(Received for publication, September 9, 1994; and in revised form, November 8, 1994)
From the
Elevation of cytosolic free Ca inhibits the
type VI adenylyl cyclase that predominates in C6-2B cells. However, it
is not known whether there is any selective requirement for
Ca
entry or release for inhibition of cAMP
accumulation to occur. In the present study, the effectiveness of
intracellular Ca
release evoked by three independent
methods (thapsigargin, ionomycin, and UTP) was compared with the
capacitative Ca
entry that was triggered by these
treatments. In each situation, only Ca
entry could
inhibit cAMP accumulation (La
ions blocked the
effect); Ca
release, which was substantial in some
cases, was without effect. A moderate inhibition, as was elicited by a
modest degree of Ca
entry, could be rendered
substantial in the absence of phosphodiesterase inhibitors. Such
conditions more closely mimic the physiological situation of normal
cells. These results are particularly significant, in demonstrating not
only that Ca
entry mediates the inhibitory effects of
Ca
on cAMP accumulation, but also that diffuse
elevations in [Ca
]
are
ineffective in modulating cAMP synthesis. This property suggests that,
as with certain Ca
-sensitive ion channels,
Ca
-sensitive adenylyl cyclases may be functionally
colocalized with Ca
entry channels.
A rise in cytosolic free calcium,
[Ca]
, (
)has
been associated with the inhibition of cAMP accumulation in various
tissues and cell
lines(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) .
In a number of these situations, a Ca
-dependent
stimulation of phosphodiesterase (PDE) was the apparent mechanism for
the inhibition of cAMP accumulation(1, 3) . However,
in many of these cases, a Ca
stimulation of PDE has
been specifically precluded and a direct inhibitory effect of elevated
[Ca
]
on adenylyl
cyclase has been demonstrated to be the likely
mechanism(2, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
A number of the sources displaying this behavior also express either
adenylyl cyclase activity that is inhibited by submicromolar
[Ca
] in in vitro assays(2, 6, 7, 8, 9) ,
or mRNAs corresponding to either types V or VI adenylyl
cyclase(13, 14, 15, 16, 17, 18, 19) ;
the latter are recently cloned species that can be inhibited by
Ca
when their cDNAs are transfected into HEK 293
cells(13, 19, 20, 21) . Such
negative influences of [Ca
]
on cAMP synthesis have been speculated to represent a useful
feedback in systems where cAMP controls
[Ca
]
, such as cardiac
myocytes(8, 22, 23) . However, an unresolved
issue, which may cast light on the organization of
Ca
-sensitive adenylyl cyclases within cells, is
whether there is any selective ability of Ca
released
from intracellular pools or Ca
entry to inhibit
adenylyl cyclase.
The C6-2B glioma cell line is a useful model
system for evaluating the efficacy of Ca entry and
internal release on the inhibition of cAMP synthesis. In these
nonexcitable cells, agonist stimulation of
[Ca
]
elevation
involves an initial release of Ca
from inositol
1,4,5-trisphosphate (IP
)-sensitive stores, accompanied by
Ca
entry(7, 11, 24) .
Strikingly, this cell line expresses almost exclusively Type VI
adenylyl cyclase, along with trace amounts of type III(14) .
Previous studies in this cell line have established an inhibition of
cAMP accumulation that is compatible with direct effects of
[Ca
]
on type VI
adenylyl cyclase(7, 11, 14) . The focus of
the present study was to determine whether there was a selective role
for either Ca
entry or release in the inhibition of
cAMP accumulation in the C6-2B cell line. Three independent tools were
utilized to manipulate
[Ca
]
: the purinergic
agonist UTP(25) , the Ca
ionophore ionomycin
(IM)(26, 27, 28) , and the microsomal
Ca
-ATPase inhibitor thapsigargin
(TG)(29, 30) . These three agents provoke
[Ca
]
rises and
capacitative Ca
entry by different
mechanisms(31, 32) . Subsequent experiments with these
agents clearly demonstrated that Ca
entry, rather
than a simple rise in
[Ca
]
, exclusively
inhibits cAMP accumulation in C6-2B cells. The results suggest that, as
with certain Ca
-sensitive ion channels(33) ,
Ca
-inhibitable adenylyl cyclase is functionally
co-localized with sites of Ca
entry.
Resting
[Ca]
in C6-2B cell populations
was approximately 90 nM. TG evoked a large and sustained
elevation in [Ca
]
in
Ca
-containing Krebs buffer (Fig. 1A).
The addition of EGTA rapidly reduced the
[Ca
]
rise to below basal
levels, suggesting that the sustained
[Ca
]
rise was largely due to
Ca
entry. Adding Ca
to the cells
after EGTA caused a dramatic, concentration-dependent Ca
entry (Fig. 1A). These data are consistent with
the premise that the status of intracellular Ca
stores dictates the degree of capacitative Ca
entry, as initially proposed by Putney (31, 32) .
Figure 1:
Effect of TG on
[Ca]
and cAMP
accumulation in C6-2B cells in Krebs-HEPES buffer. A,
[Ca
]
was determined as
described under ``Experimental Procedures.'' TG (100
nM) was added at 60 s. Ca
was chelated with
1.1 mM EGTA at 480 s and 3.0 mM (a), 1.0
mM (b), or 0.5 mM (c) CaCl
was added at 540 s. The results are representative of at least
three experiments with similar results. B, cAMP production
stimulated by isoproterenol (10 µM) and forskolin (10
µM) was measured for 1 min in the presence or absence of
additional 1 mM CaCl
, as indicated, following a
10-min preincubation in the presence of 100 nM TG alone (TG), or 1.1 mM EGTA + TG (EGTG). Control cAMP was determined in the absence
of TG pretreatment and represented 9.4 ± 0.9% ATP converted (n = 11). The results shown represent mean ±
S.E. from at least three separate experiments; *, significantly
different from controls (p < 0.05);**, significantly
different from TG preincubation (p < 0.05);***,
significantly different from EGTA/TG preincubation (p <
0.05).
The consequences of the foregoing
TG-induced [Ca]
rises on cAMP
accumulation are explored in the experiments represented in Fig. 1B. In Ca
-containing Krebs
buffer, TG pretreatment decreased cAMP production by 20% (Fig. 1B).)
The inhibitory effect of TG was
eliminated by EGTA in excess of extracellular Ca
(Fig. 1B). Reintroduction of CaCl
,
following a 10-min incubation with TG and EGTA, inhibited cAMP
production by 36%. These results implicate a significant role for
Ca
entry in the inhibition that is evoked by TG;
however, they do not evaluate the potential of the intracellular
release that is evoked by TG to cause inhibition. That issue is more
effectively addressed in a Ca
-free buffer, as
outlined in the following series of experiments.
The effect of TG on
[Ca]
in a nominally
Ca
-free Krebs buffer is shown in Fig. 2A. TG evokes a transient
[Ca
]
elevation that slowly
declines to basal values, which reflects the emptying and extrusion of
intracellular Ca
stores. Prominent Ca
entry can be demonstrated upon introduction of CaCl
to the extracellular medium (3 mM, trace a; 1
mM, traceb). This Ca
entry is blocked by LaCl
(tracesc and d, Fig. 2A).
Figure 2:
Effect of TG on
[Ca]
and cAMP
accumulation in C6-2B cells in Ca
-free Krebs buffer. A, [Ca
]
was
determined as described under ``Experimental Procedures.'' TG
(100 nM) was added at 60 s and 3.0 mM (a) or
1.0 mM (b) CaCl
was added at 420 s. In tracesc and d, 50 µM LaCl
was added at 360 s, prior to the addition of 3.0
mM or 1.0 mM CaCl
, respectively. The
results are representative of at least three experiments with similar
results. B, production of cAMP was determined for 2 min, in
the presence of isoproterenol (80 µM), either acutely upon
the addition of 100 nM TG (left) or in a parallel
analysis, after preincubation with 100 nM TG for 15 min (right), as indicated. Where CaCl
(3.0
mM) or LaCl
(200 µM) were used, they
were added acutely for the 2-min incubation, as indicated. The control
cAMP levels were 3.38 ± 0.07% ATP converted. The data shown are
means of triplicates and S.E. values from a representative of three
similar experiments; *, significantly different from controls (p < 0.05);**, significantly different from CaCl
(p < 0.05);***, significantly different from
LaCl
(p < 0.05).
In parallel
experiments, the consequences of these
[Ca]
rises on cAMP accumulation
are explored (Fig. 2B). In calcium-free Krebs buffer,
acute incubation with TG causes no effect on cAMP accumulation (Fig. 2B, compare with Fig. 1B).
However, upon the introduction of 3 mM CaCl
to the
extracellular medium, a prominent inhibition of cAMP production is
achieved. This inhibition is blocked by extracellular (200
µM) LaCl
, which suggests that Ca
entry exclusively mediates the inhibition of cAMP synthesis that
is achieved by TG.
The acute consequences on cAMP synthesis of the
various changes in [Ca]
evoked
by TG are explored in a time-course experiment, where all of the
elements are added simultaneously to cells in a nominally
Ca
-free Krebs buffer (Fig. 3). The time
courses were designed to measure the effects of these compounds prior
to the initial [Ca
]
rise,
during the Ca
spike and during the sustained second
phase of the [Ca
]
rise. TG,
without extracellular Ca
, did not affect cAMP
accumulation at any time point examined (Fig. 3), even at early
times e.g. 1 min, where (from Fig. 2A) a
considerable elevation in [Ca
]
is achieved. However, a slow onset inhibition is apparent when
CaCl
is added along with TG (Fig. 3). Presumably
this reflects the TG-promoted emptying of pools and the entry that is
associated with this process. These data strongly suggest that only the
Ca
entry promoted by TG, and none of the
Ca
release, can inhibit cAMP synthesis.
Figure 3:
Time course of the effect of TG alone and
in the presence of Ca on cAMP accumulation stimulated
by isoproterenol (10 µM) and forskolin (10
µM) in C6-2B cells in nominally Ca
-free
Krebs buffer. TG (100 nM) and CaCl
(1 mM)
were added simultaneously where indicated and incubated for the
specified time period. The control cAMP accumulation was determined in
the absence of TG and Ca
. The results represent the
mean ± S.E. from at least five experiments done in triplicate;
*, significantly different from TG alone.
Figure 4:
Effect of ionomycin on
[Ca]
and cAMP
accumulation in C6-2B cells in Krebs-HEPES buffer. A,
[Ca
]
was determined as
described under ``Experimental Procedures.'' Ionomycin (400
nM) was added at 60 s. Ca
was chelated with
1.1 mM EGTA at 480 s and 3.0 mM (a), 1.0
mM (b), or 0.5 mM (c) CaCl
was added at 540 s. The data are representative of at least three
experiments with similar results. B, cAMP production was
measured in the presence of isoproterenol (10 µM) and
forskolin (10 µM), along with 400 nM ionomycin
alone (IM), 1.1 mM EGTA + IM (EGIM) or EGTA + IM + 1.0 mM CaCl
(EGIMCA). Where
indicated, EGTA (EG) was present in the preincubation medium, i.e. for 10 min prior to initiation of cAMP production. The
control cAMP was determined in the absence of IM and represented 9.4
± 0.9% ATP converted (n = 11). The results shown
represent mean ± S.E. from at least three separate experiments;
*, significantly different from controls (p < 0.05);**,
significantly different from IM (p < 0.05);***,
significantly different from EGTA + IM (p <
0.05).
In Ca-containing Krebs buffer, a
3-min incubation with IM alone led to a modest but significant
(
12%) inhibition of cAMP production (Fig. 4B). No
such inhibition was observed when EGTA was included with the IM (Fig. 4B). A subsequent addition of excess CaCl
(1 mM) in the presence of IM and EGTA caused a 25%
inhibition of cAMP accumulation. These data again suggest an important
role for Ca
entry in cAMP inhibition, but do not
exclude possible contributions from intracellular release.
In
nominally calcium-free Krebs buffer, IM produced only half the
[Ca]
rise compared to that
observed in normal Krebs buffer (cf. Fig. 5A and 4A). The elevation in
[Ca
]
returned rather rapidly to
base-line values. These responses reflect the preclusion of
Ca
entry in nominally Ca
-free Krebs
buffer. The introduction of extracellular CaCl
yielded
concentration-dependent Ca
entry (Fig. 5A, tracesa and b),
which was effectively blocked by LaCl
(Fig. 5A, tracesc and d).
Figure 5:
Effect of ionomycin on
[Ca]
and cAMP
accumulation in C6-2B cells in Ca
-free Krebs buffer. A, [Ca
]
was
determined as described under ``Experimental Procedures.''
Ionomycin (400 nM) was added at 60 s and 3.0 mM (a) or 1.0 mM (b) CaCl
was
added at 360 s. In tracesc and d, 50
µM LaCl
was added at 300 s, prior to the
addition of 3.0 mM or 1.0 mM CaCl
,
respectively. The results are representative of at least three
experiments with similar results. B, production of cAMP was
determined in a 2-min assay in the presence of isoproterenol (80
µM), either acutely upon the addition of 400 nM IM (left) or in a parallel analysis, after preincubation
with 400 nM IM for 10 min (right), as indicated.
Where CaCl
(3.0 mM) or LaCl
(200
µM) were used, they were added acutely for the 2-min
incubation, as indicated. The control cAMP levels were 3.12 ±
0.02% ATP converted. The data shown are means and S.E. values of
triplicate determinations from a representative of three similar
experiments; *, significantly different from controls (p <
0.05);**, significantly different from Ca (p <
0.05).
Acute incubation with IM in Ca-free
Krebs buffer did not affect cAMP production (Fig. 5B).
However, the addition of CaCl
following incubation with IM
resulted in a 27% decrease in cAMP accumulation. This decrease was
blocked by LaCl
. These results confirm that Ca
entry mediates all of the IM-induced inhibition of cAMP
accumulation.
In a time-course study, analogous to that performed
with TG, IM elicited no inhibition of cAMP synthesis at any time, in
the nominal absence of extracellular Ca (Fig. 6), despite the rather substantial
[Ca
]
rise that is evoked,
particularly in the first minute of its action (cf. Fig. 5A). However, the presence of extracellular
Ca
permits substantial and significant inhibition of
cAMP accumulation from 30 s onward (Fig. 6). These results are
important in demonstrating, not only that Ca
entry
mediates the inhibitory effects of IM on cAMP accumulation, but also
that the diffuse elevation in [Ca
]
caused by IM is ineffective in modulating cAMP synthesis.
Figure 6:
Time
course of the effect of ionomycin alone and in the presence of
Ca on cAMP accumulation in C6-2B cells in nominally
Ca
-free Krebs buffer. IM (400 nM) and
CaCl
(1 mM) were added simultaneously where
indicated and incubated for the specified time period. cAMP
accumulation stimulated by isoproterenol (10 µM) and
forskolin (10 µM) was measured using the method described
under ``Experimental Procedures.'' The control cAMP
accumulation was determined in the absence of IM and
Ca
. The results represent the mean ± S.E. of
triplicates from at least five experiments; *, significantly different
from IM alone (p < 0.05).
Figure 7:
Effect of UTP on
[Ca]
and cAMP
accumulation in C6-2B cells in Ca
-containing
Krebs-HEPES buffer. A,
[Ca
]
was determined as
described under ``Experimental Procedures.'' UTP (100
µM) was added at 60 s. Ca
was chelated
with 1.1 mM EGTA at 240 s and 3.0 mM (a),
1.0 mM (b), or 0.5 mM (c)
CaCl
was added at 300 s. The results are representative of
at least three experiments with similar results. B, cAMP
production, stimulated by isoproterenol (10 µM) and
forskolin (10 µM), was measured for 1 min as described
under ``Experimental Procedures,'' in the presence of 100
µM UTP (UTP), 1.1 mM EGTA + UTP (EGU), or EGTA + UTP + 1.0 mM CaCl
(EGUCA). Where
indicated, EGTA (EG) was present in the preincubation medium
for 10 min prior to the measurement of cAMP production. The control
cAMP was determined in the absence of UTP and represented 9.4 ±
0.9% ATP converted (n = 11). The results shown
represent mean ± S.E. from at least four separate experiments;
*, significantly different from controls (p < 0.05);**,
significantly different from EGTA + UTP (p <
0.05).
The effects of UTP on cAMP
accumulation under analogous conditions to those used to manipulate
[Ca]
are presented in Fig. 7B. UTP, which causes release of intracellular
Ca
, along with Ca
entry (see Fig. 7A) resulted in a significant (
16%)
inhibition of cAMP synthesis. This inhibition was abolished in the
presence of EGTA. Readdition of excess CaCl
with UTP in the
presence of EGTA restored the inhibition of cAMP production. Since EGTA
eliminates the Ca
influx contribution to the
[Ca
]
rise elicited by UTP (Fig. 7A), these observations support a role of
Ca
entry in inhibition of cAMP accumulation caused by
UTP.
In nominally Ca-free medium, stimulation with
UTP resulted in an initial spike due to Ca
released
from intracellular stores, without a Ca
entry phase (Fig. 8A). The initial spike due to UTP was smaller
than in Fig. 7A. Capacitative Ca
entry is evident upon the subsequent introduction of CaCl
(Fig. 2A, tracesa and b). This entry could be eliminated by LaCl
(tracesc and d).
Figure 8:
Effect of UTP on
[Ca]
and cAMP
accumulation in C6-2B cells in Ca
-free Krebs buffer. A, [Ca
]
was
determined as described under ``Experimental Procedures.''
UTP (100 µM) was added at 60 s and 3.0 mM (a) or 1.0 mM (b) CaCl
was
added at 300 s. In traces c and d, 50 µM LaCl
was added at 240 s, prior to the addition of 3.0
mM or 1.0 mM CaCl
, respectively. The
results are representative of at least three experiments with similar
results. B, production of cAMP stimulated by isoproterenol (80
µM) was determined for 90 s, either acutely upon the
addition of 100 µM UTP (left) or in a parallel
analysis, after preincubation with 100 µM UTP for 4 min (right), as indicated. Where CaCl
(3.0
mM) or LaCl
(200 µM) were used, they
were added acutely for the 90-s incubation, as indicated. The control
cAMP levels were 2.23 ± 0.12% ATP converted. The data shown are
means and S.E. values from a representative of three similar
experiments; *, significantly different from controls (p <
0.05);**, significantly different from Ca (p <
0.05).
The effects of
UTP and UTP-induced Ca entry on cAMP accumulation in
a nominally Ca
-free Krebs buffer are explored in Fig. 8B. UTP in the absence of extracellular
Ca
did not affect cAMP production. However, the
addition of extracellular Ca
(3 mM CaCl
) subsequent to a UTP treatment decreased cAMP
accumulation by 25%; this inhibition was fully blocked by
LaCl
. The essential contribution of extracellular
Ca
to the inhibition of cAMP production elicited by
UTP, clearly demonstrates the required role of Ca
entry in regulating adenylyl cyclase in C6-2B cells. (
)
the concentration of cAMP relates to these synthetic and
degradation rates and the K of PDE, as follows:
where K is the affinity of the PDE for
cAMP; ATP
cAMP can be considered to be a first order reaction,
since the substrate, ATP, is in great excess(43) . Consider the
outcome for steady state [cAMP] following a 33% inhibition of
adenylyl cyclase (V
), where [cAMP] is
calculated based on the previous formula: 1) at initial state, V
= 9, V
= 10,
[cAMP] = 9 K
units (arbitrary
units, adapted from (43) ); 2) at 33% inhibition of adenylyl
cyclase, V
= 6; V
= 10; [cAMP] = 1.5 K
units.
This modest inhibition of cAMP synthesis leads to 83%
inhibition in cAMP accumulation. Thus, theoretically, a modest
inhibition of cAMP synthesis, even against an unchanging background of
PDE, can lead to a drastic loss in cAMP accumulation; if this is
combined with a stimulation of PDE, loss in cAMP can be profound. The
following experiment explores the impact of PDE activity on the
inhibition of cAMP levels caused by a rise in
[Ca]
.
C6-2B cells were
pretreated with either TG or UTP in the absence of extracellular
Ca to deplete stores and prime the cells for a
substantial or modest inhibition of cAMP accumulation (based on the
results presented in Fig. 2and Fig. 8) in the absence or
presence of PDE inhibitors (Table 1). After 10 min, cAMP
accumulation, stimulated by isoproterenol, was measured in the absence
or presence added extracellular CaCl
to provoke
Ca
entry as in Fig. 2. The absence of PDE
inhibitors resulted overall in approximately 2-fold less cAMP
accumulation (Table 1). However, the degree of inhibition was
increased from 40 and 16% to almost 80 and 33%, respectively, in
response to the substantial or modest degree of entry evoked by TG and
UTP (Table 1). These data emphasize the significance of a
background PDE activity in a normal physiological setting, i.e. a partial inhibition of adenylyl cyclase can result in almost
total inhibition of cAMP accumulation.
In the present study, the efficacy of Ca entry versus release from intracellular stores at
inhibiting cAMP accumulation was compared in C6-2B cells. Previous
studies in the C6-2B glioma cell line had already established that an
increase in [Ca
]
was
accompanied by inhibition of cAMP
accumulation(7, 11) . This inhibition was independent
of PDE, protein kinase C, and Bordetella pertussis toxin (PTX)
effects. However, it was not possible from the experiments performed to
evaluate the efficacy of Ca
entry versus release, at inhibiting cAMP synthesis. (
)A selective
requirement for Ca
emanating from a particular
source, would suggest a functional co-localization of the adenylyl
cyclase with the source of the [Ca
]
rise. We have adopted strategies that elevate
[Ca
]
independent of receptor
activation and second messenger pathways, since certain adenylyl
cyclase species are subject to modulation by protein kinase C and
subunits of G-proteins, whose activation can arise from
receptor occupancy(38, 39) . Each of the strategies
applied provides convincing evidence that only Ca
entry modulates cAMP synthesis. Thus, in the presence of
extracellular Ca
, TG, IM, and UTP inhibited cAMP
synthesis to varying extents, but in the absence of extracellular
Ca
, the intracellular release induced by each of
these treatments was incapable of eliciting inhibition. The
capacitative Ca
entry that was provoked by these
various means of depleting Ca
stores could inhibit
cAMP synthesis and this effect was fully blocked by extracellular
LaCl
. The importance of entry versus release is
further emphasized when the ineffective, but substantial, rise in
[Ca
]
due to release that was
elicited by TG, IM, or UTP (peak increments of 240, 400, and 90
nM, respectively; see Fig. 2, Fig. 5, and Fig. 8) is compared with the inhibitory action of the modest
degree of entry (100 nM over basal; Fig. 8) evoked by
UTP. In other words, even though the elevation in
[Ca
]
arising from intracellular
release in response to TG, IM, or UTP is substantially greater than, or
equal to, the elevation in [Ca
]
arising from entry in response to UTP, only the
[Ca
]
rise due to entry caused
inhibition of cAMP synthesis. The time-course studies reinforce the
fact that it is the onset of Ca
entry, rather than
the net elevation in [Ca
]
, that
correlates with inhibition of cAMP synthesis.
The increasing
instances in which [Ca]
elevation is associated with inhibition of cAMP synthesis,
apparently as a result of a direct inhibition by Ca
of a type V or VI adenylyl
cyclase(2, 4, 5, 6, 7, 8, 9, 10, 11, 12) hints at
potentially important physiological roles for
Ca
-inhibitable adenylyl cyclases. For instance, in
the case of cardiac contractility, it has been proposed that feedback
inhibition by Ca
on the cAMP signal may provide a
delicate mechanism for fine-tuning cAMP control of
[Ca
]
elevation(8, 22, 23) . However, the
magnitude of the inhibition often observed in studies such as those
described presently may cast doubt on whether such effects can be
sensed physiologically. A number of technical issues are relevant in
this context. (i) for ease of measurement of cAMP levels, PDE is
normally pharmacologically inhibited, unlike the situation in normal
cells; consequently, a modest inhibition of cAMP synthesis, coupled
with (possibly, a Ca
-stimulated) PDE action, could
result in dramatic inhibition of cAMP
accumulation(1, 3) . Indeed, in the present studies,
when PDE was not inhibited, a modest inhibition of adenylyl cyclase was
converted into a dramatic loss in steady state cAMP levels. (ii) cAMP,
rather than the activity state of its major cellular target,
cAMP-dependent protein kinase, is measured in most studies; the kinase,
or its targets, may be very sensitive to small changes in ambient cAMP
concentrations, as is the case in adipocytes, where a 25% change in
cAMP-dependent protein kinase activity is accompanied by a 90%
reduction in lipolysis(45) . (iii) cAMP is measured on a very
gross time scale, relative to some of the events that it regulates,
such as the opening of ion channels(46) . Measurements of cAMP
formation with high temporal resolution would significantly clarify the
kinetic features of the regulation of its synthesis. Thus, our present
limited ability to measure these phenomena should not imply that
cellular elements cannot respond to and exploit such regulatory events.
In conclusion, the present results convincingly demonstrate a
selective requirement for Ca entry, and not release
or a diffuse elevation in [Ca
]
,
to inhibit adenylyl cyclase in C6-2B cells. The fact that inhibition is
observed even when cytosolic Ca
concentrations
arising from release are greater than the concentrations deriving from
entry, implies that the entry sites and adenylyl cyclase must be
relatively close inside the cell, i.e. they must be
sufficiently close that, regardless of the diffusion of Ca
away from entry channels, Ca
concentrations
must remain high enough to inhibit adenylyl cyclase. This situation is
directly analogous to the requirement for localized Ca
entry for neurotransmitter and exocytotic secretion (47, 48) in which diffuse
[Ca
]
rises fail to mimic the
actions of Ca
entry through discrete channels. An
extension of this speculation is that the density of Ca
entry channels relative to adenylyl cyclases will dictate whether
adenylyl cyclases can respond to Ca
. It will be
interesting to determine whether any cell type can release sufficient
Ca
from intracellular stores so that efficacious
concentrations reach the plasma membrane, or whether it is always
necessary that Ca
-sensitive adenylyl cyclases and
Ca
entry channels are functionally colocalized for
cAMP synthesis to be regulated by
[Ca
]
. Given the limited range
of Ca
diffusion through cells because of mobile and
immobile buffers(49) , it is possible that unless release sites
can be very closely apposed to adenylyl cyclase, released
Ca
will never reach adenylyl cyclase in sufficient
concentration. For the present, these studies establish that
Ca
-sensitive adenylyl cyclase in C6-2B cells is
regulated only by the entry of Ca
. Whether this
functional colocalization is coincidental or directed will be an
interesting avenue for future research.