serine 1177 has been associated with increased synthesis of nitric oxide (Butt et al, 2000), and we found that it was markedly increased in EPCs treated with 20-HETE just as others reported in ECs (Cheng et al, 2008;Bodiga et al, 2010), pointing out the close similarities between these progenitor cells and mature ECs. Both the proliferative and the migratory effects of 20-HETE could be directly due to stimulation of some unknown receptor (Miyata and Roman, 2005), or they might be secondary to increases in nitric oxide and VEGF (Sautina et al, 2010).…”

Loss of TIMP3 selectively exacerbates diabetic nephropathy

“…NADPH oxidase activity in kidney cortex (homogenized in PBS containing a phosphatase inhibitor cocktail from Roche Applied Sciences) of mice was quantified using a lucigenin-enhanced chemiluminescence assay using a single-tube luminometer (Berthold FB12, Berthold Technologies) with NADPH (1 mM) and lucigenin (50 M) at 37°C as previously described (39). The specific peptide inhibitor of NOX2, gp91phox ds tat (50 M), was used to confirm superoxide generation from NADPH oxidase (5,40). Dihydroethidium (DHE), an oxidative fluorescent dye, was used to measure superoxide levels in kidney samples as previously described (39).…”

Stimulation of cardiomyogenesis from mouse embryonic stem cells by nuclear translocation of cardiotrophin-1

“…In NADPH oxidaseknockout (p47(phox−/−)) mice reduced NOS3 phosphorylation was observed and VEGF-mediated coronary vasodilatation was impaired [15]. Moreover, the addition of 20-hydroxyeicosatetraenoic acid (20-HETE) to endothelial cells activated NOS3 through generation of ROS and PI3Kinase/Akt phosphorylation [6]. The sequential generation of ROS and NO observed in the present study may represent the physiological condition of NOS3 activation and may circumvent the uncoupling of NOS3 with the result of oxidative stress which occurs under pathophysiological conditions, e.g.…”