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+

Abstract

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 Ca(2+)-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 A1, 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+.

Calmodulin and calmodulin-dependent protein kinase II inhibit hormone secretion in human parathyroid adenoma

“…Calmidazolium induced an increase in [Ca 2C ] i , which is in agreement with previous findings reported in other cell types and species, such as endothelial cells (Watanabe et al 1999) and hepatoma cells (Schlatterer & Schaloske 1996, Liao et al 2009). As shown in Fig.…”

Endogenously Bound Calmodulin Is Essential for the Function of the Inositol 1,4,5-Trisphosphate Receptor

“…This approach, however, resulted in different observations and led to some confusion. W-7, calmidazolium, trifluoperazine, chlorpromazine, and fendiline caused Ca 2ϩ release from the endoplasmic reticulum (33)(34)(35), whereas it was also reported that they directly inhibited IICR without interacting with CaM (36).…”

Computational modelling suggests dynamic interactions between Ca2+, IP3 and G protein-coupled modules are key to robust Dictyostelium aggregation

“…We then used the model to provide a mechanistic interpretation of the results of experiments with the application of the CaM inhibitor, calmidazolium (R24571), which causes a transient surge in cytosolic free Ca 2+ concentration. 26,28 It has been shown that one of the targets for the R24571 action is the CaM that regulates PMCA. 55,56 PMCA pumps Ca 2+ out of the cytoplasm and compensates for the constant Ca 2+ leak through the plasma membrane.…”

“…22 and 23. While the above computational studies have significantly advanced our understanding of various aspects of Dictyostelium behaviour, there are a range of questions that still require further elucidation. It has been shown experimentally that the levels of intracellular Ca 2+ and cAMP in Dictyostelium are tightly interconnected [24][25][26][27] and a number of experiments involving the application of calmidazolium (R24571), a calmodulin (CaM) inhibitor, to Dictyostelium have demonstrated a dramatic impact on the light scattering oscillations 26,28 which are one of the major characteristics of aggregation. 26,29 Ca 2+ has also been shown to be directly involved in cell migration via the stretch-activated Ca 2+ channels (SAC)s. 1 Previously developed models for Dictyostelium signalling and aggregation do not allow these phenomena to be investigated, however, as they all omit one or more of the networks involved.…”

“…Eqn (28) and (29) have been used in our model to describe the modulation of the ACA and ERK2 activities when phosphorylated by PKA. 22 Procedure for comparing the robustness of the proposed model with the G protein-coupled receptor model…”