Nitric oxide-dependent beta2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt.
Vasorelaxation to beta(2)-adrenoceptor stimulation occurs through both endothelium-dependent and endothelium-independent mechanisms, and the former is mediated through Ca(2+)-independent activation of endothelial-type nitric oxide synthase (NOS-3). Since Ca(2+)-independent NOS-3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of beta(2)-adrenergic relaxation of rat aorta. Rat aortic rings were pre-incubated with the PKA inhibitor H-89 (10(-7) m), the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (5 x 10(-7) m), Akt inhibitor (10(-5) m), or vehicle, in the absence or presence of the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10(-4) m). Rings were then contracted with phenylephrine (10(-7) m), and concentration-relaxation responses determined to the beta(2)-adrenoceptor agonist albuterol. Rings exhibited a concentration-dependent relaxation to albuterol: pEC(50) 6.9+/-0.2, E(max) 88.2+/-4.0%. l-NAME attenuated E(max) to 60.2+/-3.5% (P<0.001). In the presence of l-NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H-89 reduced E(max) further, to 27.5+/-2.2% (P<0.001). In the absence of l-NAME, E(max) to albuterol was reduced by H-89, wortmannin or Akt inhibitor, to 56.2+/-2.2, 56.0+/-1.6 and 55.4+/-1.8%, respectively (P<0.001 for each); the combinations H-89 plus wortmannin or H-89 plus Akt inhibitor reduced E(max) further still. Western blotting of NOS-3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS-3, and this increase was attenuated by H-89 or Akt inhibitor. Our results indicate that beta(2)-adrenoceptor stimulation relaxes rat aorta through both NO-dependent and independent mechanisms. The latter is predominantly PKA-mediated, whereas the former occurs through both PKA and PI3K/Akt activation.
Ferro
Albert
A
Department of Clinical Pharmacology, Division of Cardiovascular Science, GKT School of Medicine, King's College London (Guy's Campus), 2.36B New Hunts House, London Bridge, London SE1 1UL.
Coash
Marcy
M
Yamamoto
Takahiro
T
Rob
Jubli
J
Ji
Yong
Y
Queen
Lindsay
L
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000420:Albuterol / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011412:Propanolamines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D000077191:Wortmannin
D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D000420:Albuterol; D000730:Androstadienes; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007545:Isoproterenol; D007546:Isoquinolines; D019331:NG-Nitroarginine Methyl Ester; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D010656:Phenylephrine; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011412:Propanolamines; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D018343:Receptors, Adrenergic, beta-2; D013449:Sulfonamides; D000077191:Wortmannin; C026777:ICI 118551; C494920:Akt1 protein, rat; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide