The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse.

Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP), but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor-A (NPR-A) generating cGMP and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye extravasation and wet weight in the reperfused left lung, suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient sevenfold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration, and lung protein kinase G (PKG(I)) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKG(I) activation but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.

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(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.