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.
Elliott
S J
SJ
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Doan
T N
TN
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001692:Biological Transport; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D006873:Hydroquinones / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010545:Peroxides / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D020122:tert-Butylhydroperoxide
D000975:Antioxidants; D006873:Hydroquinones; D016877:Oxidants; D010545:Peroxides; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D015759:Ionomycin; D019284:Thapsigargin; D020122:tert-Butylhydroperoxide; D001920:Bradykinin; D002118:Calcium