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 microg/ml) and lower doses were without effect. By contrast, LPS more potently (1.0 pg to 1.0 microg/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 > or = 1.0 microg/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.
Department of Medicine, University of Chicago, Illinois 60637, USA. jumans@medicine.bsd.uchicago.edu
J G
JG
Umans
D
D
Salvi
P T
PT
Murray
M E
ME
Wylam
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III; D015398:Signal Transduction / Q000187:drug effects
D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D001920:Bradykinin; D019331:NG-Nitroarginine Methyl Ester; D007213:Indomethacin