Effect of diamide‐induced oxidative stress on Ca2+ signaling in cultured bovine aortic endothelial cells (BAEC)

Abstract

Diamide is a thiol‐oxidizing agent commonly used to rapidly and reversibly oxidize intracellular glutathione (GSH) to its disulfide (GSSG) and to increase the formation of protein‐glutathione mixed disulfides (P‐SSG). In the present study we examined the acute effects of diamide on free cytosolic Ca2+ ([Ca2+]i) of cultured BAECs loaded with fura‐2. Diamide increased spontaneous Ca2+ oscillations and elevated basal [Ca2+]i when compared to vehicle‐treated controls. Low concentrations (50, 100 μM) progressively increased the number of cells spontaneously oscillating as well as the oscillation frequency of individual cells, whereas high concentrations (250, 500 μM) caused a synchronized oscillation of almost all cells immediately upon diamide exposure followed by a significant rise in basal [Ca2+]i. Similar dose‐ and time‐dependent effects of diamide were observed in the absence of extracellular Ca2+. Pretreatment with the phospholipase C inhibitor U73122, but not the inactive analog U73343, prevented the observed changes in [Ca2+]i. These results indicate that diamide promotes Ca2+ release from the inositol 1,4,5‐trisphosphate (IP3)‐sensitive internal Ca2+ store and is capable of elevating basal [Ca2+]i in the absence of extracellular Ca2+, effects which may be related to a diamide‐induced glutathionylation of the IP3 receptor and the plasmalemmal Ca2+ pump, respectively. Supported in part by HL097355