LPS-induced NF-kappa B activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat.
OBJECTIVE
To investigate the effect of Ca(2+) on lipopolysaccharide (LPS)-induced NF-kappa B activation in pancreatic acinar cells and the role of NF-kappa B in LPS-induced acinar cell injury.


METHODS
Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-kappa B's subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-kappa B binding to the DNA sequence containing the recognition site of NF-kappa B.


RESULTS
LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P < 0.05). NF-kappa B p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-kappa B DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-kappa B activation preceded the pathological alteration of pancreatic acinar cells. The Ca(2+) chelator EGTA inhibited LPS-induced NF-kappa B activation.


CONCLUSIONS
NF-kappa B activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca(2+) is an important mediator in the process of LPS-induced NF-kappa B activation.
Zhang
Hong
H
Department of Pathophysiology, Medical College of Tongji University, Shanghai 200331, China.
Li
Yongyu
Y
Wang
Shengnian
S
Zhang
Konghua
K
Li
Lijuan
L
Wu
Xianzhong
X
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D016328:NF-kappa B / Q000502:physiology*; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
D008070:Lipopolysaccharides; D016328:NF-kappa B; D002118:Calcium