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

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 (CIC-3) by treatment with siRNA. Extracellular O-2(-) triggered Ca 21 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 21 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 CIC-3 channels and induces intracellular Ca2+ release, which activates mitochondrial O-2(-) generation.