Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Sebastien Banquet, Estelle Delannoy , Abdelali Agouni, Chantal Dessy , Sabrina Lacomme , Fabien Hubert, Vincent Richard , Bernard Muller , Véronique Leblais

Research output: Contribution to journalArticle

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Abstract

Activation of the β2-adrenoceptor (β2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β2-AR-mediated eNOS activation, with special focus on Gi/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β2-AR agonist procaterol was reduced by inhibitors of Gi/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser1177, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr14, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β2-AR is coupled to a Gi/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser1177 leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr14, through a Gi/o-Src kinase pathway. Since pulmonary β2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.
Original languageEnglish
Pages (from-to)1136-1143
Number of pages8
JournalCellular Signalling
Volume23
Issue number7
Early online date6 Mar 2011
DOIs
Publication statusPublished - 23 Jul 2011

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Caveolin 1
src-Family Kinases
Nitric Oxide Synthase Type III
Procaterol
Phosphatidylinositol 3-Kinases
Nitric Oxide Synthase
Adrenergic Receptors
Pulmonary Artery
Pertussis Toxin
Caveolae
Phosphorylation
Gene Deletion
Cyclodextrins
Muscarinic Receptors
Pulmonary Hypertension
Lung
Phosphatidylinositol 3-Kinase
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Transmission Electron Microscopy
Vasodilator Agents

Keywords

  • pulmonary artery
  • endothelium
  • β2-adrenoceptor
  • nitric oxide
  • caveolin

Cite this

Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery. / Banquet, Sebastien; Delannoy , Estelle ; Agouni, Abdelali; Dessy , Chantal ; Lacomme , Sabrina; Hubert, Fabien; Richard , Vincent ; Muller , Bernard ; Leblais , Véronique .

In: Cellular Signalling, Vol. 23, No. 7, 23.07.2011, p. 1136-1143.

Research output: Contribution to journalArticle

Banquet, S, Delannoy , E, Agouni, A, Dessy , C, Lacomme , S, Hubert, F, Richard , V, Muller , B & Leblais , V 2011, 'Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery', Cellular Signalling, vol. 23, no. 7, pp. 1136-1143. https://doi.org/10.1016/j.cellsig.2011.02.008
Banquet, Sebastien ; Delannoy , Estelle ; Agouni, Abdelali ; Dessy , Chantal ; Lacomme , Sabrina ; Hubert, Fabien ; Richard , Vincent ; Muller , Bernard ; Leblais , Véronique . / Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery. In: Cellular Signalling. 2011 ; Vol. 23, No. 7. pp. 1136-1143.
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abstract = "Activation of the β2-adrenoceptor (β2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β2-AR-mediated eNOS activation, with special focus on Gi/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β2-AR agonist procaterol was reduced by inhibitors of Gi/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser1177, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr14, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β2-AR is coupled to a Gi/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser1177 leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr14, through a Gi/o-Src kinase pathway. Since pulmonary β2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.",
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note = "Acknowledgements The authors would like to thank Mrs. M. Lacayrerie for excellent animal care, Dr. MC. Mart{\'i}nez (UMR INSERM 771-CNRS6214, Angers, France) for providing assistance with Western blotting experiments, Mrs. F. Lefebvre (UMR-S 769, Ch{\^a}tenay-Malabry, France) and Mrs. V. Domergue-Dupont (IFR141, Ch{\^a}tenay-Malabry, France) for assistance in animal care and Dr. R. Fischmeister (UMR-S 769, Ch{\^a}tenay-Malabry, France) for critical reading of the manuscript. This work was partially supported by the “Fondation de France” [grants 2006005603, 2008002719]; and the “Agence Nationale de la Recherche” [grant ANR06 Physio 015 01]. The authors thank the “Association des Enseignants de Pharmacologie des Facult{\'e}s de Pharmacie” for partially supporting the collaboration between Unit of Pharmacology and Therapeutics (Brussels) and INSERM U1045 (Bordeaux).",
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T1 - Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

AU - Banquet, Sebastien

AU - Delannoy , Estelle

AU - Agouni, Abdelali

AU - Dessy , Chantal

AU - Lacomme , Sabrina

AU - Hubert, Fabien

AU - Richard , Vincent

AU - Muller , Bernard

AU - Leblais , Véronique

N1 - Acknowledgements The authors would like to thank Mrs. M. Lacayrerie for excellent animal care, Dr. MC. Martínez (UMR INSERM 771-CNRS6214, Angers, France) for providing assistance with Western blotting experiments, Mrs. F. Lefebvre (UMR-S 769, Châtenay-Malabry, France) and Mrs. V. Domergue-Dupont (IFR141, Châtenay-Malabry, France) for assistance in animal care and Dr. R. Fischmeister (UMR-S 769, Châtenay-Malabry, France) for critical reading of the manuscript. This work was partially supported by the “Fondation de France” [grants 2006005603, 2008002719]; and the “Agence Nationale de la Recherche” [grant ANR06 Physio 015 01]. The authors thank the “Association des Enseignants de Pharmacologie des Facultés de Pharmacie” for partially supporting the collaboration between Unit of Pharmacology and Therapeutics (Brussels) and INSERM U1045 (Bordeaux).

PY - 2011/7/23

Y1 - 2011/7/23

N2 - Activation of the β2-adrenoceptor (β2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β2-AR-mediated eNOS activation, with special focus on Gi/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β2-AR agonist procaterol was reduced by inhibitors of Gi/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser1177, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr14, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β2-AR is coupled to a Gi/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser1177 leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr14, through a Gi/o-Src kinase pathway. Since pulmonary β2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.

AB - Activation of the β2-adrenoceptor (β2-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β2-AR-mediated eNOS activation, with special focus on Gi/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β2-AR agonist procaterol was reduced by inhibitors of Gi/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser1177, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr14, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β2-AR is coupled to a Gi/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser1177 leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr14, through a Gi/o-Src kinase pathway. Since pulmonary β2-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.

KW - pulmonary artery

KW - endothelium

KW - β2-adrenoceptor

KW - nitric oxide

KW - caveolin

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DO - 10.1016/j.cellsig.2011.02.008

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VL - 23

SP - 1136

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JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

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