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cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233–238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura 2–dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

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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+.

cAMP-induced Ca2+ influx in Dictyostelium follows two pathways: a G-protein-dependent pathway where influx is reduced by 50-70% in Galpha2 and Gbeta-negative strains and a heterotrimeric G-protein-independent pathway. Using a pharmacological approach, we found that phospholipase A2 (PLA2) is the target of both pathways. The products of PLA2 activity, arachidonic acid (AA) and palmitic acid, induced Ca2+ influx to a similar extent as cAMP. Half-maximal activation occurred at 3 microM AA and saturation at 10 microM AA. The response to AA was quantitatively similar throughout early differentiation and thus independent of cAMP-receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The PLA2-activating peptide, a peptide with sequence similarity to the G-protein beta-subunit, activated Ca2+ influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C and other kinases, suggesting that tyrosine kinase may directly or indirectly activate PLA2 in this case.