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

Abstract

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.

Figure 1

Early light scattering oscillations are enhanced by W-7. Free running oscillations of a cell suspension at 2 × 10⁷ cells per ml were recorded using the light scattering technique as described in Material and Methods 4 h after induction of differentiation. 30 μM W-7 was added where indicated. One out of ten independent experiments is shown.

Figure 2

Just starting cAMP oscillations are upregulated by W-7. cAMP concentrations were determined as described in Materials and Methods. 80 μl samples were withdrawn from the cell suspension before and after addition of 30 μM W-7. The peak cAMP value corresponds to 89 pmol/10⁷ cells. One out of three independent experiments is shown.

Figure 3

Dose response curve for inhibition of cAMP-induced Ca²⁺-influx in the presence of W-7. Each data point represents a separate experiment. Ca²⁺-influx was determined as described in Materials and Methods. The mean control influx was 165 ± 68 pmol per10⁷ cells. The extracellular Ca²⁺-concentration was about 4 μM.

Figure 4

Light scattering oscillation of the IplA minus strain and its response to W-7. The experiment was performed as described in the legend of figure 1.

Figure 5

Induction of vesicular Ca²⁺-release by W-7. Vesicular Ca²⁺-uptake was measured as described in Materials and Methods. Addition of 400 μM ATP initiated Ca²⁺-uptake. Following a Ca²⁺ calibration pulse W-7 was added. One out of ten independent experiments is shown.

Figure 6

Dose response curve for W-7-induced vesicular Ca²⁺-release. The experiment was performed as described in the legend of figure 5. One out of two independent experiments is shown.

Figure 7

Dose response curve for inhibition of W-7-induced vesicular Ca²⁺-release by CaMBP. The experiment was performed as described in the legend of figure 4. 100 μM W-7 was added after preincubation of CaMBP with the vesicles for 10 to 15 min to allow for Ca²⁺-release elicited by CaMBP. 100% Ca²⁺-release amounted to 6 nmol Ca²⁺ per mg protein. One out of three independent experiments is shown.

Figure 8

Inhibition of ATP-dependent vesicular acidification by CaM antagonists. Determination of ATP-dependent acidification was performed as described in Material and Methods. Control acidification amounted to 20 relative fluorescence units. One out of three independent experiments is shown.

Figure 9

Vesicular Ca²⁺-uptake and W-7-induced Ca²⁺-release in the absence or presence of CMA. The experiment was performed as described in the legend of figure 5. 30 μM CMA was added 2 min before ATP addition (right panel) the control received the solvent DMSO (left panel). One out of three independent experiments is shown.

Figure 10

Light scattering and cAMP oscillation in the presence of CMA and W-7. The experiment was performed as described in the legend of figure 2. cAMP concentration were measured before the addition of CMA and after the addition of W-7. The results are expressed as xfold increase in cAMP concentration. One out of three independent experiments is shown.

Figure 11

Cytosolic Ca²⁺-concentration in the presence of CMA or W-7. Single cells were loaded with Fura-2-Dextran and the fluorescence ratio was measured as described in Materials and Methods. One out of three independent experiments is shown.

Figure 12

Light scattering and cAMP-oscillation of the phospholipase C-null-strain, HD10. W-7 was added were indicated. Note that the light scattering change underlying cAMP relay is negative. The experiment was performed as described in the legend of Fig. 2.

Figure 13

Hypothetical scheme for Ca²⁺-oscillations in Dictyostelium and a link to adenylylcyclase. Adenylylcyclase was shown to be activated by G_βγ, rasC, and CRAC. PI₃K activity provides for PIP₃ allowing cytosolic CRAC to bind to the plasma membrane. Aimless and RipA are thought to act in concert with rasC. The direct target of pianissimo is not yet known nor the detailed sequence of events leading to cAMP synthesis. ERK2 stimulation by cAR1 is independent of rasC [8]. ERK2 inhibits RegA, a cytosolic phosphodiesterase. cAR1 also activates phospholipase A₂ that liberates fatty acids (FA) and elicits Ca²⁺-influx [17,26]. Ca²⁺ released by fatty acids from the acidic store activates a positive feedback loop via PLC activity. IP₃ stimulates Ca²⁺-release from the ER, thereby raising cytosolic Ca²⁺ and enhancing PLC activity. Ca²⁺ sinks are provided by Ca²⁺-uptake into the ER and the acidic Ca²⁺-store. Inhibition of either pump, the Ca²⁺-pump by XeC or the H⁺-pump by CMA leads to an increase of [Ca²⁺]_i and to a blockade of Ca²⁺-oscillation as well as cAMP relay [48]. ACA is suggested to be activated by a CalDAG stimulated exchange factor acting on RasC. cAMP released to the outside of the cell elicits feed back stimulation during oscillation in cell suspension. Extracellular phosphodiesterase (PdsA) provides for a low extracellular cAMP level allowing sensitive detection of the next wave.