Oxidative stress inhibits bradykinin-stimulated 45Ca2+ 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 45Ca2+. Bradykinin-stimulated uptake of 45Ca2+ was determined after cells were incubated with the membrane-permeant oxidant t-butylhydroperoxide (0.4 mM) for various durations. t-Butylhydroperoxide increased uptake of 45Ca2+ 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 45Ca2+ and 86Rb+ was examined in control and oxidant-stressed endothelial cells. t-Butylhydroperoxide initially decreased bradykinin-stimulated efflux of 45Ca2+ but had no effect on 86Rb+ efflux. After more prolonged incubation with the oxidant, stimulated 45Ca2+ efflux was further inhibited, and basal efflux of 86Rb+ was increased to a rate similar to that observed with bradykinin stimulation. Elevated basal 86Rb+ 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.
Elliott
S J
SJ
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Schilling
W P
WP
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002132:Calcium Radioisotopes; D002330:Carmustine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010545:Peroxides / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D013312:Stress, Physiological / Q000378:metabolism*; D020122:tert-Butylhydroperoxide
D002132:Calcium Radioisotopes; D010545:Peroxides; D000644:Quaternary Ammonium Compounds; D020122:tert-Butylhydroperoxide; C009405:tetrabutylammonium; D012413:Rubidium; D001920:Bradykinin; D002118:Calcium; D002330:Carmustine