cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum
© Lusche et al; licensee BioMed Central Ltd. 2005
Received: 07 December 2004
Accepted: 07 March 2005
Published: 07 March 2005
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© Lusche et al; licensee BioMed Central Ltd. 2005
Received: 07 December 2004
Accepted: 07 March 2005
Published: 07 March 2005
Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i.
We analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced.
We conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.
Starving Dictyostelium discoideum amoebae form a multicellular organism by chemotactic aggregation. The signaling molecule that mediates aggregation and development is cAMP. Aggregation proceeds in a rhythmic fashion; cAMP is secreted periodically by cells in the center of the aggregate. Cells in the neighbourhood respond by an oriented inward movement and secrete cAMP themselves to relay the signal. In cell suspensions periodic synthesis and release of cAMP leads to rhythmic shape changes that cause alterations in light transmittance and spike-shaped and sinusoidal light scattering oscillations . The marked rhythmic behaviour of the cell population is also apparent by oscillations of other parameters, e.g. extracellular concentrations of Ca2+, K+ or H+ (for review see ). Recently, changes in [Ca2+]i were postulated to comprise the (or at least a part of the) master oscillator controlling oscillation patterns [3, 4]. A short [Ca2+]i-transient induced by addition of CaCl2 or calmodulin antagonists alters light scattering oscillations and can even reset the oscillation phase . The height of the [Ca2+]i-increase determines whether light scattering and the accompanying cAMP oscillations are abolished or augmented: large [Ca2+]i-transients inhibit cAMP and light scattering oscillations  whereas small [Ca2+]i-elevations enhance oscillations of both parameters . From these experiments it was concluded that Ca2+ exerts a dual control over the production of the first messenger cAMP (for a detailed model see ). cAMP controls its own synthesis as binding of the agonist to cell surface receptors induces a transient [Ca2+]i-elevation [5–7]. However, until now the question as to whether there is an interaction between cAMP acting intracellularly as second messenger and [Ca2+]i in D. discoideum has not been resolved. In other cell systems such as nerve cells crosstalk between the cAMP and the Ca2+ signaling pathway exists that is necessary to generate oscillations of both parameters .
In order to gain insight into a possible connection between intracellular cAMP and [Ca2+]i we used a mutant defective in the phosphodiesterase RegA. RegA is one out of two cAMP-specific phosphodiesterases (for an overview of classes of PDEs in Dictyostelium see ) that is inhibited by IBMX and comprises part of an eukaryotic phospho-relay system [10, 11]. RegA- mutants are rapid developers; their differentiation is shifted towards the stalk pathway [12, 13]. Chemotactic migration is characterized by an increased frequency of lateral pseudopod extension as compared to wild type amoebae . We found that the mutant displayed an altered [Ca2+]i-response pattern upon stimulation with cAMP with an augmentation of Ca2+-release from stores and a concomitant decrease of extracellular Ca2+-entry. Light scattering oscillations and the underlying cAMP oscillations were drastically reduced in regA- cells. Chemotaxis was influenced by the extracellular presence of EGTA. We conclude that indeed, intracellular cAMP signaling and the regulation of [Ca2+]i are linked at the level of Ca2+-storage compartments.
Basal [Ca2+]i and the increase over basal [Ca2+]i after cAMP-addition in wild type and regA- cells in the absence and presence of IBMX. 1 μM cAMP was added to wild type at t7–t8 and to regA- at t4 because the mutant develops more rapidly. [Ca2+]i was determined by ratiometric imaging in single cells either in nominally Ca2+-free buffer (- Ca2+) or in buffer containing 1 mM Ca2+. Values are mean ± s.e.m. and numbers in brackets indicate the numbers of cells tested in at least 3 determinations in at least 2 independent experiments each.
55 ± 1 (131)
97 ± 1 (111)
71 ± 8 (30)
81 ± 9 (25)
+ 1 mM Ca2+
54 ± 1 (85)
96 ± 2 (66)
79 ± 6 (52)
85 ± 8 (35)
53 ± 1 (94)
98 ± 1 (148)
132 ± 9 (58)
+ 1 mM Ca2+
50 ± 1 (185)
53 ± 1 (127)
125 ± 6 (83)
155 ± 10 (55)
The cytosolic concentration of Ca2+ was demonstrated to control light scattering oscillations by affecting the synthesis of cAMP; depending on the height of an artificial [Ca2+]i-transient the production of cAMP which in this case serves as first messenger was either augmented or blocked [3, 4]. The results presented in this study provide evidence for a reciprocal influence of the second messengers cAMP and Ca2+ in Dictyostelium cells. We observed altered agonist-induced Ca2+-fluxes and [Ca2+]i-transients in the regA- mutant cell line where the absence of the main cAMP-hydrolyzing PDE led to a fourfold increased basal cAMP level. One could argue that the effect on [Ca2+]i was not a consequence of the increased basal concentration of cAMP but rather due to a potentially altered pattern of gene expression in the mutant strain. Indeed, this is possible and could result in a different signal perception and/or processing. However, we consider an alteration in gene expression unlikely to be responsible for the augmented [Ca2+]i-transients upon cAMP-stimulation since the same effect could be evoked in wild type amoebae by loading of the PDE inhibitor IBMX into the cytosol. In addition, IBMX evoked an increase in basal [Ca2+]i in both, wild type and mutant cells. In regA- the inhibitor should act on the additional cAMP-PDE (PDE-E) [16, 17] and therefore increase cAMP levels even further. In wild type amoebae hydrolysis of cAMP should be retarded as well. Yet, the threshold of the cAMP concentration required to increase basal [Ca2+]i might not be achieved as consistently as in the mutant since IBMX must act on both PDEs.
The sensitizing effect of the increased amount of cAMP on [Ca2+]i could be caused by several factors. Ca2+-flux characteristics can be changed by influencing Ca2+-channels and/or Ca2+-ATPases located on both, the plasma membrane and membranes of internal stores. When we analyzed Ca2+-fluxes with a Ca2+-sensitive electrode influx was reduced in the mutant while the rates of influx and efflux were unchanged. If the activity of the plasma membrane Ca2+-ATPase (PMCA) was altered then flux rates should be affected. Moreover, the reduced amount of Ca2+-influx precludes activation of a plasma membrane Ca2+-channel. In other cell systems activation of the PMCA and of Ca2+-channels by an increase in cAMP levels was shown [22–24] but our data argue against a stimulating effect on plasma membrane Ca2+-channel or PMCA activity in Dictyostelium amoebae.
The second target of action of cAMP are intracellular stores. Indeed, we showed for the first time that in Dictyostelium a cAMP-activated [Ca2+]i-elevation occurred in the extracellular presence of the Ca2+-chelator BAPTA. This argues for an alteration of Ca2+-uptake into and/or Ca2+-release from stores. An as yet unknown negative regulation of Ca2+-sequestration could cause accumulation of Ca2+ in the cytosol; until now, however, activation of SERCA-type Ca2+-ATPases was found only (for review see ). On the other hand, release of Ca2+ could have been augmented by the high basal cAMP level in the mutant. cAMP-dependent phosphorylation of the IP3-receptor by PKA results in increased sensitivity for IP3 in pancreatic acinar cells ; the same holds true for the ryanodine receptor . Stimulation of PKA activity is plausible since pretreatment with the PKA-activator Sp-5,6-DCl-cBIMPS elevated basal [Ca2+]i and reduced agonist-evoked Ca2+-entry. Membrane permeable Sp-5,6-DCl-cBIMPS was shown to be virtually ineffective in inducing gene expression and to be highly selective for PKA vs cAMP receptor activation at the concentration employed . In summary, we propose the following model: in the mutant sensitivity of the Ca2+-release system is enhanced by an augmented PKA-mediated phosphorylation which is due to increased basal cAMP levels. This results in larger amounts of Ca2+ being liberated upon stimulation. In Dictyostelium release of Ca2+ from stores was also found after addition of calmidazolium  which was shown to inhibit calmodulin-dependent and independent activity of calcineurin . Calcineurin in turn was proposed to be responsible for termination of Ca2+-release by dephosphorylating the IP3-receptor . In regA- augmented release of Ca2+ leads to a reduction of Ca2+-entry across the plasma membrane as a negative feedback.
We suggest the alteration in [Ca2+]i to be responsible for the irregular light scattering and extracellular [Ca2+]-oscillations of regA- cells. Previously, Wessels et al.  have shown that the mutant cannot propagate a cAMP wave since wild type amoebae no longer aggregated correctly when mixed with mutant cells. Indeed, we found that peak cAMP levels during light scattering oscillations were very low in regA- as compared to wild type. This effect is plausible, as the increased sensitivity of the Ca2+ second messenger system exerts a negative feedback on cAMP synthesis: large [Ca2+]i-transients inhibit production of cAMP . An interplay of cAMP and [Ca2+]i-oscillations and their mutual dependence has also been shown in neurons: absence of either, cAMP or [Ca2+]i-oscillations resulted in failure of the other component to oscillate . In Dictyostelium the strong decrease in peak cAMP oscillation levels affected [Ca2+]e-oscillations which were irregular. The basis is probably an influence on [Ca2+]i-oscillations. Such oscillations were suggested to occur but have not been demonstrated in single cells until now, presumably due to the small size of the amoebae and the characteristics of the wave .
With respect to chemotaxis, reduced suppression of lateral pseudopod formation was shown in regA- cells and an essential role of RegA for a correct response in a natural cAMP wave and chemotactic migration was assigned ; subsequently, a similar result was found in a mutant expressing a constitutively active PKA . When we analyzed chemotaxis towards a cAMP-filled glass capillary we observed the same behaviour as described by Wessels et al. . In principle, it is possible that the reduced capacity of regA- cells to polarize was due to a difference in the developmental stage as compared to wild type cells. However, regA- develops much faster than wild type which suggests an even more efficient chemotaxis as this response increases during differentiation to aggregation competence. Alternatively, an altered or dampened signaling response caused by a lower number of cAMP receptors present on the cell surface could have caused the reduced chemotactic response. We consider this to be unlikely for the following reason. Aggregation-competent Dictyostelium amoebae possess roughly 50.000 cAMP receptors at the cell surface . Yet, for chemotactic orientation and polarization in a cAMP gradient the difference in receptor occupancy between the front and the rear end of the amoebae is important rather than the absolute number of stimulated receptors . So even if regA- expressed less receptors than wild type this should not influence the accuracy of the response. We propose the reduced polarization capacity of regA- amoebae to be caused by their altered [Ca2+]i-regulation. In the mutant strain the threshold for generation of an agonist-induced [Ca2+]i-increase is lower than in wild type. The [Ca2+]i-elevation is not as tightly controlled and occurs even in the presence of BAPTA. The characteristics of a [Ca2+]i-increase are important for the resulting cytoskeletal rearrangements and whether pseudopods are formed correctly. Indeed, artificial induction of a small global [Ca2+]i-transient by incubation with calmidazolium caused overall pseudopod protrusion . In migrating cells the establishment of a [Ca2+]i-gradient at the rear end was shown [5, 32] which indicates the presence of a highly organized spatial [Ca2+]i-pattern during chemotaxis. By contrast, a role of the [Ca2+]i-elevation for the chemotactic response was questioned by Traynor et al.  because a mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregated and differentiated almost normally but displayed no cAMP-activated global [Ca2+]i-change; yet, the existence of localized, small [Ca2+]i-transients in this particular mutant cell line that had escaped detection could not be excluded .
When we analyzed the influence of pretreatment with EGTA on chemotactic behaviour of wild type and regA- cells we found that the mutant was more sensitive. When compared to wild type, lower doses of EGTA were sufficient to reduce chemotactic speed. The effect of EGTA treatment is most probably due to emptying of the storage compartments ; the presence or absence of extracellular Ca2+ affects the Ca2+-content of stores [35, 36]. RegA- cells are more sensitive than wild type amoebae because of the lower threshold for Ca2+ release and thus a more rapid depletion of Ca2+ in the cells.
Abnormal basal levels of cAMP impair chemotactic performance by augmenting agonist-activated [Ca2+]i-elevations which in turn lead to uncontrolled pseudopod extension. [Ca2+]i regulates cAMP acting as first messenger in a negative feedback loop: when the [Ca2+]i response is increased the amount of cAMP synthesized upon stimulation is low as observed in regA- cells devoid of the phosphodiesterase RegA. The low level of cAMP relay results in improper light scattering oscillations. We conclude that intracellular cAMP acts on [Ca2+]i via PKA: phosphorylation of the system responsible for release of Ca2+ from stores leads to a greater sensitivity facilitating Ca2+ liberation. The cAMP activated [Ca2+]i-increase is due to Ca2+-release from internal stores which triggers subsequent extracellular Ca2+-entry. The fraction of the [Ca2+]i-elevation that is mediated by liberation of Ca2+ is thus larger in the mutant.
Fura2-dextran and BAPTA were from MoBiTec (Göttingen, FRG). IBMX was purchased from Sigma (Munich, FRG) and cAMP was from Boehringer (Mannheim, FRG). Sp-5,6-DCl-cBIMPS was from Biomol (Hamburg, FRG).
D. discoideum axenic wild type Ax2 was grown as described ; the mutant regA- (kindly provided by Dr. P. Thomason) was grown in the presence of blasticidinS. Cells were washed by repeated centrifugation and resuspension of the cell pellet in cold Sørensen phosphate buffer (17 mM Na+/K+-phosphate, pH 6.0; SP-buffer). Amoebae were shaken at 2 × 107 cells/ml, 150 rpm and 23°C until use. The time, in hours, after induction of development is designated tx.
At t2.5–t4 2 ml of cell suspension was pipetted into cuvettes and aerated. Light scattering oscillations were recorded at 500 nm with a photometer as described .
The total amount of cAMP was determined using the cAMP enzyme immuno assay (Biotrak, Amersham Pharmacia Biotech, Freiburg, FRG) according to the manufacturer's instructions. Samples were prepared as outlined previously .
The extracellular Ca2+-concentration ([Ca2+]e) was measured in 2 ml of cell suspension (5 × 107 cells/ml in 5 mM Tricine, 5 mM KCl, pH 7.0) with a Ca2+-sensitive electrode (Möller, Zürich, Switzerland) as described . [Ca2+]e-oscillations were measured at a cell density of 1 × 108 cells/ml.
Cytosolic [Ca2+]-imaging was done as outlined in . Cells (5 × 107 cells/ml; 20 μl) were loaded at t3 with the Ca2+-indicator fura2-dextran (concentration in the loading solution: 5 mg/ml SP-buffer + 1 mM CaCl2) by electroporation (0°C, 850 V, 3 μF, 200 Ω). Immediately after electroporation, 80 μl of cold 5 mM MgCl2 was added and cells were incubated for 10 min on ice. Then cells were washed 3× with 5 mM Hepes, pH 7.0 (H5-buffer). Washed cells (2–5 μl) were placed on glass coverslips and incubated in a humid chamber until use. When experiments were done in nominally Ca2+-free medium, 85–88 μl of H5-buffer was added 1 min before the [Ca2+]-imaging experiment. To test the response of amoebae in the presence of BAPTA, 75–78 μl of H5-buffer was pipetted to the cells; 10 μl of 10 mM BAPTA was added during the [Ca2+]-imaging experiment and 10–12 sec later cAMP was given. When the response of cells was to be analyzed in the presence of extracellular CaCl2, H5-buffer (85–88 μl) with 1 mM CaCl2 was added to the cells 15 min before the [Ca2+]-imaging experiment to load stores (see also ). cAMP-stimulation was done by adding 10 μl of 10 μM cAMP (± 1 mM CaCl2) to the cells. To load cells with IBMX, they were electroporated with fura2-dextran in the presence of 250 μM of the inhibitor. The cytosolic concentration of IBMX is in the range of maximally 2–5% of the concentration present during electroporation . Measurement of regA- was done at t4 and wild type [Ca2+]-imaging was done at t7–8. In another series of experiments we treated regA- cells with Sp-5,6-DCl-cBIMPS, a membrane permeant activator of PKA . Incubation was done with 37 μM of the activator for 60 min prior to the [Ca2+]-imaging experiment.
Chemotactic performance of the amoebae depends on the degree of differentiation, so their shape was checked prior to the chemotaxis assay. 200 μl of cells at 2 × 107 cells/ml were placed on a coverslip and allowed to settle for at least 30 min. The morphology of the cells was controlled microscopically: when elongated and thus aggregation competent cells were present, an aliquot of cells from the suspension was diluted for the chemotaxis assay. RegA- was tested at t4–t5, wild type was measured at t7–t10. 250 μl of cells in 5 mM Hepes, pH 7.0 (1 × 105 cells/ml) were placed on glass coverslips. After 30 min cells were challenged with a cAMP (100 μM) filled glass capillary and chemotaxis was recorded for 40–45 min either on vidoe tape or images were stored directly on a hard disk. In addition, experiments were done with cells incubated with 2–10 mM EGTA for 30 min to empty Ca2+-storage compartments. Analysis of chemotaxis was done as outlined previously .
Cytosolic free Ca2+ concentration
cAMP-dependent protein kinase
Plasma membrane Ca2+-ATPase
The authors wish to thank Dieter Malchow for many helpful discussions and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the FAZIT foundation.
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