| Popis: |
Background. Human and rat fundic mucosa contain Enterochromaffinlike(ECL) cells. We have previously shown that these cells undergo apoptosis in the presence of TNF-a, a cytokine that is released during H. pylori infection in the gastric mucosa. Of further interest is basic FGF which is detected in ECL cell tumors. The aim of our current study was to elucidate the mechanisms of TNF-a induced apoptotis in ECL-cells and the role of bFGF during this reponse. MethodslResults. ECL-cells were isolated from rat fundic mucosa to a purity> 90% and cultured up to 24 hr. Western Blotting showed a strong signal from unphosphorylated lKBa at 0 min. After 5 min, the signal was decreased and after 10 min, unphosphorylated lKBa-was no longer detectable, indicating that IKBa was strongly phosphorylated, which in tum leads to the activation of NFKB. To determine NFKB-activation more directly, we performed electromobility-shift assays (EMSA) using cell extracts pretreated with vehicle for I hr, then treated with TNF-a (25 ng/mL) or vehicle for another hour. The EMSAs revealed a specific activation of the NFKB subunits p50 and p65. In order to investigate the functional role of NFKB in the mediation of pro-apoptotic TNF-a effects, we measured ECL-cell apoptosis (programed cell death) after preincubation (I hr) with the specific NFKB-inhibitors MGl32 (5 mM) and PSI (6 mM) and subsequent incubation with TNF-a (25 ng/mL, I hr). Cells incubated without inhibitors and/or TNF-a served as controls. Using the TUNEL technique, TNF-a was found to cause apoptosis in 16% ofECL cells compared to a basal rate of6%. MGI32 and PSI alone did not induce apoptosis (total of 5-8% of ECL cells), but were able to inhibit TNF-a-induced apoptosis completely. For MG132, these results were repeated using an ELISA-assay based upon the detection of nucleosomes which also showed complete inhibition ofTNF-a effects. The observations were verified using the EMSA technique mentioned above. To elucidate the role of basic FGF, we tested bFGF effects on TNF-a induced apoptosis. Using the ELISA, we found that bFGF at 10-100 ng/mL completely blocked TNF-a induced cell death. Conclusion. TNF-a induced apoptosis of ECL cells is mediated by phosphorylation of IKBa and subsequent activation of NFKB. bFGF inhibits TNF-a induced ECL cell death, which may also playa role for the persistence of ECL cells during chronic gastric inflammation (DFG Pr 41112-2). |
