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.
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).
- pulmonary artery
- nitric oxide