LPS-induced NF-κB activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB 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-κB’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-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P<0.05). NF-κB 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-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation.

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.