title: Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O⁻₂ signaling.
abstract: To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.
authors.affiliation: Department of Physiology, Center for Developmental Lung Biology at Children's Healthcare of Atlanta, USA.
authors.forename: Preston
authors.initials: P
authors.lastname: Goodson
authors.forename: Amrita
authors.initials: A
authors.lastname: Kumar
authors.forename: Lucky
authors.initials: L
authors.lastname: Jain
authors.forename: Kousik
authors.initials: K
authors.lastname: Kundu
authors.forename: Niren
authors.initials: N
authors.lastname: Murthy
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
authors.forename: My N
authors.initials: MN
authors.lastname: Helms
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D045604:Extracellular Fluid / Q000000981:diagnostic imaging / Q000378:metabolism*; D055786:Gene Knockout Techniques; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000493:pharmacokinetics; D008168:Lung / Q000000981:diagnostic imaging / Q000201:enzymology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011650:Pulmonary Alveoli / Q000201:enzymology* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D011859:Radiography; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D015222:Sodium Channels / Q000378:metabolism*; D012965:Sodium Chloride / Q000008:administration & dosage / Q000493:pharmacokinetics; D013481:Superoxides / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020830:rac1 GTP-Binding Protein
chemical_list: D000634:Aminoquinolines; D053503:Epithelial Sodium Channels; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; C487513:NSC 23766; D009479:Neuropeptides; D011743:Pyrimidines; C484602:Rac1 protein, mouse; D017382:Reactive Oxygen Species; D015222:Sodium Channels; D013481:Superoxides; D012965:Sodium Chloride; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein