EFFECT OF THAPSIGARGIN AND CAFFEINE ON CA-2+ HOMEOSTASIS IN HELA-CELLS - IMPLICATIONS FOR HISTAMINE-INDUCED CA-2+ 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 experiment 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 Hl 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.