(Received for publication, November 16, 1995; and in revised form, February 21, 1996)
From the
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.
The regulation of adenylyl cyclase by cytosolic Ca ([Ca
]
) (
)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
-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.
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
,
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
10
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
]
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
]
(up to 600
nM). Subsequent addition of carbachol caused no additional
release in [Ca
]
(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
]
,
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
]
(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
]
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
]
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
]
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
]
rises on ACI, ACIII, and
ACVIII, which were concurrently stimulated by forskolin or
PGE
, were evaluated (Fig. 6). PGE
stimulates adenylyl cyclase via the G-protein subunit
in HEK 293 cells. [Ca
]
rises were generated using capacitative Ca
influx as described earlier, with
[Ca
]
of 3 mM.
Elevation of [Ca
]
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
stimulation (Fig. 6). It is noteworthy that the
Ca
-sensitive adenylyl cyclase most prominently
stimulated by PGE
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
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
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
]
. The
insensitivity of ACIII to elevation in
[Ca
]
is quite compatible with
its barely detectable response to supra-normal Ca
in 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
]
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
]
, 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
]
reflecting only entry in
response to IM would ensue.) Subsequently, upon the reintroduction of
graded concentrations of extracellular Ca
,
[Ca
]
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
]
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
]
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
]
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
10
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
]
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.
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
]
. 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
]
in nonexcitable cells. This approach revealed a quite unexpected
level of sophistication in the relationship between
[Ca
]
and
Ca
-sensitive adenylyl cyclases. A hormone-induced
rise in [Ca
]
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
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
]
. 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
]
in HEK 293
cells(11) , could cause no further elevation in
[Ca
]
. 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
]
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]
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]
elevation from
release sites. It has been convincingly argued that
[Ca
]
can only diffuse short
distances in the cytosol due to buffering by numerous
Ca
-binding proteins(24, 25) . Even
though higher [Ca
]
was achieved
by release than by entry in the present experiments, no spatial
information is provided in these measurements of
[Ca
]
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
]
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]
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
]
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
]
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
]
, in whichever tissues
they are expressed.
The present findings that ACIII is quite
refractory to physiological [Ca]
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
]
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
]
is elevated remains to be determined. For the present, we can
only wonder at the efficiency with which desirable cellular
interactions are ensured.