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

Key points In non‐excitable cells, oxidative stress increases inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R) activity, which can cause Ca 2+ oscillations under basal conditions and enhance agonist‐stimulated changes in cytosolic free Ca 2+ concentration. Protein S ‐glutathionylation, the reversible modification of cysteine thiols by glutathione, is elevated in response to oxidative stress, but the consequence of glutathionylation for IP 3 R function is not known. In this study we provide evidence that Ca 2+ ‐induced Ca 2+ ‐release (CICR) via the IP 3 R is enhanced by oxidant‐induced glutathionylation in cultured aortic endothelial cells. Our results suggest glutathionylation may represent a fundamental mechanism for regulating IP 3 R activity during physiological redox signalling and during pathological oxidative stress. Abstract In non‐excitable cells, thiol‐oxidizing agents have been shown to evoke oscillations in cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) by increasing the sensitivity of the inositol 1,4,5‐trisphosphate (IP 3 ) receptor (IP 3 R) to IP 3 . Although thiol modification of the IP 3 R 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 (H 2 O 2 ), on oscillations in [Ca 2+ ] 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 Ca 2+ , acute treatment with either diamide or H 2 O 2 increased the number of BAECs exhibiting asynchronous Ca 2+ oscillations, whereas HAECs were unexpectedly resistant. Diamide pretreatment increased the sensitivity of HAECs to histamine‐stimulated Ca 2+ oscillations and BAECs to bradykinin‐stimulated Ca 2+ oscillations. Moreover, in both HAECs and BAECs, diamide dramatically increased both the rate and magnitude of the thapsigargin‐induced Ca 2+ transient suggesting that Ca 2+ ‐induced Ca 2+ release (CICR) via the IP 3 R is enhanced by glutathionylation. Similar to diamide, H 2 O 2 increased the sensitivity of HAECs to both histamine and thapsigargin. Lastly, biochemical studies showed that glutathionylation of native IP 3 R 1 is increased in cells challenged with H 2 O 2 . Collectively our results reveal that thiol‐oxidizing agents primarily increase the sensitivity of the IP 3 R to Ca 2+ , i.e. enhanced CICR, and suggest that glutathionylation may represent a fundamental mechanism for regulating IP 3 R activity during physiological redox signalling and during pathologicalical oxidative stress.