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

Abstract

.؊ 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.

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

“…The specificity of mitochondrial superoxide production measured by MitoSOX Red in normal cells could be verified by SOD-PEG (a cell-permeable superoxide dismutase that scavenges superoxide; 1,000 U per 2 ml) and DETANONOate (30 μM; a nitric oxide donor that promptly reacts with superoxide to form peroxynitrite) pretreatment, which markedly reduced the intensity of MitoSOX at the FL2 channel (data not shown); furthermore, it could be verified by the use of the superoxide generator system as described 7,18,19 . The specificity of the superoxide detection by confocal microscopy can further be enhanced by using the dualwavelength excitation (at 396 and 510 nm) method proposed by Beckman and co-workers 7 .…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Similarly, transmembrane diffusion of O 2 •− is limited because it exists primarily as an anion at physiological pH (pK a of protonated form HOO • is 4.8). Intriguing recent observations suggest that NOX-generated O 2 •− can enter cells through the chloride channel-3 (ClC3) and thereby activate intracellular signaling events (77). The ClC3 channel also appears to be critical for neutrophil phagocytic activity and migration, which is linked to affects on intracellular ROS formation (78).…”

“…Accordingly, pulmonary endothelial cells have been demonstrated to generate NADPH-derived oxidants in response to pulmonary ischemia (178,179) as well as hyperoxia (180), which is associated with membrane depolarization due to the activation of K(ATP) channels, and resulting in stimulated endothelial cell proliferation and NO production (178,181,182). Although these responses were in some cases inhibited by catalase, suggesting the involvement of H 2 O 2 (182), recent studies also suggest a role for O 2 •− in endothelial NOX signaling (77). Endothelial NOX activation in response to hyperoxia was found to occur in association with the actin cytoskeleton via the activation of the non-receptor tyrosine kinase Src, and has been suggested to contribute to decreased alveolar-capillary barrier function as a mechanism of hyperoxia-induced lung injury (183,184).…”

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation

“…In non-excitable cells such as ECs, InsP 3 Rs are the major ER Ca 2+ release channels activated by InsP 3 produced when phospholipase C-coupled (PLC-coupled) receptors are activated (33). We have previously shown that ROS evokes intracellular Ca 2+ mobilization in ECs (15,34). To identify the intracellular Ca 2+ release component in LPS-induced Ca 2+ oscillations, we treated wild-type MPMVECs with LPS in the presence of the PLC inhibitor U73122 or the potent InsP 3 R inhibitor xestospongin B (XesB), and the results revealed suppression of Ca 2+ oscillations mediated by both LPS and the glutathione-depleting agent dl-buthionine sulfoximine (BSO) (our unpublished observations).…”