Protein S-glutathionylation enhances Ca2+-induced Ca2+ release via the IP3 receptor in cultured aortic endothelial cells

摘要

In non-excitable cells, thiol-oxidizing agents have been shown to evoke oscillations in cytosolic free Ca²⁺ concentration ([Ca²⁺]i) by increasing the sensitivity of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) to IP3. Although thiol modification of the IP3R is implicated in this response, the molecular nature of the modification(s) responsible for changes in channel activity is still not well understood. Diamide is a chemical oxidant that selectively converts reduced glutathione (GSH) to its disulfide (GSSG) and promotes the formation of protein–glutathione (P-SSG) mixed disulfide, i.e. glutathionylation. In the present study, we examined the effect of diamide, and the model oxidant hydrogen peroxide (H2O2), on oscillations in [Ca²⁺]i in fura-2-loaded bovine (BAECs) and human (HAECs) aortic endo-thelial cells using time-lapse fluorescence video microscopy. In the absence of extracellular Ca²⁺, acute treatment with either diamide or H2O2 increased the number of BAECs exhibiting asynchronous Ca²⁺ oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine-stimulated Ca²⁺ oscillations and BAECs to bradykinin-stimulated Ca²⁺ oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin-induced Ca²⁺ transient suggesting that Ca²⁺-induced Ca²⁺ release (CICR) via the IP3R is enhanced by glutathionylation. Similar to diamide, H2O2 increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP3R1 is increased in cells challenged with H2O₂. Collectively our results reveal that thiol-oxidizing agents primarily increase the sensitivity of the IP₃R to Ca²⁺, i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP₃R activity during physiological redox signalling and during pathologicalical oxidative stress.