Faculty Opinions recommendation of Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling (original) (raw)

2007, Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature

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. Hydroethidine (HE), MitoSOX Red, propidium iodide (PI), rhodamine 123, Fluo-4/AM, and BAPTA-AM were purchased from Invitrogen (Carlsbad, CA). The Basic Nucleofector Kit for primary mammalian endothelial cells was purchased from Amaxa Biosystems (Gaithersburg, MD). Silencer Predesigned ClC-3 (ID 60947 and 60858), negative control 1, and Cy3-labeled GAPDH small interfering RNA (siRNA) were purchased from Ambion (Austin, TX). Primers for ␤-actin, ClC-3, and ClC-4 were obtained from Operon Biotechnologies (Huntsville, AL). Rabbit polyclonal anti-ClC-3 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). pEYFP-Mito was purchased from Clontech (BD Biosciences, Mountain View, CA). KO 2 , apoc-This article was published online ahead of print in MBC in Press