Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B)

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca 2+](i)) has been implicated in cellular oxygen-sensing processes. We invest igated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustai ned increase of [C2+](i) while thapsigargin, an inhibitor of endoplasmic re ticulum calcium ATPase, only caused a 20% elevation of [Ca2+](i) within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [C2+](o) did not result in altered ETO or VEGF secretion r ates. Ionomycin decreased EPO production while the lowering of VEGF product ion was not statistically significant. In the presence of extracellular Ca2 + the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimul ated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia inducible factor-1 alpha (HIF-1 alpha). This treatment significa ntly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ conce ntration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.