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Originally published In Press as doi:10.1074/jbc.M307096200 on February 18, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18407-18414, April 30, 2004
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Cytosolic [Ca2+] Transients in Dictyostelium discoideum Depend on the Filling State of Internal Stores and on an Active Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) Ca2+ Pump*

Christina Schlatterer{ddagger}, Kathrin Happle, Daniel F. Lusche, and Jürgen Sonnemann§

From the Faculty for Biology, University of Konstanz, 78457 Konstanz, Germany and the §Institute of Pharmacology, Pediatric Oncology, and Hematology, University of Greifswald, 17487 Greifswald, Germany

Received for publication, July 3, 2003 , and in revised form, January 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of Dictyostelium discoideum with cAMP evokes a change of the cytosolic free Ca2+ concentration ([Ca2+]i). We analyzed the role of the filling state of Ca2+ stores for the [Ca2+] transient. Parameters tested were the height of the [Ca2+]i elevation and the percentage of responding amoebae. After loading stores with Ca2+, cAMP induced a [Ca2+]i transient in many cells. Without prior loading, cAMP evoked a [Ca2+]i change in a few cells only. This indicates that the [Ca2+]i elevation is not mediated exclusively by Ca2+ influx but also by Ca2+ release from stores. Reducing the Ca2+ content of the stores by EGTA preincubation led to a cAMP-activated [Ca2+]i increase even at low extracellular [Ca2+]. Moreover, the addition of Ca2+ itself elicited a capacitative [Ca2+]i elevation. This effect was not observed when stores were emptied by the standard technique of inhibiting internal Ca2+ pumps with 2,5-di-(t-butyl)-1,4-hydroquinone. Therefore, in Dictyostelium, an active internal Ca2+-ATPase is absolutely required to allow for Ca2+ entry. No influence of the filling state of stores on Ca2+ influx characteristics was found by the Mn2+-quenching technique, which monitors the rate of Ca2+ entry. Both basal and cAMP-activated Mn2+ influx rates were similar in control cells and cells with empty stores. By contrast, determination of extracellular free Ca2+ concentration ([Ca2+]e) changes, which represent the sum of Ca2+ influx and efflux, revealed a higher rate of [Ca2+]e decrease in EGTA-treated than in control amoebae. We conclude that emptying of Ca2+ stores does not change the rate of Ca2+ entry but results in inhibition of the plasma membrane Ca2+-ATPase. Furthermore, the activities of the Ca2+ transport ATPases of the stores are of crucial importance for the regulation of [Ca2+]i changes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Agonist-induced [Ca2+]i1 signaling induces liberation of stored Ca2+ and capacitative or noncapacitative Ca2+ entry in almost all nonexcitable cells (for a review, see Refs. 1–3). In Dictyostelium discoideum, the chemotactic stimulus cAMP activates a [Ca2+]i transient (4–7). There are several hypotheses concerning the participation of individual parameters in this [Ca2+]i increase. One hypothesis favors extracellular Ca2+ influx to be the sole source for the generation of cAMP-induced [Ca2+]i transients (6), since they were practically undetectable at extracellular Ca2+ levels of less than 10 µM (5, 6). Internal stores were considered to be of minor importance and to be involved secondary to influx. Indeed, Ca2+ entry occurs shortly after binding of cAMP to its receptor (8). Influx is substantial already at 2–4 µM [Ca2+]e and was calculated to result in a [Ca2+]i elevation of roughly 10 µM (8). Subsequent activation of plasma membrane Ca2+-ATPases results in efflux, which, however, decreases in the course of differentiation (8) with a concomitant increase of the level of sequestered Ca2+ (9). The discrepancy between Ca2+ flux and [Ca2+]i measurements argues for a strong local and/or temporal restriction of the resulting [Ca2+]i signal; candidates responsible for restriction are highly efficient transport ATPases leading to sequestration of Ca2+ in stores.

In contrast to the above model, a second hypothesis assigns a crucial function to internal stores as the trigger for the generation of agonist-activated [Ca2+]i transients, with liberation of sequestered Ca2+ being followed by capacitative Ca2+ entry. cAMP-induced Ca2+ influx was observed to be sensitive to agents known to empty storage compartments; it was blocked, for example, by inhibitors of H+ and Ca2+ pumps present on either the acidic, fatty acid-sensitive (10, 11) or the inositol 1,4,5-trisphosphate-sensitive store (11–14), so both types of stores were concluded to regulate Ca2+ entry via capacitative Ca2+ influx (11, 14).

In this study, we investigated the importance of Ca2+ influx and of the filling state of internal Ca2+ stores for agonist-induced [Ca2+]i transients. We found that preloading stores with Ca2+ led to an increase in the number of cells responding toward cAMP stimulation, which indicates that liberation of stored Ca2+ is the trigger for cAMP-induced Ca2+ influx. Reduction of the Ca2+ content of the stores increased the responsiveness of the cells by inhibition of the plasma membrane Ca2+ pump rather than by an increased rate of Ca2+ influx. Moreover, we found that the classical way to activate capacitative Ca2+ entry does not operate in Dictyostelium; inhibition of the internal SERCA Ca2+ pump abolished both agonist- and CaCl2-induced Ca2+ entry.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Fura2-dextran was obtained from MobiTec and cAMP from Roche Applied Science. BHQ was from Aldrich.

Cell Culture—D. discoideum strain Ax2 was cultured as described (11). Growing cells were washed by centrifugation and resuspension of the cell pellet in cold Sørensen phosphate buffer (17 mM Na+/K+-phosphate, pH 6.0). Amoebae were shaken at 2 x 107 cells/ml, 150 rpm, and 23 °C until use. Time, in hours, after induction of development is designated tx.

[Ca2+]i Measurements and Mn2+-quenching Experiments—Cells were loaded at t2–t3 with fura2-dextran as described (15). The use of fura2-dextran prevents the rapid sequestration and extrusion of the dye that occurs when either fura2 is loaded or cells are incubated with the membrane-permeant dye fura2-AM (16). Nonviable cells (typically 20–30% of the cells) and extracellular fura2-dextran were removed by repeated washing in an Eppendorf centrifuge (15). Aliquots of washed amoebae in H5-buffer (5 mM Hepes, 5 mM KCl, pH 7.0; 2–5 µl) on glass coverslips were placed in a humid chamber until use. Under control conditions, 85–88 µl of H5 buffer containing different amounts of Ca2+ (1 µM to 1 mM) were added 15 min prior to the [Ca2+]i imaging experiment.

When the effect of inhibition of intracellular pumps on agonist-activated [Ca2+]i transients was assayed, preincubation with 100–200 µM BHQ in 90 µl of H5 buffer was done for 5–15 min before the [Ca2+]i measurement. The effect of 50 µM BHQ itself on [Ca2+]i was tested in Ca2+-free H5 buffer; during the [Ca2+]i imaging experiment, 1 mM BAPTA was given to the amoebae 10 s prior to BHQ addition. In another set of experiments designed to reduce the Ca2+ content of the stores cells were preincubated for 1 h in H5 buffer plus 0.1 or 1 mM EGTA (10 µl). 10 min prior to the experiment, this solution was carefully removed and replaced by 100 µl of buffer to which variable amounts of CaCl2 had been added. This was repeated three times; final volume was 90 µl. All Ca2+-containing buffers were prepared with triple distilled H2O using plasticware throughout to avoid Ca2+ contamination by glassware. Free [Ca2+] of these buffers was measured with a [Ca2+]-sensitive electrode; the free [Ca2+] of nominally Ca2+-free H5 buffer was in the range of 150–600 nM. When 1 µM CaCl2 had been added, the free [Ca2+] ranged from 2–2.5 µM; when 3 µM CaCl2 was added, free [Ca2+] was in the range of 4–4.5 µM. External [Ca2+] values given under "Results" refer to the amounts of added CaCl2.

[Ca2+]i imaging experiments were performed at the aggregation-competent stage of development (t7–t8) as described (11); stimulation was done by adding 10 µl of cAMP (10 µM) or 10 µl of CaCl2 (30 µM to 10 mM) in H5 buffer. The values given for a [Ca2+]i change refer to the increase above basal [Ca2+]i. For Mn2+-quenching assays, cells in H5 buffer were challenged with Mn2+ or Mn2+/cAMP. Fluorescence quenching was measured at 360-nm excitation; influx rates are expressed as decrease of fluorescence units/s.

Electrode Recordings of [Ca2+]e—The extracellular free Ca2+ concentration ([Ca2+]e) in cell suspensions was measured as described (11). Fluxes were analyzed at t5.5–t7 in cells with emptied (EGTA preincubation) stores or after inhibition of Ca2+ uptake into stores by BHQ treatment. Amoebae were incubated in EGTA (5 mM; 30 min) or in BHQ (100 µM; 15 min). Then cells were washed and resuspended at 5 x 107 cells/ml in 5 mM Tricine, 5 mM KCl, pH 7.0. All measurements were done in this buffer (2-ml test volume).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
External [Ca2+] Influences Agonist-induced [Ca2+]i Responses—We investigated the dependence of cAMP-activated [Ca2+]i transients on extracellular Ca2+ levels by analysis of the reaction in buffer containing different amounts of Ca2+. At our standard condition (i.e. when incubated in 1 mM CaCl2 for 10–15 min), roughly 70% of amoebae displayed an agonist-induced global [Ca2+]i increase of 31 ± 3 nM (mean ± S.E.; Table I and Figs. 1 and 2). The average basal [Ca2+]i under this condition was 50 ± 0.3 nM, which was similar to basal [Ca2+]i of 51 ± 0.4 nM in nominally Ca2+-free buffer. Lowering external Ca2+ affected the fraction of cells reacting toward stimulation with cAMP. In the presence of 10 µM CaCl2 a [Ca2+]i elevation occurred in only 17% of amoebae; its height (34 ± 3nM; mean ± S.E.) was similar to that observed at 1 mM Ca2+. When further reduced to 1 µM CaCl2, a remainder of 7% of cells responded upon cAMP stimulation with an average [Ca2+]i change of 24 ± 3 nM. The duration of the [Ca2+]i increase was independent of the level of extracellular Ca2+ and lasted 30 ± 0.8 s (mean ± S.E.). In nominally Ca2+-free buffer, a cAMP-elicited [Ca2+]i change was not observed (29 cells tested). According to Nebl and Fisher (6), [Ca2+]i increases are mediated solely by Ca2+ influx. However, when 1 mM CaCl2 was added just prior to or concomitantly with cAMP, the percentage of responding cells was only 5% (214 cells tested in 16 independent experiments) instead of the ~70% seen at the standard condition. This indicates that the mere presence of extracellular Ca2+ does not result in sufficient influx to achieve a global [Ca2+]i elevation but that there are other components that participate in its control. We therefore sought to determine the influence of the state of internal storage compartments on the generation of the [Ca2+]i transient.


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  		TABLE I
Percentage of cells displaying a [Ca2+]i transient after cAMP addition

1 µM cAMP was given at t7–t8. After preincubation with EGTA (1 h), amoebae were washed and incubated in H5 buffer to which the indicated [Ca2+] had been added. None, the control situation where cells were kept in the respective buffer for 15 min before stimulation. The numbers in parentheses indicate the numbers of cells tested in at least four determinations in at least two independent experiments each. ND, not determined.

 



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  		FIG. 1.
The height of the cAMP-induced [Ca2+]i change at low external [Ca2+] depends on the filling state of the stores. a, control response (mean [Ca2+]i ± S.E.; 14 cells) in the continued presence of 1 mM Ca2+. b, the response of cells at 3 µM extracellular Ca2+ was small (b, mean [Ca2+]i ± S.E.; 14 cells). Emptying of stores by EGTA preincubation (0.1 mM, 1–2 h) led to a large [Ca2+]i transient at 3 µM Ca2+ (c, mean [Ca2+]i ± S.E., 20 cells). The arrows indicate the addition of 1 µM cAMP. Levels of extracellular Ca2+ refer to the amounts of CaCl2 added to H5 buffer.

 



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  		FIG. 2.
The height of an agonist-evoked [Ca2+]i increase at different external [Ca2+] is affected by the state of the stores. Amoebae were challenged with 1 µM cAMP at t7–t8. The average [Ca2+]i change above basal [Ca2+]i is shown as mean ± S.E. Values of extracellular Ca2+ refer to doses of CaCl2 added to the buffer.

 
The Filling State of the Stores Affects the Agonist-induced [Ca2+]i Response—We reasoned that when mechanisms of Ca2+ uptake in internal stores are blocked, then cAMP-induced [Ca2+]i transients might be seen also at low extracellular [Ca2+]. Inhibitors of SERCA-type Ca2+-ATPases attenuate Ca2+ entry elicited by both cAMP and fatty acids such as arachidonic acid (AA) (11), yet residual Ca2+ liberation and subsequent diminished influx might still result in a [Ca2+]i increase when sequestration is blocked. We therefore tested the effect of the inhibitor BHQ on cAMP-, AA-, and Ca2+-induced [Ca2+]i transients. The more commonly employed inhibitor thapsigargin was not used, since we found it to be effective in D. discoideum only at roughly 50–200 times higher concentrations than generally applied (17, 18). The addition of 50 µM BHQ itself to cells in Ca2+-free buffer containing 1 mM BAPTA elicited a small transient [Ca2+]i elevation (28 ± 2nM; mean ± S.E.) in 36% of the amoebae (146 cells tested in 20 determinations in four independent experiments). However, in cells preincubated with 100–200 µM BHQ, both cAMP- and AA-activated [Ca2+]i elevations were absent, showing that residual Ca2+ fluxes were not sufficient to trigger a global [Ca2+]i change (61 and 119 cells analyzed in 4 and 8 determinations in 2 and 3 independent experiments for cAMP and AA, respectively). By contrast to data from other cell systems where store emptying via pump inhibition represents the standard protocol to activate capacitative Ca2+ entry (3), application of 1 mM CaCl2 to Dictyostelium cells preincubated with BHQ resulted in no capacitative [Ca2+]i increase (275 cells tested in 32 determinations in seven independent experiments). Moreover, when Ca2+ entry was assayed by analysis of extracellular [Ca2+] changes in cell suspensions with an ion-sensitive electrode, there was a significant (p = 0.001) reduction of the rate of Ca2+ influx after preincubation with 100 µM BHQ; the rate of Ca2+ entry induced by the addition of 10 µM CaCl2 was diminished by 56% compared with control cells (rate of 0.32 ± 0.06 µM/min in control cells versus 0.14 ± 0.01 µM/min in BHQ-treated cells; mean ± S.E. of three independent experiments and seven determinations each). These results argue for a requirement of active internal pump(s) to stimulate Ca2+ entry.

Since BHQ was also reported to affect plasma membrane Ca2+ channels and thus might block Ca2+ entry directly or indirectly (19, 20), we compared basal [Ca2+]i in cells pretreated with 100–200 µM BHQ with that of untreated cells, in the presence and absence of extracellular Ca2+. We found that in nominally Ca2+-free buffer, cells incubated with BHQ had a basal [Ca2+]i that was not significantly different from that of untreated amoebae (i.e. in the range of 51 nM) (see above). By contrast, in the presence of 1 mM CaCl2, basal [Ca2+]i was significantly lower in untreated cells (average of 50 nM) (see above) than in cells incubated with BHQ (on average 86 ± 1nM after 8–15 min of incubation; mean ± S.E. of 145 cells) arguing against inhibition of Ca2+ entry by BHQ. Therefore, we conclude that in Dictyostelium BHQ acts by blockade of Ca2+ uptake into internal stores rather than by inhibiting plasma membrane Ca2+ channels.

An alternative method to manipulate the filling state of the stores without impairing uptake mechanisms is to incubate amoebae with EGTA. This should lead to a depletion of sequestered Ca2+, since the presence of the chelator in the medium should result in leakage of Ca2+ from the amoebae. Indeed, continuous efflux of 45Ca2+ was observed in amoebal suspensions after washing in Ca2+-free buffer (21), and quantitative x-ray microanalysis revealed that the amount of Ca2+ stored intracellularly critically depends on the level of extracellular Ca2+ (9, 22). Table I shows that EGTA pretreatment strongly affected the dependence of cAMP-activated [Ca2+]i transients on extracellular Ca2+ levels. Starting from an average basal [Ca2+]i of 57–59 nM, a greater portion of cells displayed an agonist-induced [Ca2+]i increase at low external [Ca2+]. In 38% of amoebae preincubated with 0.1 mM EGTA, a transient [Ca2+]i elevation was observed in the presence of 1 µM CaCl2. The [Ca2+]i increase was significantly higher than under control conditions and even augmented when cells had been treated with 1 mM EGTA (Figs. 1 and 2). However, under this experimental condition, amoebae appeared contracted and occasionally detached from the substrate after the addition of cAMP; detached amoebae were not included in the determinations. Detachment was not observed after preincubation with 0.1 mM EGTA; cells were spread and fully adherent to the substrate.

Emptying of stores by EGTA treatment also influenced the [Ca2+]i response upon the addition of CaCl2. Just 10–12% of the cells kept in nominally Ca2+-free buffer displayed a transient [Ca2+]i elevation after challenge with 1 or 0.1 mM CaCl2 (Table II). By contrast, many more amoebae reacted when preincubated with 0.1 or 1 mM EGTA (30 and 50%, respectively). [Ca2+]i transients occurred even when low doses of 10 and 3 µM CaCl2 were added, albeit in fewer cells. Again, the height of the increase was even augmented after preincubation with 1 mM EGTA (Figs. 3 and 4). The duration of the increase was also higher (25 ± 0.8 s in cells treated with 0.1 mM EGTA versus 21 ± 0.5 s in control amoebae; mean ± S.E.). These data indicate that when emptying of stores is done by EGTA incubation, capacitative Ca2+ entry mechanisms are operative.


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  		TABLE II
Percentage of cells displaying a [Ca2+]2 transient after the addition of CaCl2

Amoebae were preincubated with EGTA or kept in buffer containing 600 nM Ca2+. All cells were washed with this buffer and stimulated with CaCl2. The numbers in parentheses indicate the numbers of cells tested (at least four determinations in at least two independent experiments each).

 



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  		FIG. 3.
Store-operated Ca2+ influx evokes a [Ca2+]i elevation. Cells preincubated in EGTA (1 mM, 1 h) were kept in nominally Ca2+-free buffer. The addition of 30 µM CaCl2 and 1 µM cAMP (arrows) led to [Ca2+]i transients. Mean [Ca2+]i ± S.E. of 24 cells at t7–t8 is shown.

 



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  		FIG. 4.
The average height of a [Ca2+]i increase induced by CaCl2 depends on preincubation conditions. Different amounts of CaCl2 were added to cells in nominally Ca2+-free buffer. Values represent mean [Ca2+]i increase above basal [Ca2+]i ± S.E.

 
Analysis of Ca2+ Influx by Mn2+ Quenching and with a Ca2+ Electrode in Cell Suspensions—The effect of EGTA treatment on [Ca2+]i changes might be due to an increased rate of Ca2+ entry. The kinetics of Ca2+ influx were measured via quenching of fura2-dextran fluorescence by Mn2+. The basal influx rate increased by a factor of 5 in the presence of cAMP (Fig. 5). However, neither basal nor cAMP-stimulated influx rates were significantly increased by pretreatment with EGTA (Table III), so this parameter does not seem to be responsible for the increased percentage of responding cells and the altered height of the [Ca2+]i increase after EGTA incubation. However, when basal Ca2+ fluxes were assayed with a Ca2+-sensitive electrode in cell suspensions, there was a significant (p < 0.001) difference between the response of control amoebae and those preincubated with EGTA (Fig. 6). In suspensions of EGTA-treated cells, the rate of the [Ca2+]e decrease observed after the addition of Ca2+ was larger than in control cells. Changes in the extracellular free [Ca2+] represent the sum of influx and efflux, whereas Mn2+ quenching measures influx only, so the difference between the results argues for an inhibition of the plasma membrane pump after preincubation with EGTA. Further analysis of Ca2+ fluxes activated by the addition of 1 µM cAMP supported the view of an inhibitory effect on plasma membrane Ca2+-ATPase (PMCA) activity. We found that the rate of Ca2+ efflux that follows cAMP-induced Ca2+ entry was significantly diminished (Fig. 6) in cells pretreated with EGTA (0.86 ± 0.06 µM/min as compared with 1.49 ± 0.07 µM/min in control amoebae; mean ± S.E. of at least 15 determinations in four independent experiments). The amount of Ca2+ released was also significantly reduced. Shortly after the removal of EGTA, Ca2+ efflux was in the range of only 20–30% of the preceding influx and gradually increased to roughly 100%; on average, it amounted to 76 ± 7% in EGTA-treated amoebae versus 116 ± 7% in control cells (mean ± S.E. of at least 11 determinations in four independent experiments).



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  		FIG. 5.
Basal and cAMP-induced Mn2+ influx. Influx was assayed by quench of fura2-dextran fluorescence. Cells in nominally Ca2+-free buffer were challenged with 1 µM Mn2+ (a) or 1 µM Mn2+ and cAMP (b). Fluorescence intensity at 360-nm excitation is shown as mean ± S.E. of 45 and 60 cells, respectively.

 


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  		TABLE III
Rate of basal and cAMP-induced Mn2+ influx

Amoebae were challenged with 1 µM Mn2+ either with or without 1 µM cAMP. Quenching of fura-2-dextran fluorescence was assayed at 360-nm excitation. Cells were preincubated with 0.1 mM EGTA as outlined under "Experimental Procedures." Mn2+ quench was tested in nominally Ca2+-free buffer and is given as decrease in fluorescence units/s (mean ± S.E.). The numbers in parentheses show number of experiments (ne), number of determinations (nd), and total number of cells (nc) tested.

 



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  		FIG. 6.
Extracellular Ca2+ fluxes proceed at an altered rate after preincubation of amoebae with EGTA. Changes in [Ca2+]e reflect the sum of Ca2+ influx into and release from cells when measured with a Ca2+-sensitive electrode in cell suspensions. a, the addition of CaCl2 evokes a decrease of [Ca2+]e, which proceeds faster in EGTA-treated than in control cells. Rates of Ca2+ influx at two different [Ca2+]e ranges (2–6 and 6–11 µM) are shown. Bars, mean ± S.E. of at least eight determinations in four independent experiments. b, cAMP induces a transient [Ca2+]e decrease caused by Ca2+ influx into and efflux from cells (see also c). The rate of Ca2+ efflux is reduced in EGTA-treated amoebae. Bars, mean ± S.E. of at least 15 determinations in 4 independent experiments. c, [Ca2+]e recording in a control cell suspension during cAMP stimulation. Subsequent to influx, Ca2+ is released back into the medium. d, [Ca2+]e recording in a suspension of cells preincubated with EGTA (5 mM, 30 min). The first and second cAMP stimulus were applied 9 and 16 min after the removal of EGTA, respectively. Both the amount and the rate of Ca2+ efflux are reduced as compared with control cells. Measurements were done at t6.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to investigate the role of extracellular Ca2+ entry and of the filling state of stores for the generation of agonist-induced [Ca2+]i changes. cAMP activated a [Ca2+]i increase in 70% of the cells under our standard conditions (i.e. when 1 mM CaCl2 had been present for roughly 15 min). The reason that a reaction was not detected in all cells tested most probably reflects an oscillatory responsiveness. Oscillations of various parameters (e.g. extracellular Ca2+ fluxes) are well known in Dictyostelium (23). In our measurements, we found a clear response pattern within the batches of cells tested; either all cells of one batch reacted or not. Thus, the 70% of responding cells are due to seven batches where all amoebae reacted and three batches where they did not. This distribution matches well with the temporal oscillation pattern (23), and we conclude that our results reflect an oscillatory behavior rather than, for example, a difference in the responsiveness of the predifferentiated prestalk and prespore cells. If this was the case, one would expect that indeed 70% of the amoebae in one batch would respond and 30% would not.

By contrast to our standard condition, a reaction was observed in only 5% of amoebae when 1 mM CaCl2 was added shortly before or concomitantly with the agonist. This result strongly argues against the notion that Ca2+ influx exclusively mediates the cAMP-induced [Ca2+]i increase as had been postulated previously (6). Continuous reduction of the extracellular Ca2+ level decreased the fraction of reacting amoebae but not the height of the [Ca2+]i elevation, which indicates an all-or-none reaction of the amoebae. Our single cell imaging results provide evidence for a functional heterogeneity of the cells that in suspension measurements yield a continuous decrease in the [Ca2+]i transient as documented by Nebl and Fisher (6). Heterogeneous response characteristics were observed in other cell types such as PC12 cells as well (24). The molecular nature of the process(es) responsible for the different reactions in Dictyostelium amoebae at low external Ca2+ is unknown. It might be due to a different sensitivity toward cAMP stimulation or to more efficient Ca2+ signaling, if, for example, the Ca2+ content of internal stores was different. Our data support the hypothesis that filled stores release Ca2+ upon cAMP activation, which causes Ca2+ entry. We postulate that the filling state of the store and the amount of Ca2+ liberated determine the characteristics of Ca2+ influx and thus the [Ca2+]i elevation, whether it is restricted locally or spread throughout the cell's cytosol.

First we manipulated the Ca2+ content of the stores by incubating amoebae with the SERCA-type Ca2+-ATPase blocker BHQ (25). Until now there has been no direct biochemical evidence that BHQ binds to internal Ca2+-ATPases in Dictyostelium. However, due to the results of earlier studies showing inhibition of Ca2+ uptake into partially purified stores by BHQ (14, 26) and the fact that in suspensions of cells with permeabilized plasma membranes BHQ dose-dependently inhibits the sequestration of Ca2+ (12), we consider the effects of BHQ to be caused by an influence on sequestration characteristics. The [Ca2+]i transient after BHQ addition was caused by liberation of stored Ca2+, since (i) it occurred in the extracellular presence of the Ca2+ chelator BAPTA and (ii) BHQ elicits release of 45Ca2+ from cells (12). Neither cAMP nor arachidonic acid induced a [Ca2+]i elevation in BHQ-treated amoebae. Unexpectedly, the addition of Ca2+ itself also resulted in no significant [Ca2+]i increase, although capacitative Ca2+ entry should have been operative. Similarly, the rate of Ca2+-activated Ca2+ influx as measured in cell suspensions with an electrode was diminished after BHQ pretreatment. These findings differ from data of other nonexcitable cells, where inhibition of internal Ca2+ pumps triggers capacitative Ca2+ entry (27). From these results, we conclude that in Dictyostelium, unlike in other cell systems, capacitative Ca2+ entry requires active pumps on internal stores. How the activity of the intracellular ATPases is coupled to influx is at present unknown. It appears that without an active pump the driving force for Ca2+ influx is missing. This seems clear in the case of low external Ca2+ concentrations; however, it seems to hold true also for elevated levels, because the cells must control their Ca2+ content at highly variable extracellular [Ca2+], which occur in their natural habitat.

Emptying of stores by incubation of amoebae with EGTA did not inhibit pumping mechanisms. Indeed, this pretreatment resulted in agonist-induced [Ca2+]i transients at low extracellular [Ca2+] and after the addition of low amounts of Ca2+. To test whether EGTA treatment directly affects regulation of the Ca2+ channel mediating influx we used the Mn2+-quenching technique. Mn2+ influx is an indirect measure of Ca2+ fluxes across the plasma membrane, since many Ca2+ channels are permeable to Mn2+ (28). In Dictyostelium, the nature of the Ca2+-channel is not yet known and electrophysiological data are not available; inhibition of Ca2+ influx by gallopamil2 indicates the presence of an L-type-like channel. We observed basal and cAMP-activated influx already at 1 µM Mn2+; in mammalian cell systems, the dose of Mn2+ is usually higher, in the range of 50–100 µM (29, 30). This indicates that (i) the amoebae can take up Ca2+ at a rather shallow concentration gradient across the plasma membrane and that (ii) their influx mechanisms are even strongly augmented by cAMP stimulation. The rate of Ca2+ influx was independent of the filling state of the stores. Both basal and agonist-activated Mn2+ influx were similar in control cells and cells preincubated with EGTA. Therefore, altered influx characteristics are unlikely to account for the fact that in cells with empty stores the addition of cAMP at low extracellular [Ca2+] or the addition of CaCl2 itself evoked a transient [Ca2+]i elevation. By contrast, in EGTA-treated amoebae, Ca2+ fluxes across the plasma membrane were augmented when assayed with a Ca2+-sensitive electrode in cell suspensions. This rather argues for an inactivation of the PMCA, resulting in a larger net amount of extracellular Ca2+ taken up and thus a greater [Ca2+]i elevation. Delayed activation of the PMCA during capacitative Ca2+ entry was observed in other cell systems such as endothelial cells (31) and Jurkat cells (18). Reduced PMCA activity could account for the observation that Ca2+-induced [Ca2+]i elevations occurred at lower doses of Ca2+, were higher, and lasted longer in EGTA-treated amoebae than in control cells. On the other hand, the activity of intracellular pumps must be high and might even increase after preincubation with EGTA. The surplus rate of Ca2+ influx of 0.19 µM/min measured with the Ca2+ electrode in suspensions of cells with empty stores compared with control cells should result in a [Ca2+]i change by 7 µM/min (calculated volume of 108 amoebae: 52 µl (radius: 5 µm); decrease by 0.38 nmol/min in test volume of 2 ml), yet the [Ca2+]i transient was increased by roughly 50–90 nM only. Indeed, Ca2+ uptake into partially purified stores is augmented by EGTA treatment of cells.2

In Dictyostelium, high affinity Ca2+-ATPases were described both in the plasma membrane (32, 33) and in intracellular compartments (34, 35). Böhme et al. (32) calculated the activity of the PMCA to be sufficient to transport Ca2+ at a rate of roughly 5 x 106 ions/cell/min; this corresponds to removal of Ca2+ from the cytosol at greater than 10 µM/min. Similarly, the rates of Ca2+ uptake into stores by intracellular pumps of roughly 0.3 and 1 nmol/mg protein observed by Milne and Coukell (34) and Moniakis et al. (35), respectively, should result in clearance of Ca2+ from the cytosol at a rate comparable with that of the PMCA. Activation of both pumps is most probably responsible for a strong local and/or temporal restriction of agonist-induced [Ca2+]i elevations at low external [Ca2+] and the fact that a cytosolic increase is detected in few cells only, although under this condition (i) stored Ca2+ is liberated (12), (ii) Ca2+ entry was calculated to be sufficient to increase [Ca2+]i by roughly 10 µM (8), and (iii) the rate of Mn2+ influx was increased 5-fold when compared with basal influx. Under conditions of reduced PMCA activity after EGTA incubation, cAMP stimulation allows a larger net amount of Ca2+ to enter the cells, which thus can spread in the interior of the amoebae resulting in a global [Ca2+]i elevation. Our finding that efflux of Ca2+ that follows cAMP-activated influx was diminished and slower in EGTA-treated amoebae supports this view. The [Ca2+]i transient is smaller than expected from the difference in the flux rates as intracellular pumps are activated. An alternative mechanism for the extrusion of Ca2+ could be the presence of a Na+/Ca2+ exchanger in the plasma membrane (for a review, see Refs. 36 and 37). However, in D. discoideum, there is no experimental evidence favoring the existence of such an exchanger. In addition, all [Ca2+]i imaging and [Ca2+]e measurements are routinely performed in the absence of Na+, which renders a role for a Na+/Ca2+ exchanger for mediation of Ca2+ fluxes unlikely. Fig. 7 depicts a model of Ca2+ fluxes in D. discoideum. In a first step (1) cAMP activates the signaling cascade, leading to liberation of stored Ca2+, which then (2) results in Ca2+ entry. Ca2+ release and activation of the SERCA pump are required to induce influx. The elevation of the cytosolic Ca2+ level is followed by activation of the PMCA and Ca2+ efflux (3). When stores are filled, the amount of released Ca2+ is expected to be greater than under conditions of reduced Ca2+ contents. Continuous emptying of the store leads to activation of the SERCA pump and inhibition of the PMCA pump in order to restore the Ca2+ level in the storage compartments. This condition, for example, by EGTA preincubation, allows for the generation of a global [Ca2+]i increase after cAMP application at low external [Ca2+] or even after the addition of low amounts of CaCl2.



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  		FIG. 7.
Model of the sequence of events leading to [Ca2+]i changes. 1, cAMP binding to its receptor triggers a signaling cascade (38), which first results in liberation of stored Ca2+. Reduction of Ca2+ in the storage compartment activates Ca2+ influx (2) by opening Ca2+ channels in the plasma membrane and activation of the SERCA Ca2+-ATPase. Finally, cytosolic Ca2+ is extruded (3) via the plasma membrane Ca2+-ATPase. See "Discussion" for further details.

 
In conclusion, store-operated Ca2+ entry in Dictyostelium cells is not regulated by an increase in Ca2+ channel permeability but rather by an inhibition of their PMCA. In addition, capacitative Ca2+ entry in Dictyostelium is different from other cell systems in that it depends on an active Ca2+ pump of internal stores. When stimulated with cAMP, liberation of stored Ca2+ activates Ca2+ entry via the channels and allows for a greater rate of influx. At the same time, the activity of intracellular pumps is augmented, thus limiting the resulting [Ca2+]i increase.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Fax: 49-7531-882966; E-mail: Christina.Schlatterer{at}uni-konstanz.de.

1 The abbreviations used are: [Ca2+]i, cytosolic free Ca2+ concentration; [Ca2+]e, extracellular free Ca2+ concentration; AA, arachidonic acid; BHQ, 2,5-di-(t-butyl)-1,4-hydroquinone; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PMCA, plasma membrane Ca2+-ATPase. Back

2 D. Malchow, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dieter Malchow, Rupert Mutzel, and Gerd Knoll for many helpful discussions and critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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