title: Mechanisms underlying beta2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells.
abstract: Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l-arginine uptake, NOS activity (from l-arginine to l-citrulline conversion), membrane potential (using [(3)H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with betaAR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased l-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of l-arginine uptake by l-lysine inhibited NOS activity and, conversely, blockade of NOS using N(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by l-NAME, suggesting that the increase in l-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l-arginine uptake resulting from NO-mediated membrane hyperpolarization.
authors.affiliation: 2.38A, New Hunts Hose, King's College London, Guy's Hospital Campus, London Bridge, London SE1 1UL, UK.
authors.forename: Lindsay R
authors.initials: LR
authors.lastname: Queen
authors.forename: Yong
authors.initials: Y
authors.lastname: Ji
authors.forename: Biao
authors.initials: B
authors.lastname: Xu
authors.forename: Lora
authors.initials: L
authors.lastname: Young
authors.forename: Kang
authors.initials: K
authors.lastname: Yao
authors.forename: Amanda W
authors.initials: AW
authors.lastname: Wyatt
authors.forename: David J
authors.initials: DJ
authors.lastname: Rowlands
authors.forename: Richard C M
authors.initials: RC
authors.lastname: Siow
authors.forename: Giovanni E
authors.initials: GE
authors.lastname: Mann
authors.forename: Albert
authors.initials: A
authors.lastname: Ferro
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*
chemical_list: D004791:Enzyme Inhibitors; D018343:Receptors, Adrenergic, beta-2; D009569:Nitric Oxide; D012694:Serine; D001120:Arginine; D000242:Cyclic AMP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D019331:NG-Nitroarginine Methyl Ester