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) (EC(50) similar to 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.