title: Endothelial nitric-oxide synthase activation generates an inducible nitric-oxide synthase-like output of nitric oxide in inflamed endothelium.
abstract: High levels of NO generated in the vasculature under inflammatory conditions are usually attributed to inducible nitric-oxide synthase (iNOS), but the role of the constitutively expressed endothelial NOS (eNOS) is unclear. In normal human lung microvascular endothelial cells (HLMVEC), bradykinin (BK) activates kinin B2 receptor (B2R) signaling that results in Ca(2+)-dependent activation of eNOS and transient NO. In inflamed HLMVEC (pretreated with interleukin-1β and interferon-γ), we found enhanced binding of eNOS to calcium-calmodulin at basal Ca(2+) levels, thereby increasing its basal activity that was dependent on extracellular l-Arg. Furthermore, B2R stimulation generated prolonged high output eNOS-derived NO that is independent of increased intracellular Ca(2+) and is mediated by a novel Gα(i)-, MEK1/2-, and JNK1/2-dependent pathway. This high output NO stimulated with BK was blocked with a B2R antagonist, eNOS siRNA, or eNOS inhibitor but not iNOS inhibitor. Moreover, B2R-mediated NO production and JNK phosphorylation were inhibited with MEK1/2 and JNK inhibitors or MEK1/2 and JNK1/2 siRNA but not with ERK1/2 inhibitor. BK induced Ca(2+)-dependent eNOS phosphorylation at Ser(1177), Thr(495), and Ser(114) in cytokine-treated HLMVEC, but these modifications were not dependent on JNK1/2 activation and were not responsible for prolonged NO output. Cytokine treatment did not alter the expression of B2R, Gα(q/11), Gα(i1,2), JNK, or eNOS. B2R activation in control endothelial cells enhanced migration, but in cytokine-treated HLMVEC it reduced migration. Both responses were NO-dependent. Understanding how JNK regulates prolonged eNOS-derived NO may provide new therapeutic targets for the treatment of disorders involving vascular inflammation.
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Lowry
authors.forename: Viktor
authors.initials: V
authors.lastname: Brovkovych
authors.forename: Yongkang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Randal A
authors.initials: RA
authors.lastname: Skidgel
mesh_terms: D001120:Arginine / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology / Q000628:therapy; D053583:Interleukin-1beta / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D043782:Receptor, Bradykinin B2 / Q000378:metabolism; D014657:Vasculitis / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology
chemical_list: D004791:Enzyme Inhibitors; C508585:IL1B protein, human; D053583:Interleukin-1beta; D043782:Receptor, Bradykinin B2; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C482128:MAP2K2 protein, human; D048056:Mitogen-Activated Protein Kinase 9; D048055:Mitogen-Activated Protein Kinase 8; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D001920:Bradykinin; D002118:Calcium