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

ABSTRACT: 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.

SUBMITTER: Hawkins BJ

PROVIDER: S-EPMC1877121 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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

Hawkins Brian J BJ Madesh Muniswamy M Kirkpatrick C J CJ Fisher Aron B AB

Molecular biology of the cell 20070314 6

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)(.-) res ...[more]

PMID: 17360969

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

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

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