A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium.

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.

Early development of Dictyostelium requires the pulsatile release of cAMP that binds to the cAMP receptor (CAR1) 1 and attracts neighboring cells to migrate to the cAMP source (for review, see Refs. [1][2][3]. This pulsatile release of cAMP underlies spikelike light scattering oscillations of Dictyostelium cells (4).
Oscillations in cAMP are thought to arise due to a positive extracellular feedback loop of cAMP (5)(6)(7). Extracellular binding of cAMP to CAR1 transiently activates adenylyl cyclase (ACA). This leads to an increase in cytosolic cAMP. One portion of cAMP is secreted and restimulates CAR1. It is inactivated thereafter by extracellular phosphodiesterases (for review, see Ref. 8). The cAMP remaining in the cytosol activates protein kinase A and is hydrolyzed by the intracellular phosphodiesterase Reg A (for review, see Ref. 9). A negative feedback loop could occur due to protein kinase A activity attenuating ACA, either directly or indirectly (5). Alternatively, a negative feedback loop of intracellular Ca 2ϩ was postulated to control cAMP synthesis (7).
Previous work in Dictyostelium discoideum has shown that besides the mitochondria two other Ca 2ϩ stores exist: acidic vesicles and the IP 3 -sensitive store. All are involved in Ca 2ϩ homeostasis, and two of them, the acidic vesicles and the IP 3 -sensitive store, in cAMP-mediated Ca 2ϩ influx (10 -16). The role of these storage compartments in differentiation and in oscillation is unclear. Unfortunately for the elucidation of their functions during development, no specific inhibitor for either store is known.
Here we used xestospongin C (XeC), a bis-1-oxaquinolizodine isolated from the marine sponge Xestospongia. XeC blocks Ca 2ϩ release from the inositol 1,4,5-trisphosphate (IP 3 )-sensitive store of PC12 cells, primary astrocytes, and rabbit cerebellum without interacting with the IP 3 -binding site (17). We showed that XeC is a suitable inhibitor of the IP 3 -sensitive store in Dictyostelium and analyzed the role of this store in early differentiation of Dictyostelium. We found that, in extension to the proposed models for cAMP oscillations (5,6), Ca 2ϩ takes part in oscillatory regulation of cAMP.
Culture of Cells and Induction of Differentiation-The axenic strain Ax2 was grown in shaking culture as described (18). Differentiation was induced by washing cells free of medium twice in ice-cold Sørensen phosphate buffer (17 mM (KH 2 /Na 2 H)PO 4 , pH 6.0). Cells were shaken on a rotary shaker at 23°C, 150 rpm at 2 ϫ 10 7 cells/ml until use.
Preparation of Vesicles-30 ml of a cell suspension differentiated for 1-2 h were washed once in ice-cold 20 mM Hepes buffer, pH 7.2, resuspended at 2 ϫ 10 8 cells/ml, and lysed by passage through nuclepore filters. Immediately, 3% sucrose, 50 mM KCl, 1 mM MgCl 2 , 20 g/ml leupeptin, 1 g/ml aprotinin, 2.5 mM dithiothreitol, and 1 M microcystin were added (final concentrations). After centrifugation for 5 min at 3000 ϫ g, the supernatant was further fractionated by centrifugation for 20 min at 12,000 ϫ g. The sediment (P1) was resuspended in 1 ml of the above buffer yielding a protein concentration of about 2 mg/ml. P1 contained the IP 3 -sensitive Ca 2ϩ store and part of the acidic vesicle fraction. For further purification, 1 ml of P1 (from 45 ml of cell suspension preincubated for 2 h in 5 mM EGTA) was centrifuged on a 30% Percoll gradient (10 ml) for 30 min at 40,000 ϫ g at 4°C.
Ca 2ϩ Transport -Ca 2ϩ transport was measured as described (15). In brief, about 70 l of P1 was added to 10 mM Hepes, pH 7.2, 50 mM KCl, 3% sucrose, 6 g/ml antimycin A, 6 g/ml oligomycin A, 100 M NaN 3 , 2 mM MgCl 2 , and about 6 M fura-2 in a total volume of 1 ml. After preincubation for 3-10 min at 23°C in the absence or presence of drugs, 1 mM ATP was added to activate Ca 2ϩ uptake. Fura-2 fluorescence was monitored at 335 and 380 nm excitation and 508 nm emission with a double wavelength fluorimeter (Sigma ZWS11, Sigma Instrumente, Berlin, Germany).
Chemotaxis and Cell Shape Recording-200 l of 4 ϫ 10 5 cells/ml in Sørensen phosphate buffer were placed on a coverslip, and a borosilicate glass capillary filled with 100 M cAMP was inserted at time zero. Cells were viewed with a 16ϫ or 25ϫ objective using an inverted Zeiss IM microscope. Cell movement was recorded using a CCD camera and a digital video recorder (Sony).
Other Measurements-Ca 2ϩ influx was determined with a Ca 2ϩsensitive electrode (14), and light scattering oscillations were recorded as described (18). Protein concentrations were determined with the Coomassie protein assay reagent (Pierce) using bovine serum albumin as standard. The amount of cAMP (intracellular and extracellular) present during spike-shaped oscillations in the absence or presence of XeC was measured using an enzyme immunoassay (Biotrak, Amersham Pharmacia Biotech). Samples were prepared as described (18).

Vesicular Uptake and IP 3 -induced Ca 2ϩ
Release-When Dictyostelium extracts were centrifuged for 20 min at 12,000 ϫ g, the supernatant (S1) and the pellet (P1) contained about equal amounts of Ca 2ϩ transport activity. The rate of Ca 2ϩ uptake for S1 was 1.25 Ϯ 0.61 nmol/min ϫ 10 8 cells and 1.24 Ϯ 0.67 nmol/min ϫ10 8 cells for P1 (Ϯ S.D. n ϭ 9). We found that XeC induced Ca 2ϩ release (see below). This effect was larger in P1 (71 Ϯ 3%, n ϭ 3) than in S1. Therefore, we used P1 in the subsequent experiments to analyze the mechanism of XeC action. We first tested whether XeC inhibited Ca 2ϩ uptake into IP 3 -sensitive Ca 2ϩ stores of Dictyostelium.
ATP induced Ca 2ϩ uptake into the microsomal fraction P1 in the presence of mitochondrial inhibitors. Addition of IP 3 caused a slow but steady release of Ca 2ϩ from the store (Fig. 1B). In the presence of 20 M XeC, Ca 2ϩ transport was strongly reduced and IP 3 -mediated Ca 2ϩ release was virtually absent (Fig. 1A). In Table I we compared the potency of XeC with that of the Ca 2ϩ -ATPase blocker BHQ. BHQ was shown to inhibit Ca 2ϩ uptake into the IP 3 -sensitive store at about 100 M concentration and into the acidic vesicles at 200 M concentration (11,12). Table I demonstrates that 20 -30 M XeC was as potent as 100 M BHQ to block Ca 2ϩ uptake and IP 3 -induced Ca 2ϩ release.
A Thapsigargin-sensitive Ca 2ϩ Store Is Inhibited by XeC-Thapsigargin is another specific blocker of the Ca 2ϩ pump of the IP 3 -sensitive store in mammalian cells. Inhibition of the pump results in leakage of Ca 2ϩ from storage compartments (20). To investigate whether Tg-and XeC-sensitive vesicles distribute similarly on a density gradient, we purified the microsomal fraction P1 on a 30% Percoll gradient (Fig. 2). Protein was distributed over two peaks, whereas Ca 2ϩ uptake activity was present predominantly in one peak at the top of the gradient. Under these conditions mitochondrial porin was found in fractions 6 -8 (data not shown). Thapsigargin and XeC elicited Ca 2ϩ release mainly in fractions 1 and 2 at the top of the gradient (Fig. 2, A and B). The amount of Ca 2ϩ released by XeC or Tg from the same fraction (fraction 2 of Fig. 2A) was in the same range (29 nmol/mg protein and 34 nmol/mg protein for XeC and Tg, respectively). This suggests that XeC acts on the same store as Tg. We therefore analyzed whether XeC blocks the Tg-sensitive store. Fig. 3 shows that 30 M XeC inhibited Tg-induced Ca 2ϩ release to a greater extent (68%) than Ca 2ϩ uptake (50%).
In Dictyostelium the target of XeC seems to be the IP 3sensitive store as in mammalian cells for the following reasons. 1) XeC inhibited IP 3 -induced Ca 2ϩ release and Ca 2ϩ uptake to a similar extent as BHQ; 2) XeC inhibited Tg-induced Ca 2ϩ release to a greater extent than Ca 2ϩ uptake in each of three independent experiments (by 19.7 Ϯ 7%); 3) XeC-and Tginduced Ca 2ϩ release activity distributed similarly on a Percoll gradient; 4) after Ca 2ϩ had been liberated from fraction 2 by Tg and IP 3 , XeC-induced Ca 2ϩ release was inhibited by 92 Ϯ 12% (n ϭ 3). Therefore, we conclude that the IP 3 -sensitive store is indeed the target of XeC action in Dictyostelium. We then aimed to elucidate the function of the IP 3 -sensitive store in early development of Dictyostelium.
cAMP-induced Ca 2ϩ Influx-Indirect evidence had pointed to the necessity of the IP 3 -sensitive store for cAMP-induced Ca 2ϩ influx (13,14). Previously, we have shown that an active phospholipase A 2 is required for cAMP-induced Ca 2ϩ influx (14). Free fatty acids like arachidonic acid cause Ca 2ϩ release from the acidic vesicles, the second Ca 2ϩ store in Dictyostelium besides the IP 3 -sensitive Ca 2ϩ store (15). We reasoned that the increase in cytosolic Ca 2ϩ then either activates phospholipase C or IP 5 phosphatase. Both events lead to IP 3 formation and subsequent release of Ca 2ϩ from the IP 3 -sensitive store that in turn elicits capacitative Ca 2ϩ entry (21).
Here we used XeC to address the question of whether the IP 3 -sensitive store was indeed required for capacitative Ca 2ϩ entry. cAMP-induced Ca 2ϩ influx was reduced immediately after XeC application, and maximal inhibition occurred within 25 min. 36 M XeC caused nearly complete inhibition of cAMPinduced Ca 2ϩ influx, demonstrating that the IP 3 -sensitive store is required to induce capacitative Ca 2ϩ entry (Table II).
Spike-shaped Oscillations and Chemotaxis-Spikelike light scattering oscillation start spontaneously in Dictyostelium cell suspensions after about 4 h of differentiation (21). In the pres-  ence of 30 M XeC, free running spikes were gradually abolished over a period of about 30 min. Before addition of XeC, spikes were symmetric. Afterward they became more and more asymmetric. The declining phase of the spike receded earlier than the rising phase and finally did not return to the base line anymore (Fig. 4). Cells treated with XeC during oscillations, when plated on agar, underwent morphogenesis and gave rise to normal fruiting bodies (data not shown).
XeC affected the morphology of the amoebae. Aggregationcompetent control cells displayed an elongated, polarized cell shape with many protrusions at the front and laterally (Fig.  5A). In the presence of 8 M XeC, cells rounded up. After about 30 min they began to extend pseudopods at one end, thereby forming pointed ends in the direction of migration. Little or no pseudopods were observed along their sides (Fig. 5B). Within 60 min these cells recovered to control cell behavior (data not shown). Cells treated with 15-20 M XeC behaved in the same way, except that extension of pseudopods and migration of the cells began about 90 min later.
As soon as the migration stage was reached, XeC-treated cells were able to orient and to migrate to a cAMP-source (Fig.  6). In contrast to control cells, which exhibited multiple pseudopods at the front and laterally (Fig. 6A), XeC-treated cells predominantly extended pseudopods at the front (Fig. 6B). Only in rare cases were pseudopods formed along their sides. We conclude that formation of lateral protrusions is suppressed transiently in XeC-treated cells.

Basal [Ca 2ϩ ] i Increases during XeC Treatment-Determination of [Ca 2ϩ
] i in single cells in the presence of 20 M XeC revealed a time-dependent increase in the cytosolic Ca 2ϩ concentration (Fig. 7). The basal concentration of 56 Ϯ 9 nM (65 cells) increased within 30 min to 230 Ϯ 11 nM (15 cells). Concomitantly, the percentage of cells that exhibited a cAMP-dependent transient [Ca 2ϩ ] i increase dropped to zero. In five experiments basal cytosolic Ca 2ϩ increased 3-fold to 188 Ϯ 58 nM in the presence of 15-20 M XeC, whereas the percentage of responding cells decreased to 12 Ϯ 8%.
cAMP Oscillations-Although we found that spike formation was attenuated in the presence of XeC, periodic cAMP synthesis could still occur. Therefore, we measured cAMP concentrations during free running spikes before and after XeC addition. Fig. 8 shows that the cAMP concentration periodically in-  creased beneath the spikes before and after XeC addition, but finally dropped to basal levels when light scattering oscillations had completely stopped. The rather long time required for inhibition of cAMP oscillations may indicate that a threshold concentration of [Ca 2ϩ ] i controls cAMP synthesis. Inspection of Fig. 7 reveals a steep 2-fold [Ca 2ϩ ] i increase about 20 min after addition of XeC. DISCUSSION In the first part of this study we have shown that XeC is an efficient inhibitor of the IP 3 -sensitive Ca 2ϩ store in Dictyostelium. XeC had a similar potency in Dictyostelium as compared with PC12 cells or primary astrocytes, where 20 M XeC was used to block bradykinin-induced Ca 2ϩ release or Ca 2ϩ oscillations, respectively. By contrast, we found that the IP 3 -sensitive store displayed a low affinity for Tg in Dictyostelium as opposed to mammalian cells. 2 Previously, Tg-induced Ca 2ϩ release from internal stores of this organism escaped detection (12,23). Only recently, a [Ca 2ϩ ] i increase in response to Tg was reported for aequorin-expressing cells (24).
The site of XeC binding to the IP 3 -sensitive store is not known, only that [ 3 H]IP 3 binding was not antagonized by XeC (17). The authors suggested that XeC binds to the Ca 2ϩ release channel and blocks the channel because of its rodshaped structure. However, we found that XeC not only inhibited Ca 2ϩ release by Tg and IP 3 , but also was a potent inhibitor of Ca 2ϩ uptake into the IP 3 -sensitive store. Moreover, XeC by itself induced Ca 2ϩ release. This indicates that in Dictyostelium XeC does not act as a plug of the Ca 2ϩ release channel as suggested previously (17). It seems instead to open the channel counteracting Ca 2ϩ uptake. In agreement with this assumption, basal [Ca 2ϩ ] i increased 4-fold during the course of incubation with XeC for 30 min.
In the second part, the use of XeC allowed us to examine the role of the IP 3 -sensitive store in cell motility and cellular oscillations. Incubation of cells with XeC had a profound effect on 2 D. Malchow, manuscript in preparation. cell motility and cell shape. It is known that in Dictyostelium the Ca 2ϩ -dependent actin-binding proteins ␣-actinin and severin are involved in cytoskeletal rearrangements (25,26). Furthermore, the time of the maximal rate of Ca 2ϩ influx, 20 s after stimulation with cAMP, corresponds to the association of myosin II with the plasma membrane (27) and to the cringing response, where the cell adopts a rounded shape and contracts (28). A small artificial elevation of [Ca 2ϩ ] i by calmidazolium causes an extension of pseudopods over the whole cell's circumference, whereas a larger [Ca 2ϩ ] i elevation induces full contraction (19). In the presence of XeC, the cells first assumed a round, contracted state. Afterward the cells resumed migration, where visible extensions were largely confined to the front. This altered type of behavior can be explained by (a) the increased [Ca 2ϩ ] i of XeC-treated cells and (b) a Ca 2ϩ gradient with the maximum at the rear end reported for amoeboid movement (29,30). As long as the [Ca 2ϩ ] i remains high, the cells display a contracted round shape. As soon as the Ca 2ϩ concentration drops below a critical level at a particular site, pseudopod extensions become possible and a front end is generated. Since the front displays the lowest Ca 2ϩ concentration, pseudopods occur predominantly at the front. The rear remains inactive due to the still elevated Ca 2ϩ -level. Our results show that the IP 3 -sensitive store is crucially involved in the generation of Ca 2ϩ fluxes required to regulate cell shape and motility.
A principal event during early differentiation is the periodic synthesis and release of cAMP. However, it is still an open question how cAMP oscillations evolve during differentiation. Spike-shaped and sinusoidal light scattering oscillations exist (22), but only the former are accompanied by cAMP oscillations (31). Do cAMP oscillations arise independently, or are they coupled to a cellular oscillator? Phase shift experiments have shown that light scattering spikes and cAMP oscillations are coupled (18). However, Wurster and Mohn described a mutant, agip 43, that displayed spike-shaped light scattering oscillations without elevation of the cAMP concentration. This result indicated that, despite the coupling of both oscillations, cAMP is not the pacemaker for cellular oscillations (32). We found that light scattering oscillations were attenuated in the presence of XeC. Therefore, we conclude that the IP 3 -sensitive Ca 2ϩ store is part of this unknown pacemaker.
The finding that periodic cAMP pulses also were inhibited by XeC could result from (i) the requirement of the pacemaker for cAMP oscillations or (ii) suppression of cAMP-production by the cytosolic Ca 2ϩ elevation. The first possibility becomes plausible if we assume that store-operated Ca 2ϩ oscillations serve as a pacemaker. In the presence of XeC, Ca 2ϩ uptake by the IP 3 -sensitive store was inhibited and basal [Ca 2ϩ ] i increased. Therefore, ongoing Ca 2ϩ oscillations should be perturbed. With respect to the second possibility, an inhibition of the increase of the concentration of cAMP by Ca 2ϩ under various conditions has been shown (18,33,34). It is unknown, however, whether adenylyl cyclase activity is inhibited or hydrolysis of cAMP is activated by Ca 2ϩ . Schaap and co-workers (35) reported a 47% inhibition of adenylyl cyclase activity in Dictyostelium homogenates at 10 M Ca 2ϩ . However, the experiment was performed at pH 8.0 and not at physiological pH, where the activity is reduced by a factor of 4. Moreover, high concentrations of dithiothreitol were used, which has been reported to activate the enzyme by a receptor-independent mechanism (36). Regulation of several mammalian adenylyl cyclase subtypes by Ca 2ϩ or Ca 2ϩ /calmodulin has been described (37,38), as well as regulation via phosphorylation by calmodulin kinases II and IV (37). It has also been shown that type VI adenylyl cyclase was insensitive to ionomycin-induced intracellular Ca 2ϩ release, whereas capacitative Ca 2ϩ entry inhibited cyclase activity (39). This suggests an organized co-localization of adenylyl cyclase with capacitative Ca 2ϩ entry channels.
An interference by XeC with other components of the cAMP oscillatory cycle (5) besides the IP 3 -sensitive Ca 2ϩ store cannot be excluded. However, a direct inhibition of ACA or of CAR1 should have resulted in a faster reduction of cAMP synthesis and thus seems unlikely. cAMP synthesis proceeded unabridged for 20 min in the presence of XeC, indicating that an indirect effect, namely the increase of [Ca 2ϩ ] i or the inactivation of the IP 3 -sensitive store, was the primary cause for the oscillatory stop.