Effect of t-butyl-hydroperoxide on bradykinin-stimulated changes in cytosolic calcium in vascular endothelial cells.

The effect of oxidant stress on agonist-induced changes in endothelial cell cytosolic free Ca2+ (Ca2+i) was measured using the fluorescent probe, fura-2. Cultured vascular endothelial cells were loaded with fura-2 via the acetoxymethyl ester form, fura-2/AM, before incubation with t-butyl-hydroperoxide (0.4 mM). Bradykinin-stimulated changes in (Ca2+i) were measured in cells exposed to the hydroperoxide for 0, 30, 60, 120, and 180 min. Incubation of cells with the oxidant initially (within 30 min) diminished the peak rise in (Ca2+i) that occurs after stimulation with bradykinin. Experiments conducted with cells in a Ca2+-free buffer indicated that t-butyl-hydroperoxide inhibited bradykinin-stimulated Ca2+ influx from the extracellular space and had little effect on agonist-induced release of Ca2+ from internal stores. At the later incubation periods (greater than 60 min), basal (Ca2+i) progressively rose and the peak response to bradykinin progressively decreased. After 180 min, the cells appeared unable to maintain steady-state with respect to Ca2+ flux. These alterations in Ca2+ homeostasis occurred before detectable changes in the ability of the cells to exclude trypan blue. These results suggest that oxidant stress alters the change in Ca2+i of vascular endothelial cells following stimulation with vasoactive agents.