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 ([Ca²⁺](i)) has been implicated in cellular oxygen-sensing processes. We investigated 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 sustained increase of [Ca²⁺](i) while thapsigargin, an inhibitor of endoplasmic reticulum 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 [Ca²⁺](o) did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production 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 stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1α (HIF-1α). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca²⁺ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.