Hormones – Cytokines – Signaling
Kidney International (1998) 54, 1063–1069; doi:10.1046/j.1523-1755.1998.00090.x
Selectivity of endotoxin-induced defect in endothelial calcium mobilization
Jason G Umans, Debra Salvi, Patrick T Murray and Mark E Wylam
Departments of Medicine, Pediatrics, Anesthesia and Critical Care, and the Committee on Clinical Pharmacology, Division of the Biological Sciences, University of Chicago, Chicago, Illinois, USA
Correspondence: Jason G. Umans, Ph.D., M.D., Section of Nephrology, Department of Medicine, University of Chicago, 5841 S. Maryland Avenue, MC-5100, Chicago, Illinois 60637, USA. E-mail: jumans@medicine.bsd.uchicago.edu
Received 24 February 1998; Revised 22 April 1998; Accepted 23 April 1998.
Abstract
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 
g/ml) and lower doses were without effect. By contrast, LPS more potently (1.0 pg to 1.0 g/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 g/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.
Keywords:
endotoxin, endothelial cells, bradykinin, intracellular calcium, vascular, sepsis
Abbreviations:
ADP, adenosine 5'-diphosphate; BK, bradykinin; EDRF, endothelium-derived relaxing factor; FBS, fetal bovine serum; HBS, HEPES buffered saline; LDL, low density lipoprotein; L-NAME, N
-nitro-L-arginine methyl ester; LPS, lipopolysaccharide; NO, nitric oxide; PBS, phosphate buffered saline; PMT, photomultiplier tube
