OXIDATIVE STRESS INHIBITS BRADYKININ-STIMULATED CA-45(2+) FLUX IN PULMONARY VASCULAR ENDOTHELIAL-CELLS

The effects of oxidant stress and altered glutathione reductase activity on agonist-induced flux of Ca2+ were studied in cultured calf pulmonary artery endothelial cells using radioisotopic Ca-45(2+). Bradykinin-stimulated uptake of Ca-45(2+) was determined after cells were incubated with the membrane-permeant oxidant t-butylhydroperoxide (0.4 mM) for various durations. t-Butylhdroperoixde increased uptake of Ca-45(2+) under basal conditions and significantly decreased bradykinin-stimulated uptake in a time-dependent manner through incubation periods of 2 h. Preincubation of cells with 1,3-bis(chloroethyl)-1-nitrosourea markedly reduced bradykinin-stimulated uptake in cells subsequently treated with t-butylhydroperoxide. Bradykinin-stimulated efflux of Ca-45(2+) and Rb-86+ was examined in control and oxidant-stressed endothelial cells. t-Butylhydroperoxide initially decreased bradykinin-stimulated efflux of Ca-45(2+) but had no effect on Rb-86+ efflux. After more prolonged incubation with the oxidant, stimulated Ca-45(2+) efflux was further inhibited, and basal efflux of Rb-86+ was increased to a rate similar to that observed with bradykinin stimulation. Elevated basal Rb-86+ efflux was blocked by tetrabutylammonium chloride, a selective inhibitor of Ca2+- dependent K+ channels in endothelial cells. These findings, together with our previously described results using fura-2, suggest that oxidant stress initially inhibits bradykinin-stimulated Ca2+ influx and later inhibits stimulated Ca2+ efflux. Finally, cytosolic free Ca2+ concentration becomes persistently elevated and is associated with elevated basal efflux of K+ via the Ca2+ -dependent K+ channel.