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Vol. 18, Issue 6, 2002-2012, June 2007
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*Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA 19104-6068; and Institute of Pathology, Johannes-Gutenberg University, D-55101 Mainz, Germany
Submitted September 18, 2006;
Revised January 10, 2007;
Accepted March 2, 2007
Monitoring Editor: John Cleveland
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.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Aron B. Fisher (abf{at}mail.med.upenn.edu).
Abbreviations used: ROS, reactive oxygen species; SOD, superoxide dismutase; ClC-3, chloride channel-3; 
m, mitochondrial membrane potential; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; HE, hydroethidium; HPMVEC, human pulmonary microvascular endothelial cells; Ang II, angiotensin II; MPMVECs, murine pulmonary microvascular endothelial cells; Apo, apocynin; FCCP, carbonyl cyanide p[trifluoromethoxy]-phenyl-hydrazone; Tg, thapsigargin.
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