Extracellular Ca2+-sensing receptor is a promiscuous divalent cation sensor that responds to lead

Mary E. Handlogten1, Naoki Shiraishi2, Hisataka Awata1, Chunfa Huang1, and R. Tyler Miller1

1 Division of Nephrology, Department of Medicine, University of Florida, Gainesville, Florida 32610; and 2 Fourth Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The extracellular Ca2+-sensing receptor (CaR) responds to polycations, including Ca2+ and neomycin. This receptor is a physiological regulator of systemic Ca2+ metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb2+, Co2+, Cd2+, and Fe2+, are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A2 (PLA2) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca2+, Ba2+, Cd2+, Co2+, Fe2+, Gd3+, Ni2+, Pb2+, and neomycin activated the CaR, but Hg2+ and Fe3+ did not. We analyzed the kinetics of activation of p42ERK and PLA2 by the CaR in response to Ca2+, Co2+, and Pb2+. The EC50 values ranged from ~0.1 mM for Pb2+ to ~4.0 mM for Ca2+. The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca2+ and Pb2+ were additive for activation of the CaR. The EC50 for Ca2+ or Pb2+ was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA2 via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb2+, Cd2+, Co2+, and Fe2+ for the kidney and other tissues where it is expressed.

G protein-coupled receptor; divalent cation; mitogen-activated protein kinase; phospholipase A2; cell signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE EXTRACELLULAR Ca2+-sensing receptor (CaR) was cloned using an expression strategy by virtue of its ability to signal in response to changes in extracellular Ca2+ concentrations in the millimolar range (8). This receptor is expressed in the parathyroid glands and kidney, where it regulates Ca2+ balance by reducing parathyroid hormone (PTH) secretion and increasing salt, water, and Ca2+ excretion in the urine. The CaR is also expressed in the brain, stomach, colon, and several epithelial tissues where the role for a Ca2+ sensor is not as clear (12, 19).

In addition to participating in the normal regulation of Ca2+ metabolism, the CaR also appears to mediate many of the adverse effects of hypercalcemia. In humans and animal models, hypercalcemia reduces glomerular filtration rate (GFR), induces an Na+, Cl-, Ca2+, and H2O diuresis, and leads to altered mental status and gastrointestinal disturbances. Chronic hypercalcemia leads to interstitial fibrosis in the kidney, but effects on other organs are not well defined (29). If some of the toxic effects of Ca2+ are mediated by the CaR, other ligands for the CaR may also cause injury to the kidney and other tissues where the CaR is expressed.

The CaR is a member of the G protein-coupled receptor family. Within this large family, it has structural similarity and limited homology to the metabotropic glutamate receptors. These receptors have large extracellular domains made up of ~650 amino acids that contain multiple cysteines (5). The CaR signals as a dimer, with dimerization occurring through interactions of the extracellular domains (15, 25, 36). The CaR presumably contains multiple cooperative binding sites for Ca2+ and its other ligands, because activation occurs with a Hill coefficient >1 (30).

The CaR signals through pertussis toxin-sensitive and -insensitive G proteins (Gi and Gq/11 families) to regulate second messengers that include, but may not be limited to, cAMP, inositol trisphosphate, diacylglycerol, intracellular Ca2+, and arachidonic acid (AA) metabolites (2, 7, 21). These second messengers presumably regulate kinases, phosphatases, and other signaling molecules. However, the known signaling pathways may not explain all the biological functions of the CaR, because they do not fully account for inhibition of PTH secretion by the CaR (7). Consequently, the CaR may regulate signaling pathways that involve other G proteins (e.g., G12/13) or G protein-independent signaling pathways (5). In addition to reducing PTH secretion and reducing Na, Cl, Ca2+, and H2O reabsorption, activation of the CaR in epithelial cells inhibits their growth while stimulating the growth of fibroblasts (10, 35).

The CaR responds to divalent cations and polycations in addition to Ca2+, but the full range of ligands for this receptor, including many toxic divalent cations, is not defined (8, 30). To determine whether potentially toxic divalent cations could activate the CaR, we expressed a cDNA encoding the human CaR in HEK-293 cells and measured its ability to activate two downstream enzymes, phospholipase A2 (PLA2) and mitogen-activated protein (MAP) kinase (p42ERK), in response to a range of divalent cations, including Cd2+, Co2+, and Pb2+. Activation of the CaR by these divalent cations suggests a new mechanism by which these agents could cause injury to the kidney or nervous system.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Materials. The cDNAs coding for the wild-type human CaR and its inactive mutant R796W, both in pCDNA-3 (3), were a generous gift from Edward Brown and Mai Bai (Harvard Medical School, Boston, MA), and the influenza virus hemaglutinin antigen (HA)-tagged cDNA coding for p42ERK in pCEPH4L (16) was a generous gift from Melanie Cobb (University of Texas Southwestern Medical School, Dallas, TX). HEK-293 and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection. Tissue culture medium, serum, and G418 were obtained from Life Technologies; [3H]AA from NEN Life Sciences; the chemiluminescent system for visualizing immunoblots from Pierce; and the other reagents from Fisher or Sigma Chemical. The anti-active extracellular signal-regulated kinase (ERK) antibody was obtained from Promega, and the monoclonal anti-HA antibody was obtained from the University of Florida Hybridoma Core.

cDNA constructions. To add HA tags to the COOH-termini of the CaR, we amplified part of the 3'-coding region of the human CaR cDNA using a 57-bp sense oligonucleotide coding for the last six amino acids of the CaR, the nine amino acids of the HA epitope, a stop codon, an Xba I restriction site, a 3-bp tail (5' CGC TCT AGA CTA AGC GTA GTC TGG GAC GTC GTA TGG GTA TGA ATT CAC TAC GTT TTC 3'), and a 20-bp antisense oligonucleotide that is 5' of a unique BamH I site in the COOH-terminal region of the receptor (5' ACC TTT ACC TGT CCC CTG AA 3'). The sequence of the amplified segment was verified with dideoxy sequencing. The PCR products were digested with BamH I and Xba I, purified, and ligated into the expression vector (pCDNA3) that contained the remainder of the cDNA for the wild-type and nonfunctional mutant (R796W) CaRs.

Cell culture and expression of cDNAs. HEK-293 and MDCK cells were cultured in DMEM, 25 mM HEPES, and 10% calf serum in a 90% air-10% CO2 incubator. For transient transfections, cells were split so that they were ~70% confluent at the time of transfection. Cells in 60-mm dishes were fed fresh medium 4 h before transfection and were transfected by addition of 500 µl of CaPO4 solution containing 3 µg of DNA (31). After 16 h the medium was replaced with fresh medium, and experiments were performed after ~48 h. For stable expression, the cells were split after 48 h and grown in medium containing G418 (500 µg/ml). Individual colonies were isolated by limiting dilution, or single colonies from large plates were grown and tested for expression of the receptor with the anti-HA antibody by Western blotting and Ca2+-stimulated [3H]AA release.

Measurement of p42ERK activity. Cells were deprived of serum overnight before experiments. At the time of experiments, the medium was replaced with a solution containing 150 mM NaCl, 5 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2, and 0.5 mM MgCl2. At time 0, reagents were added at the concentrations indicated and incubated with the cells at 37°C for the times indicated. Commonly, the cells were exposed to activators of the receptor for 5 min, at which time the reactions were stopped by rinsing the cells at 4°C in buffer containing 50 mM NaF, 100 mM NaCl, 0.1 mM sodium orthovanadate, and 20 mM sodium phosphate, pH 7.4-7.5, and placing the dishes on a dry ice-and-ethanol bath. The cells were scraped in iced buffer that contained 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 40 mM beta -glycerophosphate, 50 mM NaF, 50 nM okadaic acid, 5 mM sodium pyrophosphate, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 40 mM pNPP, 4 µg/ml each of pepstatin, aprotinin, and leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged at 15,000 g for 10 min in a refrigerated microfuge. Triton-soluble extracts normalized for protein were size fractionated on a 9% SDS-polyacrylamide gel (30:0.6 acrylamide-bisacrylamide with 0.75 M Tris, pH 8.8) and immunoblotted with the anti-HA monoclonal antibody (12CA5). The bands, shifted (activated) and total p42ERK, were quantified with scanning densitometry and analyzed with NIH Image software (Scion Image). The activity was determined as the ratio of the shifted (activated) band to the total p42ERK.

Measurement of AA release. Cells grown in collagen-coated 24-well trays were deprived of serum and labeled for 6 h or overnight with [3H]AA. The medium was removed, and the cells were washed with fresh medium containing 130 mM NaCl, 5.4 mM KCl, 10 mM HEPES, pH 7.4, 10 mM glucose, 0.5 mM CaCl2, 0.8 mM MgCl2, and 0.5 mg/ml BSA. Cells were treated with reagents as indicated. AA release was linear over 30 min. The reactions were stopped at 15 min by removal of the medium. The radioactivity remaining in the cells was solubilized with 0.2 N NaOH and 0.2% SDS and counted, and the results are presented as percentage of [3H]AA released.

Western blotting. Cellular proteins in the membrane or cytosolic fractions were size fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose. The nitrocellulose membranes were blocked with Blotto and blotted with a monoclonal antibody to the HA antigen (12CA5) at a dilution of 1:40. The blots were visualized with the enhanced chemiluminescence system (Pierce) using the horseradish peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:40,000.

Statistical analysis. Concentration-response relationships (EC50 values and Hill coefficients) were determined using GraphPad prism software, and significance was calculated using the Instat two-tailed, unpaired t-test statistics program.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To characterize the response of the CaR to a range of polycations, some of which are nephrotoxic, we expressed the human CaR in HEK-293 cells and studied CaR-stimulated activation of MAP kinase (p42ERK) and PLA2. To be certain that the effects we studied required an active CaR, we also expressed the nonfunctional mutant CaR R796W in HEK-293 cells to control for receptor-independent effects of the polycations (3). We added HA tags to the wild-type and mutant receptors to be certain of protein expression and document similar levels of protein expression. Figure 1A shows a Western blot of membranes from cells that express the expression vector pcDNA3, the wild-type and mutant CaRs, and the HA-tagged wild-type and mutant CaRs. The anti-HA antibody recognizes only the HA-tagged receptors. A nonspecific band from the anti-HA antibody is present at ~75 kDa in all lanes. The wild-type and mutant receptors were expressed at comparable levels in each of the two expression systems. To be certain that the HA-tagged and untagged receptors behave in a similar fashion, we compared activation of p42ERK by HA-tagged and untagged wild-type and mutant (R796W) receptors. We coexpressed the CaRs with an HA-tagged p42ERK and measured ERK activation with a gel-shift assay. Figure 1B shows the response of the HA-tagged p42ERK to the untagged CaR wild-type and mutant R796W (top) and the HA-tagged wild-type and mutant CaR (bottom). Ca2+ and neomycin activated p42ERK through the HA-tagged and untagged CaR in a dose-dependent manner. Neither receptor agonist activated p42ERK when expressed with the mutant receptor R796W (response to neomycin not shown), demonstrating that the effects of these agonists require the active receptor. Figure 1C shows a time course for p42ERK activation in cells that express the HA-tagged CaR by 5 mM Ca2+ or epidermal growth factor as a positive control. For both stimuli, the maximum activation occurred between 5 and 10 min.


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Fig. 1.   A: expression of wild-type (CaRWt) and mutant Ca2+ receptors (CaRmut) in HEK-293 cells. Membranes from cells expressing the vector (pcDNA3), the untagged wild-type CaR, the hemaglutinin antigen (HA)-tagged CaR, the untagged CaR R796W, and the HA-tagged CaR R796W (15 µg/lane) were immunoblotted with the anti-HA antibody 12CA5. The bottom band (~75 kDa) is a nonspecific band due to the anti-HA antibody and is present in all lanes. B: gel-shift assay demonstrating activation of HA-tagged p42ERK via untagged (top) and HA-tagged CaRs (bottom) in HEK-293 cells. Wild-type receptor was expressed in the first 5 lanes; mutant (R796W) receptor was expressed in the last 2 lanes. Ca2+ or neomycin (Neo) concentration to which the cells were exposed for 5 min is shown. After treatment with agonists, cell extracts were prepared and immunoblotted (15 µg/lane) for the HA epitope. In B, the top band represents the active, "shifted" form of p42ERK and the bottom band represents the inactive form. The HA-tagged CaR is not seen in this blot, because its size is greater than the top of each panel. C: time course of stimulation of p42ERK activity by Ca2+ or epidermal growth factor (EGF). Cells were treated with 5 mM extracellular Ca2+ or 10-7µg/ml EGF for the times shown. p42ERK activity was measured by a gel-shift assay (B). Activity of p42ERK was calculated as the ratio of the density of the shifted band to the total p42ERK density and normalized for the response to 1 mM Ca2+.

Table 1 shows the effect of CaR activation on the activities of p42ERK and PLA2 in response to a variety of polycations, including Ba2+, Cd2+, Co2+, Fe2+, Fe3+, Gd3+, Hg2+, Ni+2, Pb2+, and neomycin3+. p42ERK activity increased minimally in response to Fe3+ but did not increase in response to Hg2+. The true concentration of Fe2+ is not certain, because it was converted rapidly to Fe3+ on addition to the cells. In the cells treated with Fe3+, we cannot exclude the possibility that some Fe2+ was also present. In experiments using the R796W mutant, no activation of p42ERK or PLA2 was detected, indicating that these effects require a functional CaR and do not occur through other effects of the cations on the cells. The kinetics of activation of the CaR by Pb2+, Co2+, and Ca2+ were determined as shown in Fig. 2. Dose-response curves for activation of p42ERK (Fig. 2A) and PLA2 (Fig. 2B) by Pb2+, Co2+, and Ca2+ demonstrate that these cations have the same rank order for activation of the CaR. For activation of p42ERK by Pb2+, Co2+, and Ca2+, the EC50 values were 0.14, 0.39, and 3.3 mM, respectively, and for activation of PLA2 the EC50 values were 0.1, 0.7, and 4.2 mM. The Hill coefficients for all polycations were >3. The EC50 for AA release for neomycin was 0.14 mM, and the Hill coefficient was 3.4. 

                              
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Table 1.   Activation of p42ERK or PLA2 by the CaR in response to various divalent cations in HEK-293 cells



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Fig. 2.   Kinetics of activation of p42ERK (A) or phospholipase A2 (PLA2, B) by the CaR in HEK-293 cells in response to Ca2+ (), Co2+ (black-lozenge ), and Pb2+ (). Activity of p42ERK was measured as described in Fig. 1 legend, except it was expressed as a percentage of p42ERK activity stimulated by 5 mM Ca2+. PLA2 activity was measured as [3H]arachidonic acid release in HEK-293 cells that stably express the CaR as described in Table 1.

To determine whether the effects of Ca2+ and another divalent cation, Pb2+, are additive for activation of the CaR at submaximal concentrations, we measured activation of PLA2 by the CaR at four different concentrations of Ca2+, 0.5, 2, 3, and 5 mM, with increasing concentrations of Pb2+ from 17.5 to 500 µM (Fig. 3A). The different Ca2+ concentrations progressively shifted the dose-response curves for Pb2+ to the left. At 5 mM Ca2+, activation was maximal, with no further increase by Pb2+, indicating that Pb2+ and Ca2+ activate the CaR by the same mechanisms and probably through the same sites. With increasing Ca2+ concentrations, the EC50 for Pb2+ was reduced from 0.1 mM at 0.5 mM Ca2+ to 0.018 mM at 3 mM Ca2+. The Hill coefficient was reduced from 4 to 1.2, which may reflect partial activation by 3 mM Ca2+. In similar experiments, we studied the dose-response relationship of the CaR to Ca2+ at four different Pb2+ concentrations (17.5-500 µM). In these experiments (Fig. 3B), increasing concentrations of Pb2+ progressively shifted the EC50 for Ca2+ to lower values, from 4.2 mM in the absence of Pb2+ to 1.1 mM in the presence of 75 µM Pb2+. The Hill coefficient was progressively reduced with increasing concentrations of Pb2+. These results suggest that Ca2+ and Pb2+ activate the CaR via the same mechanism and possibly at the same sites. Their effects are additive in the sense that submaximal concentrations of the two cations combine to produce a greater level of activation than either cation alone, but the maximum level of activation with both cations does not exceed the maximal level of activation with a single cation.


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Fig. 3.   Additive effects of Ca2+ and Pb2+ to activate the CaR. A: activation of PLA2 over a range of concentrations of Pb2+ at 5 mM Ca2+ (black-triangle), 3 mM Ca2+ (), 2 mM Ca2+ (black-lozenge ), and 0.5 mM Ca2+ (). B: activation of PLA2 over a range of Ca2+ concentrations at 500 µM Pb2+ (black-lozenge ), 75 µM Pb2+ (black-triangle), 50 µM Pb2+ (), 17.5 µM Pb2+ (), and 0 µM Pb2+ (). Cells stably expressing the CaR were plated in 24-well trays, deprived of serum, and labeled with [3H]arachidonic acid. Cells were washed and incubated with medium containing 0.5 mg/ml BSA and the indicated concentrations of Ca2+ and Pb2+ for 15 min. Arachidonic acid release was calculated as described in Table 1.

To determine whether cellular responses to activation of the CaR by divalent cations depend solely on expression of the receptor or possibly on other cell-specific factors, we tested the ability of several divalent cations to activate PLA2 in MDCK cells that stably express the CaR. As shown in Table 1, Ca2+, Cd2+, Co2+, and Pb2+ activate the CaR in HEK-293 cells. Figure 4A shows that, in MDCK cells, Ca2+, Cd2+, Co2+, and Pb2+ also activate PLA2 in the cells that express the CaR. Figure 4B shows concentration-response curves for CaR-stimulated PLA2 activity by the CaR in MDCK cells in response to Ca2+, Cd2+, and Pb2+. The curve for Co2+ is not shown, because the magnitude of the response was low (Fig. 4A), and the values are not accurate. The EC50 values for Ca2+ and Pb2+ are comparable to those in the HEK-293 cells: 4.5 mM for Ca2+ and 72 µM for Pb2+. The EC50 for Cd2+ is 75 µM. These ions also activate ERK in a pattern that is similar to that for activation of PLA2 (Fig. 4C). The maximum level of activation is similar for 5 mM Ca2+ and Cd2+ and 0.5 mM Pb2+ but is reduced by approximately one-half for 5 mM Co2+. As shown in Fig. 4D, the CaR is expressed in the MDCK cells, but at a lower level per milligram of membrane protein than in the HEK-293 cells. In the HEK-293 cells the maximum response to all these divalent cations was similar (Table 1), but in the MDCK cells the maximum response to Co2+ is approximately one-half that of the other ions. These results indicate that the CaR responds to a variety of divalent cations in different epithelial cell types and suggest that cell-specific factors in addition to the receptor may determine the specificity of the receptor for ligands and the magnitude of its responses (5, 22).


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Fig. 4.   CaR-dependent activation of the PLA2 and ERK in Madin-Darby canine kidney (MDCK) cells that stably express the CaR and relative levels of expression of the HA-tagged CaR in HEK-293 and MDCK cells. A: activation of PLA2 by 7.5 mM Ca2+, 0.15 mM Cd2+, 5 mM Co2+, and 0.5 mM Pb2+. Open bars, cells that express the CaR; solid bars, cells that express pcDNA3. Cells were treated with the divalent cations for 15 min, and [3H]arachidonic acid release was measured over 15 min as described in Fig. 2B legend. Bars represent means ± SD of triplicate samples and are representative of >= 3 experiments. *Statistically different from 0.5 mM Ca2+ control (P < 0.05) by 2-tailed t-test. B: kinetics of activation of PLA2 by the CaR in MDCK cells in response to Ca2+ (), Cd2+ (black-triangle), and Pb2+ (). Cells were treated with the divalent cations shown, and [3H]arachidonic acid release was measured over 15 min as described in Fig. 2B legend. Values were plotted as a percentage of the [3H]arachidonic acid. C: activation of ERK by Ca2+, Cd2+, Co2+, and Pb2+. MDCK cells were treated with 5 mM Ca2+, Cd2+, or Co2+ or 0.5 mM Pb2+ for 5 min, cell extracts were prepared for measurement of ERK activity, and extracts were immunoblotted with an anti-active ERK antibody and quantitated with densitometry. Bars represent means ± SE of triplicate samples. D: relative levels of expression of the HA-tagged CaR in HEK-293 and MDCK cells. Membrane fractions were prepared from HEK-293 cells that express pcDNA3 (HEK-293), the HA-tagged wild type (CaRWT in HEK-293 cells) or mutant (CaRR796W in HEK-293 cells), MDCK cells that express pcDNA3 (MDCK), and MDCK cells that express the HA-tagged wild-type CaR (MDCK-CaR). Samples (15 µg/lane) were immunoblotted with the anti-HA antibody 12CA5 as described above and visualized with enhanced chemiluminescence.

The only divalent cation tested that did not stimulate the CaR to activate p42ERK or PLA2 was Hg2+. To determine whether Hg2+ was simply inactive as an agonist or reacted with cysteines and prevented activation of the receptor, we tested the effects of other sulfhydral-reactive reagents on CaR-stimulated ERK activity in response to extracellular Ca2+. Figure 5A shows the effects of increasing concentrations of HgCl2, p-chloromercuribenzene sulfonate (PCMBS), and N-ethylmaleimide (NEM) on activation of p42ERK by the CaR. Addition of all three reagents blocked the ability of 5 mM Ca2+ to activate p42ERK with EC50 values of 10-75 µM. After treatment of cells expressing the receptor with PCMBS (a cell-impermeant, reversible agent), activity of the receptor could be restored by removal of the PCMBS and addition of 5 mM Ca2+ in the presence of 1 mM dithiothreitol (DTT; Fig. 5B). However, inhibition of CaR activation by NEM was not reversible with DTT, as would be expected, because NEM covalently modifies free sulfhydryl groups. In similar experiments, treatment of cells with PCMBS or NEM also inhibited CaR-dependent stimulation of ERK activity by 0.5 mM Pb2+. In additional studies not shown, addition of up to 10 mM DTT or 5 mM mercaptoethanol did not activate the receptor and had no effect on the ability of Ca2+ to activate the CaR. These results indicate that the CaR requires free sulfhydryl groups to respond to extracellular Ca2+, Pb2+, and presumably its other ligands but that reducing agents do not interfere with its activation.


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Fig. 5.   Effects of sulfhydral-reactive reagents on activation of p42ERK by the CaR. A: inhibition of CaR-stimulated activation of p42ERK by HgCl2 (), p-chloromercuribenzene sulfonate (PCMBS, black-triangle), and N-ethylmaleamide (NEM, ). HEK-293 cells transiently expressing the CaR and p42ERK-HA were treated with the agents at the concentrations indicated for 5 min, exposed to 5 mM Ca2+, and assayed for p42ERK activity by gel-shift assay as described above. Results are shown as percent stimulation of p42ERK activity above basal (1 mM Ca2+). B: reversal of PCMBS- or NEM-dependent inhibition of CaR activation by dithiothreitol. black-lozenge , Cells treated with 5 mM Ca2+ beginning at 0 min; black-triangle, cells treated with 1 mM Ca2+ beginning at 0 min; , cells treated with medium containing 5 mM Ca2+ and 0.05 mM PCMBS from 0 to 5 min; , cells treated with medium containing 5 mM Ca2+ and 0.1 mM NEM from 0 to 5 min. At 5 min (arrow), medium was replaced with medium containing 5 mM Ca2+ and 1 mM DTT. p42ERK activity was measured by gel shift at 0, 5, and 10 min as described above and is expressed as percentage of basal p42ERK activity.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although cloned by expression as an extracellular Ca2+ sensor, the CaR is not specific for Ca2+ and responds to numerous divalent cations and polycations. We found that all the divalent cations tested except Hg2+ activated the CaR. These divalent cations also appear to act through the same mechanism or possibly the same site on the receptor, because their effects are additive at submaximal, but not maximal, concentrations, and they have similar Hill coefficients. The fact that the effects of the divalent cations were not found in cells expressing the nonfunctional mutant CaR (R796W) indicates that the cellular responses observed are specific for the CaR and do not represent effects of the cations interacting with other proteins or entering the cells. Finally, since two different cell types that express the CaR respond to these divalent cations, these responses represent a general characteristic of the CaR. These findings suggest a novel mechanism by which a number of divalent cations may damage the kidney, nervous system, and other tissues where the CaR is expressed.

Studies over the last 10 years have demonstrated that, in addition to Ca2+ and Mg2+, the parathyroid Ca2+ receptor can be activated by Ba2+, Cr2+, and neomycin (7). Ruat et al. (30) tested a number of the same divalent cations we studied (Ni2+ and Cd2+) in a Chinese hamster ovary cell expression system but found that they either did not activate the receptor (Cd2+) or did so weakly (Ni2+) (30). This apparent discrepancy may result from the use of different concentrations of divalent cations, cell types, or media. The data from Table 1 and Fig. 4 demonstrate that the same CaR responds differently to Co2+ in HEK-293 and MDCK cells. Precedents exist for accessory proteins affecting the ligand specificity of G protein-coupled receptors (5, 22).

Understanding the range of compounds that activates the CaR and the mechanisms by which they do so is important, because many of these compounds are environmental toxins that can cause human disease. Clear nephrotoxicity for Pb2+, Cd2+, and Hg2+ has been described, while nephrotoxic effects of Co2+ and Ni2+ are suspected although not fully documented (37). Because it is a strong oxidizing agent, Fe2+ may also play a role in renal injury in proteinuric states (1). The fact that a number of divalent cations that cause human disease affect tissues that express the CaR, including the kidney, brain, gastrointestinal tract, and bone, suggests that a relationship might exist between exposure to these ions, activation of the CaR, and disease (9).

The pathology of chronic nephrotoxicity from Ca2+ and other divalent cations including Pb2+ and Cd2+ is similar, in that they all lead to interstitial fibrosis and hypertension. Since these ions also activate the CaR, activation of this receptor may be a common mechanism by which they may cause tissue injury. Polycations that activate the CaR could cause toxicity though several mechanisms. Activation of kinase pathways and increased levels of AA metabolites could contribute to the injury (29). Activation of the CaR activates nonselective cation channels that carry Ca2+, Mn2+, and Ba2+ and that could also carry ions such as Pb2+, Cd2+, or Co2+, leading to increases in the intracellular concentrations of Ca2+ or other ions (34, 39). Recent studies suggest the intriguing possibility that long-term activation of the CaR could lead to renal injury by inhibiting growth of epithelial cells and stimulating growth of fibroblasts (10, 13, 35). These effects could lead to loss of organ function with fibrosis, such as is found in kidneys of patients with chronic hypercalcemia or Pb2+ exposure (37). Other mechanisms such as epithelial transdifferentiation certainly contribute to renal interstitial injury and fibrosis, but mechanisms by which the CaR could participate in these processes are not defined (18).

The CaR is expressed in regions of the kidney that are affected by acute and chronic hypercalcemia. The CaR is expressed on the basolateral cell surface of the medullary thick ascending limb of Henle (MTAL) and distal convoluted tubule, in the distal nephron on the apical surface, and in the macula densa (11, 26, 32, 38). Acute nephrotoxicity from hypercalcemia may be caused by a combination of tubular and vascular effects. Tubular effects include inhibition of Na-K-2Cl cotransport in the MTAL and inhibition of vasopressin action in the distal nephron leading to Na+, Cl-, and H2O wasting and volume depletion. The vascular effects could be mediated by CaRs in the macula densa and lead to renal vasoconstriction with a decrease in GFR. Although the mechanism by which Ca2+ reduces GFR is not fully established, 1-10 mM Ca2+ increases renin release from macula densa cells (33). Additionally, patients who are hypercalcemic with hyperparathyroidism have increased levels of renin (27). Hypercalcemia results in increased renal prostanoid production (29). On the basis of the present studies, the increased prostanoids may be attributable to activation of PLA2 by the CaR. Over time, hypercalcemia causes atrophy of the MTAL and interstitial fibrosis, particularly in the inner stripe of the outer medulla (29).

Pb2+ is the best-studied heavy metal that causes renal injury. However, the mechanisms by which it injures the kidney and other tissues are not completely understood (14, 23, 37). In many situations, Pb2+ can bind to cellular proteins in place of Ca2+ and may antagonize the effects of Ca2+, altering cell metabolism and energetics (23). Some of the toxic effects of Pb2+, such as acute lead exposure on the proximal tubule and chronic effects on urate metabolism, are distinct from those of hypercalcemia. However, the chronic effects of Pb2+ exposure and hypercalcemia are similar. Chronic Pb2+ exposure leads to interstitial fibrosis via a mechanism that may involve renal vasoconstriction (37). The Pb2+-induced increase in renin activity could be mediated via Ca2+ receptors in the macula densa (23). The complex relationship among Pb2+ toxicity, dietary Ca2+, and vitamin D suggests that chronic Pb2+ nephrotoxicity involves mechanisms that are involved in the regulation of systemic Ca2+ metabolism (23, 37).

Our data demonstrate that the CaR is highly sensitive to activation by Pb2+, with EC50 values for Pb2+ of ~0.1 mM in HEK-293 cells and 0.072 mM in MDCK cells. This finding is important because of environmental Pb2+ exposure and its potential connection with renal disease. The Pb2+- and Ca2+-binding characteristics of the CaR appear to be similar to those of the intestinal Ca2+-binding protein. The vitamin D-induced intestinal Ca2+-binding protein has a higher affinity for Pb2+ than for Ca2+: ~1.5 × 107 M-1 for Pb2+ and 2 × 106 M-1 for Ca2+ (17). Like our EC50 values for Pb2+ and Ca2+, these values differ by ~10-fold. The intestinal Ca2+-binding protein also binds many of the same divalent cations as the CaR, so promiscuous binding of divalent cations by Ca2+-binding proteins may be a common phenomenon (17).

Although the concentrations of ions such as Pb2+, Cd2+, Co2+, and Fe2+ that are required to activate the CaR are generally higher than would be expected under physiological conditions, the fact that their effects are additive with those of Ca2+ and Mg2+ suggests that even small concentrations could activate the CaR. As demonstrated in Fig. 3, the EC50 for Pb2+ was reduced approximately fivefold from 0.1 to 0.018 mM by increasing the concentration of Ca2+ from 0.5 to 3 mM. Conversely, in the presence of 0.075 mM Pb2+, the EC50 for Ca2+ was reduced from 4.2 to 1.1 mM. The additivity of the effects of Ca2+ and Pb2+ for activation of the CaR appears to be a general property of this receptor, because additivity with similar kinetics was demonstrated for Ca2+ and Mg2+ (30). Normal physiological fluids contain Ca2+, Mg2+, and possibly other polycations. Under these circumstances, small concentrations of Pb2+, Co2+, Cd2+, or Fe2+ could increase the state of activation of the CaR.

Pb2+ toxicity occurs with whole blood Pb2+ concentrations as low as 1.5 µM, and metabolic evidence of toxicity is universally present with whole blood levels of 5 µM. The concentrations of Pb2+ and other metals including Co2+, Cd2+, or Fe2+ in the extracellular fluid are not known with certainty for any organ, and their concentrations could vary within an organ. For example, concentrations could be higher in the medulla than in the cortex of the kidney, or they could be concentrated in synaptic vesicles in the brain or peripheral nervous system, resulting in levels that could activate the CaR. Increased concentrations of free Fe2+ and possibly other ions can be found in biological fluids such as the urine when the pH is reduced (1). In the presence of physiological concentrations of Ca2+, Mg2+, and other polycations, low micromolar concentrations of Pb2+ or other divalent cations could activate the CaR.

Hg2+ inhibited CaR activation, suggesting a critical role for cysteines in activation of the CaR. The extracellular domain of the CaR contains numerous cysteines, some of which are involved in receptor dimerization and some of which may contribute to the tertiary structure of the extracellular domain (4, 15, 24, 28, 36). Mutation of individual cysteines to serine resulted in a variety of phenotypes. These include receptors that had little effect on function, receptors that were not expressed at the cell surface, receptors that were not expressed efficiently, or receptors that did not signal (6). These studies demonstrated that many of the cysteines are important in receptor function but did not address the specific function of the cysteine or whether it was disulfide bonded or reduced (free S-H) (15). Our findings that agents that bind to free cysteines, including Hg2+, PCMBS, and NEM, block activation of the CaR indicate that although some of the cysteines may be bound to other cysteines covalently and confer structure to the receptors, others must be in the reduced form for the receptor to be activated by its ligands. The findings that DTT or mercaptoethanol did not activate the receptor suggest that activation of the receptor does not involve reduction of cysteines. Our data do not exclude the possibility that some cysteines or S-H groups may be inaccessible to the reducing agents. However, this possibility seems unlikely, because PCMBS and NEM could react with critical groups to block receptor activation.

These studies demonstrate that the CaR is a relatively nonspecific polycation sensor that requires free S-H groups for activity. The rank order of EC50 values for the different cations that were studied does not clearly correspond to their rank order for oxidation-reduction potential, size, or solubility. However, these characteristics are normally determined in nonbiological systems, so failure to identify a relationship may not be meaningful. Nevertheless, the ability of many polycations to activate the CaR coupled with its ability to suppress growth of epithelial cells suggests that inopportune activation of the CaR by low levels of these agents could adversely affect the tissues and organs in which it is expressed. These possibilities and the potential contributions of the CaR to disease can be tested using transgenic animals that lack the CaR (20).


    ACKNOWLEDGEMENTS

The authors thank Stephen P. Baker, Raymond C. Harris, and Jeff Sands, and I. David Weiner for helpful discussions.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-41726, the American Heart Association with funds from the Florida Affiliate (R. T. Miller and C. Huang), and the National Kidney Foundation (N. Shiraishi and H. Awata).

Address for reprint requests and other correspondence: R. T. Miller, Nephrology, University of Florida, Box 100224 JHMHC, 1600 SW Archer Rd., Gainesville, FL 32610 (E-mail: millert{at}medicine.ufl.edu).

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.

Received 26 January 2000; accepted in final form 3 August 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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