The role of endoplasmic reticular Ca2+ stores in cell viability and tumor necrosis factor-α production of the murine macrophage RAW 264.7 cell line

Thapsigargin (TG), an endoplasmic reticular (ER) Ca2+-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca2+ pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell tine. We found that treatment with TG (10-800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC50, 200 nM). Lipopolysaccharide (LPS, 1 mu g/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC50, 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca2+ concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca2+ stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca2+ stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line. Copyright (C) 2000 National Science council, ROC and S. Karger AG, Basel.