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Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 μM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H2O2-challenged mouse lung microvascular (MLMVEC) monolayers. 8pCPT-cGMP also decreased perfusate LDH release in isolated mouse lungs exposed to H2O2or ischemia-reperfusion. The protective effect of increasing cGMP in MLMVECs was accompanied by enhanced endothelial H2O2scavenging (measured by H2O2electrode) and decreased intracellular ROS concentration (measured by 2′,7′-dichlorofluorescin fluorescence) as well as decreased phosphorylation of p38 MAPK and Akt. The cGMP-mediated cytoprotection and increased H2O2scavenging required >2 h of 8pCPT-cGMP incubation in wild-type MLMVEC and were absent in MLMVEC from protein kinase G (PKGI)−/− mice suggesting a PKGI-mediated effect on gene regulation. Catalase and glutathione peroxidase 1 (Gpx-1) protein were increased by cGMP in wild-type but not PKGI−/− MLMVEC monolayers. Both the cGMP-mediated increases in antioxidant proteins and H2O2scavenging were prevented by inhibition of translation with cycloheximide. 8pCPT-cGMP had minimal effects on catalase and Gpx-1 mRNA. We conclude that cGMP, through PKGI, attenuated H2O2-induced cytotoxicity in MLMVEC by increasing catalase and Gpx-1 expression through an unknown posttranscriptional effect.

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 (PKGI) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKGIactivation 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.

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca2+concentration. Membrane-permeable analogs of guanosine 3′,5′-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential ( E m), which influences the electrochemical gradient for Ca2+ influx, is regulated by K+ channels, we investigated the effect of 8-bromo-cGMP on E m and inwardly rectifying K+ (KIR) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to −50 mV that were abolished by application of 10 μM Ba2+, consistent with KIR current. Ba2+ also depolarized both cell types. 8-Bromo-cGMP (10−3 M) depolarized BPAEC and BMVEC and inhibited KIR current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of KIR current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca2+ entry.