Selectivity of endotoxin-induced defect in endothelial calcium mobilization

Background. We hypothesized that endotoxin (LPS) would impair bradykinin (BK)-induced calcium (Ca²⁺) mobilization in aortic endothelial cells, perhaps due to cytotoxicity or via stimulation of nitric oxide (NO) synthesis. As well, we sought to define contributions of LPS-stimulated Ca²⁺ mobilization to these effects. Methods. LPS- or BK-induced increments of intracellular Ca²⁺ were assessed by microspectrofluorimetry with fura-2 in passaged bovine aortic endothelial cells. Time- and dose-dependent effects of LPS exposure (± inhibitors of NO or prostaglandin synthesis) on subsequent BK-induced Ca²⁺ mobilization and on attached cell counts were determined. Results. LPS (0.1 to 1.0 mg/ml) led to rapid increments of Ca²⁺, while Ca²⁺ responses were delayed following LPS (1 to 10 μg/ml) and lower doses were without effect. By contrast, LPS more potently (1.0 pg to 1.0 μg/ml) led to dose- and time-dependent impairment of subsequent BK- induced Ca²⁺ mobilization, with peak effect at four to six hours, persisting for at least 18 hours. This delayed effect on BK-response was unaltered by inhibition of either NO synthase or cyclooxygenase. The effect of LPS on BK-responsivity depended importantly on cell confluence, as it was not observed in subconfluent cells. By contrast, LPS-induced cell detachment, which was observed only at doses ≤ 1.0 μg/ml, did not depend on confluence. Conclusions. Different mechanisms lead to endothelial cytotoxicity and to impaired BK-response following LPS. Only the former effect, occurring at higher doses, might depend on initial LPS-induced Ca²⁺ mobilization.