Differential Tyrosine Phosphorylation of Plasma Membrane
Ca2+-ATPase and Regulation of Calcium Pump Activity by
Carbachol and Bradykinin*
György
Babnigg
,
Tatiana
Zagranichnaya§,
Xiaoyan
Wu§, and
Mitchel L.
Villereal§¶
From the § Department of Neurobiology, Pharmacology, and
Physiology, The University of Chicago, Chicago, Illinois 60637 and
Argonne National Laboratory,
Argonne, Illinois 60439
Received for publication, October 10, 2002, and in revised form, February 19, 2003
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ABSTRACT |
We investigated the effects of thapsigargin (TG),
bradykinin (BK), and carbachol (CCh) on Ca2+ entry
via endogenous channels in human embryonic kidney BKR21 cells. After
depletion of Ca2+ stores by either TG, BK, or CCh, the
addition of Ca2+ gave a much larger rise in
Ca2+ levels in CCh-treated and TG-treated cells than in
cells treated with BK. However, in experiments performed with
Ba2+, a cation not pumped by Ca2+-ATPases, only
a modest difference between CCh- and BK-stimulated Ba2+
entry levels was observed, suggesting that the large difference in the
Ca2+ response is mediated by a differential regulation of
Ca2+ pump activity by CCh and BK. This hypothesis is
supported by the finding that when Ca2+ is removed during
the stable, CCh-induced Ca2+ plateau phase, the decline of
cytosolic Ca2+ is much faster in the absence of CCh than in
its presence. In addition, if Ca2+ is released from a caged
Ca2+ compound after a UV pulse, the resulting
Ca2+ peak is much larger in the presence of CCh than in its
absence. Thus, the large increase in Ca2+ levels observed
with CCh results from both the activation of Ca2+ entry
pathways and the inhibition of Ca2+ pump activity. In
contrast, BK has the opposite effect on Ca2+ pump activity.
If Ca2+ is released from a caged Ca2+ compound,
the resulting Ca2+ peak is much smaller in the presence of
BK than in its absence. An investigation of tyrosine phosphorylation
levels of the plasma membrane Ca2+-ATPase (PMCA)
demonstrated that CCh stimulates an increase in tyrosine
phosphorylation levels, which has been reported to inhibit Ca2+ pump activity, whereas in contrast, BK stimulates a
reduction of PMCA tyrosine phosphorylation levels. Thus, BK and CCh
have a differential effect both on Ca2+ pump activity and
on tyrosine phosphorylation levels of the PMCA.
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INTRODUCTION |
When cells are stimulated with agonists for
G-protein-coupled receptors
(GPCRs)1 coupled to
Gq, the resulting Ca2+ response is generally
biphasic in nature. The initial peak of the response is because of the
rapid release of Ca2+ from internal stores in response to
inositol 1,4,5-trisphosphate generation. This initial peak is followed
by a lower but longer lasting plateau phase that results from
Ca2+ entry via plasma membrane Ca2+ channels. A
substantial portion of this Ca2+ entry is via capacitative
calcium channels, also called store-operated calcium channels, as
initially described by Putney (1). Since the original description of
store-operated calcium channels, much work has been done to investigate
what proteins mediate store-operated Ca2+ entry (SOCE) and
how these channels are regulated. There is substantial evidence that
the Drosophila Trp protein can function as a
store-operated Ca2+ channel (2, 3). Recently a number of
papers have described investigations into whether mammalian Trp
homologs can also function as store-operated Ca2+ channels,
reviewed by Minke (4). Although some of the early papers supported the
hypothesis that mammalian Trp homologs mediate store-operated calcium
entry (5-7), a number of other papers argued that these Trp homologs
mediate other types of Ca2+ entry. For example, several
recent papers report that overexpression of either human TrpC3 (hTrpC3)
or murine TrpC6 gives a low level of Ca2+ entry in
response to depletion of internal Ca2+ stores by
thapsigargin (TG) but gives a much larger Ca2+ entry in
response to carbachol (CCh) (8, 9). These observations have led some to
the interpretation that hTrpC3 and murine TrpC6 may code for
receptor-operated rather than store-operated Ca2+ channels.
From our previous investigations of endogenous Ca2+
channels in HEK-293 cells, we knew that the level of Ca2+
entry in response to depletion of internal Ca2+ stores by
TG was always significantly larger than the level of Ca2+
entry in response to stimulating the G-protein-coupled receptor for
bradykinin (BK). Because this result was in contrast to the CCh results
reported for HEK-293 cells overexpressing various Trp isoforms, we were
interested in determining whether there was something fundamentally
different about the stimulation of Ca2+ entry by CCh in
comparison to BK. For example, does CCh release more Ca2+
from internal stores than BK, thereby giving a higher store-operated Ca2+ entry, or does CCh stimulate a receptor-operated
Ca2+ channel that for some reason is not activated by BK?
On the other hand, perhaps the exogenously expressed hTrpC3 and murine
TrpC6 channels behave differently than the channels endogenous to
HEK-293 cells. To investigate these questions, we performed a detailed comparison of the effects of CCh, BK, and TG on Ca2+ entry
via endogenous channels in HEK-293 cells, which we have previously
demonstrated express mRNA coding for endogenous hTrpC1, hTrpC3,
hTrpC4, and hTrpC6 channels (10). In addition, we have recently
confirmed that HEK-293 cells express endogenous hTrpC1, hTrpC3, and
hTrpC4 proteins (11).
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EXPERIMENTAL PROCEDURES |
Materials--
HEK-293 cells were obtained from the Richard
Miller laboratory (Northwestern University, Chicago, IL). The cDNA
encoding for the hB2-BKR was obtained from Fred Hess (Merck).
Fura-2 acetoxymethyl ester (Fura-2-AM), Fura-2 free acid, Fluo-3-AM,
nitrophenyl-EGTA-AM (NP-EGTA-AM), and Pluronic F-127 were purchased
from Molecular Probes, Inc. (Eugene, OR). All other chemicals were
purchased from Sigma.
Cell Culture--
HEK-293 cells were cultured in Dulbecco's
minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin at 37 °C in a humidified atmosphere
containing 5% CO2 and 95% air. Cells were subcultured
onto 25-mm round coverslips 1 day before experiments. HEK-293 cells
were stably transfected with the hB2-BKR cDNA construct using the
calcium phosphate method. The cDNA for the B2 receptor was
originally cloned from the lung fibroblast cell line CCD-16Lu into the
eukaryotic expression vector pcDNA I-Neo (12). G418-resistant clones were selected, and 48 clones were assayed for B2-BKR expression by Fura-2 imaging. A clone (HEK-BKR21) was selected for use in all
experiments because it expresses a similar number of BK binding sites
to that expressed in normal human fibroblasts. These cells, however,
express only endogenous TrpC channels and carbachol receptors. Clones
were cultured in the presence of 400 µg/ml G418 and used for 20-30 passages.
Calcium Imaging--
Cells were loaded with 5 µM Fura-2-AM in HEPES-buffered Hanks' balanced salt
solution (HHBSS) plus 1 mg/ml bovine serum albumin plus 0.025%
Pluronic F127 detergent for 30 min at room temperature and incubated
without Fura-2-AM in HHBSS for 30 min. To monitor intracellular
[Ca2+], the glass coverslips were placed in a perfusion
chamber and mounted onto the stage of a Nikon inverted epifluorescence
microscope. The cells were excited with alternating 340- and 380-nm
light, and the emission was measured at 510 nm. The images were
captured on an InCyt Im2 imaging system from Intracellular Imaging,
Inc. (Cincinnati, OH), and the data were analyzed by their InCyt
software. The ratios of the 340- and 380-nm images were determined, and the [Ca2+]i values were calculated for each cell
using a calibration curve established with Fura-2 potassium salt. In
some experiments, to distinguish between changes in Ca2+
entry and Ca2+ removal processes, we used Ba2+
to monitor Ca2+ entry pathways, since Ba2+ is
not pumped by Ca2+ ATPases. The changes in
[Ba2+]i are monitored by Fura-2 and shown on a
scale of ratio 340/380 (R340/380), since the calibration for
Ba2+ differs from that for Ca2+. All
experiments were conducted at room temperature. Nominally Ca2+-free solutions were prepared by treating
Ca2+-free, Mg2+-free, bicarbonate-free HBSS
with 10 g of Chelex-100, filtering out the Chelex-100 beads, and
then adding MgCl2 to a final concentration of 1 mM. Some traces show the response of individual
representative cells, whereas others show the average response of
300-600 cells on a representative coverslip.
For the experiments using NP-EGTA "caged calcium," HEK-BKR21 cells
were plated on coverslips 1 day before the experiment. The next
morning, cells were preincubated with NP-EGTA-AM (6 µM, dissolved in loading buffer) at 37 °C for 1.5 h. Cells were
then removed from the incubator, and they were loaded with fluorescent indicator Fluo-3-AM for 30 min at room temperature (5 µM,
added into the NG-EGTA-AM loading buffer). Cells were then incubated with HEPES-buffered HBSS for a 30-min unloading period. The coverslips were mounted at the bottom of a perfusion chamber that was placed on
the stage of a Nikon Diaphot inverted epifluorescence microscope. An
InCyt Im1_UN uncoupling module, and software (Intracellular Imaging
Inc., Cincinnati, OH) was used to uncage the Ca2+ from the
cytosolic NP-EGTA during the experiment. The UV light for uncaging is
delivered via the microscope objective. The uncaging module works with
two excitation filters, the uncaging filter and a Fluo-3 excitation
filter. The uncaging signal was initiated by moving the filter wheel to
the uncaging filter position (330 nm) to deliver a burst of UV light
(from a 300 watt xenon lamp), and then the filter wheel was switched to
the Fluo-3 excitation filter position (485 nM) for
excitation of the Fluo-3 to continue the Ca2+ measurement.
The UV photolysis, achieved by a 1-s exposure followed by a 1-s
interval, repeated 10 times, resulted in a rapid, transient increase in
[Ca2+]i. Fluo-3-AM fluorescence intensity was
measured for each coverslip as an average for ~400 cells. The
background-corrected fluorescence intensity was reported in the
figures. Control studies were carried out on cells with and without
preincubation in NP-EGTA-AM, which demonstrated that the presence of
the caged calcium compound did not change the basal level of
[Ca2+]i or the maximal response to CCh or BK
stimulation (data not shown). Also, the fact that the BK and CCh peak
heights were the same before and after flash photolysis demonstrated
that the repeated, brief (1 s) UV exposures did not lead to
photo-dynamic damage of the cells during the experimental period.
Immunoprecipitations and Immunoblotting--
Cells were grown on
10-cm dishes to confluence. Experiments were performed as follows.
Cells were incubated at 37 °C for 2 h in HBSS then incubated
with 100 nM BK for 2 min or with 100 µM CCh
for 2 min. Cells were lysed in modified radioimmune precipitation assay
buffer (10 mM Tris-HCl, pH 7.5, 500 mM NaCl,
0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mM
EDTA, 2 mM Na2VO4, 2 mM
Na4P2O7, 2 mM NaF). Two
milligrams of total protein from each lysate were used for each
immunoprecipitation reaction as well as 5 µg of anti-phosphotyrosine
antibody, clone PY20 (Upstate), or 2 µg of anti-plasma membrane
Ca2+-ATPase (anti-PMCA) antibody, clone 5F10 (Affinity
Bioreagents). Lysates plus antibody were incubated overnight at 4 °C
with continuous rotation, then 100 µl of protein A-Sepharose (50%
solution) was added and incubated again for 1 h at room
temperature with continuous rotation. Then the Sepharose was washed
with cold radioimmune precipitation assay buffer 4 times, and proteins
were eluted from Sepharose by adding 2× Laemmli buffer (plus 100 mM dithiothreitol) and heated for 5 min at 95 °C, and
samples were loaded on 7.5% SDS-PAGE. Electrophoresis was performed,
and then proteins were electrotransferred onto polyvinylidene
difluoride Immobilon membranes (Millipore). The membranes were blocked
with 5% milk solution in Tris-buffered saline, 0.1% Tween 20 for
1 h and were then incubated with primary antibodies raised against
PMCA (clone 5F10) overnight at room temperature. The antibodies were
diluted 1:3000 in the blocking solution. Membranes were washed 4 × 15 min with Tris-buffered saline, 0.1% Tween 20, incubated for 30 min at room temperature with secondary anti-mouse antibody (1:10000 in
Tris-buffered saline, 0.1% Tween 20), washed under the same
conditions, and developed with SuperSignal chemiluminescent substrate
(Pierce) at a suitable time so as not to saturate the film. The films
were digitized on a flatbed scanner, and the relative spot intensities
were determined in Photoshop 6.0. The bands were outlined, and a
measure of the average gray level and the number of pixels in the spot
were obtained within the histogram function. The product of the average
intensity and the pixel number was used as a measure of the integrated
spot intensity. Because the basal integrated intensity can be
influenced by a number of factors that might vary from experiment to
experiment, data were expressed for each experiment in terms of a ratio
of the stimulated to the basal value for comparisons to other experiments.
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RESULTS |
Although we had never compared them in the same experiment, our
previous experience using BK and TG to stimulate Ca2+ entry
in HEK-BKR21 cells indicated that depleting Ca2+ stores
with TG would give a more robust Ca2+ entry than activating
the GPCR for BK. This result was in stark contrast to those reported in
HEK-293 cells overexpressing hTrpC3, where stimulation of the CCh GPCR
gave a more robust Ca2+ entry than did stimulation of cells
with TG (9). Thus, we were interested in determining whether there is a
fundamental difference between the way BK and CCh stimulate
Ca2+ entry in HEK-BKR21 cells. We began our study by
comparing the Ca2+ response initiated by BK and CCh in
HEK-BKR21 cells that were expressing only endogenous calcium channels.
We incubated cells in HBSS and added agonist to determine whether the
plateau phase differed in cells stimulated with either BK or CCh. As
seen in Fig. 1, CCh stimulated a
significantly higher plateau phase of [Ca2+]i
than did BK. The mean value for the CCh-stimulated plateau was
138.4 ± 13.6 (n = 5) with a basal level of
53.6 ± 8.5 (n = 5) compared with the mean value
for the BK stimulated plateau of 71.5 ± 8.6 (n = 4) with a basal level of 48.8 ± 9.5 (n = 4).
These CCh and BK plateau levels values are significantly different
(p < 0.006). When cells were rinsed with
Ca2+-free HBSS at the end of the experiments, the
Ca2+ returned to the basal level (data not shown),
indicating that Ca2+ entry from the extracellular space
mediates the sustained elevation of Ca2+ under both CCh-
and BK-stimulated conditions. These results suggested that CCh might be
more effective than BK in stimulating Ca2+ entry via
endogenous HEK-BKR21 Ca2+ channels.

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Fig. 1.
Comparison of Ca2+ plateaus in
HEK-BKR21 cells stimulated with either BK or CCh. HEK-BKR21 cells
were perfused with HBSS, and the intracellular Ca2+
concentration ([Ca2+]i) was monitored with Fura-2
imaging. After the establishment of a base line, cells were stimulated
with 100 nM BK (panel A) or 100 µM
CCh (panel B), both saturating concentrations. After 5 min
of treatment plateau levels were measured.
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To take a closer look at the comparative ability of CCh and BK to
stimulate Ca2+ entry, we used another protocol to monitor
the activation of Ca2+ entry pathways. In Fig.
2, we treated cells with either BK, CCh, or TG in a Ca2+-free HBSS solution, and after internal
pools were empty, we added HBSS containing Ca2+. The
initial slope of the Ca2+ uptake curve was taken as a
measure of the rate of Ca2+ accumulation. It is even more
clear in this protocol that CCh stimulates Ca2+ uptake much
better than does BK (CCh slope = 5.63 ± 0.51, n = 5, versus BK slope = 0.96 ± 0.11, n = 9; significantly different, p < 0.001). TG also was able to stimulate the uptake of Ca2+
much more effectively than BK (TG slope = 5.28 ± 0.65, n = 5; significantly higher than BK slope,
p < 0.003). There was not a statistically significant
difference between the Ca2+ uptake stimulated by CCh and
TG.

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Fig. 2.
Calcium entry in cells stimulated with TG,
BK, or CCh. HEK-BKR21 cells were perfused with HBSS followed by
Ca2+-free HBSS. Intracellular Ca2+ stores were
emptied with 1 µM TG (panel A), 100 nM BK (panel B), or 100 µM CCh
(panel C). Calcium entry was initiated by the addition of
HBSS in the continued presence of the agonists.
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Based on the observation that CCh and TG both stimulated
Ca2+ uptake much more effectively than BK, it seemed that
one possibility was that CCh and TG both give a more complete depletion
of internal Ca2+ stores, thereby producing more SOCE than
seen with BK. Therefore, we next wanted to compare the ability of BK
and CCh to deplete intracellular pools to determine whether CCh was
simply more effective than BK at emptying the TG-sensitive
Ca2+ pools. To test the relative ability of the two
agonists to release stored Ca2+, we stimulated with one
agonist in Ca2+-free medium and then came back and
stimulated with the other agonist and monitored how much additional
Ca2+ could be released by the second agonist. The data in
Fig. 3 indicate that there is
considerable overlap between the BK-sensitive and CCh-sensitive
Ca2+ pools. We see that CCh treatment releases most but not
all of the BK-sensitive intracellular calcium pool. Likewise, BK
treatment releases a significant portion of the CCh-sensitive
intracellular calcium pool. In a set of three experiments, we observed
that the area under the curve for the CCh response was 26,511 ± 1697 compared with 19,139 ± 2340 for BK, (significantly different
p < 0.05). It also appears that CCh is more effective
than BK at releasing the Ca2+ pool sensitive to the other
agonist. When CCh is the first agonist, the area under the BK curve is
15.4 ± 0.02% (n = 4) of the area under the first
peak. When BK is the first agonist, the area under the CCh peak is
60.1 ± 0.01% (n = 3) of the area under the BK peak. These values are significantly different (p < 0.00002). We will return later to the question of whether this
difference in Ca2+ release is of sufficient magnitude to
explain the differential activation of Ca2+ entry. The data
in Fig. 4 shows that the intracellular
Ca2+ pool emptied by TG includes both the BK- and
CCh-sensitive Ca2+ pools.

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Fig. 3.
The relationship between BK- and
CCh-responsive intracellular Ca2+ pools. HEK-BKR21
cells were perfused in Ca2+-free HBSS, and the
intracellular Ca2+ pools were emptied with either 100 µM CCh (panel A) or 100 nM BK
(panel B). After the [Ca2+]i returned
to base line the agonists were washed away, and the cells were treated
with 100 nM BK (panel A) or 100 µM
CCh (panel B) in the absence of extracellular
Ca2+.
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Fig. 4.
TG depletes both the BK- and CCh-responsive
intracellular Ca2+ pools. HEK-BKR21 cells were
incubated in Ca2+-free HBSS and then treated with
Ca2+-free medium containing 1 µM TG. When the
intracellular [Ca2+]i returned to base line,
cells were stimulated with Ca2+-free medium containing
either no addition (panel A), 100 nM BK
(panel B), or 100 µM CCh (panel
C).
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One consistent observation about the CCh-stimulated release of internal
Ca2+ pools led us to examine the possibility that a subtle
difference in the way CCh releases pool Ca2+ might provide
an explanation for the difference in activation of Ca2+
entry between BK and CCh. We find that when we monitor the
Ca2+ response to CCh in a Ca2+-free medium and
average that response over a large number of cells, we always see a
double peak for the CCh response. Our initial question was whether this
double peak was the result of two different populations of cells with
slightly different time courses of Ca2+ release or the
result of each individual cell responding with a double peak.
Therefore, we monitored the release of Ca2+ in individual
cells. In Fig. 5A, we see that
individual HEK-BKR21 cells stimulated with 100 µM CCh
show a double peak that is typical of the whole population of cells.
This suggested that CCh might stimulate Ca2+ release from
two separate intracellular Ca2+ pools with different
kinetics of release and perhaps this could explain why CCh releases
more Ca2+ than BK.

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Fig. 5.
CCh induced Ca2+ release
from individual HEK-BKR21 cells. Cells were loaded with
either Fura-2 (A) or Fluo-3 (B and C)
to monitor the Ca2+ response in individual CCh-treated (100 µM) cells. The responses were monitored at our normal
sampling rate (85 image pairs/min) with Fura-2 or a faster sampling
rate (207 captures/min) with Fluo-3. The traces in A and
B are representative of single cell traces, whereas the
trace in C is the average of the separately measured
responses of 11 individual HEK-BKR21 cells.
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In an attempt to better resolve the two peaks of CCh-stimulated
Ca2+ release, we went to a more rapid data acquisition
protocol utilizing the non-ratio calcium dye Fluo-3. Without delays for
filter switching, we obtained a more rapid data acquisition in
individual cells, which enabled us to observe that the notched peak was
not due to release from two separate Ca2+ pools but was
more likely due to the repetitive release from the same pool. As seen
in Fig. 5B, at faster rates of image capture, we could
resolve a series of damped oscillations, which at slower rates of
capture had fused into a declining plateau. One can see from the
average response of 11 individual HEK-BKR21 cells in Fig.
5C that such a response averaged over 300-500 cells would mask the damped oscillations and give the dual peak seen earlier.
Because the comparison of Ca2+ pool depletion by BK and CCh
suggested that a difference in pool depletion by CCh versus
BK might explain the differences in Ca2+ uptake on the
basis of differential activation of store-operated channels, we
performed one more set of experiments to make sure the difference in
Ca2+ uptake was due to increased channel activity and not
due to a change in Ca2+ pump activity. Although an initial
ion uptake rate such as we measured in Fig. 2 is normally considered to
be independent of ion efflux, this may not be the case for
Ca2+. First, the cytosolic Ca2+ concentration
when external Ca2+ is added is not very different from the
steady state basal Ca2+ concentration for cells in HBSS,
where Ca2+ influx is equal to Ca2+ efflux.
Second, it is likely that the Ca2+ concentration near the
membrane rises much more rapidly than in the general cytosol and that
the PMCA may kick in very early. This would be especially true if
HEK-BKR21 cells express those PMCA isoform splice variants that have
sufficiently rapid Ca2+/calmodulin activation kinetics
(13-15) to allow the pumps to respond rapidly to a rise in
Ca2+ with a much steeper dependence than predicted from
pump kinetics alone. Thus, we may be measuring a combination of influx
and Ca2+ pump efflux very early in the Ca2+
uptake curve in Fig. 2. Thus, we went back to the type of experiment seen in Fig. 2, but instead of monitoring Ca2+ entry, we
chose to monitor Ba2+ entry, since it is known that
Ba2+ is not pumped by Ca2+-ATPases either into
internal stores or out of the cell (16, 17). The data in Fig.
6 show that when we use Ba2+
uptake to monitor channel activity, we observed a dramatically different order of efficacy. Although CCh was still the most effective agonist, BK was now more effective than TG at stimulating
Ba2+ entry (CCh slope = 0.0038 ± 0.0001, n = 6; BK slope = 0.00265 ± 0.0001, n = 12; TG slope = 0.00147 ± 0.00003, n = 6). These values are statistically different,
p < 0.0001. Note that the scale of these slopes is
different from those reported earlier because we are plotting 340/380
ratios for Ba2+ experiments and Ca2+ values for
Ca2+ experiments. At this point, the data suggest that, for
some reason, BK stimulates Ca2+ uptake much less
effectively than CCh, which contrasts with the relatively minor
differences in their effect on Ba2+ uptake. This could
indicate either a differential permeability of Ca2+
versus Ba2+ through channels activated by store
depletion, or by CCh and BK, or it could indicate that BK stimulates
removal of cytosolic Ca2+ via Ca2+- ATPases. We
will return to this issue later in the results section.

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Fig. 6.
Effect of TG, BK, and CCh on Ba2+
entry. Cells were incubated in Ca2+-free medium and
then stimulated with either 1 µM TG (A), 100 nM BK (B), or 100 µM CCh
(C). After the Ca2+ returned to basal levels,
Ca2+-free medium containing 5 mM
Ba2+ was added in the continued presence of the stimulating
agent.
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When we were performing the experiments in Fig. 1, to examine the
height of the plateau phase of Ca2+ in response to CCh, we
noted one very interesting phenomenon that suggested that CCh also may
modify the Ca2+ pump activity. The data in Fig.
7 show a comparison of the rates of
decline of cytosolic Ca2+ after removal of Ca2+
either in the continued presence of CCh or in the absence of CCh. The
data in Fig. 7A show that removal of Ca2+ in the
absence of CCh results in a much steeper decline of cytosolic levels
than seen in the presence of CCh. The data in Fig. 7B show that the order in which the media changes are performed is not important. The return to basal after the removal of external
Ca2+ is always faster in the absence of CCh. The rate
constant for the first-order decline in 0 Ca2+ medium was
0.094 ± 0.007, which was significantly different
(p < 0.00002, n = 9) from the rate
constant for the decline in 0 Ca2+/CCh, which was
0.039 ± 0.003 (n = 9). To rule out the
possibility that the Na+/Ca2+ exchanger could
play an important role in the Ca2+ efflux process, we
performed experiments similar to the ones in Fig. 7, except in the
absence of external Na+. We found that CCh still modified
the rate constant for Ca2+ removal even when
n-methyl-D-glucamine was substituted for
the external Na+ ions. The rate constants for
Ca2+ decline were 0.147 ±0.015 (n = 6) in
0 Ca2+, Na+-free compared with 0.074 ± 0.006 (n = 6) in CCh/0
Ca2+/Na+-free (significantly different,
p < 0.001). Although both the rate constants were
higher than those observed in Na+-containing medium, there
was still a 2-fold difference in rate constants in response to CCh.

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Fig. 7.
Kinetics of the Ca2+ decline
after the removal of Ca2+ in the presence or absence of
CCh. HEK-BKR21 cells were stimulated with 100 µM CCh
in the presence of HBSS. The cells were then perfused with
Ca2+-free HBSS, incubated again in 100 µM
CCh/HBSS, and then perfused with Ca2+-free HBSS in the
presence of 100 µM CCh (panel A). The
reciprocal experiment is shown in panel B.
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A trivial explanation for the slow return to basal levels in the
presence of CCh could be that, in Ca2+-free medium, the
Ca2+ is pumped both out of the cell and into the ER,
whereas in the presence of CCh the impact of the ER pump on the removal
of Ca2+ from the cytosol is reduced due to the continual
loss of Ca2+ from the ER via the inositol
1,4,5-trisphosphate receptor. To test this possibility, we performed a
similar experiment where the pump capacity of the ER is compromised by
the presence of TG. A similar approach to quantifying the
Ca2+ efflux rate in platelets has been reported previously
(18). In the initial part of Fig. 8, we
see the previously described slower decline of Ca2+ in the
presence of CCh, as compared with the very rapid decline in
Ca2+-free medium in the absence of CCh. We then added TG to
inhibit pumping into the ER store, resulting in store depletion as well as establishment of a plateau level due to activation of the SOCE pathway. Now if the sole effect of CCh on Ca2+ removal were
to prevent accumulation of Ca2+ into the ER, we should see
the same rate of decline of Ca2+ to basal levels in
Ca2+-free medium containing TG as we saw in the CCh + Ca2+-free medium. However, we observe that although the
rate of decline is slower in TG (rate constant was 0.0642 ± 0.005, n = 3) than what was seen in
Ca2+-free medium earlier in the experiment (rate constant
was 0.118 ± 0.004, n = 3; significantly different
from the TG condition, p < 0.001), it was nowhere near
as slow as the decline seen in the Ca2+-free medium
containing CCh (rate constant was 0.0391 ± 0.005, n = 3; significantly different from the TG condition,
p < 0.01). This suggests that a large portion of the
reduction of Ca2+ pump activity induced by CCh may be due
to inhibition of the plasma membrane Ca2+ pump.

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Fig. 8.
Kinetics of the Ca2+ decline
after the removal of Ca2+ in CCh-stimulated
versus TG-stimulated cells. HEK-BKR21 cells were
stimulated with 100 µM CCh in the presence of HBSS. The
cells were then perfused with Ca2+-free HBSS in the
presence of 100 µM CCh, incubated again in 100 µM CCh/HBSS, and then perfused with Ca2+-free
HBSS. The cells were then incubated with HBSS containing 1 µM TG for a sufficient time to completely empty the
internal stores, and then the cells were perfused with
Ca2+-free HBSS.
|
|
Earlier under "Results" we mentioned that perhaps BK might be
stimulating plasma membrane pump activity. The initial data supporting
this hypothesis are shown in Fig. 9. In
Fig. 9A, the data indicate that there is a dramatic
difference in the levels of Ca2+ uptake stimulated by low
versus high doses of BK. Although stimulation of cells with
1 nM BK releases much less intracellular Ca2+
than stimulation with 100 nM BK, the initial
Ca2+ uptake stimulated by 1 nM BK is much
higher (~6.6-fold) than that stimulated by 100 nM BK (the
slope for 1 nM BK = 6.32 ± 1.6, n = 7; the slope for 100 nM BK = 0.96 ± 0.11, n = 9; statistically different,
p < 0.01). However, if we measure Ba2+
entry in response to low and high doses of BK we see just the opposite.
The high dose of BK stimulates both a higher level of Ca2+
release and a slightly higher level of Ba2+ entry
(~1.3-fold) than the low dose of BK (slope for 1 nM
BK = 0.0020 ± 0.0002, n = 8; slope for 100 nM BK = 0.00265 ± 0.0001, n = 12; significantly different, p < 0.03). Note again
that the units for slope are quite different for Ba2+
versus Ca2+ entry due to expression of
Ba2+ data in 340/380 ratio and the Ca2+ data as
nM Ca2+. The dramatic difference between the
Ca2+ and the Ba2+ response to high dose BK
suggests that BK may be activating a Ca2+ pump in addition
to stimulating Ca2+ entry.

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Fig. 9.
The effect of concentration of BK on calcium
entry. HEK-BKR21 cells were stimulated with either 1 nM BK (light trace) or 100 nM BK
(dark trace) in the absence of extracellular
Ca2+. When the [Ca2+]i returned to
base line, calcium entry was initiated by the addition of HBSS
(panel A) or 5 mM Ba2+ (panel
B) in the continued presence of agonist.
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|
We sought to provide independent evidence that CCh and BK affect
Ca2+ pump activity in opposite ways. To do this, we
utilized a caged Ca2+ compound to elevate the cytosolic
Ca2+ concentration in the absence of receptor activation.
In Fig. 10, we show that in cells
loaded with NP-EGTA a train of pulses of UV light results in a
substantial peak of Ca2+ that rapidly declines with time
when the train of pulses is stopped. The magnitude of the peak is
reproducible as seen in Fig. 10A. However, if cells are
stimulated with either CCh or BK in between light pulses, then the peak
heights from the UV-induced release of Ca2+ are
dramatically different. Stimulation of the cells with CCh in between
the two peaks results in a peak area for the second peak that is
significantly higher than the one seen before CCh stimulation (Fig.
10C). This suggests that CCh slows the removal of
Ca2+ from the cytosol and, therefore, must inhibit some
pump activity. In contrast to CCh, the effect of BK is to stimulate the
pump. The addition of 100 nM BK between the two light
pulses dramatically reduces the Ca2+ peak area in response
to the second pulse (Fig. 10B). This suggests that BK
increases the rate of removal of Ca2+ from the cytosol. A
statistical analysis of the ratio of the area under the curves for the
first peak and second peak is shown in Fig. 10D. Based on 15 separate experiments CCh produces a second peak that is 2.05 ± 0.24 (n = 15) times larger than the first peak
(significantly different than a ratio of 1, p < 0.0003), whereas BK results in a second peak that is 0.61 ± 0.12 times as large as the first peak (significantly different from 1, p < 0.004).

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Fig. 10.
Effect of CCh or BK on the peak
height of [Ca2+]i after flash photolysis of
NP-EGTA-AM. HEK-BKR21 cells grown on coverslips were preincubated
with 6 µM caged calcium compound NP-EGTA-AM before
loading with fluorescence indicator Fluo-3-AM. UV exposure was utilized
to release Ca2+ from the caged calcium compound.
A, Ca2+ response to repeated, brief UV
exposures. B, Ca2+ response to repeated, brief
UV exposures before and after CCh (100 µM) stimulation.
C, Ca2+ response to repeated, brief UV exposures
before and after BK (100 nM) stimulation. D, the
statistical analysis of 15 separate experiments comparing the ratio of
the area under the second peak to the area under the first 1.
|
|
Because the results of Fig. 8 indicate that CCh must have an effect
beyond reducing Ca2+ removal via the ER pump, we chose to
focus further investigations on the PMCA. Previously, work from Vanaman
and coworkers (19) indicates that tyrosine phosphorylation of PMCA can
significantly inhibit its pump activity. Thus, we investigated whether
the levels of tyrosine phosphorylation of PMCA changed in response to
CCh and BK. The data in Fig.
11A are from a
representative experiment that shows a substantial basal level of
tyrosine phosphorylation of PMCA. The PMCA tyrosine phosphorylation
level is greatly enhanced by the addition of CCh and significantly
reduced by the addition of BK. The data in Fig. 11B show the
statistics from a total of seven phosphorylation experiments. Because
of the substantial variation in the amount of basal phosphorylation
from experiment to experiment, we expressed the data in Fig.
11B as the ratio of the stimulated tyrosine phosphorylation
level to the basal tyrosine phosphorylation level for each individual
experiment. We found that CCh greatly increased the level of tyrosine
phosphorylation of PMCA, with the mean fold increase over basal levels
being 8.8 ± 3.1 (this value is significantly higher than a ratio
of 1, predicted for no stimulation, p < 0.015). In
contrast, BK reduced the level of tyrosine phosphorylation of PMCA to a
fold basal value of 0.23 ± 0.09 (this value is significantly lower
than a ratio of 1 predicted for no inhibition, p < 0.00002). Thus, the differential response of tyrosine phosphorylation
of PMCA fits well with the differential Ca2+ pump activity
monitored in Figs. 7 and 10.

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Fig. 11.
Tyrosine phosphorylation of PMCA in control
and CCh- and BK-stimulated HEK-BKR21 cells. In the control study,
HEK-BKR21 cells were incubated with HBSS at 37 °C for 2 h, and
total protein was extracted with modified radioimmune precipitation
assay buffer. In the CCh or the BK study, HEK-BKR21 cells were
incubated with HBSS at 37 °C for 2 h and stimulated with 100 µM CCh or 100 nM BK at room temperature for 2 min before total protein was extracted. Two mg of total protein was
used for each immunoprecipitation reaction in which 5 µg of PY20 or 2 µg of 5F10 antibody was added for an overnight incubation at 4 °C.
Western blots were performed with a 1:3000 dilution of anti-PMCA
antibody. The blot shown in panel A is representative of
seven experiments. In panel B, the statistical analysis of
seven experiments is shown comparing the CCh-stimulated-to-basal ratio
or the BK-stimulated-to-basal ratio of tyrosine phosphorylation.
|
|
 |
DISCUSSION |
It has been recognized for some time that GPCRs can couple to a
variety of different G proteins and thereby initiate a variety of
signaling pathways. Thus, some GPCRs will couple to Gs or
Gi and have opposing effects on cAMP production, whereas
others will couple to Gq and initiate signaling via the
phospholipase C pathway. Some receptors may even couple to multiple G
proteins and, thereby, produce a more complex signaling response.
However, it generally has been assumed that GPCRs that couple to
Gq will produce a Ca2+ response that is at
least qualitatively similar, i.e. the size of the response
might differ due to variations in receptor number, but in general they
will stimulate the same downstream pathways. Given this ongoing
assumption, the results from some of the early Trp overexpression
studies were difficult to understand. These studies demonstrated that
CCh was much more effective stimulating Ca2+ entry via
hTrpC3 than was TG, a result that is in direct contrast to our early
observations that TG was more effective in stimulating Ca2+
entry than BK. This was rather puzzling since both BK and CCh are known
to couple to Gq and should therefore stimulate similar pathways. A direct comparison showed that CCh stimulated a higher Ca2+ plateau level than BK in HEK-BKR21 cells (Fig. 1) and
after depletion of Ca2+ in a Ca2+-free medium
CCh was much more effective in stimulating Ca2+ entry than
was BK. However, in contrast to studies overexpressing hTrpC3 in
HEK-293 cells, CCh was not significantly better than TG at stimulating
Ca2+ entry via endogenous channels in our HEK-BKR21 cells
(Fig. 2).
The initial hypothesis tested was that CCh was more effective at
emptying internal Ca2+ stores and, thereby, more effective
at stimulating SOCE, and studies to investigate the level of
Ca2+ release in Ca2+-free medium soon indicated
that CCh is better at releasing Ca2+ stores (Fig. 3).
Although both BK and CCh released significant amounts of the internal
Ca2+ stores, the addition of TG after either BK or CCh
stimulation in Ca2+-free medium resulted in a substantial
release of internal Ca2+ (data not shown). On the other
hand, TG could fully deplete both the BK- and CCh-sensitive
Ca2+ pools. Thus, if we were looking solely at SOCE, one
would predict that TG might be much more effective at stimulating
Ca2+ entry than CCh as well as being more effective than
BK. Thus, the differential activation of SOCE by CCh and BK does not
appear to be sufficient to explain the large difference in activation of Ca2+ entry. There are, however, several problems with
this argument. First, results in other cell types suggest that TG
depletes Ca2+ stores in addition to those residing in the
inositol 1,4,5-trisphosphate-sensitive store (20), which is presumably
the one coupled to SOCE. Second, activating these GPCRs could activate
receptor-stimulated Ca2+ entry pathways in addition to
store-operated channels.
We next investigated whether CCh might release Ca2+ from a
pool in addition to the Ca2+ pool that was accessed by BK.
This possibility was suggested by the double peak observed when CCh is
added to cells in a Ca2+-free medium (Fig. 5A).
Perhaps the two peaks resulted from release from two kinetically
distinct pools. More rapid data acquisition using a non-ratio
Ca2+ indicator demonstrated that the double peak we had
previously seen was simply the result of averaging between cells whose
individual response showed damped oscillations (Fig. 5, B
and C). Thus, rather than indicating release from two
separate pools, the double peak was more likely to be the result of
"re-release" from the same pool.
Before launching an intense investigation of whether multiple
Ca2+ entry pathways were stimulated by CCh but not by BK,
we first investigated whether some differences in pump activity might
explain the higher CCh- than BK-stimulated Ca2+ entry. To
begin, we examined whether the differential effects of CCh and BK on
Ca2+ entry could also be observed when Ba2+ is
used to trace channel activity. Although Ba2+ passes
through many Ca2+ channels, it is not pumped by
Ca2+-ATPases, thereby allowing us to separate effects on
Ca2+ pump activity from the regulation of channel activity.
We observed that with Ba2+ as a tracer, the CCh-stimulated
entry was only 1.4 times the level of BK-stimulated entry (Fig. 6).
This was in dramatic contrast to the Ca2+ results, where
the CCh-stimulated entry was 5.5 times the BK-stimulated entry. Both
the BK-stimulated and the CCh-stimulated Ba2+ entry were
higher than the TG-stimulated Ba2+ entry, indicating that
both CCh and BK stimulate entry pathways in addition to the
TG-stimulated entry pathway. Furthermore, the large difference in CCh-
and BK-stimulated Ca2+ uptake must be due to effects on
Ca2+ pumping rather than Ca2+ entry via
channels; that is, either CCh must inhibit Ca2+ pump
activity or BK must stimulate Ca2+ pump activity.
When we were comparing the effects of BK and CCh on Ca2+
plateaus in the Ca2+-containing medium, we observed another
phenomenon that seemed to support the hypothesis that CCh has an
inhibitory effect on Ca2+ pump activity. We found that
different results were obtained when Ca2+ was removed in
the continued presence of CCh versus when Ca2+
was removed in the absence of CCh. When we switched the medium to
Ca2+-free in the absence of CCh we observed a very sharp
decline of [Ca2+]i. This was in contrast to the
very slow decline observed when Ca2+ was removed in the
continuing presence of CCh (Fig. 7). It did not matter in which order
the experiment was performed, the decline was always slower in the
presence of CCh. We ruled out the simple explanation that in the
continued presence of CCh the ER could not effectively pump
Ca2+ out of the cytosol. Although the decline of cytosolic
Ca2+ was measurably slower when TG was added to inhibit the
sarco(endo)plasmic reticulum calcium ATPase pump, the rate of decline
was still much more rapid than that observed in the presence of CCh.
This suggested that a significant portion of CCh effect is likely to be
on the PMCA rather than on the sarco(endo)plasmic reticulum calcium ATPase.
Although the data appear to support an inhibitory effect of CCh on
Ca2+ pump activity, the evidence points to BK having a
stimulatory effect on Ca2+ pump activity. Because BK can
stimulate the release of a large fraction of the TG-sensitive
Ca2+ store one would expect that the effect of BK on
Ca2+ entry would be close to that seen with TG. However,
instead we see that 100 nM BK stimulates only 18% of the
Ca2+ uptake stimulated by TG (Fig. 2). This was the first
indication that BK might stimulate Ca2+ pump activity. This
hypothesis was further supported by the observation that BK actually
stimulated a level of Ba2+ entry that was 1.8 times that
seen with TG (Fig. 6), arguing that the lower BK effect on
Ca2+ entry must be via a Ca2+ pump effect. In
addition, the effect of BK on Ca2+ pump activity seemed to
be more visible at higher BK doses. The initial Ca2+ entry
was 6.5-fold higher at 1 nM BK than at 100 nM
BK despite the fact that the higher dose of BK released more
Ca2+ from internal stores (Fig. 9). In contrast, the
Ba2+ entry was 1.3-fold higher at 100 nM BK
than at 1 nM BK. These data indicate that one effect of BK
on Ca2+ uptake results from a stimulation of
Ca2+ pump activity at higher BK doses.
The hypothesized effect of BK and CCh on Ca2+ pump activity
was further investigated by producing test pulses of Ca2+
in the cytosol and monitoring the way this Ca2+ was handled
in the presence and absence of CCh or BK. When Ca2+ was
released from a caged Ca2+ compound, we could obtain a
reproducible spike level (Fig. 10A). If cells were treated
with CCh in between the two spikes, the area under the second spike
curve was approximately doubled in the presence of CCh (Fig. 10,
B and D). In contrast, if the cells were treated
with BK between the two spikes, the area under the second spike curve
was decreased by ~40% (Fig. 10, C and D).
Although our expectations were that BK and CCh would modify the rate of decline and not the peak height after the UV pulses, this expectation ignored two factors. First, the UV pulses are given over a finite period of time, thereby allowing time for the Ca2+ pumps to
modulate the extent of the peak height. Second, with changes in peak
height came another form of modulation of the Ca2+ pump
activity, namely Ca2+ activation of the PMCA via a
calmodulin-dependent mechanism. In our early experiments to
calibrate the size and number of UV pulses required to provide a
measurable Ca2+ spike, we determined that there was a
significant Ca2+ dependence to the rate of decay from the
peak. Thus, because BK and CCh both change the peak height, the
Ca2+-dependent form of PMCA regulation is
superimposed on the regulation of PMCA by tyrosine phosphorylation and
complicates the analysis of the rate of decline. For those reasons, we
chose the area under the curve as the best measure of the effect of CCh
and BK in these experiments.
Because the data in Na+-free medium ruled out participation
of the Na+/Ca+2 exchanger and the data in Fig.
8 suggested that a significant portion of the CCh effect was not
mediated by the sarco(endo)plasmic reticulum calcium ATPase pumps, we
began to consider how BK and CCh might differentially affect the PMCA.
Because a previous publication reports that the tyrosine
phosphorylation of PMCA led to the inhibition of its pump activity
(19), we investigated the effects of BK and CCh on the tyrosine
phosphorylation of PMCA. We consistently observed a dramatic increase
(~8-fold) in tyrosine phosphorylation of the PMCA in response to the
addition of CCh (Fig. 11). Based on results from Vanaman and co-workers
(19), the increased PMCA tyrosine phosphorylation would be consistent
with an inhibitory effect of CCh on the PMCA. In contrast, we
consistently observed a decrease in tyrosine phosphorylation (to
~20% of control level) after stimulation of cells by BK (Fig. 11),
an observation that would be consistent with a stimulation of PMCA activity.
In summary, we have demonstrated that two compounds which are agonists
for GPCRs coupled to Gq have opposite effects on PMCA activity, and these effects appear to be mediated via a differential tyrosine phosphorylation of PMCA. These observations clearly point out
the fact that we cannot treat all agonists of GPCR linked to
Gq as if they will all regulate cytosolic Ca2+
levels in a similar manner. There clearly are subtle differences in the
way they regulate [Ca2+]i, and it is clear that
we must learn more about these subtle differences and the mechanisms
for such differential regulation before we will fully understand the
regulation of cytosolic Ca2+ levels. Studies are under way
to investigate the tyrosine kinases involved in the tyrosine
phosphorylation of PMCA and the mechanism by which they are regulated.
 |
ACKNOWLEDGEMENT |
We acknowledge the efforts of our research
assistant, Arpad Danos, for efforts in curve fitting and image analysis.
 |
FOOTNOTES |
*
This work was supported by NIGMA, National Institutes of
Health Grant GM-54500 (to M. L. V.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Neurobiology, Pharmacology and Physiology, Abbott 532, The University of Chicago, 947 E. 58th St., Chicago, IL 60637. E-mail:
mitch@drugs.bsd.uchicago.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M210418200
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G-protein-coupled receptor;
h-, human;
TG, thapsigargin;
BK, bradykinin;
CCh, carbachol;
AM, acetoxymethyl ester;
HEK-293 cells, human embryonic kidney cells;
NP, nitrophenyl;
HBSS, Hanks' balanced
salt solution;
PMCA, plasma membrane Ca2+-ATPase;
ER, endoplasmic reticulum;
SOCE, store-operated Ca2+
entry.
 |
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.