©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Co-localization of Transfected Ca-stimulable Adenylyl Cyclases with Capacitative Ca Entry Sites (*)

(Received for publication, November 16, 1995; and in revised form, February 21, 1996)

Kent A. Fagan Rajesh Mahey Dermot M. F. Cooper (§)

From the Department of Pharmacology and Neuroscience Program, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Three adenylyl cyclases (ACI, ACIII, and ACVIII) have been described, which are putatively Ca-stimulable, based on in vitro assays. However, it is not clear that these enzymes can be regulated by physiological rises in [Ca] when expressed in intact cells. Furthermore, it is not known whether transfected adenylyl cyclases might display the strict requirement for capacitative Ca entry that is shown by the Ca-inhibitable ACVI, which is indigenous to C6-2B glioma cells (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F.(1995) J. Biol. Chem. 270, 1149-1155). In the present study, ACI, ACIII, and ACVIII were heterologously expressed in HEK 293 cells, and conditions were devised that distinguished capacitative Ca entry from both internal release and nonspecific elevation in [Ca] around the plasma membrane. Remarkably, not only were ACI and ACVIII largely insensitive to Ca release from stores, but they were robustly stimulated only by capacitative Ca entry and not at all by a substantial increase in [Ca] at the plasma membrane elicited by ionophore. (ACIII, reflecting its feeble in vitro sensitivity to Ca, was unaffected by any [Ca] rise.) These results suggest a quite unsuspected, essential association of Ca-sensitive adenylyl cyclases with capacitative Ca entry sites, even when expressed heterologously.


INTRODUCTION

The regulation of adenylyl cyclase by cytosolic Ca ([Ca]) (^1)could provide cells with an early opportunity to integrate the activity of the two major second messengers, cAMP and Ca. Eight adenylyl cyclase isoforms are described currently (reviewed in (1, 2, 3) ), of which five are reported to be sensitive to Ca, based on in vitro assays; ACI(4) , ACIII(5) , and ACVIII (6) are stimulated by Ca, whereas ACV and ACVI are inhibited by Ca(7, 8, 9) . The physiological potential of this Ca sensitivity is being suggested by accumulating data, which indicate that some of these enzymes can, in fact, be regulated by cellular transitions in [Ca]. For instance, in HEK 293 cells transiently transfected with ACI and ACVI cDNAs, Ca entry provoked by depletion of Ca stores (i.e. capacitative Ca entry(10) ) caused a stimulation and inhibition, respectively, of cAMP accumulation(11) . In HEK 293 cells stably transfected with ACVIII cDNA, activation of purinergic receptors (which elevate [Ca]) markedly stimulated adenylyl cyclase activity(6) . Recently, it was shown that the Ca-inhibitable ACVI, which is the predominant indigenous adenylyl cyclase species in C6-2B cells, is selectively inhibited by capacitative Ca entry rather than by Ca released from internal stores(12) . However, it is not known whether Ca-sensitive adenylyl cyclases are intrinsically dependent on Ca entry for their regulation and whether this property is retained even when adenylyl cyclase cDNAs are transfected. In this study, we examine all of the reportedly Ca-stimulable adenylyl cyclases (i.e. ACI, ACIII, and ACVIII) transfected into HEK 293 cells and separately assess the impact of Ca entry versus release on their activities. When all three adenylyl cyclases are expressed, the enzymes display some characteristic differences in their responsiveness to G(s)-mediated stimulation(6, 7, 13) . However, very strikingly, ACI and ACVIII are prominently stimulated by capacitative Ca entry, whereas there is no significant effect of ionophore-mediated Ca release on their activity. ACIII is quite refractory to any physiological elevation in [Ca]. When the Ca sensitivity of these cyclases is examined in vitro, again a robust stimulation by Ca of ACI and ACVIII is elicited by [Ca] in the low micromolar range. By contrast, ACIII is only marginally stimulated by very high [Ca], although, curiously, it is resistant to inhibition by supramicromolar Ca, which is a hallmark of all other adenylyl cyclases(14) . The predilection of the Ca-stimulable adenylyl cyclases for Ca entry, rather than release, suggests a discrete localization of adenylyl cyclases in areas where high [Ca]levels can be sustained. Furthermore, the studies demonstrate that HEK 293 cells possess the ability to localize even transfected adenylyl cyclases appropriately, which suggests that the ``targeting'' information is encoded within the proteins' sequences.


EXPERIMENTAL PROCEDURES

Materials

Thapsigargin and ionomycin were from L C Services Corp. (Woburn, MA) and Calbiochem, respectively. [2-^3H]Adenine, [^3H]cAMP, and [alpha-P]ATP were obtained from Amersham Corp. Fura-2/AM and pluronic F-127 were from Molecular Probes, Inc. (Eugene, OR). Other reagents were from Sigma.

Plasmids

The mammalian expression vector pCMV-5 was used for the expression of ACI and ACIII. The construction of the bovine type I construct, designated as pCMV-ACI, was described previously(4) . The rat adenylyl cyclase ACIII cDNA clone in pBluescript was obtained from Dr. R. R. Reed (Johns Hopkins University, Baltimore, MD). This cDNA was excised using EcoRI and ligated into pCMV-5 for expression. The ACVIII cDNA was provided by Dr. J. Krupinski (Weis Center for Research, Danville, PA) and was introduced into the expression vector pcDNA3 (Invitrogen) using KpnI and NotI sites. Both vectors drive expression using the cytomegalovirus promoter. Control experiments were conducted using pcDNA3 vector alone.

Cell Culture and Transfection of HEK 293 Cells

HEK 293 cells were maintained in 13 ml of minimal essential medium with 10% (v/v) fetal bovine serum, penicillin (50 µg/ml), streptomycin (50 µg/ml), and neomycin (100 µg/ml) in 75-cm^2 flasks at 37 °C in a humidified atmosphere of 95% air and 5% CO(2). Transfection was performed on HEK 293 cells at about 50% confluency, using the calcium phosphate method described by Chen and Okayama(15) . 26 µg of plasmid DNA was used for each transfection. Seventeen hours after transfection, the cells from 75-cm^2 flasks were harvested using phosphate-buffered saline containing 0.06% EDTA, plated onto 12-well culture plates, and incubated for 2 days before cAMP measurements were made.

Measurement of cAMP Accumulation

cAMP accumulation in intact cells was measured according to the method of Evans et al.(16) as described previously (12) with some modifications. HEK 293 cells on 12-well plates were incubated in minimal essential medium (60 min, 37 °C) with [2-^3H]adenine (1.5 µCi/well) to label the ATP pool. The cells were then washed twice and incubated with a nominally Ca-free Krebs buffer containing 120 mM NaCl, 4.75 mM KCl, 1.44 mM MgSO(4), 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 Ca-free Krebs buffer in experiments denotes the addition of 0.1 mM EGTA to the nominally Ca-free Krebs buffer. All experiments were carried out at 37 °C in the presence of the 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. In order to minimize the influence of basal [Ca](i), the cells were preincubated for 10 min with the Ca-ATPase inhibitor thapsigargin (TG) at a final concentration of 100 nM. (This has the effect of passively emptying the Ca stores and establishing a low basal [Ca](i);(10) .) Assays were terminated by addition of 5% (w/v final) trichloroacetic acid. Unlabeled cAMP (100 µl, 10 mM), ATP (10 µl, 65 mM), and [alpha-P]ATP (7000 cpm) were added to monitor recovery of cAMP and ATP. After pelleting, the [^3H]ATP and [^3H]cAMP content of the supernatant were quantified according to the standard Dowex/alumina methodology(17) . Accumulation of cAMP is expressed as the percent conversion of [^3H]ATP into [^3H]cAMP; means ± S.D. of triplicate determinations are indicated.

Adenylyl Cyclase Activity Measurements

Determination of adenylyl cyclase activity in vitro was performed as described previously (18) on isolated transfected HEK 293 cell membranes. Crude membranes were prepared following mechanical shearing of the cells by passage through a 22-gauge needle, 10 times, in homogenization buffer containing 2 mM MgCl(2), 1 mM EDTA, 50 mM Tris buffer, pH 8.0, 1 mg/50 ml of DNase and protease inhibitors as described (18) with the addition of 52.3 µg/ml phenylmethylsulfonyl fluoride, 52.4 µg/ml benzamidine, and 2 µg/ml pepstatin A. The supernatant from a low speed centrifugation (1,000 rpm, 1 min, SS-34 Sorvall) was pelleted (12,000 rpm, 10 min, SS-34 Sorvall), resuspended in assay buffer (40 mM Tris buffer, pH 7.4, 800 µM EGTA and 0.25% bovine serum albumin), and used immediately. The adenylyl cyclase activity of the HEK 293 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 MgCl(2), 0.1 mM ATP, 70 mM Tris buffer, pH 7.4, 0.04 mM GTP, 1 µCi [alpha-P]ATP, calmodulin (3 µM) and forskolin (10 µM), as indicated. Free Ca concentrations were established from a series of CaCl(2) solutions buffered with 200 µM EGTA in the assay(18) . The reaction mixture (final volume, 100 µl) was incubated at 30 °C for 20 min. Reactions were terminated with sodium lauryl sufate (0.5%); [^3H]cAMP was added as a recovery marker, and the [P]cAMP formed was quantitated as described previously (17) . Data points are presented as mean activities ± S.D. of triplicate determinations. Protein concentrations were determined by the Lowry et al. (19) method.

[Ca](i) Measurements

[Ca](i) was measured in populations of HEK 293 cells, using fura-2 as the Ca-indicator, exactly as described previously for C6-2B cells(12) .


RESULTS

Transient expression of HEK 293 cells transfected with Ca-sensitive adenylyl cyclase cDNAs was assessed by comparing cAMP accumulation in cells transfected with the expression vector alone (pcDNA3) or the vector encoding adenylyl cyclase ACI, ACIII, or ACVIII. Significant expression of ACI and ACVIII was apparent even in the unstimulated state. The detection of ACIII expression required stimulation by forskolin (Fig. 1; or PGE(1), see Fig. 5). Even in this relatively undiscerning comparison, it is interesting to note the different activity levels and stimulability by forskolin. ACVIII is remarkable by the high levels of activity it displays; both ACI and ACIII are much more modest in their forskolin-stimulated activity, as noted previously(5, 6, 20) .


Figure 1: Expression of adenylyl cyclases in HEK 293 cells. cAMP accumulation was measured in intact HEK 293 cells transiently expressing either vector alone (Control) or ACI, ACIII, or ACVIII cDNAs (as indicated) in the absence (open bars) or presence (hatched bars) of 50 µM forskolin. Assays were carried out over 1 min in nominally Ca-free Krebs buffer. Asterisks denote values that differ significantly from the relevant controls, as judged by Student's t test (p < 0.05).




Figure 5: Response of transiently expressed adenylyl cyclases in HEK 293 cells to varying degrees of capacitative [Ca] entry. [Ca] ranging from 0 to 10 mM was used to promote capacitative Ca entry as described in Fig. 2b. cAMP measurements were performed identically to those depicted in Fig. 3a for 1 min, with forskolin (10 µM) present throughout the experiments. Data shown are representative of three similar experiments.




Figure 2: a, Ca release; b, capacitative Ca entry in HEK 293 cells. [Ca] was determined in aliquots of 4 times 10^6 fura-2 loaded cells as described under ``Experimental Procedures.'' a, IM, in increasing concentrations (0.1, 0.2, 0.3, and 1 µM, in lowest to highest curves) was added at 60 s; carbachol (CCh; 100 µM) was added 5 min later to assess the extent of Ca store depletion. b, intracellular Ca stores were depleted by adding 100 nM TG at 60 s. Capacitative Ca entry was initiated by adding the indicated [Ca] 4 min later. All experiments were conducted in Ca-free Krebs buffer. The data shown are representative of five similar experiments.




Figure 3: Effect of capacitative Ca entry (a) or release from intracellular stores on the activity of expressed adenylyl cyclases (b). Vector alone (Control), ACI, ACIII, and ACVIII were expressed in HEK 293 cells as in Fig. 1. a, capacitative Ca entry was evoked by depleting Ca stores with TG (100 nM) in a Ca-free medium (cf. Fig. 2b). After 10 min, 4 mM Ca was added (where indicated) to the medium to promote Ca influx (analogous to Fig. 2b), and cAMP accumulation was measured over the subsequent minute. b, Ca was released from intracellular stores using IM (100 nM) in Ca-free media (cf. Fig. 2a), and cAMP accumulation was measured during the first minute of the treatment (all assays were conducted in the presence of 10 µM forskolin and in Ca-free Krebs buffer). The results shown are representative of five experiments that yielded similar results. Asterisks denote values that differ significantly from the relevant controls as judged by Student's t test (p < 0.05).



In order to compare the sensitivity of the adenylyl cyclases to capacitative Ca entry versus release from internal stores, conditions were established to separately observe these two normal constituents of a physiological [Ca](i) rise. In the absence of extracellular Ca, ionophores are well-suited to cause a maximal release of Ca from intracellular Ca stores(21) . Therefore, a range of concentrations of ionomycin (IM) was explored to determine an optimal dose for releasing intracellular stores. As shown in Fig. 2, 0.3 µM IM caused a substantial and rapid rise in [Ca](i) (up to 600 nM). Subsequent addition of carbachol caused no additional release in [Ca](i) (Fig. 2a) confirming that IM could release all of the mobilizable Ca.

Capacitative Ca entry could be separated from release by first treating cells with the microsomal Ca-ATPase inhibitor, thapsigargin (TG). By inhibiting this ATPase, TG causes the passive emptying of intracellular stores(22) . If such emptying is carried out in the absence of extracellular Ca, Ca is extruded and the cells are depleted of Ca. Under such conditions, the cells are primed to permit the rapid, capacitative influx of external Ca(10, 23) . In the present experiments, cells were first treated with TG (0.1 µM) in the absence of extracellular Ca. This resulted in a slow transient rise in [Ca](i), which returned to base-line level, reflecting the emptying of Ca stores and their extrusion. (Note also that carbachol treatment following such an exposure to TG results in no further release(11) , which confirms that the stores are empty.) After 4 min, increasing concentrations of Ca were added, which resulted in a rapid, concentration-dependent entry of Ca, achieving values of 180-280 nM [Ca](i) (Fig. 2b). Thus, experimental conditions are provided to assess separately the impact of release versus entry on Ca-stimulable adenylyl cyclases. It is worth noting that the peak (released) [Ca](i) achieved by IM was approximately 600 nM compared with only 270 nM entering in response to store depletion. It is also notable that both experimental paradigms achieve their maximum [Ca](i) rise rapidly and within the time frame (i.e. 1 min) of the cAMP measurements that are to follow.

The consequences of Ca release from intracellular stores and capacitative Ca influx are compared in the experiments depicted in Fig. 3. Addition of 4 mM [Ca] following a TG pretreatment in Ca-free Krebs buffer caused a pronounced increase in cAMP accumulation in HEK 293 cells transfected with ACI and ACVIII (Fig. 3a). Cells expressing ACI increased cAMP accumulation from 0.57 to 1.56%, and in those expressing ACVIII, the increase was from 4.72 to 15.52%. Neither the control nor ACIII transfected cells showed a significant increase in cAMP accumulation. The elevation in cAMP accumulation caused by Ca influx in cells transfected with ACI or ACVIII is in sharp contrast with the effects of Ca release on the same cells (Fig. 3b). In this case, cells transfected with ACI, ACIII, or ACVIII cDNAs showed no significant increase in cAMP in response to 100 nM IM in the absence of extracellular Ca. All of the above experiments were carried out with 10 µM forskolin present in the assay mixture to enhance the detection of signals and to facilitate Ca stimulation of ACIII, which has been reported only to be stimulated by Ca when activated by other factors(5) . In the absence of forskolin, the -fold stimulation by Ca entry of ACI and ACVIII was approximately the same, and again no stimulation of cells expressing ACIII was detected (not shown). These data indicate that Ca entry rather than Ca release from stores selectively regulates the Ca-stimulable ACI and ACVIII.

The foregoing experiment was designed to distinguish unambiguously the effects of Ca release versus capacitative entry on Ca-sensitive adenylyl cyclases. However, it might be argued that a phospholipase C-coupled receptor agonist rather than an ionophore could elicit a Ca release of more relevant spatial origins (and consequences) for a Ca-sensitive adenylyl cyclase. Of course, effects other than Ca release can arise from receptor occupancy and G-protein activation, including triggering of additional signal transduction pathways, which can cloud the interpretation of any such effects. Nevertheless, the manifest ability of carbachol to cause Ca release and thereby stimulate transfected ACVIII was explored. In the absence of extracellular Ca, carbachol (1 mM) elicited a significant 70% increase in cAMP accumulation (Fig. 4). This action of carbachol was likely dependent on intracellular Ca release, since prior emptying of the stores by TG treatment eliminated the effect (Fig. 4). By contrast, in the same experiment, TG- and CCh-mediated capacitative Ca entry yielded about a 5- and 4-fold increase, respectively, in cAMP accumulation (Fig. 4).


Figure 4: Effect of carbachol-induced [Ca] elevations on transiently expressed ACVIII activity in HEK 293 cells. Vector alone (Control; open bars) and ACVIII (hatched bars) were expressed in HEK 293 cells as in Fig. 1. Cells were pretreated 7 min before a 1-min assay with either vehicle alone (dimethyl sulfoxide), TG (100 nM), or CCh (1 mM) as indicated in Ca-free Krebs buffer. Acute treatments with either CCh (1 mM) or Ca (5 mM), as indicated, were carried out for a 1-min assay period. All experiments were conducted in the presence of forskolin (10 µM). Four combinations of pretreatments and acute treatments are shown, which depict, from left to right: CCh-mediated Ca release from intracellular stores; TG-mediated capacitative Ca entry; CCh-mediated capacitative Ca entry; and CCh-mediated Ca release from intracellular stores following depletion of the stores by TG pretreatment. Asterisks denote values that differ significantly from the relevant controls, as judged by Student's two-tailed t test (p < 0.05). The data shown are representative of four similar experiments.



The sensitivity of transfected adenylyl cyclases to different degrees of capacitative Ca influx was compared (Fig. 5). By using differing [Ca], following TG treatment, a dose-response curve was generated for the transfected adenylyl cyclases. HEK 293 cells expressing ACI or ACVIII showed a maximal Ca stimulation of cAMP accumulation at 5 mM [Ca]. The cells expressing ACI were consistently (in each of 3 experiments) about 2 times more sensitive to [Ca](i) rises than cells expressing ACVIII. Cells expressing ACIII again showed no increase in activity at any [Ca]. The refractoriness of the presumed Ca-stimulable ACIII to this in vivo assessment of Ca sensitivity should probably be viewed in the context of its reportedly (5) low sensitivity to Ca in in vitro assays. Consequently, in later experiments, we compared the sensitivity of the cyclases to extremely high [Ca] in in vitro experiments.

In a further attempt at eliciting in vivo stimulation of ACIII by Ca entry, the effects of [Ca](i) rises on ACI, ACIII, and ACVIII, which were concurrently stimulated by forskolin or PGE(1), were evaluated (Fig. 6). PGE(1) stimulates adenylyl cyclase via the G-protein subunit alpha(s) in HEK 293 cells. [Ca](i) rises were generated using capacitative Ca influx as described earlier, with [Ca] of 3 mM. Elevation of [Ca](i) had negligible effects on cAMP accumulation in cells transfected with either vector alone or ACIII, regardless of the assay conditions. This lack of effect contrasted with the robust effect of Ca entry on ACI and ACVIII under any assay conditions, which ranged from an approximately 1.5-fold stimulation of basal activity to 2.5-fold in forskolin-stimulated and 2-fold with PGE(1) stimulation (Fig. 6). It is noteworthy that the Ca-sensitive adenylyl cyclase most prominently stimulated by PGE(1) is ACIII (an observation that agrees with other studies of ACIII(13) ). This confirms that ACIII is well-expressed, but again draws attention to the insensitivity of this species to physiological Ca entry in these cells. (A lesser stimulation of transfected ACIII activity was elicited by isoproterenol (50 µM), but this also failed to elicit any stimulation by Ca entry; not shown.)


Figure 6: Stimulation of transiently expressed adenylyl cyclases in HEK 293 cells by multiple factors. Assays were conducted on HEK 293 cells transiently transfected with either vector alone (Control), ACI, ACIII, or ACVIII. Depletion of intracellular Ca stores with TG (100 nM) pretreatment was used to promote capacitative Ca entry as shown in Fig. 2b. Assays were conducted for 1 min following the addition of 3 mM [Ca], 50 µM forskolin, and/or 80 µM PGE(1) as indicated. Data shown are representative of three similar experiments. Asterisks denote values that differ significantly from the relevant controls as judged by Student's t test (p < 0.05).



The ability of ACI, ACIII, and ACVIII to be stimulated by Cain vitro was compared in plasma membranes from transfected HEK 293 cells. An extensive range of Ca concentrations was employed to address the reported low sensitivity of ACIII to Ca(5) . Plasma membrane preparations from cells transfected with ACI and ACVIII showed a strong stimulation of adenylyl cyclase activity by [Ca] in the range up to 1 µM (Fig. 7), agreeing with previous data from broken cell preparations(4, 6) . (The stimulation observed was strictly dependent on the presence of calmodulin, since removal of endogenous calmodulin by EGTA washes (14) largely eliminated the stimulation by low [Ca]; not shown.) Plasma membranes from ACIII-transfected cells showed an unremarkable 50% enhancement in activity at very high Ca (Fig. 7) which agreed with previously published data(5) . No manipulation of the assay conditions, with regard to forskolin or PGE(1) concentration, could elicit a more robust effect. Thus, it seems fair to conclude that the in vitro responsiveness of ACI, ACIII, and ACVIII to Ca is a fair predictor of their responses to physiological elevations in [Ca](i). The insensitivity of ACIII to elevation in [Ca](i) is quite compatible with its barely detectable response to supra-normal Cain vitro.


Figure 7: In vitro regulation by Ca of adenylyl cyclases expressed in HEK 293 membranes. Adenylyl cyclase activity was determined in plasma membranes prepared from cells transfected with either vector alone (Control), ACI, ACIII or ACVIII or rat brain suspended in EGTA-containing buffer. Activity was measured in the presence of forskolin (10 µM), calmodulin (3 µM), and the indicated free Ca concentrations. Values shown are from an experiment that was repeated five times with similar results. Values from control transfected cells (a) have been subtracted from ACI (c), ACIII (d) and ACVIII (e) values.



The previous experiments led to the conclusion that the Ca-stimulable ACI and ACVIII were predominantly regulated by Ca entry arising in response to store depletion. However, the possibility exists that Ca entering the cell, by virtue of its proximity to the plasma membrane, has a greater likelihood of regulating a plasma membrane-bound enzyme than Ca released from intracellular stores. It has been estimated that Ca diffuses only limited distances within the cytoplasm(24, 25) . To address the possibility that the sensitivity of the Ca-stimulable adenylyl cyclases to Ca entry merely reflected selective access of Ca entering the cell to the adenylyl cyclase, conditions were developed that would allow ionophore to yield a substantial [Ca](i) rise at the plasma membrane, arising largely from entry through ionophore-generated pores. Ionophore molecules insert readily in any lipid bilayer, including mitochondrial, endoplasmic reticular, or other internal membranes, as well as the plasma membrane(26) . Consequently, to study the effects of Ca ions flowing largely through plasma membrane pores, intracellular stores were first emptied with TG (in the absence of extracellular Ca) and then cells were exposed to IM. This resulted in two successive, transient elevations in [Ca](i), which was extruded from the cells, returning levels to base-line values (Fig. 8a). (It was important to empty internal stores of Ca, prior to the introduction of external Ca, so that an elevation in [Ca](i) reflecting only entry in response to IM would ensue.) Subsequently, upon the reintroduction of graded concentrations of extracellular Ca, [Ca](i) rose, reflecting both entry through the ionophore pores and limited capacitative Ca entry (to replenish the empty stores; Fig. 8a). A parallel series of [Ca](i) measurements were performed in the same time frame, including the TG treatment but omitting IM, to reveal the contribution of capacitative entry to the observed [Ca](i) rises (Fig. 8b). When the peak entry values in response to these [Ca] are plotted, it can be seen that IM allows substantial influx of Ca at relatively low [Ca]; values between 500 and 2300 nM are achieved with [Ca] of 0.2-0.8 mM, respectively (Fig. 8a). As [Ca] is raised, capacitative entry becomes evident, which from the data using TG alone, without a subsequent IM treatment (Fig. 8b), is a low affinity process that reaches its maximum [Ca](i) of 500 nM at 4 mM [Ca] and its half-maximum at 0.8 mM. Thus, conditions were generated to rigorously compare the impact of Ca entry through ionophore pores versus capacitative Ca entry channels.


Figure 8: Effects of capacitative versus IM-mediated Ca entry on ACVIII activity. [Ca] levels were measured in a suspension of 4 times 10^6 fura-2 loaded HEK 293 cells, in Ca-free Krebs buffer, as described under ``Experimental Procedures.'' a, cells were treated with TG (100 nM) and IM (4 µM) at 60 and 240 s, respectively, to release and deplete both mobilizable and nonmobilizable Ca stores. The addition of [Ca] ranging from 0 to 800 µM at 400 s yields predominantly unregulated ionophore-mediated Ca entry overlaid on small amounts of capacitative Ca entry (as can be deduced from b). b, cells were treated with TG (100 nM) at 60 s followed at 400 s by varying [Ca] ranging from 0 to 4000 µM (cf. Fig. 2b). a and b are representative data of three similar experiments. c, plot of the maximum [Ca]achieved following the addition of a range of [Ca] as in a (TG/IM, squares) and b (TG, circles). d, cAMP accumulation measured in HEK 293 cells transiently transfected with ACVIII in response to the Ca entry shown in a (squares) and b (circles). Forskolin (10 µM) was present throughout. Inset, comparison of cAMP accumulation in three different conditions highlighted in c. Condition 1 shows cAMP accumulation following TG treatment with the addition of 2.5 µM [Ca]. Conditions 2 and 3 compare cAMP accumulation in response to TG/IM treatment with addition of 200 µM [Ca] (largely ionophore-mediated Ca entry) and TG treatment with addition of 4000 µM [Ca] (capacitative Ca entry only), respectively (note the [Ca] produced by conditions 2 and 3 are virtually identical (c, 500 nM)).



HEK 293 cells transfected with ACVIII were subjected to both of these regimens, viz. IM-mediated entry or capacitative (TG-mediated) entry (with no prior exposure to IM) at the same extensive range of [Ca], cf.Fig. 8, a and b. Clearly, there is no effect of low [Ca] on cAMP accumulation following IM treatment (Fig. 8d and inset). This lack of effect is consistent with the modest degree of capacitative Ca entry occurring at these concentrations, even though there is an enormous degree of Ca entry occurring through the ionophore pores at these concentrations (Fig. 8c). At higher [Ca], substantial stimulation of cAMP accumulation is observed. However, this stimulation can be completely ascribed to capacitative Ca entry. There is no discernible difference that can be traced to the prior treatment with IM at any [Ca], viz. the effects of the full range of [Ca] on cAMP accumulation in transfected cells are indistinguishable whether or not the cells were exposed to IM in addition to TG. Thus a nonspecific, albeit substantial, elevation of [Ca](i) around the plasma membrane is not adequate to regulate the adenylyl cyclase. Therefore, the efficacy of capacitative Ca entry over release cannot be explained simply by the inability of Ca to diffuse in adequate concentrations from release sites to the plasma membrane. Instead, a more intimate relationship between capacitative entry sites and the adenylyl cyclase is strongly suggested.


DISCUSSION

The present study has explored the ability of three adenylyl cyclase isoforms, which are considered to be Ca-stimulable based on in vitro measurements, to be regulated by physiological transitions in [Ca](i). At the outset, it was instructive to consider both the limitations of some strategies to what might appear to be a straightforward issue and the elements of a physiological elevation in [Ca](i) in nonexcitable cells. This approach revealed a quite unexpected level of sophistication in the relationship between [Ca](i) and Ca-sensitive adenylyl cyclases. A hormone-induced rise in [Ca](i) in nonexcitable cells comprises a release from internal stores coupled with the entry of Ca from outside the cell(10, 23) . Although hormones might therefore seem to be an appropriate physiological tool, the possibility of activating additional signaling mechanisms, along with the liberation of beta subunits of G-proteins, which have type-specific effects on adenylyl cyclases(2) , renders interpretation of hormone effects somewhat problematic. Another option, the use of Ca ionophores, requires caution both experimentally and in interpretation. For instance, in the presence of normal extracellular concentrations of Ca, ionophores can elicit a profusion of effects, including entry of very high [Ca] into the cell through the ionophore pores, release from internal mobilizable and non-mobilizable (e.g. mitochondrial) stores, overlaid on capacitative entry in response to the depletion of the mobilizable stores(26) . Alteration of adenylyl cyclase activity as a result of such a treatment is certainly not interpretable within the context of normal cellular transitions in [Ca](i). When such treatments are protracted, e.g. for 30 min, as are sometimes used(13, 27, 28) , the range of possible outcomes defies interpretation.

Against that background, the present study first examined the effects of ionophore-mediated release of all of the internal Ca stores on transfected ACI, ACIII, and ACVIII. Whereas each of the adenylyl cyclases was expressed, as detected basally or upon stimulation, none could be significantly stimulated by such release. The experimental treatment used to provoke release had certainly been effective, in that a subsequent addition of the muscarinic agonist, carbachol, which mobilizes [Ca](i) in HEK 293 cells(11) , could cause no further elevation in [Ca](i). By contrast, when Ca was allowed to enter the cells in response to the emptying of the mobilizable stores, a striking stimulation of both ACI and ACVIII was achieved. The efficacy of entry compared with release is particularly striking in the light of the higher [Ca](i) that is achieved by release compared with entry (600 versus 270 nM; see Fig. 2). Thus, it could be concluded that ACI and ACVIII were exclusively regulated by capacitative Ca entry and not at all by release.

Notwithstanding the potential ambiguities raised above in ascribing changes in cAMP accumulation to [Ca](i) rises generated by receptor agonists, in the present study, CCh produced some intriguing results. Ca release stimulated by CCh was accompanied by a stimulation of transfected ACVIII that was 14 and 17%, respectively, of the stimulation caused by the capacitative influx elicited by TG and CCh (see Fig. 4). This modest stimulation of cAMP accumulation associated with CCh-stimulated Ca release did seem to depend on Ca, since emptying of the Ca stores by TG eliminated the effect (Fig. 4).

These findings provoke two complementary questions. (i) Why is release so ineffective and (ii) why is entry efficacious? The answer to the first may have to do with the limited diffusion of a sustained [Ca](i) elevation from release sites. It has been convincingly argued that [Ca](i) can only diffuse short distances in the cytosol due to buffering by numerous Ca-binding proteins(24, 25) . Even though higher [Ca](i) was achieved by release than by entry in the present experiments, no spatial information is provided in these measurements of [Ca](i) in populations of cells. Thus, the concentration of Ca reaching adenylyl cyclase at the plasma membrane may be inadequate to regulate the adenylyl cyclases. This interpretation could explain why Ca entering the cells could regulate adenylyl cyclase in the subplasmalemmal domain. Other examples are available; for instance, in chromaffin cells, secretion occurs in response to nicotine, which activates Ca influx, but not to muscarinic agonist-mediated Ca release(29) . Numerous other examples are available in which diffuse rises in [Ca](i) do not mimic the effects of the entry of Ca through voltage-gated channels (e.g.(30) ).

In order to pursue the possibility that an elevation in [Ca](i) around the plasmalemma had a greater likelihood of regulating adenylyl cyclase located in the plasma membrane, conditions were devised that permitted the effects of noncapacitative entry of Ca to be compared with capacitative Ca entry. Remarkably, when the same level of Ca entry was achieved by both mechanisms, only capacitative entry regulated the transfected ACVIII. These data provide compelling evidence that not only is a Ca-stimulable adenylyl cyclase regulated predominantly by entry and not release but also that the adenylyl cyclase is compartmentalized in the same domain as are capacitative entry channnels and that Ca entering the cell by other means does not have free access to these domains.

The present findings should also be viewed along with recent immunohistochemical studies, which demonstrate that, in neurons, adenylyl cyclases are concentrated in dendritic spines(31) . These regions are areas of high concentration of Ca channels and pumps, as well as cyclic AMP-dependent protein kinase anchoring proteins(32, 33) . Dendritic spines are increasingly being viewed as areas of independent neuronal activity and [Ca](i) homeostasis(34, 35) . It appears as though neurons possess the ability to place adenylyl cyclases where they are most likely to be regulated by [Ca](i) rises and to propagate their signals efficiently. This placement suggests that the structure of these adenylyl cyclases encodes the information that targets them to such microdomains. Structure-function studies directed toward such issues might be profitable. Whether subdomains functionally analogous to dendritic spines exist in nonneuronal cells is not clear; such clearly defined morphological entities do not exist in nonexcitable cells, although caveolae have been speculated to provide possible domains where signal transducing elements concentrate(36) .

We had previously shown that the indigenous (Ca-inhibitable) ACVI of C6-2B glioma cells was exclusively regulated by capacitative Ca entry(12) . However, this exclusive dependence on entry could have been a feature conferred by factors peculiar to the organization of ACVI within C6-2B cells. Whether a heterologously expressed adenylyl cyclase would have been placed in the appropriate cellular subdomains to render it similarly sensitive to Ca entry might not have been anticipated. Therefore, the present findings on ACI and ACVIII suggest that it is a property of the adenylyl cyclases to be placed in appropriate subcellular domains. An obvious corollary from these observations is that these Ca-sensitive adenylyl cyclases are poised to play significant roles in cell physiology in response to physiological rises in [Ca](i), in whichever tissues they are expressed.

The present findings that ACIII is quite refractory to physiological [Ca](i) rises, coupled with its marginal stimulability by extremely high [Ca] in vitro, suggest limited usefulness as an integrator of Ca signals. Conceivably, if this species were located very close to ion channels, sufficient Ca might be achieved transiently to yield a small burst in cAMP formation. The elevation in cAMP would be expected to dissipate rapidly, since even around the pores of ion channels the high concentrations of Ca are only estimated to persist for a few milliseconds (37) . Nevertheless, this might be a useful burst for some signaling purposes. An alternative perspective on the insensitivity of ACIII to high Ca is that most cyclases are inhibited by very high [Ca](14) , ACIII is rather unique in its insensitivity. This then could be a useful property that allows cAMP synthesis to persist even in the presence of very high Ca.

In conclusion, the present studies convincingly demonstrate that transfected Ca-stimulable adenylyl cyclases respond predominantly to Ca entry and not to release from stores. Remarkably, they also demonstrate a strict requirement for capacitative Ca entry in the face of much more substantial nonspecific [Ca](i) elevation around the plasma membrane. This indicates a high degree of spatial colocalization of adenylyl cyclases and Ca entry channels. Whether there is a clear structural basis for such aggregations or whether there is an additional passive component, simply reflecting the concentrating of Ca-responsive proteins in domains where [Ca](i) is elevated remains to be determined. For the present, we can only wonder at the efficiency with which desirable cellular interactions are ensured.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 32483. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 East Ninth Ave., Denver, CO 80262. Tel.: 303-270-8964; Fax: 303-270-7097; cooperd{at}essex.hsc.colorado.edu.

(^1)
The abbreviations used are: [Ca], cytosolic Ca; AC, adenylyl cyclase; TG, thapsigargin; IM, ionomycin; [Ca], extracellular Ca concentration; CCh, carbachol; PGE(1), prostaglandin E(1); IBMX, 3-isobutyl-1-methylxanthine.


ACKNOWLEDGEMENTS

We thank Drs. A. Gilman, R. Reed, and J. Krupinski for the cDNAs used in this study and Dr. R. A. Harris for continuing use of his spectrofluorimeter.


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