Effect of proteinS-glutathionylation on Ca²⁺homeostasis in cultured aortic endothelial cells

Abstract

Diamide is a membrane-permeable, thiol-oxidizing agent that rapidly and reversibly oxidizes glutathione to GSSG and promotes formation of protein-glutathione mixed disulfides. In the present study, the acute effect of diamide on free cytosolic Ca2+ concentration ([Ca2+]i) was examined in fura-2-loaded bovine aortic endothelial cells. At low concentrations (50, 100 μM), diamide reversibly increased spontaneous, asynchronous Ca2+ oscillations, whereas, at higher concentrations (250, 500 μM), diamide caused an immediate synchronized Ca2+ oscillation in essentially all cells of the monolayer, followed by a time-dependent rise in basal [Ca2+]i. The effects of diamide on [Ca2+]i dynamics were independent of extracellular Ca2+. Inhibition of phospholipase C by U-73122 prevented the observed changes in [Ca2+]i. Additionally, the diamide-induced oscillations, but not the rise in basal [Ca2+]i, were blocked by inhibition of the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) by 2-aminoethyl diphenyl borate. However, diamide failed to alter the plasmalemmal distribution of a green fluorescent protein-tagged phosphatidylinositol-4,5-bisphosphate binding protein, demonstrating that diamide does not activate phospholipase C. Inhibition of glutathione reductase by N,N'-bis(2-chloroethyl)-N-nitrosourea or depletion of glutathione by l-buthionine-sulfoximine enhanced the effects of diamide, which, under these conditions, could only be reversed by addition of dithiothreitol to the wash buffer. Biochemical assays showed that both the IP3R and the plasmalemmal Ca2+-ATPase pump could be reversibly glutathionylated in response to diamide. These results demonstrate that diamide promotes Ca2+ release from IP3-sensitive internal Ca2+ stores and elevates basal [Ca2+]i in the absence of extracellular Ca2+, effects that may be related to a diamide-induced glutathionylation of the IP3R and the plasmalemmal Ca2+-ATPase Ca2+ pump, respectively.

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

“…Similarly, depletion of glutathione using BSO to increase ROS gave rise to a sustained pattern of Ca 2+ oscillations (our unpublished observations). Consistent with this, it has been proposed that cytosolic Ca 2+ oscillations in ECs are induced by exogenous delivery of oxidants, hypoxia/ reoxygenation, or S-glutathionylation (16,47,48).…”

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…While the former induces Ca 2+ release likely via receptor S-nitrosylation [120,121] , the latter inhibits InsP3-induced Ca 2+ discharge probably through receptor S-sulfhydration [122,123] . An additional post-translational modification of endothelial InsP3Rs is provided by S-glutathionylation, which might sensitize the receptor to the basal level of InsP3 [124] . In this view, the InsP3R may be directly regulated by ROS, which may oxidize the two highly conserved thiol groups located in the COOH-tail of the protein [102] .…”

“…In addition to extruding Ca 2+ across the plasma membrane, PMCA suppresses both eNOS activity and the subsequent NO production by promoting Thr-495 phosphorylation in resting and activated ECs [381] . PMCA activity in vascular ECs is, in turn, inhibited by S-glutathionylation, which leads to a progressive raise in [Ca 2+ ]i [124] . The elucidation of the role served by PMCA in endothelial signaling might benefit from the recent discovery of caloxin 1b3, a specific PMCA1 inhibitor [382] .…”

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

“…Various exogenously added oxidants stimulate IP 3 R-mediated Ca 2ϩ release. This includes thimerosal (12)(13)(14), t-butylhydroperoxide (15), and diamide (16,17). In the case of thimerosal, the proposed mechanism involves an increased sensitivity of the receptor to lower [IP 3 ], which in some cells is sufficient to trigger Ca 2ϩ oscillations at the ambient [IP 3 ] present in unstimulated cells (11).…”