title: Up-regulation of endothelial nitric-oxide synthase by endothelium-derived hyperpolarizing factor involves mitogen-activated protein kinase and protein kinase C signaling pathways.
abstract: Cytochrome P450 (P450)-dependent metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are proposed to be endothelium-derived hyperpolarizing factors (EDHF) that affect vascular tone; however, the effects of EDHF on endothelial-derived nitric oxide biosynthesis remain unknown. We examined the regulation of endothelial nitric-oxide synthase (eNOS) by EDHF and investigated the relevant signaling pathways involved. The P450 epoxygenases CYP102 F87V mutant, CYP2C11-CYPOR, and CYP2J2 were transfected into cultured bovine aortic endothelial cells, and the effects of endogenously formed or exogenously applied EETs on eNOS expression and activity were assessed. Transfection with the P450 epoxygenases led to increased eNOS protein expression, an effect that was attenuated by cotreatment with the P450 inhibitor 17-ODYA. Northern analysis demonstrated that P450 transfection led to increased eNOS mRNA levels consistent with an effect at the pretranslational level. P450 epoxygenase transfection resulted in increased eNOS activity as measured by the conversion of L-arginine to L-citrulline. Addition of synthetic EETs (50-200 nM) to the culture media also increased eNOS expression and activity. Treatment with mitogen-activated protein kinase (MAPK), MAPK kinase, and protein kinase C inhibitors apigenin, 2'-amino-3'-methoxyflavone (PD98059), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), respectively, significantly inhibited the effects of P450 transfection on eNOS expression. Overexpression of P450 epoxygenases or addition of synthetic EETs increased Thr495 phosphorylation of eNOS, an effect that was inhibited by both apigenin and PD98059. Overexpression of P450 epoxygenases in rats resulted in increased aortic eNOS expression, providing direct evidence that EDHF can influence vascular eNOS levels in vivo. Based on this data, we conclude that EDHF up-regulates eNOS via activation of MAPK and protein kinase C signaling pathways.
authors.affiliation: Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, #1095 Jie Fang Da Dao Avenue, Wuhan 430030, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Lin
authors.forename: Jiangang
authors.initials: J
authors.lastname: Jiang
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Zai Ying
authors.initials: ZY
authors.lastname: Lu
authors.forename: J Alyce
authors.initials: JA
authors.lastname: Bradbury
authors.forename: Fred Bjørn
authors.initials: FB
authors.lastname: Lih
authors.forename: Dao Wen
authors.initials: DW
authors.lastname: Wang
authors.forename: Darryl C
authors.initials: DC
authors.lastname: Zeldin
mesh_terms: D000818:Animals; D001685:Biological Factors / Q000378:metabolism*; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation
chemical_list: D001685:Biological Factors; C067410:endothelium-dependent hyperpolarization factor; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases