title: Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells.
abstract: Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)(I), human pulmonary artery endothelial cells (HPAEC) lacking PKG(I) expression were infected with a recombinant adenovirus encoding PKG(Ibeta) (Ad.PKG) and compared with uninfected and control-infected (Ad.betagal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H(2)O(2) (250 microM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.betagal, expressed PKG(I) protein and demonstrated Ser(239) and Ser(157) phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.betagal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 microM) caused a PKG(I)-independent decrease in permeability (8.2 +/- 0.6%). In all three groups, H(2)O(2) (250 microM) caused a similar approximately 35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). In uninfected and Ad.betagal-infected HPAEC, pretreatment with CPT-cGMP (100 microM) partially blocked the increased permeability induced by H(2)O(2). In Ad.PKG-infected HPAEC, CPT-cGMP (50 microM) prevented the H(2)O(2)-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca(2+)](i) caused by H(2)O(2) similarly (23%) in Ad.betagal- and Ad.PKG-infected HPAEC, indicating a PKG(I)-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKG(I)-independent process, whereas the ability of cGMP to prevent H(2)O(2)-induced barrier dysfunction was predominantly mediated by PKG(I) through a Ca(2+)-independent mechanism.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Hopkins Bayview Medical Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Aigul
authors.initials: A
authors.lastname: Moldobaeva
authors.forename: Laura E
authors.initials: LE
authors.lastname: Welsh-Servinsky
authors.forename: Larissa A
authors.initials: LA
authors.lastname: Shimoda
authors.forename: R Scott
authors.initials: RS
authors.lastname: Stephens
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Verin
authors.forename: Rubin M
authors.initials: RM
authors.lastname: Tuder
authors.forename: David B
authors.initials: DB
authors.lastname: Pearse
mesh_terms: D000256:Adenoviridae; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D011651:Pulmonary Artery / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP