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

2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês

10.1074/jbc.m210418200

1083-351X

G. Babnigg, Tatiana K. Zagranichnaya, Xiaoyan Wu, Mitchel L. Villereal,

Ion Transport and Channel Regulation

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. 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. G-protein-coupled receptor human thapsigargin bradykinin carbachol acetoxymethyl ester human embryonic kidney cells nitrophenyl Hanks' balanced salt solution plasma membrane Ca2+-ATPase endoplasmic reticulum store-operated Ca2+entry 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 (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2109) Google Scholar). 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 (2Vaca L. Sinkins W.G. Hu Y. Kunze D.L. Schilling W.P. Am. J. Physiol. 1994; 267: C1501-C1505Crossref PubMed Google Scholar, 3Petersen C.C. Berridge M.J. Borgese M.F. Bennett D.L. Biochem. J. 1995; 311: 41-44Crossref PubMed Scopus (172) Google Scholar). Recently a number of papers have described investigations into whether mammalian Trp homologs can also function as store-operated Ca2+ channels, reviewed by Minke (4Minke B. Cook B. Physiol. Rev. 2002; 82: 429-472Crossref PubMed Scopus (529) Google Scholar). Although some of the early papers supported the hypothesis that mammalian Trp homologs mediate store-operated calcium entry (5Philipp S. Cavalie A. Freichel M. Wissenbach U. Zimmer S. Trost C. Marquart A. Murakami M. Flockerzi V. EMBO J. 1996; 15: 6166-6171Crossref PubMed Scopus (258) Google Scholar, 6Wes P.D. Chevesich J. Jeromin A. Rosenberg C. Stetten G. Montell C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9652-9656Crossref PubMed Scopus (515) Google Scholar, 7Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar), 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) (8Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 9Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). 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 (10Wu X. Babnigg G. Villereal M.L. Am. J. Physiol. Cell Physiol. 2000; 278: 526-536Crossref PubMed Google Scholar). In addition, we have recently confirmed that HEK-293 cells express endogenous hTrpC1, hTrpC3, and hTrpC4 proteins (11Wu X. Babnigg G. Zagranichnaya T. Villereal M.L. J. Biol. Chem. 2002; 277: 13597-13608Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). 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. HEK-293 cells were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 2 mml-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 (12Hess J.F. Borkowski J.A. Young G.S. Strader C.D. Ransom R.W. Biochem. Biophys. Res. Commun. 1992; 184: 260-268Crossref PubMed Scopus (448) Google Scholar). 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. 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. 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 mmEDTA, 2 mm Na2VO4, 2 mmNa4P2O7, 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. 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 (9Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). 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+]ithan 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. 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. 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 differentp < 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.Figure 4TG 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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 versusBK 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 (13Caride A.J. Elwess N.L. Verma A.K. Filoteo A.G. Enyedi A. Bajzer Z. Penniston J.T. J. Biol. Chem. 1999; 274: 35227-35232Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 14Caride A.J. Filoteo A.G. Penheiter A.R. Paszty K. Enyedi A. Penniston J.T. Cell Calcium. 2001; 30: 49-57Crossref PubMed Scopus (79) Google Scholar, 15Bautista D.M. Hoth M. Lewis R.S. J Physiol. 2002; 541: 877-894Crossref PubMed Scopus (111) Google Scholar) 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 (16Schilling W.P. Rajan L. Strobl-Jager E. J. Biol. Chem. 1989; 264: 12838-12848Abstract Full Text PDF PubMed Google Scholar, 17Kwan C.Y. Putney Jr., J.W. J. Biol. Chem. 1990; 265: 678-684Abstract Full Text PDF PubMed Google Scholar). 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. 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 whenn-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. 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 (18Rosado J.A. Sage S.O. J. Biol. Chem. 2000; 275: 19529-19535Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 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.