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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+.

SUBMITTER: Schlatterer C

PROVIDER: S-EPMC1216958 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

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Project description:BackgroundStimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.ResultsWe investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae.ConclusionThese results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.

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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+.

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