Dependence of the Ca2+-inhibitable Adenylyl Cyclase of C6-2B Glioma Cells on Capacitative Ca2+ Entry*

Kent A. Fagan, Nicole MonsDagger , and Dermot M. F. Cooper§

From the Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262 and Dagger  URA-CNRS 339, University of Bordeaux I, Talence F-33405, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration ([Ca2+]i) and cAMP signaling. Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, require Ca2+ entry for their regulation, rather than Ca2+ release from intracellular stores (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444). The present study compared the regulation by capacitative Ca2+ entry versus ionophore-mediated Ca2+ entry of an endogenously expressed Ca2+-inhibitable adenylyl cyclase in C6-2B cells. Even in the face of a dramatic [Ca2+]i rise generated by ionophore, Ca2+ entry via capacitative Ca2+ entry channels was solely responsible for the regulation of the adenylyl cyclase. Selective efficacy of BAPTA over equal concentrations of EGTA in blunting the regulation of the cyclase by capacitative Ca2+ entry defined the intimacy between the adenylyl cyclase and the capacitative Ca2+ entry sites. This association could not be impaired by disruption of the cytoskeleton by a variety of strategies. These results not only establish an intimate spatial relationship between an endogenously expressed Ca2+-inhibitable adenylyl cyclase with capacitative Ca2+ entry sites but also provide a physiological role for capacitative Ca2+ entry other than store refilling.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Ca2+-sensitive adenylyl cyclases provide a key point for integration of signaling by [Ca2+]i1 and cAMP (1). Their likely contribution to cellular regulation is underscored by the fact that whether they are expressed heterologously or endogenously, these cyclases are regulated by physiological transitions in [Ca2+]i (2-9). Somewhat unexpectedly, the Ca2+-sensitive adenylyl cyclases, whether they are expressed endogenously or heterologously, show a preference for regulation by Ca2+ entering the cell over Ca2+ released from intracellular stores (7, 10). Even more strikingly, Ca2+ entry promoted by ionophore is unable to regulate transfected Ca2+-stimulable adenylyl cyclases (7). Consequently, we had proposed that Ca2+-stimulable adenylyl cyclases and capacitative Ca2+ entry channels (ICRACs)2 were functionally colocalized (7). However, it is always conceivable that when they are transfected, adenylyl cyclases are expressed in discrete cellular domains, which reflects the response of the cell to overexpression of signaling molecules. It is therefore of considerable interest to determine whether similar colocalization is encountered with endogenously expressed adenylyl cyclase in continuous cell lines, which are more appropriate models of a normal signaling repertoire. Previous studies have established that the endogenous Ca2+-inhibitable adenylyl cyclase, which is the predominant form in C6-2B glioma cells (11), is also regulated by the entry of Ca2+ rather than release from intracellular stores, which was triggered by a variety of treatments (10). However, it is not known whether such endogenous adenylyl cyclases show a similar absolute dependence on capacitative versus any other form of entry. Such a requirement would predict a close association between entry sites and the cyclases. The potential intimacy of an endogenous Ca2+-inhibitable adenylyl cyclase and Ca2+ entry channels is explored in the present study by assessing (i) the sensitivity of the cyclase to various types of [Ca2+]i rise, (ii) whether this action can be differentially modulated by fast versus slow chelators of Ca2+, (iii) the role of the cytoskeleton, and (iv) whether the simple activity of the ICRAC, independent of the ion being transported, can modulate the enzyme.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Thapsigargin, ionomycin, forskolin, and Ro 20-1724 were from Calbiochem. [2-3H]Adenine, [3H]cAMP and [alpha -32P]ATP were obtained from Amersham Pharmacia Biotech. Fura-2/AM, pluronic F-127, EGTA-AM, and BAPTA-AM were from Molecular Probes, Inc. (Eugene, OR). Other reagents were from Sigma.

Cell Culture-- C6-2B rat glioma cells were maintained in 13 ml of F-10 medium (Life Technologies, Inc.) with 10% (v/v) bovine calf serum (Gemini) in 75-cm2 flasks at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Cells were plated at approximately 70% confluency in 24-well plates for cAMP accumulation experiments.

Measurement of cAMP Accumulation-- cAMP accumulation in intact cells was measured according to the method of Evans et al. (12) as described previously (7) with some modifications. C6-2B cells on 24-well plates were incubated in F-10 medium (60 min at 37 °C) with [2-3H] adenine (1.5 µCi/well) to label the ATP pool. The cells were then washed once and incubated with a nominally Ca2+-free Krebs buffer containing 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO4, 11 mM glucose, 25 mM HEPES, and 0.1% bovine serum albumin (fraction V) adjusted to pH 7.4 with 2 M Tris base (900 µl/well). The use of Ca2+-free Krebs buffer in experiments denotes the addition of 0.1 mM EGTA to the nominally Ca2+-free Krebs buffer. For experiments in which Ba2+ was added to the medium, MgSO4 was replaced with MgCl2. All experiments were carried out at 30 °C in the presence of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine (500 µM), and Ro 20-1724 (100 µM), which were preincubated with the cells for 10 min prior to a 1-min assay.

Adenylyl Cyclase Activity Measurements-- The adenylyl cyclase activity of the C6-2B and rat brain membranes was measured in the presence of the following components: 12 mM phosphocreatine, 2.5 units of creatine phosphokinase, 0.1 mM cAMP, 1 mM MgCl2, 0.1 mM ATP, 70 mM Tris buffer, pH 7.4, 0.04 mM GTP, 1 µCi [alpha -32P]ATP, and 20 µM forskolin. Rat brain membrane assays also contained 1 µM calmodulin. Free Ca2+ and Ba2+ concentrations were established from a series of CaCl2 and BaCl2 solutions buffered with 200 µM EGTA in the assay (13). The reaction mixture (final volume, 100 µl) was incubated at 30 °C for 20 min. Reactions were terminated with sodium lauryl sulfate (0.5%); [3H]cAMP was added as a recovery marker, and the [32P]cAMP formed was quantified as described previously (14). Data points are presented as mean activities ± S.D. of triplicate determinations. Protein concentrations were determined by the Lowry method (15).

[Ca2+]i Measurements-- [Ca2+]i was measured in populations of C6-2B cells, using fura-2 as the Ca2+ indicator, exactly as described earlier (10).

Immunofluorescence Staining-- C6-2B cells were grown on ECL coverslips coated with attachment matrix (Upstate Biotechnology) to the desired density. Some cells were incubated with cystochalasin D (1 µM) for 60 min at 37 °C before processing for immunofluorescence microscopy. Control and treated cells were fixed with a mixture of 3.7% fresh paraformaldehyde and 0.05% glutaraldehyde diluted in 0.1 M phosphate buffer, pH 7.0, for 20 min at room temperature. Cells were washed with phosphate-buffered saline (PBS) (1.8 mM KH2PO4, 10 mM Na2HPO4, 2.7 mM KCl, and 137 mM NaCl), treated with 0.1% sodium borohydride, and permeabilized for 7 min with 0.1% Triton X-100 in PBS. The nonspecific reactive sites were blocked with 2% bovine serum albumin in PBS for 30 min at room temperature. Permeabilized cells were incubated with primary antibody (rabbit anti-ACV/VI (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:50; this antibody is directed against the C-terminal decapeptide of ACV/VI) in PBS-bovine serum albumin overnight at 4 °C. Cells were washed three times with PBS and incubated for 1 h with species-specific secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma)). For F-actin visualization, cells were incubated as above except that an incubation of 1 µM rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR) in PBS-bovine serum albumin was substituted for treatments with primary and secondary antibodies. Cells were then washed five times in phosphate buffer and once with distilled water and mounted with 0.1% p-phenylenediamine (Sigma) in Fluoromount G (Southern Biotechnology Associates, Inc.). Cells were observed on a fluorescence microscope (Nikon) with an 100× oil immersion objective set with the appropriate excitation/emission viewing.

Preparation of Rat Brain Plasma Membranes-- Rat brain plasma membranes were prepared from a continuous sucrose gradient and washed three times in a buffer containing 1 mM EGTA, as described previously (16).

Preparation of Plasma Membranes from C6-2B Cells-- Cultured C6-2B cells were detached from flasks with PBS containing 0.03% EDTA. Membranes were prepared using a method described previously (17). Cell suspensions were centrifuged, washed with Phillip's buffer containing protease inhibitors (20 µg/ml soybean trypsin inhibitor, 4 µg/ml leupeptin, 12 units/ml kallikrein inactivator, 4 µg/ml antipain, 52.4 µg/ml benzamidine, 52.3 µg/ml phenylmethylsulfonyl fluoride, and 2 µg/ml pepstatin A). Following centrifugation and subsequent lysis of cells in hypo-osmotic buffer containing protease inhibitors, the lysate was centrifuged at 270 × g for 10 min. The supernatant was fractionated on a continuous gradient of 5-50% sucrose in lysis buffer. Material collected at ~35% was removed, washed, and resuspended in lysis buffer to a final protein concentration of 1.0 mg/ml (as determined by the method of Lowry et al. (15)) and stored in liquid nitrogen.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Capacitative versus Ionophore-mediated Ca2+ Entry-- We have previously shown that Ca2+-stimulated adenylyl cyclases, when transiently expressed in HEK 293 cells, are selectively regulated by capacitative Ca2+ entry (CCE) and not at all by nonspecific, ionophore-facilitated entry of Ca2+ across the plasma membrane (7). The argument could be made, however, that transiently overexpressed adenylyl cyclases are restricted to a selective domain of the cell, a situation that does not occur with endogenously expressed adenylyl cyclases. To address this issue, we investigated the Ca2+-inhibitable adenylyl cyclase VI, which is the predominant species expressed endogenously in C6-2B cells (11). We had earlier shown that this cyclase is regulated by physiological modes of Ca2+ entry and not at all by release from any intracellular sites (10). However, we had not compared the ability of CCE versus nonregulated ionomycin-mediated Ca2+ entry to regulate such an endogenously expressed adenylyl cyclase. To address this issue, we established conditions to distinguish these two modes of [Ca2+]i elevation. For CCE, the cells were first treated with the intracellular Ca2+ store Ca2+-ATPase inhibitor thapsigargin (TG) (100 nM), which depletes the intracellular Ca2+ stores (18). The cells were initially maintained in a Ca2+-free Krebs buffer (for composition, see "Experimental Procedures") so that after depletion of the intracellular Ca2+ stores by TG, the cells are primed for CCE (19). Various concentrations of extracellular Ca2+ were added at 400 s, which elicited graded [Ca2+]i rises (Fig. 1a). The peak intracellular level of [Ca2+]i ranged from approximately 200 nM with 100 µM [Ca2+]ex to approximately 830 nM with 4 mM [Ca2+]ex. Note that the peak [Ca2+]i rise occurred within 1 min, which is the assay period used in later cAMP measurements. The modest capacitative [Ca2+]i rise is in stark contrast to the dramatic [Ca2+]i entry produced by addition of 4 µM ionomycin to the cells prior to introduction of [Ca2+]ex (Fig. 1b). The ionomycin-treated cells yielded a [Ca2+]i rise that ranged from 300 nM with 100 µM [Ca2+]ex to approximately 3300 nM with 800 µM [Ca2+]ex. It should be noted, of course, that the [Ca2+]i rise produced by ionomycin comprised both ionophore-mediated Ca2+ entry and an underlying CCE, because ionomycin can deplete intracellular Ca2+ stores (20, 21). Therefore, to detect additional effects, if any, of ionomycin-mediated entry over those of CCE, TG (100 nM) was added to both experimental conditions. The peak [Ca2+]i produced by CCE alone and ionomycin-mediated Ca2+ entry are compared in Fig. 1c. It is readily apparent that CCE gave a modest [Ca2+]i rise, whereas ionophore-mediated Ca2+ entry gave an extremely robust [Ca2+]i rise, which at 600 µM [Ca2+]ex produced a [Ca2+]i peak that was more than 4-fold the [Ca2+]i rise produced by CCE with the same [Ca2+]ex. In Fig. 2a, the effects of the two Ca2+ entry protocols are compared on the cAMP accumulation by the endogenous Ca2+-inhibitable adenylyl cyclase. In all conditions, the adenylyl cyclase was stimulated with forskolin (10 µM) and isoproterenol (10 µM) during the 1-min assay period during which [Ca2+]ex was also added. Given the dramatically different Ca2+ rises induced by the two protocols, noted above, it is striking that the two Ca2+ entry protocols elicited a quite similar profile of inhibition of cAMP accumulation as a function of [Ca2+]ex, with a maximum of approximately 40% (Fig. 2b). For instance, 600 µM [Ca2+]ex for both Ca2+ entry protocols inhibited cAMP accumulation by approximately 26%, even though the [Ca2+]i rise produced by CCE was approximately 540 nM as opposed to approximately 2200 nM for ionomycin-mediated Ca2+ entry. Because the [Ca2+]i rise produced by ionomycin is composed of both capacitative and ionophore-mediated Ca2+ entry and yet gives a similar inhibition profile of cAMP accumulation to CCE, the large additional [Ca2+]i rise contributes nothing to adenylyl cyclase regulation. These data strongly suggest that the increased [Ca2+]i rise produced by ionomycin does not have access to the adenylyl cyclase and, by inference, that the CCE sites are located in the same microdomain as adenylyl cyclase and that this domain is not accessible to ionomycin.


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Fig. 1.   Intracellular Ca2+ rise produced by capacitative versus ionomycin-mediated Ca2+ entry. [Ca2+]i was determined in aliquots of 4 × 106 fura-2 loaded C6-2B cells as described under "Experimental Procedures." a, capacitative Ca2+ entry was evoked by depleting intracellular Ca2+ stores with TG (100 nM) in a Ca2+-free medium at 60 s. At 400 s, various [Ca2+]ex, ranging from 100 to 4000 µM, were added, which results in the depicted rises in [Ca2+]i. b, cells were treated with TG (100 nM) and ionomycin (IM) (4 µM) at 60 and 270 s, respectively, to release and deplete both the mobilizable and nonmobilizable Ca2+ stores. The addition of [Ca2+]ex ranging from 0 to 800 µM at 400 s yields predominantly unregulated ionophore-mediated Ca2+ entry overlaid on capacitative Ca2+ entry (as deduced from a). a and b are representative data of four similar experiments. c, plot of peak [Ca2+]i achieved following the addition of [Ca2+]ex shown in a (circles, TG P.T.) and b (squares, TG/ionomycin (TG/IM P.T.)). P.T., pretreated.


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Fig. 2.   Effects of capacitative versus ionomycin-mediated Ca2+ entry on ACVI activity in C6-2B cells. cAMP accumulation was measured in intact C6-2B cells as described under "Experimental Procedures." All conditions include forskolin (10 µM) and isoproterenol (10 µM) to stimulate adenylyl cyclase activity. a, TG-mediated (open bars) or ionomycin/TG-mediated (hatched bars) Ca2+ entry was evoked followed by the addition of various [Ca2+]ex with the resultant effect on ACVI activity shown. The cAMP accumulation was measured over a 1-min period beginning with the addition of [Ca2+]ex, forskolin, and isoproterenol. b, effects produced in a expressed as the percentage of inhibition compared with the 0 [Ca2+]ex condition. a and b are representative data of four similar experiments. c, effect of cAMP elevation on TG-mediated Ca2+ release and subsequent CCE. Trace a shows the effect of 3-isobutyl-1-methylxanthine (500 µM) and Ro 20-1724 (100 µM) pretreatment (P.T., 9 min before addition of [Ca2+]ex (4 mM)) on the [Ca2+]i rise produced by TG (100 nM) and CCE. Trace b shows the effect of adding forskolin (10 µM) 1 min prior to [Ca2+]ex. Trace c is the control condition.

Both the elevation of intracellular cAMP and the direct introduction of the catalytic unit of cyclic AMP-dependent protein kinase sensitize the inositol trisphosphate receptor to injected inositol trisphosphates in hepatocytes (22, 23), with the result that somewhat higher levels of Ca2+ release arise than in the absence of cAMP elevation. CCE was not examined in these earlier studies. Because in the present experiments, comparisons were being made between effects of [Ca2+]i as measured by fura-2 fluorescence on cAMP accumulation under various conditions, it seemed important to determine whether elevation of cAMP might perturb the [Ca2+]i measurements. We had noted earlier that elevation of cAMP by a variety of means exerted minimal effects on Ca2+ release in C6-2B cells (10). However, we had not looked in detail at the effects of the conditions of cAMP elevation being used in the present series of experiments on CCE. Therefore, as used in standard cAMP assays, we exposed cells either to a preincubation with the phosphodiesterase a inhibitors 3-isobutyl-1-methylxanthine and Ro 20-1724 for 9 min, or to a pulse of forskolin for 1 min, and measured CCE in response to store depletion mediated by TG. An insignificant enhancement in CCE is associated with conditions that elevate cAMP (Fig. 2c), which is quite unlikely to complicate the effects of any comparisons between [Ca2+]i rises and cAMP accumulation. Therefore, in subsequent [Ca2+]i measurements, cells were not exposed to these agents.

Time Dependence of Store Depletion-- As outlined earlier, CCE is stimulated by the depletion of intracellular Ca2+ stores, but is the ability of CCE to regulate adenylyl cyclase predicated on complete store depletion? To examine this possibility, experimental conditions were employed in which [Ca2+]ex was added at various time points following TG depletion of Ca2+ stores. Following the addition of TG (100 nM) at 60 s, [Ca2+]ex (1 mM) was added at increasing intervals, which produced progressively increasing [Ca2+]i rises (Fig. 3). It is important to note that 1 mM [Ca2+]ex, which gives a submaximal inhibition of cAMP accumulation (see Fig. 2), was chosen to allow Ca2+ regulation of the adenylyl cyclase to be either increased or decreased on varying the TG-Ca2+ interval. Addition of [Ca2+]ex, 30 s following TG, a period that shows only limited depletion of intracellular Ca2+ stores, gave a modest and slower rise in [Ca2+]i compared with longer TG-Ca2+ intervals (peak [Ca2+]i rise was 56% of that of the 4-min condition). (A TG-Ca2+ interval of 4 min was employed in the experiment shown in Fig. 2.) Increasing the TG-Ca2+ interval to 7 min produced a larger [Ca2+]i rise, which was approximately 145% of that of the 4-min condition. (Note that TG-Ca2+ intervals greater than 1 min had similar rates of [Ca2+]i rise, but yielded increasing peak [Ca2+]i (Fig. 3).) The effect of varying the TG-Ca2+ interval was then examined with respect to the effect on cAMP accumulation in these cells. Quite unexpectedly, there was no difference in the ability of CCE to regulate adenylyl cyclase activity with different TG-Ca2+ intervals (Fig. 4), even though there was a striking difference in the peak [Ca2+]i. The simplest explanation for these data is that the first stores to be depleted after a short TG exposure are those near the plasma membrane and that this depletion is sufficient to trigger adequate Ca2+ entry to regulate the cyclase. More extensive depletion, after longer TG exposure, stimulates further CCE, which is ineffectual at regulating the cyclase.


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Fig. 3.   Effect of varying the time interval between TG treatment and addition of [Ca2+]ex on [Ca2+]i rise. [Ca2+]i was measured in aliquots of 4 × 106 fura-2 loaded C6-2B cells in Ca2+-free Krebs buffer as described under "Experimental Procedures." Addition of TG (100 nM) at 60 s was followed by the addition of [Ca2+]ex (1 mM) at time points ranging from 30 s to 7 min post-TG treatment. The resultant rise in [Ca2+]I is shown. Note that the 4-min time interval between TG and [Ca2+]ex addition was employed in Fig. 1 and 2. Data are representative of two similar experiments.


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Fig. 4.   Effect of varying the time interval between TG treatment and addition of [Ca2+]ex on cAMP accumulation. The time interval between TG (100 nM) and [Ca2+]ex (1 mM) addition was varied similarly to those employed in Fig. 3 with the resultant inhibition in cAMP accumulation, as compared with the [Ca2+]ex-free condition (left-most bar), shown. cAMP accumulation was measured in intact C6-2B cells exactly as in Fig. 2, with forskolin (10 µM) and isoproterenol (10 µM) present during the 1-min period. Data are representative of two similar experiments.

The Role of the Cytoskeleton-- Whether the cytoskeleton played a role in maintaining or underpinning the functional interaction between the CCE site and the adenylyl cyclase established in the foregoing studies was explored using a variety of cytoskeletal disrupting agents. Concentrations of cystochalasin D that were effective at disrupting the cytoskeleton were established by rhodamine-phalloidin staining of actin filaments. Thus, cystochalasin D (1 µM) treatment for 1 h at 37 °C significantly disrupted both actin filaments, as well as adenylyl cyclase distribution, compared with control, untreated cells (Fig. 5). As a prelude to detecting the effects of cytoskeletal disrupters on adenylyl cyclase regulation, it was also critical to determine whether CCE was itself perturbed by cytoskeletal disruption. As shown in Fig. 6, cystochalasin D (1 µM) treatment did not affect either the release of intracellular Ca2+ promoted by TG or the subsequent CCE. These data agree with recent findings in NIH3T3 cells, which showed that although Ca2+ mobilization in response to agonists was abolished, TG-mediated Ca2+ release and CCE were unaffected by cytoskeletal disruption (24). Treatment of C6-2B cells with these concentrations of cystochalasin D, which fully disrupted the cytoskeleton, had no effect on the ability of CCE to inhibit cAMP accumulation; following store depletion, [Ca2+]ex (4 mM) yielded approximately 40% inhibition of cAMP accumulation with or without cystochalasin D (1 µM) pretreatment (n = 4). This indicates that the adenylyl cyclase is still functionally associated with CCE sites. Other cytoskeletal disrupters, namely nocodazole and colchicine, which target microtubular cytoskeletal structures, were similarly ineffective at uncoupling CCE from adenylyl cyclase (data not shown). These data indicate that an intact cytoskeleton is not required to maintain or support the functional colocalization of the adenylyl cyclase and CCE channels.


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Fig. 5.   Ability of cystochalasin D treatment to disrupt actin filaments and adenylyl cyclase localization in C6-2B cells. Immunofluorescence labeling of either actin (A and C) or ACVI (B and D) in C6-2B cells that were either untreated (A and B) or treated for 1 h at 37 °C with cystochalasin D (1 µM) (C and D). Cells were fixed and stained as described under "Experimental Procedures." Actin filaments were visualized by staining with rhodamine-conjugated phalloidin, and the ACVI distribution was detected using rabbit anti-ACVI antibodies and fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies. Preblocking the primary antibody with the immunogenic peptide eliminated the signal (not shown). Following treatment with cystochalasin D, the cytoskeletal network collapsed and ACVI localization was also grossly altered.


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Fig. 6.   Effect of cystochalasin D treatment on TG-meditated Ca2+ release and subsequent CCE in C6-2B cells. Aliquots of 4 × 106 C6-2B cells were first treated with vehicle (MeSO2) or cystochalasin D (1 µM) (CytoD) for 60 min at 37 °C in nominally Ca2+-free Krebs buffer, followed by fura-2/AM loading as described under "Experimental Procedures." Addition of TG (100 nM) at 60 s was followed by the addition of [Ca2+]ex (4 mM) at 270 s to the cells in Ca2+-free Krebs buffer. The resultant [Ca2+]i rise produced by TG (Ca2+ store depletion, open bars) and subsequent [Ca2+]ex addition (CCE, hatched bars) are shown for control and cystochalasin D-treated cells, as indicated.

The Efficacy of BAPTA and EGTA-- The intimacy of the adenylyl cyclase and CCE functional colocalization was next probed using the Ca2+ chelators BAPTA and EGTA. A comparison of the effects of EGTA and BAPTA has been used by several groups (25, 26) to estimate the distance between a Ca2+ source and a Ca2+-sensitive target molecule. This is possible because of the different Ca2+ binding kinetics of these two reagents. Both EGTA and BAPTA have a similar affinity (KD) for Ca2+, but the on-rate for Ca2+ of BAPTA is much faster (approximately 400-fold) than that of EGTA. Therefore, at equivalent concentrations, the two chelators have differential abilities to block the regulation of target molecules by rapid rises in [Ca2+]i. To be able to compare the effects of the two chelators on the regulation of cAMP accumulation by CCE, it would have been desirable if equivalent external concentrations of the acetomethoxy esters of the chelators achieved equivalent intracellular concentrations of the free acids. Indeed, given the close structural relatedness of the two compounds, it seemed likely that equivalent concentrations would be achieved. Nevertheless, to confirm this objective, C6-2B cells were incubated with a range of BAPTA-AM and EGTA-AM concentrations and the consequences for muting the responses of fura-2 (a related, but weaker Ca2+ chelator) to various [Ca2+]i rises were compared. Cells were loaded with various concentrations of BAPTA-AM and EGTA-AM for 22 min at room temperature before measurements of CCE using 4 mM [Ca2+]ex were made (Fig. 7). At all chelator concentrations, BAPTA and EGTA had similar effects on the [Ca2+]i rise produced by CCE. Indeed, a more extensive series was performed, up to 50 µM. At no concentration tested was there any difference between BAPTA and EGTA (data not shown). This lack of difference between BAPTA and EGTA reflects their effective out-competition of fura-2 for global Ca2+ rises. The complementary cAMP accumulation experiment on cells loaded with exogenous Ca2+ chelators is shown in Fig. 8. As described earlier, the cells were treated with TG (100 nM) prior to a 1-min assay in which the cells were treated with forskolin (10 µM) and isoproterenol (10 µM) and either 0 or 4 mM [Ca2+]ex. Quite remarkably, EGTA was ineffective at blocking regulation of adenylyl cyclase by CCE with 4 mM [Ca2+]ex at external pretreatment concentrations up to 20 µM. This contrasts with the effect of BAPTA, which completely abolishes the Ca2+ inhibition of adenylyl cyclase by CCE at 20 µM [BAPTA-AM]ex (Fig. 8).3 This difference in effect is particularly striking given that both chelators at 20 µM external concentration equally perturbed the CCE profiles, as measured by fura-2 (see Fig. 7), and yet 20 µM [EGTA-AM]ex had only a very slight effect on Ca2+ inhibition of cAMP accumulation, whereas 20 µM [BAPTA-AM]ex completely abolished the Ca2+ inhibition (Fig. 8). These data lead to two complementary conclusions: (i) the faster on-rate of BAPTA for Ca2+ allows it to compete successfully with the adenylyl cyclase for Ca2+, whereas EGTA, with its slower on-rate, cannot out-compete adenylyl cyclase for Ca2+; and (ii) the site of CCE is located very close to the Ca2+-sensitive adenylyl cyclase, such that entering Ca2+ does not diffuse very far to regulate the enzyme. However, BAPTA, because of its faster on-rate, reduces the diffusion of Ca2+ entering via CCE, whereas EGTA, even at very high concentrations, is unable to limit Ca2+ diffusion sufficiently to prevent Ca2+ from reaching a regulatory concentration at the adenylyl cyclase.


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Fig. 7.   Comparison of the effects of increasing BAPTA-AM and EGTA-AM concentrations on [Ca2+]i rises produced by capacitative Ca2+ entry. Aliquots of 4 × 106 C6-2B cells were loaded with both fura-2 and the indicated chelator-AM concentration (5-20 µM) for 22 min at room temperature as described under "Experimental Procedures." The four panels compare the effect of increasing chelator-AM concentrations on capacitative Ca2+ entry promoted in the cells with TG (100 nM) addition at 60 s with [Ca2+]ex of 4 mM added at 270 s in Ca2+-free Krebs buffer. The [Ca2+]i rise produced in EGTA-AM-treated (E) and BAPTA-AM-treated (B) cells are shown along with a control trace in which no exogenous Ca2+ chelator was added. Data shown are representative of two similar experiments.


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Fig. 8.   Effect of BAPTA-AM and EGTA-AM loading on the ability of capacitative Ca2+ entry to regulate ACVI in C6-2B cells. cAMP accumulation was measured in C6-2B cells loaded with the indicated chelator-AM concentration for 22 min at room temperature as described under "Experimental Procedures." The cells were treated with TG (100 nM) at 60 s followed by the addition of either 0 or 4 mM [Ca2+]ex at 270 s with the cAMP accumulation measured over the subsequent 1 min. Forskolin (10 µM) and isoproterenol (10 µM) are present throughout the 1-min assay period. Panels a and b show the ability of EGTA-AM and BAPTA-AM loading, respectively, to perturb the ability of capacitative entry [Ca2+] to regulate ACVI in C6-2B cells, expressed as the percentage of cAMP accumulation following Ca2+ entry as compared with the Ca2+-free condition. Data are representative of three similar experiments.

Effect of Ba2+ Conductance through CCE Channels-- The exclusive dependence of the Ca2+-sensitive adenylyl cyclase of C6-2B cells on CCE for its regulation leads to the formal possibility that Ca2+ conductance through the CCE channel is sensed by the adenylyl cyclase, possibly by direct protein-protein interactions between the cyclase and the CCE channel. A precedent for this type of interaction occurs in smooth muscle, where direct conformational coupling occurs between the L-type voltage-gated Ca2+ channels and the sarcoplasmic reticulum ryanodine receptor (27). To explore this possibility, Ca2+ could be replaced by another cation that would be conducted by the channel but that was inactive at regulating adenylyl cyclase in vitro. The ICRAC characterized by Hoth and Penner (28) and Zweifach and Lewis (29) conducts Ba2+; therefore, if Ba2+ was unable to regulate the cyclase in vitro, the possibility that channel activity per se could regulate the cyclase could be evaluated. The effects of Ba2+ and Ca2+ were compared on adenylyl cyclase activity in C6-2B plasma membranes (Fig. 9). A Ca2+ dose-response curve on adenylyl cyclase activity in rat brain membranes was also performed to corroborate the estimated free [Ca2+]. Brain adenylyl cyclase activity is stimulated maximally by approximately 1 µM free Ca2+, owing to the predominance of Ca2+-stimulated adenylyl cyclase isoforms in the brain, with an inhibition of activity at greater free Ca2+ concentrations (Fig. 9a). The falling phase of the Ca2+ dose response curve is a property of all adenylyl cyclase isoforms (apart from AC3), regardless of their Ca2+ regulation at submicromolar concentrations of Ca2+ (7). A comparable experiment with the same Ca2+ solutions on adenylyl cyclase activity in C6-2B membranes shows two falling phases in adenylyl cyclase activity (Fig. 9b). These data are fit best by a two-site model in which one site has submicromolar affinity that corresponds exactly to the affinity for Ca2+ stimulation in brain membranes and the second, in the supramicromolar range, is the non-isoform-dependent Ca2+ inhibition that is also seen in the rat brain membranes (Fig. 9).4 When the effects of Ba2+ on adenylyl cyclase activity in C6-2B membranes were examined, it was seen that increasing [Ba2+] from 0 to 233 µM did not affect the adenylyl cyclase activity (Fig. 9c). Therefore, one element for determining whether ion conductance through the CCE channel itself has a regulatory effect on the adenylyl cyclase is satisfied.


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Fig. 9.   In vitro effects of Ca2+ on adenylyl cyclase activity in rat brain (a) and C6-2B plasma membranes (b) and the effect of Ba2+ on C6-2B plasma membranes (c). Adenylyl cyclase activity was determined in plasma membranes prepared from C6-2B cells or rat brain suspended in EGTA-containing buffer as described under "Experimental Procedures." Activity was measured in the presence of forskolin (10 µM) and the indicated free Ca2+ or free Ba2+ concentration. Free [Ca2+] and [Ba2+] was established with EGTA-buffered solutions as described under "Experimental Procedures." Panel a depicts the effect of increasing free Ca2+ (ranging from 0 to 39.5 µM) on rat brain membrane adenylyl cyclase activity. The effect of the same free Ca2+ concentrations on C6-2B plasma membranes is shown in panel b. Panel c shows the effect of increasing free Ba2+ concentrations (ranging from 0 to 233 µM) on adenylyl cyclase activity in C6-2B plasma membranes. Values shown are from an experiment that was repeated four times with similar results.

Of course, it must also be determined that Ba2+ can enter the cells that have been stimulated to activate CCE. To address this issue, C6-2B cells were pretreated with TG to provoke store depletion, and 1 mM Ba2+ was added to the medium, which elicited an increase in fura-2 fluorescence, demonstrating Ba2+ entry into the cells (Fig. 10). These results agree with whole-cell electrophysiological experiments, which showed that Ba2+ is conducted through CCE channels (30). Therefore, Ba2+ ions do pass through CCE channels and are not able to directly regulate adenylyl cyclase activity. In Fig. 11, cAMP accumulation was measured in C6-2B cells that had been treated with TG (100 nM) to evoke CCE with a subsequent addition of either Ca2+ or Ba2+ to the medium. Ca2+ gives a characteristic inhibition of adenylyl cyclase with a final inhibition of 44% and [Ca2+]ex of 4 mM, whereas Ba2+ was unable to affect adenylyl cyclase activity at any concentration tested. Therefore, it appears that activation of the CCE channel and inert ion conductance by the channel is not sufficient to regulate the adenylyl cyclase.


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Fig. 10.   Ba2+ entry into C6-2B cells through capacitative Ca2+ entry channels. Aliquots of 4 × 106 C6-2B cells were fura-2-loaded as described under "Experimental Procedures." Cells were TG (100 nM)-treated at 60 s in Ca2+-free Krebs buffer that was devoid of SO4, PO4, and HCO3 to prevent Ba2+ precipitation, followed at 270 s with [Ba2+]ex (1 mM). The increase in fluorescence (340/380 ratio) following TG addition represents the rise in [Ca2+]i produced by Ca2+ release from intracellular stores, which returns to baseline. The addition of Ba2+ shows a subsequent increase in fluorescence, indicating Ba2+ entry into the cell. Data are representative of three similar experiments.


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Fig. 11.   In vivo effects of Ba2+ entry through capacitative Ca2+ entry channels on ACVI activity in C6-2B cells. cAMP accumulation was determined in intact C6-2B cells that were TG (100 nM) treated in Ca2+-free Krebs (also devoid of SO4, PO4, and HCO3 to preclude the possibility of Ba2+ ion precipitation) to evoke capacitative Ca2+ entry. After 4 min, increasing concentrations of extracellular Ca2+ (circles) or Ba2+ (squares) were added to the medium to promote ion influx, along with forskolin (10 µM) and isoproterenol (10 µM). cAMP accumulation was measured over the subsequent minute, with Ca2+ entry showing the characteristic inhibition of cAMP accumulation, whereas Ba2+ entry was ineffectual. Data are representative of three experiments with similar results.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The present study examined the Ca2+ regulation of the Ca2+-inhibitable adenylyl cyclase that is endogenously expressed in C6-2B cells. Previously, we had shown that Ca2+-stimulable adenylyl cyclases that were heterologously expressed in HEK 293 cells showed a strict requirement for Ca2+ regulation via CCE over an ionophore-mediated Ca2+ entry (7). However, it might be argued that this might be a compensatory cellular response to transiently over-expressing adenylyl cyclases. Therefore, CCE and ionophore-mediated Ca2+ entry were compared as to their ability to regulate an endogenously expressed Ca2+-inhibitable adenylyl cyclase. Remarkably, even though ionomycin-mediated Ca2+ entry produced a [Ca2+]i rise that was several times greater than that of CCE, similar levels of adenylyl cyclase inhibition were seen with both types of Ca2+ entry mechanisms with the same [Ca2+]ex. Ionomycin-mediated Ca2+ entry is composed of two mechanisms, one that is produced directly by the ionophore, which facilitates Ca2+ movement across the plasma membrane (21), and the second, CCE, that is owing to the ability of ionomycin to deplete intracellular Ca2+ stores (20). When the underlying CCE, which accompanies ionophore-mediated Ca2+ entry, is taken into account, it is clear that the large additional Ca2+ entry promoted by the ionophore is quite unable to regulate the adenylyl cyclase. These data provide compelling evidence that an endogenously expressed adenylyl cyclase is tightly colocalized with CCE sites and is not accessible to ionophore-mediated Ca2+ entry. The ensuing experiments probed the intimacy of this interaction by a variety of strategies.

Although the adenylyl cyclase shows a strict requirement for CCE to be regulated by Ca2+ in vivo, maximal depletion of stores is not required for this regulation. This point was established by varying the interval between Ca2+ store depletion caused by TG treatment and addition of [Ca2+]ex. An intermediate [Ca2+]ex (1 mM) was chosen in these experiments because it yields a submaximal inhibition of cAMP accumulation and, therefore, would allow either increased or decreased Ca2+ inhibition to be observed. It is quite surprising, then, that given the large differences in [Ca2+]i achieved with longer time intervals between TG and [Ca2+]ex addition, there was no additional Ca2+ inhibition of cAMP accumulation (Fig. 4). This finding is somewhat unexpected, given that by increasing the [Ca2+]ex with the same TG-Ca2+ interval, which also gives increasing [Ca2+]i, further inhibition of cAMP accumulation can be elicited (Fig. 2). The simplest interpretation of these data is that maximal store depletion is not necessary for full regulation of the adenylyl cyclase. Ca2+ entry is the product of the channel mean open time probability and the conductance through the channel. Thus, upon substantial store depletion, increasing [Ca2+]ex will lead to increasing Ca2+ conductance through the channel and thus to graded regulation of the cyclase. However, with increasing degrees of store depletion, the mean open time probability of the channel may be increasing. The adenylyl cyclase may not respond to such changes in channel mean open time probability but only to the transient local elevation in [Ca2+]i around the channel pore. This notion is substantially supported by the BAPTA/EGTA data (see below). It is also conceivable that there may be different CCE mechanisms (31) or isoforms of CCE channels expressed in C6-2B cells that possess different gating and/or permeability characteristics and that the adenylyl cyclase may be preferentially colocalized with a particular subtype of CCE channels. For instance, candidate CCE channels have been cloned from Drosophila, and their mammalian counterparts are believed to make up a family with potentially differing conductance and gating properties (for review, see Ref. 32). Whatever the precise mechanism, these data reinforce the concept of spatial colocalization of adenylyl cyclase with CCE mechanisms that cannot be identified by global [Ca2+]i measurements.

An obvious inference from the intimate association between CCE and the Ca2+-sensitive adenylyl cyclase would be that the cytoskeleton might be playing some supporting role in maintaining this association. However, when the cytoskeleton was disrupted by a variety of agents that targeted either actin filaments or microtubules, no effects on the ability of CCE to regulate the adenylyl cyclase were observed even though the cellular distribution of the adenylyl cyclase had been altered and the cytoskeleton had collapsed. This implies that the cytoskeleton is not involved with maintaining the functional colocalization and invokes other mechanisms, such as compartmentalization to particular membrane-lipid containing subdomains or direct protein-protein interactions. The observation that the link between store depletion and activation of CCE remains following cytoskeletal disruption agrees with a recent paper from the Putney laboratory (24), in which cystochalasin D and nocodazole, at similar concentrations, had no effect on TG-mediated Ca2+ release and the subsequent CCE in NIH 3T3 cells. The retention of the latter linkage following cytoskeletal disruption is probably more surprising than the association between the adenylyl cyclase and the CCE, which at least are both localized in the plasma membrane.

The functional and spatial relatedness between CCE sites and the Ca2+-sensitive adenylyl cyclase was further reinforced in the experiments using the Ca2+ chelators EGTA and BAPTA. Differences in the on-rate of these two compounds for Ca2+, and therefore in their ability to differentially perturb Ca2+ diffusion, have allowed their use by several groups to dissect the diffusional distances between a Ca2+ source and a Ca2+ target (25, 26). In the present case, the cell permeant acetoxymethyl forms of the chelators were used in cell population studies. The differences seen on Ca2+ inhibition of the adenylyl cyclase with the same extracellular chelator concentration were quite dramatic. Whereas EGTA-AM had very little effect on perturbing the ability of CCE to inhibit cAMP accumulation, BAPTA-AM completely abolished the inhibition. This is particularly striking given that there are no differences seen with these two chelators on global [Ca2+]i, as reported by fura-2. These data support the assertion that the CCE site and the adenylyl cyclase must be co-localized, because if they were randomly distributed, these two chelators, with similar KD values for Ca2+, would be equally able to blunt the ability of CCE to inhibit cAMP accumulation.

A final series of experiments explored the possibility that the intimacy of the CCE and adenylyl cyclase might be such that the mere conductance of Ca2+ through the CCE channel may regulate the adenylyl cyclase. If the adenylyl cyclase was physically coupled to the CCE channel or was a functional component of the channel, then the conformational changes that would occur in the channel by ion conductance might translate to altered adenylyl cyclase activity. This possibility was assessed by substituting Ba2+ for Ca2+. Ba2+ does enter the cell by CCE and does not have a direct effect on the adenylyl cyclase activity in in vitro membrane assays. However, the passage of Ba2+ through the channel does not affect cAMP accumulation. Therefore, activation and divalent cation conductance by the CCE channels is insufficient to regulate the adenylyl cyclase activity; Ca2+ must be the ion being carried.

In conclusion, the present study convincingly establishes an exquisite and intimate spatial relationship between CCE and an endogenous Ca2+-sensitive adenylyl cyclase. Earlier studies had established the concentration of adenylyl cyclase immunoreactivity in dendritic spines of hippocampal neurons (33), domains of the cell that are also endowed with voltage-gated Ca2+ channels and cAMP-dependent protein kinase anchoring proteins (34-36). Thus, compartmentalization of adenylyl cyclase may be a general finding in both excitable and nonexcitable cells. However, how this organization is achieved is quite unknown at present. One possible lead may be found in the Drosophila visual system, in which a scaffolding protein, InaD, recruits three interacting proteins, the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC), and the Trp channel (a putative capacitative Ca2+ entry channel) to the identical cellular subdomains (37). Future studies may reveal an analogous protein in mammalian cells, which serves to maintain a similar relationship between the CCE and Ca2+-sensitive adenylyl cyclases.

    ACKNOWLEDGEMENT

We thank Dr. R. A. Harris for the continuing use of his spectrofluorimeter.

    FOOTNOTES

* These studies were supported in part by National Institutes of Health Grant GM 32483 (to D. M. F. C.) and a NATO collaborative research grant (to N. M. and D. M. F. C).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: Dept. of Pharmacology, Box C-236, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-8964; Fax: 303-315-7097; E-mail: cooperd{at}essex.uchsc.edu.

1 The abbreviations used are: [Ca2+]i, cytosolic Ca2+ concentration; [Ca2+]ex, extracellular Ca2+ concentration; CCE, capacitative Ca2+ entry; PBS, phosphate-buffered saline; TG, thapsigargin; ICRAC, calcium release-activated current; ACVI, adenylyl cyclase, type VI.

2 ICRAC is used not in the strict sense of the mast cell channel first characterized by Hoth and Penner (30) and Zweifach and Lewis (29) but in a generic sense to indicate any channels that are activated by store depletion (32).

3 Experiments were also conducted to examine whether very high [EGTA-AM]ex (up to 200 µM) could effectively abolish Ca2+ inhibition of cAMP accumulation. Even at the highest EGTA-AM concentration examined, there was still approximately 14% inhibition of cAMP accumulation by CCE (data not shown). It was also interesting that this treatment with 200 µM EGTA-AM resulted in a barely detectable rise in [Ca2+]i as reported by fura-2, although this Ca2+ rise could inhibit the adenylyl cyclase by 14%.

4 It is important to distinguish these two effects of Ca2+ on adenylyl cyclase activity, because the submicromolar Ca2+ effects are adenylyl cyclase isoform-dependent, whereas the supramicromolar inhibition is common to most of the isoforms. The non-isoform-dependent inhibition has been speculated to reflect Ca2+ competing for Mg2+ in MgATP2+ binding to the catalytic site of the adenylyl cyclase (38).

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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