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J Biol Chem, Vol. 275, Issue 12, 8404-8408, March 24, 2000

A Xestospongin C-sensitive Ca²⁺ Store Is Required for cAMP-induced Ca²⁺ Influx and cAMP Oscillations in Dictyostelium^*

Ralph Schaloske, Christina Schlatterer, and Dieter Malchow

From the Faculty of Biology, University of Konstanz, Postfach 55 60, 78457 Konstanz, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Xestospongin C (XeC) is known to bind to the inositol 1,4,5-trisphosphate (IP₃)-sensitive store in mammalian cells and to inhibit IP₃- and thapsigargin-induced Ca²⁺ release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca²⁺ uptake into purified vesicle fractions and induced Ca²⁺ release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP₃ 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 Ca²⁺ store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca²⁺]_i and inhibited cAMP-induced Ca²⁺ influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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. 1-3). 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 (5-7). 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 Ca²⁺ was postulated to control cAMP synthesis (7).

Previous work in Dictyostelium discoideum has shown that besides the mitochondria two other Ca²⁺ stores exist: acidic vesicles and the IP₃-sensitive store. All are involved in Ca²⁺ homeostasis, and two of them, the acidic vesicles and the IP₃-sensitive store, in cAMP-mediated Ca²⁺ 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 Ca²⁺ release from the inositol 1,4,5-trisphosphate (IP₃)-sensitive store of PC12 cells, primary astrocytes, and rabbit cerebellum without interacting with the IP₃-binding site (17). We showed that XeC is a suitable inhibitor of the IP₃-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), Ca²⁺ takes part in oscillatory regulation of cAMP.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Xestospongin C, inositol 1,4,5-trisphosphate, microcystin LR and thapsigargin were purchased from Calbiochem (Bad Soden, Germany). Fura-2 and fura-2-dextran were obtained from MobiTec (Göttingen, Germany) and 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) from Aldrich (Steinheim, Germany).

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 (KH₂/Na₂H)PO₄, pH 6.0). Cells were shaken on a rotary shaker at 23 °C, 150 rpm at 2 × 10⁷ 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 × 10⁸ cells/ml, and lysed by passage through nuclepore filters. Immediately, 3% sucrose, 50 mM KCl, 1 mM MgCl₂, 20 µg/ml leupeptin, 1 µg/ml aprotinin, 2.5 mM dithiothreitol, and 1 µM microcystin were added (final concentrations). After centrifugation for 5 min at 3000 × g, the supernatant was further fractionated by centrifugation for 20 min at 12,000 × 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 IP₃-sensitive Ca²⁺ 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 × g at 4 °C.

Ca²⁺ Transport-- Ca²⁺ transport was measured as described (15). In brief, about 70 µl of P1 was added to 10 mM Hepes, pH 7.2, 50 mM KCl, 3% sucrose, 6 µg/ml antimycin A, 6 µg/ml oligomycin A, 100 µM NaN₃, 2 mM MgCl₂, and about 6 µM 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 Ca²⁺ 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).

Determination of [Ca²⁺]_i-- [Ca²⁺]_i was determined as described (15). Cells were electroporated at 2-3 h after induction of differentiation with fura-2-dextran, and [Ca²⁺]_i imaging was carried out at 5-7 h of differentiation. 10 min before [Ca²⁺]_i recording buffer covering the cells was replaced by 90 µl of fresh buffer (5 mM Hepes, 5 mM KCl, pH 7.0) containing 1 mM Ca²⁺. [Ca²⁺]_i imaging was performed with an Axiovert T100 microscope (Zeiss, Jena, Germany). The cells were viewed with a 100× Fluar objective (numeric aperture, 1.3); 340 and 380 nm excitation was performed with a mercury lamp and a rotating filter wheel. Images of the cells were recorded with an ICCD camera (HL-A; Proxitronic, Bensheim, Germany). Image digitization and calibration were as described previously (19).

Chemotaxis and Cell Shape Recording-- 200 µl of 4 × 10⁵ cells/ml in Sørensen phosphate buffer were placed on a coverslip, and a borosilicate glass capillary filled with 100 µM cAMP was inserted at time zero. Cells were viewed with a 16× or 25× objective using an inverted Zeiss IM microscope. Cell movement was recorded using a CCD camera and a digital video recorder (Sony).

Other Measurements-- Ca²⁺ influx was determined with a Ca²⁺-sensitive 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vesicular Uptake and IP₃-induced Ca²⁺ Release-- When Dictyostelium extracts were centrifuged for 20 min at 12,000 × g, the supernatant (S1) and the pellet (P1) contained about equal amounts of Ca²⁺ transport activity. The rate of Ca²⁺ uptake for S1 was 1.25 ± 0.61 nmol/min × 10⁸ cells and 1.24 ± 0.67 nmol/min ×10⁸ cells for P1 (± S.D. n = 9). We found that XeC induced Ca²⁺ release (see below). This effect was larger in P1 (71 ± 3%, n = 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 Ca²⁺ uptake into IP₃-sensitive Ca²⁺ stores of Dictyostelium.

ATP induced Ca²⁺ uptake into the microsomal fraction P1 in the presence of mitochondrial inhibitors. Addition of IP₃ caused a slow but steady release of Ca²⁺ from the store (Fig. 1B). In the presence of 20 µM XeC, Ca²⁺ transport was strongly reduced and IP₃-mediated Ca²⁺ release was virtually absent (Fig. 1A). In Table I we compared the potency of XeC with that of the Ca²⁺-ATPase blocker BHQ. BHQ was shown to inhibit Ca²⁺ uptake into the IP₃-sensitive store at about 100 µM concentration and into the acidic vesicles at 200 µM concentration (11, 12). Table I demonstrates that 20-30 µM XeC was as potent as 100 µM BHQ to block Ca²⁺ uptake and IP₃-induced Ca²⁺ release.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   XeC inhibits Ca2+ uptake into the IP3-sensitive Ca2+ store. ATP-induced Ca2+ uptake into the microsomal fraction P1 was measured in the absence (B) or presence of 20 µM XeC (A). 200 nM IP3 was added where indicated. Ca2+ concentrations were determined with fura-2 as described under "Experimental Procedures." Bars indicate extravesicular [Ca2+]. One out of three independent experiments is shown.

A Thapsigargin-sensitive Ca²⁺ Store Is Inhibited by XeC-- Thapsigargin is another specific blocker of the Ca²⁺ pump of the IP₃-sensitive store in mammalian cells. Inhibition of the pump results in leakage of Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ release mainly in fractions 1 and 2 at the top of the gradient (Fig. 2, A and B). The amount of Ca²⁺ 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 µM XeC inhibited Tg-induced Ca²⁺ release to a greater extent (68%) than Ca²⁺ uptake (50%).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Percoll gradient fractionation of Ca2+ uptake activity and Ca2+ release by XeC (A) or Tg (B). P1 was separated on a 30% Percoll gradient. The fractions were assayed for ATP-activated Ca2+ uptake (), Ca2+ release induced by XeC (A, 10 µM, ) or Tg (B, 40 µM, ), respectively, and for protein content (). The top of the gradient is to the left. One out of two experiments is shown in A and one out of three experiments in B. A and B are from separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   XeC blocks Tg-induced Ca2+ release. ATP-induced Ca2+ uptake into fraction 2 of the Percoll gradient of Fig. 2B was measured in the absence (B) or presence of 30 µM XeC (A). 40 µM Tg was added where indicated. One out of three independent experiments is shown.

In Dictyostelium the target of XeC seems to be the IP₃-sensitive store as in mammalian cells for the following reasons. 1) XeC inhibited IP₃-induced Ca²⁺ release and Ca²⁺ uptake to a similar extent as BHQ; 2) XeC inhibited Tg-induced Ca²⁺ release to a greater extent than Ca²⁺ uptake in each of three independent experiments (by 19.7 ± 7%); 3) XeC- and Tg-induced Ca²⁺ release activity distributed similarly on a Percoll gradient; 4) after Ca²⁺ had been liberated from fraction 2 by Tg and IP₃, XeC-induced Ca²⁺ release was inhibited by 92 ± 12% (n = 3). Therefore, we conclude that the IP₃-sensitive store is indeed the target of XeC action in Dictyostelium. We then aimed to elucidate the function of the IP₃-sensitive store in early development of Dictyostelium.

cAMP-induced Ca²⁺ Influx-- Indirect evidence had pointed to the necessity of the IP₃-sensitive store for cAMP-induced Ca²⁺ influx (13, 14). Previously, we have shown that an active phospholipase A₂ is required for cAMP-induced Ca²⁺ influx (14). Free fatty acids like arachidonic acid cause Ca²⁺ release from the acidic vesicles, the second Ca²⁺ store in Dictyostelium besides the IP₃-sensitive Ca²⁺ store (15). We reasoned that the increase in cytosolic Ca²⁺ then either activates phospholipase C or IP₅ phosphatase. Both events lead to IP₃ formation and subsequent release of Ca²⁺ from the IP₃-sensitive store that in turn elicits capacitative Ca²⁺ entry (21).

Here we used XeC to address the question of whether the IP₃-sensitive store was indeed required for capacitative Ca²⁺ entry. cAMP-induced Ca²⁺ influx was reduced immediately after XeC application, and maximal inhibition occurred within 25 min. 36 µM XeC caused nearly complete inhibition of cAMP-induced Ca²⁺ influx, demonstrating that the IP₃-sensitive store is required to induce capacitative Ca²⁺ 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 presence of 30 µM 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).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Light scattering spikes are attenuated by XeC. Light scattering was measured as described under "Experimental Procedures." To a cell suspension displaying free running spikes 28 µM XeC was added as indicated. One out of three independent experiments is shown. Control cells continued spiking when spikes were abolished in XeC-treated suspensions. The solvent ethanol did not significantly affect spike amplitude in controls.

XeC affected the morphology of the amoebae. Aggregation-competent control cells displayed an elongated, polarized cell shape with many protrusions at the front and laterally (Fig. 5A). In the presence of 8 µM 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 µM XeC behaved in the same way, except that extension of pseudopods and migration of the cells began about 90 min later.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Shape of cells following XeC treatment. Aggregation-competent cells (4 ×105 cells/ml) were incubated in the absence (A) or presence of 8 µM XeC (B) on a coverslip. The reproduction shown was obtained after 37 min of incubation at 23 °C. One out of three independent experiments is shown.

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.

View larger version (98K): [in this window] [in a new window]   Fig. 6.   Chemotaxis in the presence of XeC. A cell suspension of 4 ×105 aggregation-competent cells/ml was incubated for 30 min in the absence (panel A) or presence of 8 µM XeC (panel B). A capillary containing 0.1 mM cAMP was inserted at 0 min into the field. The time in minutes at which the frames were taken is indicated at the top. One out of five independent experiments is shown.

Basal [Ca²⁺]_i Increases during XeC Treatment-- Determination of [Ca²⁺]_i in single cells in the presence of 20 µM XeC revealed a time-dependent increase in the cytosolic Ca²⁺ concentration (Fig. 7). The basal concentration of 56 ± 9 nM (65 cells) increased within 30 min to 230 ± 11 nM (15 cells). Concomitantly, the percentage of cells that exhibited a cAMP-dependent transient [Ca²⁺]_i increase dropped to zero. In five experiments basal cytosolic Ca²⁺ increased 3-fold to 188 ± 58 nM in the presence of 15-20 µM XeC, whereas the percentage of responding cells decreased to 12 ± 8%.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   XeC causes a time-dependent increase in [Ca2+]i. Aggregation competent cells were incubated with 20 µM XeC. [Ca2+]i was determined as described under "Experimental Procedures" with fura-2-dextran at the indicated times. We also measured the transient [Ca2+]i increase in response to 1 µM cAMP, due to Ca2+ influx, in each of the cells. The result was expressed as the percentage of reacting cells at the indicated times. The cytosolic Ca2+ increase after cAMP addition in the responding cells was similar to the controls, ranging from 30 to 100 nM. Data represent means ± S.E. One out of five independent experiments is shown.

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 increased 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 [Ca²⁺]_i controls cAMP synthesis. Inspection of Fig. 7 reveals a steep 2-fold [Ca²⁺]_i increase about 20 min after addition of XeC.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   cAMP oscillations cease in the presence of XeC. cAMP concentrations were determined as described under "Experimental Procedures." 30-µl samples were withdrawn from the cell suspension, where indicated, before and after application of 30 µM XeC. One out of two independent experiments is shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the first part of this study we have shown that XeC is an efficient inhibitor of the IP₃-sensitive Ca²⁺ store in Dictyostelium. XeC had a similar potency in Dictyostelium as compared with PC12 cells or primary astrocytes, where 20 µM XeC was used to block bradykinin-induced Ca²⁺ release or Ca²⁺ oscillations, respectively. By contrast, we found that the IP₃-sensitive store displayed a low affinity for Tg in Dictyostelium as opposed to mammalian cells.² Previously, Tg-induced Ca²⁺ release from internal stores of this organism escaped detection (12, 23). Only recently, a [Ca²⁺]_i increase in response to Tg was reported for aequorin-expressing cells (24).

The site of XeC binding to the IP₃-sensitive store is not known, only that [³H]IP₃ binding was not antagonized by XeC (17). The authors suggested that XeC binds to the Ca²⁺ release channel and blocks the channel because of its rodshaped structure. However, we found that XeC not only inhibited Ca²⁺ release by Tg and IP₃, but also was a potent inhibitor of Ca²⁺ uptake into the IP₃-sensitive store. Moreover, XeC by itself induced Ca²⁺ release. This indicates that in Dictyostelium XeC does not act as a plug of the Ca²⁺ release channel as suggested previously (17). It seems instead to open the channel counteracting Ca²⁺ uptake. In agreement with this assumption, basal [Ca²⁺]_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 IP₃-sensitive store in cell motility and cellular oscillations. Incubation of cells with XeC had a profound effect on cell motility and cell shape. It is known that in Dictyostelium the Ca²⁺-dependent actin-binding proteins -actinin and severin are involved in cytoskeletal rearrangements (25, 26). Furthermore, the time of the maximal rate of Ca²⁺ 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 [Ca²⁺]_i by calmidazolium causes an extension of pseudopods over the whole cell's circumference, whereas a larger [Ca²⁺]_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 [Ca²⁺]_i of XeC-treated cells and (b) a Ca²⁺ gradient with the maximum at the rear end reported for amoeboid movement (29, 30). As long as the [Ca²⁺]_i remains high, the cells display a contracted round shape. As soon as the Ca²⁺ 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 Ca²⁺ concentration, pseudopods occur predominantly at the front. The rear remains inactive due to the still elevated Ca²⁺-level. Our results show that the IP₃-sensitive store is crucially involved in the generation of Ca²⁺ 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 IP₃-sensitive Ca²⁺ 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 Ca²⁺ elevation. The first possibility becomes plausible if we assume that store-operated Ca²⁺ oscillations serve as a pacemaker. In the presence of XeC, Ca²⁺ uptake by the IP₃-sensitive store was inhibited and basal [Ca²⁺]_i increased. Therefore, ongoing Ca²⁺ oscillations should be perturbed. With respect to the second possibility, an inhibition of the increase of the concentration of cAMP by Ca²⁺ 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 Ca²⁺. Schaap and co-workers (35) reported a 47% inhibition of adenylyl cyclase activity in Dictyostelium homogenates at 10 µM Ca²⁺. 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 Ca²⁺ or Ca²⁺/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 Ca²⁺ release, whereas capacitative Ca²⁺ entry inhibited cyclase activity (39). This suggests an organized co-localization of adenylyl cyclase with capacitative Ca²⁺ entry channels.

An interference by XeC with other components of the cAMP oscillatory cycle (5) besides the IP₃-sensitive Ca²⁺ 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 [Ca²⁺]_i or the inactivation of the IP₃-sensitive store, was the primary cause for the oscillatory stop.

	ACKNOWLEDGEMENTS

We thank R. Mutzel for stimulating discussions and J. Breed for critically reading the manuscript.

	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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Faculty of Biology, University of Konstanz, Postfach 55 60, D-78457 Konstanz, Germany. Tel.: 49-7531-88-2114; Fax: 49-7531-88-2966; E-mail: ralph.schaloske@uni-konstanz.de.

² D. Malchow, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: CAR, cAMP receptor; XeC, xestospongin C; Tg, thapsigargin; BHQ, 2,5-di-(tert-butyl)-1,4-hydroquinone; IP₃, inositol 1,4,5-trisphosphate; ACA, adenylyl cyclase A; [Ca²⁺]_i, intracellular Ca²⁺ concentration.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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