We 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 (H(2)O(2)) 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 H(2)O(2) 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.