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Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

¹Division of Pulmonary and Critical Care Medicine, Department of Medicine, and ²Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland

Submitted 11 October 2005 ; accepted in final form 2 December 2005

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_I (Ad.PKG) and compared with uninfected and control-infected (Ad.gal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H₂O₂ (250 µM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.gal, expressed PKG_I protein and demonstrated Ser²³⁹ and Ser¹⁵⁷ phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.gal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 µM) caused a PKG_I-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H₂O₂ (250 µM) caused a similar 35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca²⁺ concentration ([Ca²⁺]_i). In uninfected and Ad.gal-infected HPAEC, pretreatment with CPT-cGMP (100 µM) partially blocked the increased permeability induced by H₂O₂. In Ad.PKG-infected HPAEC, CPT-cGMP (50 µM) prevented the H₂O₂-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca²⁺]_i caused by H₂O₂ similarly (23%) in Ad.gal- 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 H₂O₂-induced barrier dysfunction was predominantly mediated by PKG_I through a Ca²⁺-independent mechanism.

cAMP; pulmonary edema; acute lung injury; hydrogen peroxide

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