Regulation of endothelial nitric-oxide synthase activity through phosphorylation in response to epoxyeicosatrienoic acids.
Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.
Jiang
Jian Gang
JG
The Institute of Hypertension and Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
Chen
Rui Juan
RJ
Xiao
Bin
B
Yang
Shilin
S
Wang
Jia Ning
JN
Wang
Yong
Y
Cowart
L Ashley
LA
Xiao
Xiao
X
Wang
Dao Wen
DW
Xia
Yong
Y
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D047310:Apigenin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004852:Epoxy Compounds / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000276:immunology / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D051381:Rats; D012694:Serine / Q000378:metabolism
C477931:1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate; D002867:Chromones; D004852:Epoxy Compounds; D005419:Flavonoids; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010769:Phosphothreonine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D007294:Inositol; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D047310:Apigenin; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D052250:Nitric Oxide Synthase Type III; D000090762:Cytochrome P-450 CYP2J2; D051057:Proto-Oncogene Proteins c-akt; D015126:8,11,14-Eicosatrienoic Acid; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one