From the Department of Pharmacology and Neuroscience Program,
University of Colorado Health Sciences Center, Denver, Colorado 80262 and
URA-CNRS 339, University of Bordeaux I, Talence
F-33405, France
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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
Materials--
Thapsigargin, ionomycin, forskolin, and Ro
20-1724 were from Calbiochem. [2-3H]Adenine,
[3H]cAMP and [
-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 [
-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.
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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.
We thank Dr. R. A. Harris for the
continuing use of his spectrofluorimeter.