Differential Tyrosine Phosphorylation of Plasma Membrane Ca2+-ATPase and Regulation of Calcium Pump Activity by Carbachol and Bradykinin*

György BabniggDagger , Tatiana Zagranichnaya§, Xiaoyan Wu§, and Mitchel L. Villereal§

From the § Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637 and Dagger  Argonne National Laboratory, Argonne, Illinois 60439

Received for publication, October 10, 2002, and in revised form, February 19, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (14K):
[in this window]
[in a new window]
 
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.

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.


View larger version (16K):
[in this window]
[in a new window]
 
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.

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.


View larger version (20K):
[in this window]
[in a new window]
 
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+.


View larger version (18K):
[in this window]
[in a new window]
 
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).

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.


View larger version (19K):
[in this window]
[in a new window]
 
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.

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.


View larger version (19K):
[in this window]
[in a new window]
 
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.

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.


View larger version (18K):
[in this window]
[in a new window]
 
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.

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.


View larger version (12K):
[in this window]
[in a new window]
 
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.


View larger version (22K):
[in this window]
[in a new window]
 
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.

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).


View larger version (22K):
[in this window]
[in a new window]
 
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.


View larger version (15K):
[in this window]
[in a new window]
 
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[Medline] [Order article via Infotrieve]
2. Vaca, L., Sinkins, W. G., Hu, Y., Kunze, D. L., and Schilling, W. P. (1994) Am. J. Physiol. 267, C1501-C1505[Medline] [Order article via Infotrieve]
3. Petersen, C. C., Berridge, M. J., Borgese, M. F., and Bennett, D. L. (1995) Biochem. J. 311, 41-44[Medline] [Order article via Infotrieve]
4. Minke, B., and Cook, B. (2002) Physiol. Rev. 82, 429-472[Abstract/Free Full Text]
5. Philipp, S., Cavalie, A., Freichel, M., Wissenbach, U., Zimmer, S., Trost, C., Marquart, A., Murakami, M., and Flockerzi, V. (1996) EMBO J. 15, 6166-6171[Abstract]
6. Wes, P. D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., and Montell, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9652-9656[Abstract]
7. Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., and Birnbaumer, L. (1996) Cell 85, 661-671[Medline] [Order article via Infotrieve]
8. Boulay, G., Zhu, X., Peyton, M., Jiang, M., Hurst, R., Stefani, E., and Birnbaumer, L. (1997) J. Biol. Chem. 272, 29672-29680[Abstract/Free Full Text]
9. Zhu, X., Jiang, M., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 133-142[Abstract/Free Full Text]
10. Wu, X., Babnigg, G., and Villereal, M. L. (2000) Am. J. Physiol. Cell Physiol. 278, 526-536
11. Wu, X., Babnigg, G., Zagranichnaya, T., and Villereal, M. L. (2002) J. Biol. Chem. 277, 13597-13608[Abstract/Free Full Text]
12. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D., and Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, 260-268[Medline] [Order article via Infotrieve]
13. Caride, A. J., Elwess, N. L., Verma, A. K., Filoteo, A. G., Enyedi, A., Bajzer, Z., and Penniston, J. T. (1999) J. Biol. Chem. 274, 35227-35232[Abstract/Free Full Text]
14. Caride, A. J., Filoteo, A. G., Penheiter, A. R., Paszty, K., Enyedi, A., and Penniston, J. T. (2001) Cell Calcium 30, 49-57[CrossRef][Medline] [Order article via Infotrieve]
15. Bautista, D. M., Hoth, M., and Lewis, R. S. (2002) J Physiol 541, 877-894[Abstract/Free Full Text]
16. Schilling, W. P., Rajan, L., and Strobl-Jager, E. (1989) J. Biol. Chem. 264, 12838-12848[Abstract/Free Full Text]
17. Kwan, C. Y., and Putney, J. W., Jr. (1990) J. Biol. Chem. 265, 678-684[Abstract/Free Full Text]
18. Rosado, J. A., and Sage, S. O. (2000) J. Biol. Chem. 275, 19529-19535[Abstract/Free Full Text]
19. Dean, W. L., Chen, D., Brandt, P. C., and Vanaman, T. C. (1997) J. Biol. Chem. 272, 15113-15119[Abstract/Free Full Text]
20. Baumgarten, L. B., Lee, H. C., and Villereal, M. L. (1995) Cell Calcium 17, 41-52[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.