(Received for publication, December 26, 1995; and in revised form, February 19, 1996)
From the
Streptolysin O-permeable pancreatic acini were used to study the
regulation of the inositol 1,4,5-trisphosphate
(IP)-activated Ca
channel (IPACC) by
agonists and antagonists. Measurements of the apparent affinity for
IP
(K
IP
) showed that the
IPACC is dynamically controlled during cell stimulation and inhibition, i.e. agonists decreased and antagonists increased K
IP
. K
IP
was also independently regulated
by thimerosal, Ca
content of the stores, the
incubation temperature, activation of protein kinases, and inhibition
of protein phosphatases, but none of these mechanisms contributed to
the regulation by agonists and antagonists. Incubating the cells with
low concentration of GTP
S or AlF
reproduced the effect
of the agonist on K
IP
. Moreover, low
[GTP
S] allowed activation of the IPACC by agonists at
basal levels of IP
and markedly impaired channel
inactivation by antagonists. Channel sensitization by GTP
S also
restored the ability of thimerosal to mobilize Ca
from internal stores with no change in cellular IP
levels. The combination of low [GTP
S] and
thimerosal locked the channel in an open, antagonist-insensitive state.
All modulatory effects of GTP
S are independent of phospholipase C
activation and IP
production. We propose that the dynamic
regulation of the IPACC by a G protein-dependent mechanism can play a
major role in triggering and maintaining Ca
oscillations at low agonist concentrations when minimal or no changes
in IP
level take place.
Ca mobilizing agonists stimulate the
production of inositol 1,4,5-triphosphate (IP
), (
)which releases Ca
from intracellular
stores located in the endoplasmic reticulum (ER)(1) .
Stimulation with high agonist concentration leads to a persistent
activation of the IP
-dependent Ca
channel, which results in a single transient change in free
cytosolic Ca
concentration
([Ca
]
)(2) . On
the other hand, at low agonist concentrations usually oscillations in
[Ca
]
are
observed(1, 2) . Only partial correlation exists
between the type of the [Ca
]
signal generated and IP
production. In most
cases, high agonist concentration increases IP
to a
supermaximal concentration, while at low agonist concentration it is
difficult to demonstrate stimulated production of
IP
(1, 2) . In addition, several agonists
such as parathyroid hormone acting on osteoblasts (3, 4) or bradykinin acting on 3T6 fibroblasts (5) cause substantial Ca
release from
intracellular stores with no apparent increase in IP
.
However, basal levels of IP
, although varying widely among
different cell types, are almost always higher than that needed for
maximal Ca
release(1, 6, 7, 8, 9) .
A plausible explanation for Ca release in the
absence of IP
production and in the face of high basal
levels of IP
is that low [agonists] trigger
Ca
release by regulating the affinity of the
IP
-activated Ca
channel (IPACC) for
IP
. Studies in several cellular systems and the isolated
and reconstituted IPACC showed the presence of multiple mechanism of
channel regulation. Besides activation by IP
, the channel
can be regulated by Ca
in a biphasic
manner(10, 11, 12, 13) . ATP can
regulate the channel directly (10, 11, 14) or
by an indirect mechanism(13, 15) , which may involve
phosphorylation/dephosphorylation reactions(13) . Recent
elegant work by Cameron et al.(16) showed the
intimate association of the cerebellar IPACC with calcineurin. The
channel's affinity to IP
was regulated by the
combined action of protein kinase C and calcineurin(16) . The
same system was used to show that protein kinase A reduces the apparent
affinity of the IPACC to IP
(14, 16) . In
hepatocytes, protein kinase A increased the affinity for IP
in inducing Ca
release(17, 18) , which was further augmented by
inhibition of protein phosphatases with okadaic acid(18) . The
affinity of the IPACC for IP
was also shown to be regulated
by thiol-reactive agents such as thimerosal (TMS)(19, 20, 21, 22, 23, 24) and by
temperature(25) . The relationship between all the different
modes of regulating IP
affinity and Ca
release are not known.
The above regulatory modes offer many
mechanisms for the agonists to effect Ca release and
initiate Ca
oscillations without the need to increase
IP
levels. Indeed, in rat liver, prestimulation with
agonists was shown to modify the behavior and/or affinity for
IP
(26) . More recently we showed that agonists can
activate and antagonists inactivate the IPACC independent of IP
metabolism(27, 28) . How such a regulation is
achieved, what mechanism the agonists use to modulate Ca
release and the antagonists to terminate the release is not
known.
In the present studies we used agonist/antagonist responsive,
streptolysin O (SLO)-permeabilized pancreatic acini to demonstrate that
in the same cells the IPACC can be regulated by multiple and
independent mechanisms. More importantly, a G protein-dependent
mechanism is used by agonists to activate the channel at basal levels
of IP. Such activation dramatically impairs the ability of
the antagonist in inactivating the IPACC. Once activated by the G
protein-dependent mechanism, the channel can be locked in the open
state by TMS. These findings point to the possible mechanism by which
agonists can initiate Ca
release and Ca
oscillations with minimal changes in IP
concentrations.
Figure 1:
Effect of agonist stimulation and
antagonist inhibition on KIP
.
Pancreatic acini were allowed to reduce
[Ca
] of the permeabilization medium to
about 60 nM (a) before stimulation with 10 µM (b) or 2 mM (c) carbachol. In
experiment c, where indicated, the cells were inhibited with
0.1 mM atropine. After stabilization of medium
[Ca
] in a-c, the K
IP
was measured by incremental
additions of [IP
] between 0.05 and 2.55
µM. The numbers shown in trace a also
apply for experiments b and c. The bottom panel shows the relationship between [IP
] and the
increase in medium [Ca
] above that measured
before the additions of IP
. The results of this and all
other similar experiments are summarized in Table 1.
To study the
possible mechanism by which the agonist and antagonist regulate the KIP
, we characterized the effect of
different compounds reported to affect IP
-mediated
Ca
release and the relationship between them.
Examples of individual experiments are shown in Fig. 2and Fig. 3, and the combined results are summarized in Table 1. Treating the cells with relatively low concentrations of
GTP
S, which by itself caused no or minimal Ca
release, decreased K
IP
in a
concentration- and time-dependent manner (Fig. 2b, Table 1). Incubation with 10 µM GTP
S for 30 s
increased the affinity for IP
by about 2.4-fold. Thimerosal
(TMS) was shown to induce [Ca
]
oscillations in pancreatic acini (21) and increase the
affinity for IP
in several cell
types(19, 20, 21, 22, 23, 24) . Fig. 2c shows that unlike the case in intact cells, TMS
(up to 500 µM) alone was unable to cause Ca
release in permeable cells. However, as little as 100 µM TMS decreased K
IP
by about
4-fold (Table 1). Fig. 2c and Table 1show
the results with 100 µM TMS since this concentration was
sufficient to maximally modify the effects of the agonist and GTP
S
on IPACC (see below).
Figure 2:
Effect of GTPS, TMS,
Ca
load, and temperature on K
IP
. After completion of
Ca
uptake and stabilization of medium
[Ca
] at about 55 nM (a),
the cells were treated with 5 µM GTP
S (b) or
100 µM TMS (c). In experiment d, where
indicated, two additions of 5 µM Ca
to
the incubation medium were made. In experiment e, the acini
were first incubated for 2.5 min at 37 °C, then transferred to a
thermostated cuvette and incubated for 3 min at 0 °C before the
first addition of IP
. The trace shows the last
part of the incubation at 0 °C under control conditions. After each
treatment, the K
IP
was measured by
incremental additions of IP
. In experiments a-d, additions of IP
were as those
shown in trace a. The numbers in trace e indicate the
concentrations of IP
used for this titration. The results
of all similar experiments are summarized in Table 1.
Figure 3:
Additivity in the regulation of KIP
. Experimental protocols
were similar to those described in the legend to Fig. 2. In
experiments a and b, the stores were overloaded by
two additions of 5 µM CaCl
. For clarity, the
loading traces are shown only for experiment a. After
stabilization of [Ca
], the cells were
treated with 5 µM GTP
S (a) or 100 µM TMS (b) before additions of IP
. The numbers in trace a indicate the concentrations of IP
used for experiments a and b. In experiments c and d, the acini were incubated for 2 or 1.5 min at
37 °C before addition of 5 µM GTP
S (c,
GTP
S-treated) or 100 µM TMS (d,
TMS-treated). After an additional 0.5- or 1-min incubation at 37
°C, respectively, the cells were cooled and K
IP
was measured by the
addition of the IP
concentrations indicated by the numbers in trace c. The results of all similar experiments are
summarized in Table 1.
The effect of Ca content in
the IP
-mobilizable Ca
pool on IPACC has
been studied extensively in several cell
types(31, 32, 33, 34, 35, 36, 37, 38) . Fig. 2d shows that overloading the
IP
-sensitive pool with Ca
caused small,
but significant increase in the affinity to IP
(see also Table 1). Similar K
IP
was
measured after one (not shown), two (Fig. 2d), and four
pulses of 5 µM Ca
(not shown). Finally,
the temperature of the incubation medium had a profound effect on K
IP
but without changing the quantal
nature of Ca
release. Thus, Fig. 2e shows that after 2.5 min of cell permeabilization and
Ca
loading at 37 °C and a subsequent 3-min
incubation at 0 °C, increasing concentrations of IP
induce discrete and finite Ca
release events.
The release was much slower than that at 37 °C, which allowed a
clear resolution of each Ca
release event. In
separate experiments, Ca
release at submaximal
[IP
] was followed up to 15 min with no sign of
deviation from quantal behavior (not shown). Table 1shows that
at 0 °C K
IP
was reduced by about
8-fold.
An important question concerning the various modes of
regulation of KIP
was the
relationship between them to determine whether they regulate the IPACC
by the same or by independent mechanisms. Fig. 3and Table 1show the additive and independent effect of the different
modes. Thus, overloading the Ca
pool did not prevent
or reduce the effect of GTP
S (Fig. 3a), TMS (Fig. 3b), or low concentration of carbachol (Table 1) on the K
IP
.
Similarly, the effect of low temperature on K
IP
was additive with the effect of
GTP
S (Fig. 3c), TMS (Fig. 3d), and
carbachol (Table 1). Interestingly, addition of all agents after
cooling to 0 °C had no effect on K
IP
. The cells had to be treated
with carbachol, GTP
S, or TMS at 37 °C before cooling to
observe their effect on K
IP
,
indicating activation of biochemical pathways by all agents, including
TMS, to regulate K
IP
.
Several
previous studies reported regulation of KIP
by activation of protein
kinases(13, 14, 16, 17, 18) .
To evaluate the role of protein kinases in the SLO-permeabilized
system, we tested the effect of several protein kinase activators
(12-O-tetradecanoylphorbol 13-acetate, cAMP, cGMP), protein
kinase inhibitors (genistein, H7), and phosphatase inhibitors (KT62,
okadaic acid, cyclosporine) on K
IP
.
None of these agents affected K
IP
in
unstimulated SLO-permeabilized cells. Further, these agents did not
prevent or augment the effect of TMS, Ca
load, or low
temperature (not shown). Surprisingly, all kinase activators and
phosphatase inhibitors tested reduced or prevented the effect of
GTP
S and carbachol, whereas the kinase inhibitors augmented the
effect of the agonists. Table 1lists the effect of the most
effective compounds, okadaic acid and H7. Treatment with 0.12
µM okadaic acid largely prevented the effect of 30 s of
treatment with 10 µM GTP
S. H7 at 0.5 mM, a
concentration sufficient to inhibit most protein kinases(38) ,
had no effect in unstimulated cells but it increased the effect of
submaximal [GTP
S]. The lack of selectivity in the effect
of kinase and phosphatase activators/inhibitors raises questions as to
the physiological significance of these findings. However, their value
for the present studies is in showing the similarity between their
effects on GTP
S and carbachol, which were different from those of
TMS, Ca
load, and low temperature.
Figure 4:
Effect of carbachol and GTPS on K
IP
is independent of PLC.
Acini incubated at 37 °C were exposed to 10 µM U73122 (a-c) or 25 µg/ml heparin (d and e) before stimulation with 10 µM carbachol (b) or 5 µM GTP
S (c and e). a and d are the respective controls.
Next, K
IP
was measured by
incremental additions of the indicated [IP
]. Note
the high [IP
] used in experiments d and e.
The third and
most convincing evidence against stimulation of PLC by carbachol or
GTPS as the cause of the change in K
IP
is shown in Fig. 4, d and e. In these experiments, 25 µg/ml IP
competitive inhibitor heparin (39, 40) were
added to the incubation medium to increase the K
IP
under control conditions from
0.43 µM (Table 1) to 1.67 µM (Fig. 4d). This should dilute the effect of any
IP
generated by GTP
S or carbachol stimulation by about
4-fold (1.67/0.43) to virtually eliminate the effect of the agents on K
IP
. Fig. 4e shows
that this is not the case. Pretreatment with GTP
S in the presence
of 25 µg/ml heparin had the same effect on K
IP
as in control cells. Similar
results were obtained with heparin concentrations between 5 and 50
µg/ml and with cells stimulated with 10 µM carbachol
(not shown). Hence, together the three protocols indicate that
carbachol and GTP
S modified K
IP
independent of PLC stimulations.
More dramatic evidence for an
effect of G protein(s) activation on the activity of the IPACC was
obtained when the effect of a low concentration of GTPS on the
response to carbachol, atropine, and TMS was studied. Fig. 5a shows that exposure of control or stimulated
cells to 100 µM TMS in the absence of GTP
S augmented
the effect of low concentrations of carbachol on Ca
release (Fig. 5, b and c), without
affecting IP
levels during the first 20 s of cell
stimulation (Table 2). Performing complete concentration
dependence of both IP
production and Ca
release showed that pretreatment with 100 µM TMS had
no effect on IP
production while increasing the apparent
affinity for carbachol-mediated Ca
release by about
6.3-fold. On the other hand, GTP
S profoundly modified the effect
of carbachol. Treating the cells with as little as 2 µM GTP
S for 30 s was sufficient to cause maximal Ca
release by 10 µM carbachol (Fig. 5d).
Figure 5:
Effect of TMS and GTPS on
carbachol-triggered Ca
release. Control acini (a and b) and acini treated with 100 µM TMS (c) or 2 µM GTP
S (d) were
stimulated with 2 mM (a) or 10 µM (c and d) carbachol. The cells in b were exposed to
TMS after stimulation with 10 µM carbachol.
That the effect of GTPS was
independent of PLC activation became even more evident when the effect
of 2 µM GTP
S on the dose response to carbachol and
atropine was measured. Fig. 6shows that under control
conditions the concentration dependence curves for carbachol
stimulation of IP
production and Ca
release were identical. Half-maximal stimulation
(EC
) of Ca
release was at 210 ±
13 µM (n = 8), and the EC
for
IP
production was 236 ± 27 µM (n = 4). In the presence of 2 µM GTP
S, the
EC
for IP
production was reduced by about
2.9-fold to 82 ± 11 µM (n = 4),
whereas the EC
for Ca
release was
reduced by 90-fold to 2.35 ± 0.13 µM (n = 8).
Figure 6:
Effect of GTPS on carbachol-induced
IP
production and Ca
release. For
measurement of Ca
release (
,
), the
protocols shown in Fig. 5, a and d, were used,
except that the cells were stimulated with different
[carbachol] and in the presence (
) or absence (
)
of 2 µM GTP
S. Resting
[Ca
] was subtracted from the peak increase
in [Ca
] at each carbachol concentration.
For measurement of IP
levels (
,
), cells were
incubated for 2.5 min at 37 °C in permeabilization medium and then
stimulated for 20 s with the indicated concentration of carbachol and
in the presence (
) or absence (
) of 2 µM GTP
S. After 20 s of stimulation, the reactions were stopped
with perchloric acid, and the levels of IP
were evaluated
as described under ``Experimental
Procedures.''
The effect of low [GTPS] on signal
termination by atropine is shown in Fig. 7. In the absence of
GTP
S, atropine inhibited the IPACC of cells stimulated with 2
mM carbachol (measured from the rate of
[Ca
] reduction in carbachol-stimulated
cells, see Fig. 1and (27) ) with an IC
of
0.33 ± 0.02 µM (n = 5). Under the
same conditions, atropine accelerated IP
hydrolysis
(measured from the rate of IP
reduction relative to
continuously stimulated cells as in Fig. 11below and (27) ) with an IC
of 0.21 ± 0.03 µM (n = 3). Including 2 µM GTP
S in
the incubation medium shifted the IC
for IP
production 4-fold, to 0.83 ± 0.09 µM (n = 3), while the IC
for inhibition of
Ca
release was increased about 2600-fold to 860
± 65 µM (n = 5). The fact that
atropine inhibited PLC activation in the presence of GTP
S clearly
shows that the effect of GTP
S was independent of PLC activation,
since binding of nonhydrolyzable GTP analogues to the
subunit of
G proteins, including G
(41, 42) and
G
(43) , irreversibly stimulates the
subunits and prevents inhibition by antagonists.
Figure 7:
Effect of GTPS on atropine-dependent
channel inactivation and IP
hydrolysis. Inactivation of
Ca
uptake was measured from the initial rate of
reduction in [Ca
] measured after addition
of atropine (see Fig. 1and Fig. 8for examples) to acini
stimulated with 2 mM carbachol (
) or 2 mM carbachol and 2 µM GTP
S (
). The rationale
and procedure for calculation of the rate of reduction in
[Ca
] are detailed in (27) . For
measurement of rates of IP
hydrolysis, acini incubated in
permeabilization medium as described in the legend to Fig. 6were stimulated with 2 mM carbachol (
) or
2 mM carbachol and 2 µM GTP
S (
). After
20 s of stimulation, the cells were inhibited with the indicated
concentration of atropine, and the levels of IP
were
followed for the subsequent 3 min (see also Fig. 11).
Figure 11:
TMS accelerates atropine-induced IP hydrolysis. Acini incubated in permeabilization medium for 2.5
min at 37 °C (
) were stimulated with 100 µM carbachol and 2 µM GTP
S (
,
) and in
the presence of 100 µM TMS (
). After 20 s of
stimulation, a portion of cells stimulated in the absence (
) or
the presence of TMS (
) were transferred to vials containing
atropine to give a final concentration of 1 mM. At the
indicated times, samples were removed to assay the levels of cellular
IP
. This experiment represents 2 others with similar
results. The same behavior was found in two experiments where 10
µM carbachol was used. In the absence of GTP
S, TMS
augmented IP
hydrolysis even more than that shown in the
figure (three independent experiments).
Figure 8:
The antagonist inhibits the effect of
GTPS but not of TMS on K
IP
. In
all experiments (a-c), the acini were
stimulated with 2 mM carbachol and then inhibited with 0.1
mM atropine. After carbachol and atropine treatment, the acini
were treated with 10 µM GTP
S (b) or 100
µM TMS (c) before addition of IP
at
the concentrations indicated by the numbers next to each
trace. The results of this and all similar experiments are summarized
in Table 1.
Because the
effect of carbachol was modified by both GTPS and TMS, to
understand how they may modify channel activity we tested the effect of
TMS on the modulation of IPACC by GTP
S. Interestingly, GTP
S
markedly sensitized the effect of TMS to cause maximal discharge of the
Ca
stores. This is illustrated in Fig. 9. Fig. 9a shows that addition of 100 µM TMS
to cells treated with 5 µM GTP
S induced rapid and
maximal Ca
release. Similar results were obtained
when GTP
S was added to TMS-treated cells, but the time course of
Ca
release was significantly slower. Accordingly, as
all other effects of GTP
S, the effect shown in Fig. 9was
time- (not shown) and concentration-dependent (Fig. 9c). In the absence of TMS, high concentrations
of GTP
S could release Ca
, which was probably due
to stimulation of PLC to generate IP
. TMS actually
partially inhibited the production of IP
generated by all
concentrations of GTP
S while sensitizing activation of
Ca
release by GTP
S. It is important to note that
despite the absence of an increase in IP
the effect of TMS
and GTP
S on Ca
release was still inhibited by
heparin (Fig. 9c). This would suggest that TMS +
GTP
S sensitized the IPACC to a level that maximal and rapid
Ca
release was observed at the level of IP
present in unstimulated cells.
Figure 9:
GTPS together with TMS induces
maximal Ca
release. Experiment a in the upper panel shows that 100 µM TMS causes rapid
and maximal Ca
release from acini treated with 5
µM GTP
S for 30 s. This Ca
release
can be inhibited largely by 50 µg/ml heparin (b).
Experiment c in the lower panel shows the dependence
of Ca
release (
,
) and IP
production (
,
) on GTP
S concentration in the
absence (
,
) or presence (
,
) of 100 µM TMS. For Ca
release, the protocol in experiment a was used except that the cells were incubated with or
without 100 µM TMS for 1 min before addition of GTP
S.
IP
production was measured as described in the legend to Fig. 6except that the cells were treated with or without 100
µM TMS for 1 min before being stimulated with the
indicated concentration of GTP
S for 1
min.
In the next stage, we tested
the effect of channel sensitization by GTPS and TMS on the
inactivation induced by atropine. Fig. 10a shows that 1
mM atropine completely inactivated channels activated by 10
µM carbachol and 2 µM GTP
S. However,
treating the cells with 100 µM TMS prior to stimulation
with carbachol and GTP
S completely prevented channel inactivation
by atropine. The small reduction in medium
[Ca
] due to atropine probably reflects the
activity of channels that were not accessed by GTP
S. The channels
were permanently stabilized in an active state since medium
[Ca
] remained elevated for at least 15 min
with no sign of decline. To show that TMS and GTP
S did not inhibit
the SERCA pumps and that the maintained high level of medium
[Ca
] was due to stabilizing the IPACC in an
active state, the channel was inhibited by heparin. Addition of heparin
resulted in channel inhibition and, consequently, rapid Ca
uptake into the IP
-sensitive pool at a rate
comparable to that measured in Fig. 10a after addition
of atropine.
Figure 10:
TMS
stabilizes the IPACC in an open state. Acini incubated in
permeabilization medium were treated with (b, d) or
without (a, c) 100 µM TMS before
stimulation with 10 µM carbachol and 2 µM GTPS (a, b) or 100 µM GTP
S (c, d). Where indicated, all cells
were inhibited with 1 mM atropine. In experiments (b, d) after atropine inhibition, the cells were exposed to 100
µg/ml heparin. Results similar to those in experiment d were obtained with 2 and 5 µM GTP
S. The
experiment shown and the one using 5 µM GTP
S were
repeated at least seven times with different cell
preparations.
As indicated above, TMS alone (not shown) or GTPS
alone (Fig. 10a and Fig. 7), although reducing
the affinity for atropine, never prevented channel inactivation by
atropine. That activation of G proteins by GTP
S and channel
sensitization by TMS was required to prevent channel inactivation is
further emphasized in the experiments shown in Fig. 10, c and d. As we showed before(28) , stimulation of
Ca
release with 100 µM GTP
S did not
prevent channel inactivation by atropine (Fig. 10c),
even though IP
levels under these conditions were very
high(28) . On the other hand, treating the cells with 2
µM (not shown) or 100 µM GTP
S (Fig. 10d) and 100 µM TMS in the absence
of agonist stimulation was sufficient to prevent channel inactivation
by atropine.
An important control for the experiments in Fig. 10is to show that TMS did not prevent the hydrolysis of
IP initiated by atropine. The results of such experiments
are shown in Fig. 11. Even in the presence of 100 µM carbachol and 5 µM GTP
S, TMS accelerated, rather
than inhibited, the hydrolysis of IP
. After 2 min of
exposure to atropine, IP
was reduced to basal levels, while
the channel was fully activated (Fig. 10b).
A long standing question in understanding agonist-evoked
[Ca]
oscillations is how low
concentrations of agonists induce oscillations without an apparent or
only small change in [IP
](1) . Modulation
of the IP
-activated Ca
channel (IPACC)
can be potentially important in view of the high basal IP
levels in most
cells(1, 2, 6, 7, 8, 9) .
Although several regulatory mechanisms of IPACC have been reported (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) ,
the relationship between them and their role in agonist-dependent
regulation of Ca
release is not known. The present
study suggests the existence of multiple and independent mechanisms for
regulation of IPACC, prominent among which is regulation by G protein
activation. The latter is used by agonists and antagonists to modulate
the K
IP
in a reciprocal manner and
thus facilitate Ca
release during agonist stimulation
and impair the release during antagonist inhibition. Such a mechanism
can contribute to the cyclical activation and inactivation of the IPACC
during Ca
oscillations(44) . Below we discuss
the evidence in support of these findings.
Comparing the effect of
many agents and conditions in the same cell type and experimental
system showed that KIP
can be
modulated by several independent mechanisms (Table 1). Thus,
additive effects were found between TMS, Ca
load, low
temperature, and the agonists. The important implication of these
findings is that although the IPACC can be regulated by various
mechanisms, none of them appear to contribute to the regulation by
agonists. Of course, regulation by Ca
load can have
important physiological significance in that when the stores are
loaded, they are primed for release by small additional change in K
IP
. It is likely that in empty
stores the IPACC has the lowest affinity for IP
, which will
facilitate channel inactivation at the termination of cell stimulation.
Nonetheless, the regulation of K
IP
by Ca
content in the ER is relatively modest
and occurs by a mechanism different from that used by agonists.
Interestingly, despite the fact that variations in KIP
between compartmentalized
Ca
pools account in large part for quantal
Ca
release(28) , none of the modulators of K
IP
changed the quantal properties
of Ca
release. This includes low temperature ( Fig. 2and Fig. 3). We particularly studied the effect of
low temperature in detail since a previous report suggested that
quantal Ca
release becomes continuous at low
temperature, and this process is reversible(25) . However, we
failed to convert the quantal to a continuous Ca
release by short or long incubation at 25 or 0 °C or by any
other modulator of K
IP
. In the
earlier studies by Kindman and Meyer(25) , one concentration of
IP
was used to demonstrate submaximal Ca
release at 37 °C and maximal release at 0 °C, without
considering the effect of the temperature on K
IP
. Such an effect as demonstrated
in the present study (Table 1) can account well for the
differences between the two studies. Our results of maintained quantal
behavior under all conditions suggest that all modulators of K
IP
, including agonists, affect all
IPACC equally rather than equalize K
IP
of channels of different compartments.
The present studies
show that KIP
is dynamically
controlled. Low concentrations of agonists, which minimally activate
PLC, markedly reduced K
IP
of the
IPACC. Moreover, termination of cell stimulation with antagonists
increased K
IP
to a level above that
measured in control cells. The antagonist was effective only if the
cells were first stimulated with carbachol. The antagonist had no
effect in control cells, and, when added before GTP
S, it did not
prevent the GTP
S-dependent reduction in K
IP
. The combined effects of the
agonist and antagonist indicate that the K
IP
of the IPACC is dynamically
controlled during cell stimulation/inhibition cycles. One advantage of
such a dynamic control is that small changes in IP
levels
can lead to maximal opening or closing of the channel. This will be
translated into a high cooperativity for interaction of IP
with the IPACC and in channel activation/inactivation.
Probably the most interesting finding of the present studies is that
agonists and antagonists appear to modulate KIP
by a mechanism dependent on
activation of G proteins. The first indication of this was the
similarity between the effect of the agonist and preincubation with
GTP
S on K
IP
. Both affected K
IP
in cells treated with TMS, high
Ca
load or incubated at low temperature. Both effects
were inhibited similarly by activators of protein kinases, inhibitors
of protein phosphatases, and augmented by inhibitors of protein
kinases. Inhibition of agonist-activated cells by atropine to increase K
IP
inhibited the effect of
GTP
S, but not of any other agent or treatment. Together, these
observations strongly suggest that agonist stimulation and GTP
S
reduced the K
IP
by the same
mechanism. It is possible that the agonists and GTP
S activated
heterotrimeric, rather than small G proteins, since all effects of
GTP
S could be reproduced with low concentrations of
AlF
. The effect of GTP
S described in the present study
is different from the previously described modification of the size of
the IP
-sensitive Ca
pool by
GTP(45, 46, 47) . GTP
S inhibited the
GTP-induced expansion of the Ca
pool, whereas
GTP
S increased the K
IP
and
millimolar concentrations of GTP were required to mimic the effect of
low concentrations of GTP
S and AlF
.
That G proteins
are involved in the effect of the agonist/antagonist and their full
impact becomes more evident when the effect of their activation on
agonist/antagonist-dependent changes in Ca release
and IP
levels are considered. Even in the absence of
preincubation, low concentrations of GTP
S (0.2-2
µM) markedly increased the potency of the agonist and
decreased the potency of the antagonist in affecting Ca
release. Thus, when irreversible activation of G proteins was
allowed by the presence of GTP
S, the agonists exceedingly
sensitized the IPACC to trigger Ca
release at basal
[IP
]. Measurement of IP
levels showed
that GTP
S had a small effect on IP
production during
agonist stimulation and did not prevent initiation of IP
hydrolysis by the antagonist. The latter excludes the possibility
that the effects of GTP
S, or for that matter the agonist and the
antagonist, on Ca
release was dependent on PLC
activity (41, 42, 43) . In fact, in cells
stimulated with carbachol and GTP
S, 10-100 µM atropine completely inactivated PLC without having any
inactivation effect on the IPACC (Fig. 7). These experiments,
therefore, indicate that agonists use G protein activation to reduce K
IP
and stabilize the channel in an
open state. Once the channel is activated, complete inactivation of G
proteins by the antagonist beyond (or different from?) that required to
modulate PLC activity, is needed for channel inactivation. Indeed,
preliminary experiments showed that muscarinic, bombesin, and
cholecystokinin antagonists were between 100- and 1000-fold more
effective in preventing cell stimulation when added before the
respective agonist than in reversing agonist effects when added to
stimulated cells. (
)
Further insight into the regulation
of IPACC by G proteins was obtained when the effect of TMS on
GTPS-, agonist-, and antagonist-dependent Ca
release was studied. TMS and low GTP
S caused maximal
Ca
release at basal IP
concentration. It
is interesting that in intact cells 100 µM TMS caused
significant Ca
release (21 and data not shown),
whereas in permeabilized cells up to 500 µM TMS did not
release Ca
but only decreased K
IP
. Incubating the cells with as
little as 0.2 µM GTP
S was sufficient to restore the
ability of TMS to cause Ca
release with no change in
IP
levels. Together, these observations indicate that: (a) TMS and GTP
S modulate the IPACC by different
mechanisms, (b) the IPACC is regulated by G proteins in
permeabilized and probably in intact cells, and (c) TMS
appears to modulate the interaction of the G protein-dependent
mechanism with the IPACC to stabilize the channel in an open state.
Indeed, treating the cells with TMS and GTP
S, with or without
agonist, completely prevents channel inactivation by the antagonist.
This effect was absolutely dependent on the combined action of TMS and
GTP
S to the extent that incubating the cells with very high
GTP
S (100 µM) did not prevent the effect of atropine,
while stimulation with 2 µM GTP
S and 100 µM TMS locked the channel in an antagonist-insensitive state. It is
important to note that TMS increased, rather than decreased, the rate
of IP
hydrolysis under all conditions, including incubation
with GTP
S.
The implication of the studies with TMS, GTPS,
agonist, and antagonist is that all point to the involvement of G
proteins in the regulation of K
IP
by
agonist stimulation and antagonist inhibition. They also point to the
broad extent of such regulation. Such a regulatory mechanism can be
very attractive in explaining how agonists can stimulate Ca
oscillations in the absence of stimulated IP
production. Our results suggest that minimal activation of G
proteins (low GTP
S, AlF
, or agonist concentrations),
which is not sufficient to appreciably activate PLC, is sufficient to
markedly decrease the K
IP
and
trigger large Ca
release at basal
[IP
]. This regulation is a dynamic process as
revealed by the antagonist-induced increase in K
IP
beyond that found in resting
cells. It is well established in many cell systems that Ca
oscillations occur only at low agonist concentration, when
usually no change in IP
is observed. It is easy to see how
the dynamic regulation of K
IP
described here can contribute or even dominate the mechanism by
which agonists signal Ca
oscillations. Understanding
how activation of G proteins modulate K
IP
is essential to evaluate its role in Ca
oscillation in particular and Ca
signaling in
general. This should be the challenge for future studies.