The GTP-binding protein RhoA mediates Na,K-ATPase exocytosis in alveolar epithelial cells.
The purpose of this study was to define the role of the Rho family of small GTPases in the beta-adrenergic regulation of the Na,K-ATPase in alveolar epithelial cells (AEC). The beta-adrenergic receptor agonist isoproterenol (ISO) increased the Na,K-ATPase protein abundance at the plasma membrane and activated RhoA in a time-dependent manner. AEC pretreated with mevastatin, a specific inhibitor of prenylation, or transfected with the dominant negative RhoAN19, prevented ISO-mediated Na,K-ATPase exocytosis to the plasma membrane. The ISO-mediated activation of RhoA in AEC occurred via beta2-adrenergic receptors and involved Gs-PKA as demonstrated by incubation with the protein kinase A (PKA)-specific inhibitors H89 and PKI (peptide specific inhibitor), and Gi, as incubation with pertussis toxin or cells transfected with a minigene vector for Gi inhibited the ISO-mediated RhoA activation. However, cells transfected with minigene vectors for G12 and G13 did not prevent RhoA activation by ISO. Finally, the ISO-mediated Na,K-ATPase exocytosis was regulated by the Rho-associated kinase (ROCK), as preincubation with the specific inhibitor Y-27632 or transfection with dominant negative ROCK, prevented the increase in Na,K-ATPase at the plasma membrane. Accordingly, ISO regulates Na,K-ATPase exocytosis in AEC via the activation of beta2-adrenergic receptor, Gs, PKA, Gi, RhoA, and ROCK.
Lecuona
Emilia
E
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, Chicago, Illinois 60611, USA. e-lecuona@northwestern.edu
Ridge
Karen
K
Pesce
Liuska
L
Batlle
Daniel
D
Sznajder
Jacob I
JI
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D003001:Cloning, Molecular; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007545:Isoproterenol / Q000494:pharmacology; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000494:pharmacology; D008958:Models, Molecular; D017368:Protein Prenylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D047908:Intracellular Signaling Peptides and Proteins; D011943:Receptors, Adrenergic, beta; C012258:mevastatin; D008148:Lovastatin; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D020742:rhoA GTP-Binding Protein; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol