Oxidant stress inhibits the store-dependent Ca(2+)-influx pathway of vascular endothelial cells.

Oxidant stress induced by t-butyl hydroperoxide (t-BuOOH) inhibits bradykinin-stimulated Ca2+ signalling in vascular endothelial cells. The effect of t-BuOOH on intracellular Ca2+ pools was determined by addition of Ca(2+)-releasing agents to fura-2-loaded cells suspended in Ca(2+)-free/EGTA buffer. In control cells, sequential additions of bradykinin and ionomycin produced similar increases in cytosolic free [Ca2+] ([Ca2+]i). By contrast, incubation with t-BuOOH progressively decreased the response of [Ca2+]i to bradykinin and increased that to ionomycin, suggesting that the total (ionomycin-releasable) Ca2+ pool remains replete during oxidant stress. The effect of t-BuOOH on the InsP3-sensitive Ca2+ pool was measured by the increase in [Ca2+]i or efflux of 45Ca2+ stimulated by 2,5-di-t-butylhydroquinone (BHQ). Incubation with t-BuOOH did not inhibit BHQ-stimulated increases in [Ca2+]i or 45Ca2+ efflux, suggesting that the InsP3-sensitive Ca2+ pool remains replete and releasable. Activity of the Ca(2+)-influx pathway stimulated by release of internal Ca2+ stores was determined via re-addition of Ca2+ to BHQ-stimulated cells suspended in Ca(2+)-free/EGTA buffer and via BHQ-stimulated 45Ca2+ uptake. Incubation of cells with t-BuOOH for 1 h significantly inhibited the influx pathway. At later time points, t-BuOOH increased basal [Ca2+]i and potentiated the response of [Ca2+]i to BHQ. Similar results were demonstrated with thapsigargin. Together, these findings suggest that (1) the inhibitory effect of t-BuOOH on bradykinin-stimulated release of Ca2+ from internal stores is not related to depletion of these stores, and (2) inhibition of the store-dependent Ca(2+)-influx pathway occurs by a direct effect of the influx pathway or by inhibition of the mechanism which links the internal Ca2+ store to plasmalemmal Ca2+ influx.