A fast Ca2+-induced Ca2+-release mechanism in Dictyostelium discoideum.

In vertebrate cells calcium-induced calcium release (CICR) is thought to be responsible for rapid cytosolic Ca(2+) elevations despite the occurrence of strong Ca(2+) buffering within the cytosol. In Dictyostelium, a CICR mechanism has not been reported. While analyzing Ca(2+) regulation in a vesicular fraction of Dictyostelium rich in Ca(2+)-flux activity, containing contractile vacuoles (CV) as the main component of acidic Ca(2+) stores and ER, we detected a rapid Ca(2+) change upon addition of Ca(2+) (CIC). CIC was three times larger in active stores accumulating Ca(2+) than before Ca(2+) uptake and in inactivated stores. Ca(2+) release was demonstrated with the calmodulin antagonist W7 that inhibits the V-type H(+)ATPase activity and Ca(2+) uptake of acidic Ca(2+) stores. W7 caused a rapid and large increase of extravesicular Ca(2+) ([Ca(2+)](e)), much faster and larger than thapsigargin (Tg), a Ca(2+)-uptake inhibitor of the ER. W7 treatment blocked CIC indicating that a large part of CIC is due to Ca(2+) release. The height of CIC depended on the filling state of the Ca(2+) stores. CIC was virtually unchanged in the iplA(-) strain that lacks a putative IP(3) or ryanodine receptor thought to be located at the endoplasmic reticulum. By contrast, CIC was reduced in two mutants, HGR8 and lvsA(-), that are impaired in acidic Ca(2+)-store function. Purified Ca(2+) stores enriched in CV still displayed CIC, indicating that CV are a source of Ca(2+)-induced Ca(2+) release. CIC-defective mutants were altered in their oscillatory properties. The irregularity of the HGR8 oscillation suggests that the principal oscillator is affected in this mutant.

The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx.

BACKGROUND: cAMP-induced Ca2+-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca2+-stores: acidic stores and the endoplasmic reticulum (ER). The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated. RESULTS: Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells. CONCLUSION: The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.

Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+ signaling in Dictyostelium discoideum.

Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.

cGMP-phosphodiesterase antagonists inhibit Ca2+-influx in Dictyostelium discoideum and bovine cyclic-nucleotide-gated-channel.

We used antagonists of cGMP-phosphodiesterases to examine the role of cGMP for light-scattering oscillations and cAMP-induced Ca(2+)-influx in Dictyostelium discoideum, however, SCH 51866 (cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl-cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one) and sildenafil citrate (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1-H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazine citrate) were poor inhibitors of cGMP-hydrolysis. Instead, SCH 51866 (IC(50) = 16 microM) and sildenafil, blocked chemoattractant (cAMP)-induced Ca(2+)-influx as determined with a Ca(2+)-specific electrode. SCH 51866 (150 microM) affected neither spontaneous cGMP transients during light-scattering-oscillations nor cAMP-mediated K(+)-efflux. SCH 51866 and sildenafil are competitive inhibitors of cGMP phosphodiesterases. However, the activity of cGMP-dependent protein kinase Ialpha (PKGIalpha) was not altered by SCH 51866 (150 microM). By contrast, patch-clamp measurements of bovine cone cGMP-gated-channels (cyclic-nucleotide-gated-channel, CNGA3), stably expressed in human embryonic kidney cells, HEK 293 cells, revealed reversible, competitive and dose-dependent inhibition of sodium currents by SCH 51866 (IC(50) = 25 microM) and sildenafil, but not by another inhibitor of cGMP-phosphodiesterases, UK 114,542. The possibility that D. discoideum cells also express a cGMP-regulated channel is supported by our finding that LY 83583 (6-(phenylamino)-5,8-quinolinedione) (35 microM), known to inhibit cyclic-nucleotide-gated-channels as well as guanylyl-cyclases, reduced cAMP-induced Ca(2+)-influx in D. discoideum, but did not affect cAMP-induced cGMP accumulation. Utilizing a PDED null strain that exhibits a prolonged and elevated cGMP transient following receptor activation, we found that the inhibition of Ca(2+)-influx by SCH 51866 in the wildtype was absent in the mutant. Our results show that SCH 51866 and sildenafil are antagonists of a Ca(2+)-permeable channel (CNGA3) and that both compete with cGMP for a regulatory site of Ca(2+)-influx in D. discoideum.

Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum.

BACKGROUND: Stimulation 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. RESULTS: We 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. CONCLUSION: These 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.

A link of Ca2+ to cAMP oscillations in Dictyostelium: the calmodulin antagonist W-7 potentiates cAMP relay and transiently inhibits the acidic Ca2+-store.

BACKGROUND: During early differentiation of Dictyostelium the attractant cAMP is released periodically to induce aggregation of the cells. Here we pursue the question whether pulsatile cAMP signaling is coupled to a basic Ca2+-oscillation. RESULTS: We found that the calmodulin antagonist W-7 transiently enhanced cAMP spikes. We show that W-7 acts on an acidic Ca2+-store: it abolished ATP-dependent vesicular acidification, inhibited V-type H+ATPase activity more potently than the weaker antagonist W-5 and caused vesicular Ca2+-leakage. Concanamycin A, an inhibitor of the V-type H+-pump, blocked the Ca2+-leakage elicited by W-7 as well as cAMP-oscillations in the presence of W-7. Concanamycin A caused an increase of the cytosolic Ca2+-concentration whereas W-7 did not. In case of the latter, Ca2+ was secreted by the cells. In accord with our hypothesis that the link from Ca2+ to cAMP synthesis is mediated by a Ca2+-dependent phospholipase C we found that W-7 was not active in the phospholipase C knockout mutant. CONCLUSION: We conclude that the potentiation of cAMP relay by W-7 is due to a transient inhibition of the acidic Ca2+-store. The inhibition of the proton pump by W-7 causes a leakage of Ca2+ that indirectly stimulates adenylyl cyclase activity via phospholipase C.

Cyclic AMP- and Ins(1,4,5)P3-induced Ca2+ fluxes in permeabilised cells of Dictyostelium discoideum: cGMP regulates Ca2+ entry across the plasma membrane

Transduction of chemotactic signals in Dictyostelium discoideum apparently involves a precise regulation of the cytosolic Ca2+ concentration. Cyclic AMP stimulation causes Ca2+ influx followed by Ca2+ extrusion, the magnitude of extrusion depending on the state of differentiation. Here, we show that the cAMP receptor controls Ca2+ influx both at the level of entry across the plasma membrane and at the level of transport into Ca2+-sequestering organelles. The use of permeabilised cells allowed us to discriminate between both fluxes. Permeabilised cells still showed the cAMP-induced Ca2+ uptake. The flux across the plasma membrane was more sensitive to Ba2+ and Mn2+, respectively, than Ca2+ sequestration. We have shown previously, using stmF mutants, that cGMP regulates Ca2+ influx. We confirmed this result with the membrane-permeant cGMP-analogue, Sp-8-Br-cGMPS, which enhanced the cAMP-induced Ca2+ influx in intact cells but not the uptake in permeabilised cells, indicating that cGMP regulates Ca2+ influx across the plasma membrane. Occasionally, a fast transient Ca2+ efflux, preceding the influx, occurred in intact cells. A small cAMP-induced Ca2+ release was also found in permeabilised cells. A similarly sized Ca2+ release was elicited by Ins(1,4,5)P3 and could be substituted for by GTP or GTPgammaS. This result suggests that rapid Ca2+ release can be mediated by Ins(1,4,5)P3.