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

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 beta) (Ad. PKG) and compared with uninfected and control-infected (Ad.beta gal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2 ( 250 mu M) exposure with or without pretreatment with 8-(4-chlorophenylthio) guanosine 3', 5'-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad. PKG, but not Ad.beta gal, 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.beta gal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP ( 100 mu M) caused a PKG(I)-independent decrease in permeability (8.2 +/- 0.6%). In all three groups, H2O2 (250 mu M) caused a similar similar to 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.beta gal-infected HPAEC, pretreatment with CPT-cGMP ( 100 mu M) partially blocked the increased permeability induced by H2O2. In Ad. PKG-infected HPAEC, CPT-cGMP (50 mu M) 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.beta 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.