Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Adrenergic beta-2 Receptor Agonists
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Adrenergic beta-Agonists / pharmacology
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Adrenergic beta-Antagonists / pharmacology
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Albuterol / pharmacology
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Androstadienes / pharmacology
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Animals
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Aorta, Thoracic / drug effects
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Aorta, Thoracic / physiology*
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Blotting, Western
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Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinases / metabolism*
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Dose-Response Relationship, Drug
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Enzyme Activation / drug effects
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Enzyme Inhibitors / pharmacology
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In Vitro Techniques
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Isoproterenol / pharmacology
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Isoquinolines / pharmacology
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Male
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NG-Nitroarginine Methyl Ester / pharmacology
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Nitric Oxide / metabolism*
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Nitric Oxide Synthase / antagonists & inhibitors
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Nitric Oxide Synthase / metabolism
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Phenylephrine / pharmacology
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoinositide-3 Kinase Inhibitors
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Propanolamines / pharmacology
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Protein Kinase Inhibitors / pharmacology
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism*
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Proto-Oncogene Proteins / antagonists & inhibitors
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-akt
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Rats
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Rats, Wistar
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Receptors, Adrenergic, beta-2 / physiology*
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Sulfonamides / pharmacology
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Vasodilation / drug effects
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Vasodilation / physiology
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Wortmannin
Substances
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Adrenergic beta-2 Receptor Agonists
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Adrenergic beta-Agonists
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Adrenergic beta-Antagonists
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Androstadienes
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Enzyme Inhibitors
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Isoquinolines
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Phosphoinositide-3 Kinase Inhibitors
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Propanolamines
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Protein Kinase Inhibitors
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Proto-Oncogene Proteins
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Receptors, Adrenergic, beta-2
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Sulfonamides
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Phenylephrine
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Nitric Oxide
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ICI 118551
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Nitric Oxide Synthase
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Akt1 protein, rat
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Cyclic AMP-Dependent Protein Kinases
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Isoproterenol
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N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
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Albuterol
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NG-Nitroarginine Methyl Ester
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Wortmannin