Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling

Mol Biol Cell. 2007 Jun;18(6):2002-12. doi: 10.1091/mbc.e06-09-0830. Epub 2007 Mar 14.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetophenones / metabolism
  • Angiotensin II / metabolism
  • Animals
  • Apoptosis / physiology
  • Calcium / metabolism
  • Cells, Cultured
  • Chloride Channels / genetics
  • Chloride Channels / metabolism*
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Enzyme Inhibitors / metabolism
  • Fluorescent Dyes / metabolism
  • Humans
  • Lung / anatomy & histology
  • Membrane Potentials / physiology
  • Mitochondria / metabolism
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / metabolism
  • Oxidation-Reduction
  • Oxygen / metabolism
  • Phenanthridines / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction / physiology*
  • Superoxides / metabolism*
  • Thrombin / metabolism


  • Acetophenones
  • Chloride Channels
  • ClC-3 channel
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Phenanthridines
  • RNA, Small Interfering
  • Superoxides
  • Angiotensin II
  • hydroethidine
  • acetovanillone
  • NADPH Oxidases
  • Thrombin
  • Oxygen
  • Calcium