Abstract-In the present study, we demonstrate that lung microvascular endothelial cells express a Ca v 3.1 (␣ 1G ) T-type voltage-gated Ca 2ϩ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca 2ϩ channels. Voltage-dependent activation indicates that the Ca v 3.1 T-type Ca 2ϩ current is shifted to a positive potential, at which maximum current activation is Ϫ10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of Ϫ60 to Ϫ30 mV. Thrombin-induced transitions in membrane potential activate the Ca v 3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca 2ϩ . Furthermore, activation of the Ca v 3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Ca v 3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation.
Fragments of the mitochondrial genome released into the systemic circulation after mechanical trauma, termed mitochondrial DNA damage-associated molecular patterns (mtDNA DAMPs), are thought to mediate the systemic inflammatory response syndrome. The close association between circulating mtDNA DAMP levels and outcome in sepsis suggests that bacteria also might be a stimulus for mtDNA DAMP release. To test this hypothesis, we measured mtDNA DAMP abundance in medium perfusing isolated rat lungs challenged with an intratracheal instillation of 5 × 10(7) colony-forming units of Pseudomonas aeruginosa (strain 103; PA103). Intratracheal PA103 caused rapid accumulation of selected 200-bp sequences of the mitochondrial genome in rat lung perfusate accompanied by marked increases in both lung tissue oxidative mtDNA damage and in the vascular filtration coefficient (Kf). Increases in lung tissue mtDNA damage, perfusate mtDNA DAMP abundance, and Kf were blocked by addition to the perfusion medium of a fusion protein targeting the DNA repair enzyme Ogg1 to mitochondria. Intra-arterial injection of mtDNA DAMPs prepared from rat liver mimicked the effect of PA103 on both Kf and lung mtDNA integrity. Effects of mtDNA and PA103 on Kf were also attenuated by an oligodeoxynucleotide inhibitor of Toll-like receptor 9 (TLR-9) by mitochondria-targeted Ogg1 and by addition of DNase1 to the perfusion medium. Collectively, these findings are consistent with a model wherein PA103 causes oxidative mtDNA damage leading to a feed-forward cycle of mtDNA DAMP formation and TLR-9-dependent mtDNA damage that culminates in acute lung injury.
In cultured pulmonary artery endothelial cells and other cell types, overexpression of mt-targeted DNA repair enzymes protects against oxidant-induced mitochondrial DNA (mtDNA) damage and cell death. Whether mtDNA integrity governs functional properties of the endothelium in the intact pulmonary circulation is unknown. Accordingly, the present study used isolated, buffer-perfused rat lungs to determine whether fusion proteins targeting 8-oxoguanine DNA glycosylase 1 (Ogg1) or endonuclease III (Endo III) to mitochondria attenuated mtDNA damage and vascular barrier dysfunction evoked by glucose oxidase (GOX)-generated hydrogen peroxide. We found that both Endo III and Ogg1 fusion proteins accumulated in lung cell mitochondria within 30 min of addition to the perfusion medium. Both constructs prevented GOX-induced increases in the vascular filtration coefficient. Although GOX-induced nuclear DNA damage could not be detected, quantitative Southern blot analysis revealed substantial GOX-induced oxidative mtDNA damage that was prevented by pretreatment with both fusion proteins. The Ogg1 construct also reversed preexisting GOX-induced vascular barrier dysfunction and oxidative mtDNA damage. Collectively, these findings support the ideas that mtDNA is a sentinel molecule governing lung vascular barrier responses to oxidant stress in the intact lung and that the mtDNA repair pathway could be a target for pharmacological intervention in oxidant lung injury.
Adenosine mediates vascular smooth muscle relaxation in the pulmonary circulation. The A2 receptor has been suggested to mediate adenosine-induced vasodilation (AIV). In this study, the effect(s) of selective adenosine agonist and antagonist on the hypoxic pressor response (HPR) was assessed in the isolated blood-perfused rat lung. Adenosine (0.075-7.5 mM) infusion (0.125 ml/min) into the pulmonary artery dose dependently attenuated the HPR. AIV was mimicked by 10 microM 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine agonist. Adenosine- and NECA-induced vasodilation were attenuated by 67 microM 8-(p-sulfophenyl)theophylline. In contrast, NECA-induced vasodilation was not attenuated by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM). At 10 microM, a minimal vasodilatory effect was seen with the nonselective adenosine agonists CV-1808 and N6-(2-phenylisopropyl)adenosine (R-PIA) compared with NECA. The highly selective A2a agonist 2-[p-(2-carboxyethyl)phenyl amino]-5'-N-ethyl carboxamido adenosine (CGS-21680C, 10 microM) and A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 10 microM) had no vasodilatory effect. Neither the K+ channel blockers tetraethylammonium chloride (10 mM) and glibenclamide (100 microM) nor the NO synthase inhibitor N omega-nitro-L-arginine methyl ester attenuated NECA-induced vasodilation. These findings suggest that AIV is mediated via the A2b receptor and that AIV occurs via an NO-independent mechanism.
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