Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP₃-sensitive as well as InsP₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP₃ can be clearly documented using this experimental approach.