Calmidazolium leads to an increase in the cytosolic Ca2+ concentration in Dictyostelium discoideum by induction of Ca2+ release from intracellular stores and influx of extracellular Ca2+

The Ca2+ stores of Dictyostelium discoideum amoebae take part in control of homoeostasis of the cytosolic free Ca2+ concentration ([Ca2+](i)) and the cyclic-AMP-induced [Ca2+](i)-signalling cascade. In order to characterize regulatory mechanisms of these stores, we incubated cells with the calmodulin antagonist calmidazolium. Measurement of permeabilized and intact cells in suspension with a Ca2+-sensitive electrode revealed that calmidazolium induced Ca2+ release from intracellular stores, influx of Ca2+ across the plasma membrane and subsequent efflux. In single fura-2-loaded cells calmidazolium evoked rapid and global transient elevations of [Ca2+](i). Other calmodulin antagonists (trifluoperazine, chlorpromazine, fendiline and W7) also induced transient elevations of [Ca2+](i), which were, however, slower and observed in fewer cells. The calmidazolium-induced influx of extracellular Ca2+ was inhibited by preincubation with 2,5-di-(t-butyl)-1 ,4-hydroquinone (BHQ) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), both known to interact with pumps of the inositol 1,4,5-trisphosphate (IP3)-sensitive store, and by the V-type H+-ATPase inhibitor bafilomycin A(1), which affects the acidosomal Ca2+ store. Incubation with pump inhibitors did not itself induce changes in [Ca2+](i). We conclude that the effects of calmidazolium are, at least in part, mediated by its calmodulin-antagonizing properties, that it acts by inducing Ca2+ release from filled storage compartments, and that its target of action is both the IP3-sensitive store and the acidosome; emptying of these stores leads to influx of extracellular Ca2+.