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

doi:10.1016/j.cellsig.2011.02.008

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

Aberdeen Centre For Environmental Sustainability

Research output: Contribution to journal › Article › peer-review

45 Citations (Scopus)

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 language	English
Pages (from-to)	1136-1143
Number of pages	8
Journal	Cellular Signalling
Volume	23
Issue number	7
Early online date	6 Mar 2011
DOIs	https://doi.org/10.1016/j.cellsig.2011.02.008
Publication status	Published - 23 Jul 2011

Bibliographical note

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).

Keywords

pulmonary artery
endothelium
β2-adrenoceptor
nitric oxide
caveolin

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10.1016/j.cellsig.2011.02.008

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@article{978e9ff748f9433789914a6e3ee20d26,

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

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.",

keywords = "pulmonary artery, endothelium, β2-adrenoceptor, nitric oxide, caveolin",

author = "Sebastien Banquet and Estelle Delannoy and Abdelali Agouni and Chantal Dessy and Sabrina Lacomme and Fabien Hubert and Vincent Richard and Bernard Muller and V{\'e}ronique Leblais",

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).",

year = "2011",

month = jul,

day = "23",

doi = "10.1016/j.cellsig.2011.02.008",

language = "English",

volume = "23",

pages = "1136--1143",

journal = "Cellular Signalling",

issn = "0898-6568",

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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

U2 - 10.1016/j.cellsig.2011.02.008

DO - 10.1016/j.cellsig.2011.02.008

M3 - Article

SN - 0898-6568

VL - 23

SP - 1136

EP - 1143

JO - Cellular Signalling

JF - Cellular Signalling

IS - 7

ER -

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

Abstract

Bibliographical note

Keywords

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