title: Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism.
abstract: Adenosine is released from the myocardium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen-activated protein kinases (p42/p44MAPK) in the regulation of the l-arginine-nitric oxide (NO) signaling pathway. Expression of mRNA for the A2a-, A2b-, and A3-adenosine receptor subtypes was abundant whereas A1-adenosine receptor mRNA levels were negligible. Activation of A2a purinoceptors by adenosine (10 microM) or the A2a receptor agonist CGS21680 (100 nM) resulted in an increase in l-arginine transport and NO release that was not mediated by changes in intracellular Ca2+, pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44MAPK. l-NAME abolished the adenosine-induced hyperpolarization and stimulation of l-arginine transport whereas sodium nitroprusside activated an outward potassium current. Genistein (10 microM) and PD98059 (10 microM), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine-stimulated l-arginine transport, NO production, and phosphorylation of p42/p44MAPK. We found no evidence for activation of eNOS via the serine/threonine kinase Akt/PKB (protein kinase B) in adenosine-stimulated cells. Our results provide the first evidence that adenosine stimulates the endothelial cell l-arginine-NO pathway in a Ca2+-insensitive manner involving p42/p44MAPK, with release of NO leading to a membrane hyperpolarization and activation of l-arginine transport.
authors.affiliation: Centre for Cardiovascular Biology and Medicine, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL, UK.
authors.forename: Amanda W
authors.initials: AW
authors.lastname: Wyatt
authors.forename: Joern R
authors.initials: JR
authors.lastname: Steinert
authors.forename: Caroline P D
authors.initials: CP
authors.lastname: Wheeler-Jones
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Morgan
authors.forename: David
authors.initials: D
authors.lastname: Sugden
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Pearson
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
authors.forename: Giovanni E
authors.initials: GE
authors.lastname: Mann
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002070:Butadienes / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III; D009570:Nitriles / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002070:Butadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009025:Morpholines; D009570:Nitriles; D010627:Phenethylamines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D020033:Protein Isoforms; D058906:Purinergic P1 Receptor Agonists; D012333:RNA, Messenger; D018047:Receptors, Purinergic P1; C113580:U 0126; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D005576:Colforsin; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D001120:Arginine; D019833:Genistein; D000242:Cyclic AMP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D006152:Cyclic GMP; D000241:Adenosine; D011188:Potassium; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester; D000077191:Wortmannin