Activation of endothelial NADPH oxidase during normoxic lung ischemia is K_ATP channel dependent

Abstract

Previous studies have shown endothelial cell membrane depolarization and generation of reactive oxygen species (ROS) in endothelial cells with abrupt reduction in shear stress (ischemia). This study evaluated the role of ATP-sensitive potassium (K(ATP)) channels and NADPH oxidase in the ischemic response by using Kir6.2-/- and gp91(phox)-/- mice. To evaluate ROS generation, we subjected isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (DCF), hydroethidine (HE), or diphenyl-1-pyrenylphosphine (DPPP) to control perfusion followed by global ischemia. In wild-type C57BL/6J mice, imaging of subpleural endothelial cells showed a time-dependent increase in intensity for all three fluorescence probes with ischemia, which was blocked by preperfusion with cromakalim (a K(ATP) channel agonist) or diphenyleneiodonium (DPI, a flavoprotein inhibitor). Endothelial cell fluorescence with bis-oxonol, a membrane potential probe, increased during lung ischemia indicating cell membrane depolarization. The change in membrane potential with ischemia in lungs of gp91(phox)-/- mice was similar to wild type, but ROS generation did not occur. Lungs from Kir6.2-/- showed marked attenuation of the change in both membrane potential and ROS production. Thus membrane depolarization during lung ischemia requires the presence of a K(ATP) channel and is required for activation of NADPH oxidase and endothelial ROS generation.

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

“…Isolated Lung Perfusion-The isolated perfused mouse lung technique has been described previously (11). Briefly, mice were anesthetized with 50 mg/kg intraperitoneal sodium pentobarbital, and the lungs were cleared of blood and then removed from the thorax and placed in a perfusion chamber.…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…These NOX's appear to control several vascular processes, such as vascular tone, vascular cell growth and angiogenesis, and inflammation. Accordingly, pulmonary endothelial cells have been demonstrated to generate NADPH-derived oxidants in response to pulmonary ischemia (178,179) as well as hyperoxia (180), which is associated with membrane depolarization due to the activation of K(ATP) channels, and resulting in stimulated endothelial cell proliferation and NO production (178,181,182). Although these responses were in some cases inhibited by catalase, suggesting the involvement of H 2 O 2 (182), recent studies also suggest a role for O 2 •− in endothelial NOX signaling (77).…”

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…ROS can signal to the nucleus and control gene expression to regulate cell metabolism appropriately (Finley and Haigis, 2009). The potential effects of two major intracellular sources of ROS on generation of DAergic cells were investigated in the present study: the NADPH oxidase complex and mito-KATP channels, which have also demonstrated a ROS-mediated relationship (Kimura et al, 2005a,b;Zhang et al, 2005;Sheh et al, 2007;Alberici et al, 2009).…”

“…ROS open up KATP channels (Zhang et al, 2002;Avshalumov and Rice, 2003), and mitochondrial ROS production can be stimulated by the opening of mitochondrial mitoKATP channels (Costa and Garlid, 2008;Kimura et al, 2005b). Recent studies have shown a ROSmediated relationship between the NADPH oxidase complex and the mitoKATP channels (Kimura et al, 2005a;Zhang et al, 2005;Sheh et al, 2007;Alberici et al, 2009) and that events leading to mitochondrial dysfunction or increased cytoplasmatic ROS have a significant upstream impact on increasing NADPH activity (Li et al, 2001;Wosniak et al, 2009). This suggests cross-talk signaling between both systems (reviewed by Daiber, 2010).…”