Superoxide Flux in Endothelial Cells via the Chloride Channel-3 Mediates Intracellular Signaling□D

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 (O2 ) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2 , pulmonary microvascular endothelial cells were preloaded with the O2 -sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2 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 O2 triggered Ca2 release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2 production and cellular apoptosis. These “signaling” effects of O2 were prevented by DIDS treatment, by depletion of intracellular Ca2 stores with thapsigargin and by chelation of intracellular Ca2 . This study demonstrates that O2 flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2 release, which activates mitochondrial O2 generation.