title: Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling.
abstract: Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O(2)(.-)) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O(2)(.-), pulmonary microvascular endothelial cells were preloaded with the O(2)(.-)-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O(2)(.-) resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O(2)(.-) triggered Ca(2+) release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O(2)(.-) production and cellular apoptosis. These "signaling" effects of O(2)(.-) were prevented by DIDS treatment, by depletion of intracellular Ca(2+) stores with thapsigargin and by chelation of intracellular Ca(2+). This study demonstrates that O(2)(.-) flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca(2+) release, which activates mitochondrial O(2)(.-) generation.
authors.affiliation: Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104-6068, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Hawkins
authors.forename: Muniswamy
authors.initials: M
authors.lastname: Madesh
authors.forename: C J
authors.initials: CJ
authors.lastname: Kirkpatrick
authors.forename: Aron B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000098:Acetophenones / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000033:anatomy & histology; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010617:Phenanthridines / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: D000098:Acetophenones; D018118:Chloride Channels; C115376:ClC-3 channel; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010617:Phenanthridines; D034741:RNA, Small Interfering; D013481:Superoxides; D000804:Angiotensin II; C058428:hydroethidine; C056165:acetovanillone; D019255:NADPH Oxidases; D013917:Thrombin; D010100:Oxygen; D002118:Calcium