Xanthine oxidase inhibits transmembrane signal transduction in vascular endothelial cells.
The effect of xanthine oxidase (XO)-mediated oxidant stress on endothelial cell signal transduction was determined in bradykinin-stimulated cells loaded with the Ca+(+)-sensitive probe fura-2. Calf pulmonary artery endothelial cells were incubated with a reaction mixture containing XO (50 mU/ml) and its substrate, hypoxanthine (HX) (0.5 mM), for periods of 0.5 to 2.0 hr. HX/XO time dependently increased basal cytosolic free Ca++ ([Ca++]i) and decreased the response of [Ca++]i to bradykinin, so that incubation of cells with HX/XO for 1.5 hr or longer eliminated responsiveness to agonist. In presence of XO, HX dose dependently increased basal [Ca++]i (EC50 approximately 3 x 10(-5) M) and decreased the response of [Ca++]i to bradykinin. Sequential application of bradykinin and Ca++ to cells suspended in Ca+(+)-free/EGTA buffer was performed to characterize the effects of HX/XO on receptor-activated Ca++ entry and release of Ca++ from internal stores. HX/XO attenuated internal store Ca++ release and inhibited the bradykinin-stimulated Ca++ influx pathway in a time-dependent manner. When the HX dose was decreased by an order of magnitude, HX/XO selectively inhibited the agonist-stimulated influx pathway with little effect on internal store Ca++ release. Coincubation with superoxide dismutase tended to potentiate the effects of HX/XO, whereas catalase provided almost complete protection. Similar results to HX/XO-induced alterations in Ca++ signaling were observed when glucose-glucose oxidase (G/GO) was used as the oxidant-generating system. Inhibition of Ca++ signaling by HX/XO and G/GO occurred in the absence of decreased cell viability. Together, these results suggest that HX/XO-induced inhibition of signal transduction in endothelial cells is a function of H2O2-mediated oxidant stress and represents an early dysfunction in the process of oxidant injury.
Wesson
D E
DE
Department of Pediatrics, Baylor College of Medicine, Houston, Texas.
Elliott
S J
SJ
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane; D002478:Cells, Cultured; D004533:Egtazic Acid; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005949:Glucose Oxidase / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013482:Superoxide Dismutase / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000494:pharmacology*
D002021:Buffers; D007042:Hypoxanthines; D017382:Reactive Oxygen Species; D019271:Hypoxanthine; D004533:Egtazic Acid; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D002374:Catalase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D001920:Bradykinin; D002118:Calcium