OXIDANT STRESS INHIBITS THE STORE-DEPENDENT CA2+-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 Ca2+-releasing agents to fura-2-loaded cells suspended in Ca2+-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 Ca-45(2+) stimulated by 2,5-di-t-butylhydroquinone (BHQ). Incubation with t-BuOOH did not inhibit BHQ-stimulated increases in [Ca2+]i or Ca-45(2+) efflux, suggesting that the InsP3-sensitive Ca2+ pool remains replete and releasable. Activity of the Ca2+-influx pathway stimulated by release of internal Ca2+ stores was determined via re-addition of Ca2+ to BHQ-stimulated cells suspended in Ca2+-free/EGTA buffer and via BHQ-stimulated Ca-45(2+) uptake. Incubation of cells with t-BuOOH for 1 h significantly inhibited the influx pathway. At later time points, t-BuOOH increased basal [Ca2+], 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 Ca2+-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.