A novel pathway for receptor-mediated post-translational activation of inducible nitric oxide synthase.
Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post-translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)-mediated phosphorylation of Ser(745) . Here we identified the signalling pathway causing iNOS activation in cytokine-treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS-derived nitric oxide. G-βγ subunits were also necessary as transfection with the β-adrenergic receptor kinase C-terminus inhibited the response. B1R-dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans-fection with dominant negative Src. Other ERK-MAP kinase members were involved as the response was inhibited by dominant negative H-Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor-regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.
Brovkovych
Viktor
V
Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
Zhang
Yongkang
Y
Brovkovych
Svitlana
S
Minshall
Richard D
RD
Skidgel
Randal A
RA
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D015773:Enalaprilat / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D004789:Enzyme Activation; D005419:Flavonoids / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D043783:Receptor, Bradykinin B1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D000806:Angiotensin-Converting Enzyme Inhibitors; D016207:Cytokines; D005419:Flavonoids; D011743:Pyrimidines; D043783:Receptor, Bradykinin B1; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019061:src-Family Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins; D015773:Enalaprilat; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one