Background

Aluminum (Al³⁺) has diverse biological effects mediated through activation of a putative extracellular cation-sensing receptor. A recently identified calcium-sensing receptor (CaSR), which has been identified in target tissues for Al³⁺, may transduce some of the biological effects of Al³⁺.

Methods

To test this possibility, we transfected human embryonic kidney 293 (HEK 293) cells with a cDNA encoding the rat CaSR and evaluated CaSR expression by Western blot analysis and function by measurement of intracellular calcium ([Ca²⁺]_i) levels and inositol monophosphate (IP1) generation following stimulation with Al³⁺ and a panel of CaSR agonists.

Results

The CaSR protein was detected by immunoblot analysis in cells transfected with the CaSR cDNA but not in nontransfected HEK 293 cells. In addition, [Ca²⁺]_i levels and IP1 generation were enhanced in a dose-dependent fashion by additions of the CaSR agonists calcium (Ca²⁺), magnesium (Mg²⁺), gadolinium (Gd³⁺), and neomycin only in cells transfected with CaSR. To determine if Al³⁺ activated CaSR, we stimulated cells transfected with rat CaSR with 10 M to 1 mM concentrations of Al³⁺. Concentrations of Al³⁺ in the range of 10 M to 100 M had no effect on [Ca²⁺]_i levels or IP1 generation. In contrast, 1 mM Al³⁺ induced small but significant increases in both parameters. Whereas Gd³⁺ antagonized calcium-mediated activation of CaSR, pretreatment with Al³⁺ failed to block subsequent activation of rat CaSR by Ca²⁺, suggesting a distinct mechanism of Al³⁺ action.

Conclusion

Al³⁺ is not a potent agonist for CaSR. Because Al³⁺ affects a variety of target tissues at micromolar concentrations, it seems unlikely that CaSR mediates these cellular actions of Al³⁺.

METHODS

Creation of vectors for expressing rat calcium-sensing receptor

The rat CaSR cDNA was obtained from Drs. Snowman and Snyder¹³. To permit G418 selection of stable transfectants, a BamHI fragment containing the entire CaSR coding sequence was subcloned into the mammalian expression vector pcDNA 3 (Invitrogen, San Diego, CA, USA). The orientation and the nucleotide sequence of CaSR were confirmed by sequencing both the 5' and 3' ends of the insert.

Culture and transfection of human embryonic kidney 293 cells

Human embryonic kidney (HEK) 293 cells were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), and streptomycin (100 g/ml; all from GIBCO, Gaithersburg, MD, USA) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. HEK 293 cells were subcultured every week after becoming confluent using 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA; GIBCO). Cell viability was assessed by standard dye exclusion techniques (0.1% trypan blue) and was always greater than 95%. To create cell lines stably expressing rat CaSR, our pcDNA 3.0 expression vector containing CaSR or the pcDNA 3.0 vector was transfected into HEK cells by the calcium-phosphate method¹⁸. To enrich the population expressing rat CaSR, G418-resistant cells were selected in complete medium containing 800 g/ml G418. Following G418 selection, HEK 293 cells were evaluated for CaSR expression and function as described later here.

Immunoblotting

Membrane proteins were prepared for the culture cell lines using the method described by Bai et al¹⁹. Confluent cells in 100 mm plates were rinsed twice with phosphate-buffered saline (PBS) and treated with 0.02% ethylenediamenetetraacetic acid (EDTA) in PBS at 37°C for five minutes. The detached cells were collected by centrifugation and resuspended in buffer containing 50 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM ethyleneglycotetraacetic acid (EGTA), 1 mM EDTA, 1 mM phenylmethysulfonyl fluoride (PMSF), a protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) at 4°C. After sonication, nuclei and cell debris were removed by low-speed centrifugation (10,000 r.p.m., 10 min) at 4°C. The supernatant material was centrifuged at 30,000 r.p.m. for 30 minutes in a TLA-100 centrifuge at 4°C to collect the crude membrane proteins pellet. The membrane proteins were solubilized with a buffer containing 1% sodium dodecyl sulfate (SDS), 10 mM Tris-HCl (pH 7.4) with freshly added protease inhibitors (1 mM PMSF, protease inhibitor cocktail). The protein content of each sample was determined by the NanoOrange Protein Quantitation Kit (Molecular Probes, Eugene, OR, USA). All membrane protein extracts were stored at –70°C.

Membrane proteins were separated on 6% SDS-polyacrylamide gel. Separated proteins were transferred to nitrocellulose membrane (0.45 m; Bio-Rad, Richmond, CA, USA) over a 30-minute period at room temperature using a Milliblot semidry transfer apparatus (2.5 mA/cm²; Millipore, Bedford, MA, USA) according to the manufacturer's recommendations. To detect rat CaSR, we used a mouse monoclonal antibody raised to the peptide corresponding to amino acids 214 to 236 of the human CaSR (called ADD) made by Drs. Allen Spiegel and Paul Goldsmith²⁰ and generously supplied by NPS Pharmaceuticals (Salt Lake City, UT, USA). Immunoblotting was performed by incubating blots for 60 minutes at room temperature with SuperBlock (TBS) Blocking buffer (Pierce, Rockford, IL, USA). After washing with 1 TBS containing 0.1% Tween-20 (TBST) for one hour, blots were incubated with 1 g/ml of the ADD antibody (1:1000) overnight at room temperature. Blots were washed with TBST for 60 minutes and incubated with an antimouse Ig, horseradish peroxidase-linked whole antibody (Amersham, Little Chalfont, Buckinghamshire, UK) for 60 minutes at room temperature at a dilution of 1:2500. After washes with TBST, immunoreactivity was detected by an Western blot chemiluminescence system (NEN™ Life Science Products, Boston, MA, USA).

Signal transduction

Measurement of inositol phosphate generation.

Polyvalent cations at various concentrations were added to cell cultures at the specified times indicated later here. Al³⁺ chloride (AlCl₃ 6 H₂0) was obtained from Fisher (Fair Lawn, NJ, USA). Gadolinium chloride hexahydrate was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Calcium chloride anhydrous, neomycin sulfate, were purchased from Sigma Chemical Company (St. Louis, MO, USA). Briefly, HEK 293 cells were plated at a density 2 to 5 10⁵ cells/ml in six-well plastic culture dishes (9.5 cm²/well; Costar, Cambridge, MA, USA). After reaching confluence, cells were equilibrated for 24 hours in DMEM low inositol medium (GIBCO) with 0.1% dialyzed fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 g/ml; all from GIBCO) containing 5 Ci/ml myo-[³H]-inositol (New England Nuclear, Boston, MA, USA). The cultures were washed three times with 2 ml of buffer solution containing 20 mM HEPES, 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgSO₄, and 0.1% dextrose, pH 7.4. Ionized calcium and pH were measured after the addition of the cations using an ion detection electrode (AVL; Omni Modular System, AUL Medical Instruments, Schaffhausen, Switzerland). For studies involving IP measurements, cells were then incubated for the indicated times with the agents to be tested or their vehicle in 2 ml buffer solution at 37°C containing 20 mM lithium chloride. Lithium chloride was included in the incubation medium to inhibit breakdown of IPs. IPs were measured as previously described using anion exchange chromatography²¹. After stimulation with the various cations, the reaction was stopped by aspirating the medium, adding 240 l of 3.3 N perchloric acid, and then placing the samples on ice. After 15 minutes, cell monolayers were scraped off with a plastic spatula and transferred to a plastic microcentrifuge tube. The wells were washed with an additional 960 l of 0.55 N perchloric acid, and this rinse pooled with the initial 240 l wash. Samples were centrifuged for five minutes at 10,000 g, and exactly 1 ml of supernatant was transferred to another microcentrifuge tube. One milliliter of supernatant was neutralized by adding 55 l of 10 N KOH and placing the samples on ice. After 15 minutes, neutralized samples were centrifuged at 10,000 g for five minutes. For chromatography, 0.9 ml of supernatant was added to 9 ml of 5 mM sodium borate and applied to 1.0 ml columns packed with AG1-X8 formate anion exchange resin (Bio-Rad). Columns were washed twice with 10 ml of 5 mM sodium tetraborate containing 60 mM ammonium formate, and IPs were eluted sequentially with 3.5 ml of 0.1 M formic acid containing either 0.2 M, 0.4 M, or 1.0 M ammonium formate for inositol monophosphates (IP1), inositol bisphosphates (IP2), and inositol trisphosphates (IP3), respectively. Columns were washed with 10 ml volumes of the appropriate elution buffer between collections of eluate fractions to remove any residual radioactivity. Eluate fractions were dissolved in 17 ml of Safety-Solve (Research Products International, Mount Prospect, IL, USA) and were quantitated by liquid scintillation counting. Using this method, radioactivity recovered in the IP3, IP2, and IP1 eluate fractions typically averaged 7, 5, and 88% of the total radioactivity recovered in all eluate fractions, respectively.

Cytosolic calcium measurements

Intracellular calcium levels ([Ca²⁺]_i) were measured in confluent HEK 293 cells by fluorescence excitation of cells loaded with the fluorescent probe fura-2 as previously described²¹. Cells were grown on plastic Aclar cover slips (Pro Plastics, Linden, NJ, USA) until confluence and were then incubated for an additional day in DMEM with 0.1% FCS and antibiotics. Cells were loaded for one hour in DMEM with fura 2 acetoxymethyl ester (fura 2-AM; Sigma) added to the culture medium to a final concentration of 5 M. Cells were then washed at 37°C with a buffer solution containing 20 mM HEPES, 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgSO₄, 1 mM NaH₂PO₄, and 0.1% dextrose, pH 7.4, without fura 2-AM. After washing, cover slips were inserted diagonally into curvette and were continuously perfused with the same buffer solution. To insure adequate loading and complete hydrolysis of the methylester, emission was measured at 510 nm over the excitation spectrum from 300 to 400 nm with a Perkin-Elmer LS50 fluorescence spectrometer (Perkin-Elmer, Norwalk, CT, USA). Real-time measurements were monitored by alternating the excitation wavelength between 340 and 380 nm every 1.9 seconds with a microcomputer, and data were derived from the ratio of emission at 510 nm. [Ca²⁺]_i was calculated as described by Grynkiewicz²² using the following formula:

K_D is the dissociation constant of the Ca²⁺-fura 2 complex, and 224 nM was employed in these calculations²². R is the fluorescence emission ratio derived by dividing the fluorescence at an excitation wavelength at 340 nm by the fluorescence excitation wavelength at 380 nm. S_f2 and S_b2 are the fluorescence at an excitation wavelength of 380 nm for Ca²⁺-free dye (S_f2) and for Ca²⁺-bound dye (S_b2). R_max and R_min are the maximal and minimal fluorescence emission ratios, respectively. R_max and S_b2 were experimentally determined at 37°C using 1 M fura 2 dissolved in a solution of the following composition designed to mimic intracellular ionic conditions: 115 mM KCl, 20 mM NaCl, 1 mM MgCl₂, 10 mM HEPES, 2 mM CaCl₂, pH 7.1. This solution was supplemented with 10 mM EGTA to obtain R_min and S_f2.

Statistical analysis

Data are presented as the mean SE of the mean. Statistical significance was assessed using a paired or unpaired t-test as indicated.

RESULTS

Expression of rat calcium-sensing receptor in human embryonic kidney 293 cells

Figure 1 shows the expression of rat CaSR in cells stably transfected with the CaSR cDNA compared with nontransfected HEK 293 cells. We detected three major bands with apparent molecular masses of 140, 165, and 250 kDa in membrane protein preparations from HEK 293 cells transfected with CaSR cDNA (lane 2), which are similar to those observed in human parathyroid tissue (lane 1). These bands were not observed in nontransfected HEK 293 cells (Figure 1, lane 3) or HEK cells transfected with the vector not containing CaSR (data not shown). The 140 and 165 kDa bands may be caused by differential degrees of glycosylation, proteolytic fragmentation arising during purification, or the presence of alternatively spliced products as reported by other investigators²⁰. The 250 kDa band may represent aggregated or dimerized receptor and has also been described by other investigators²³. We did not detect smaller bands that have been observed by immunoblot analysis in other cells and tissues^20,24. These data are consistent with the absence of an endogenous CaSR protein in wild-type HEK 293 cells and the presence of high levels of exogenous CaSR protein in HEK 293 cells transfected with the CaSR cDNA.

Figure 1.

Western blot analysis of calcium sensing receptor (CaSR) expression in human embryonic kidney (HEK) 293 cells. Expression of rat CaSR was assessed by Western blotting, as described in the Methods section, in human parathyroid tissue (lane 1), in HEK 293 cells stably transfected with the CaSR cDNA (lane 2), and in nontransfected HEK 293 cells (lane 3). We detected three major bands of apparent molecular mass of 140, 165, and 250 kDa in membrane protein preparations from HEK 293 transfected with CaSR cDNA. These bands were not observed in nontransfected HEK 293 cells. The 250 and 165 kDa bands predominated in human parathyroid tissue.

Full figure and legend (34K)

Cation-induced increases in [Ca²⁺]_i and inositol phosphate generation in HEK 293 cells transfected with CaSR

Figure 2 shows the effects of extracellular calcium on [Ca²⁺]_i levels in HEK 293 cells stably expressing CaSR. We found that extracellular calcium concentrations above 2 mM were required to elicit an increase in [Ca²⁺]_i levels. A maximal response was obtained at extracellular calcium concentrations of 10 mM. In contrast, increasing extracellular calcium levels failed to elicit an increase in [Ca²⁺]_i in nontransfected cells Figure 2b or cells stably transfected with vector not containing CaSR (data not shown). To assess the response to other CaSR agonists, we examined the effects of gadolinium, neomycin, and magnesium on [Ca²⁺]_i levels in HEK 293 cells with and without CaSR Figure 3. Gadolinium, neomycin, and magnesium, at concentrations known to activate CaSR in other systems¹¹, resulted in similar increments in [Ca²⁺]_i concentrations Figure 3. No response to these agonists was detected in nontransfected HEK or cells stably transfected with vector not containing CaSR (data not shown). These data suggest that transfection of CaSR cDNA confers a functional response to extracellular cations in HEK 293 cells.

Figure 2.

Effects of extracellular calcium on [Ca²⁺]_i levels in HEK 293 cells. Calcium-induced activation of rat CaSR was assessed by monitoring [Ca²⁺]_i levels using fura 2-loaded cells as described in the Methods section. (A) Results for HEK 293 cells stably expressing the CaSR. (B) Results in nontransfected HEK 293 cells. Extracellular calcium concentrations above 2 mM were required to elicit an increase in [Ca²⁺]_i levels in cells transfected with CaSR. A maximal response was obtained at extracellular calcium concentrations of 10 mM. In contrast, increasing the extracellular calcium concentration did not enhance [Ca²⁺]_i levels in nontransfected cells, although the addition of the calcium ionophore ionomycin caused a rapid increase in [Ca²⁺]_i levels. Results are representative of three to six separate experiments.

Full figure and legend (16K)

Figure 3.

Effects of CaSR agonists on [Ca²⁺]_i levels in HEK 293 transfected with rat CaSR. The ability of CaSR agonists to activate rat CaSR was assessed by monitoring [Ca²⁺]_i levels following the addition of gadolinium (A), neomycin (B), or magnesium (C) to the extracellular fluid using fura 2-loaded cells, as described in the Methods section. Additions of gadolinium, neomycin, and magnesium at the indicated concentrations caused similar increments in [Ca²⁺]_i levels in HEK 293 cells transfected with rat CaSR. Results are representative of four to six separate experiments per compound.

Full figure and legend (14K)

We next examined the effects of extracellular cations on IP1 generation in HEK 293 cells stably expressing rat CaSR. As shown in Table 1, IP1 generation was enhanced in HEK 293 cells stably expressing rat CaSR following the addition of calcium, gadolinium, neomycin, and magnesium to the extracellular fluid at concentrations known to activate CaSR in other model systems¹¹. Addition of this same panel of cations to control cells did not significantly enhance PI hydrolysis Table 1. At the concentrations tested, the absolute magnitude to the IP1 response was less with gadolinium and neomycin compared with calcium, as has been observed by other investigators^11,24,25. These data suggest that the CaSR is coupled to PLC in HEK 293 cells, and the pattern of PLC activation induced by calcium, magnesium, gadolinium, and neomycin is similar to the pattern reported by other investigators.

Table 1 - Cation-induced IP1 generation.

Full table

Aluminum-induced increases in [Ca²⁺]_i and inositol phosphate generation in HEK 293 cells transfected with CaSR

Figure 4 compares IP1 generation induced by calcium Figure 4a and Al³⁺ Figure 4b in HEK 293 cells stably transfected with rat CaSR. PI hydrolysis was enhanced, in a dose-dependent fashion, by additions of calcium between 3 and 20 mM to the extracellular fluid. The EC₅₀ value for this response was approximately 4 mM, which is comparable to the EC₅₀ value reported for calcium by other investigators^11,24,25. Additions of Al³⁺ to the extracellular fluid also caused a dose-dependent increase in IP1 generation Figure 4b. At the 0.5 and 1 mM concentrations, Al³⁺ induced significant increases in IP1 generation in HEK 293 cells transfected with rat CaSR. The magnitude of the IP response induced by 1 mM Al³⁺, however, was approximately 10% of the maximal response induced by calcium (compare Figure 4 A and B). Because of decreased solubility at higher concentrations, we were unable to evaluate concentrations of Al³⁺ above 1 mM adequately. More importantly, no significant increase in IP1 generation was detected at the 10, 25, or 100 M concentrations of Al³⁺, which have been shown to activate a putative G-protein receptor-like mechanism in osteoblasts²⁶. Similarly, no increase in Al³⁺-induced PI hydrolysis was detected in nontransfected HEK 293 cells or HEK cells transfected with the vector not containing CaSR (data not shown).

Figure 4.

Effects of cations on inositol monophosphate (IP1) generation in HEK 293 cells transfected with rat CaSR. IP1 generation was measured as described in the Methods section 30 minutes after the addition of calcium (A) or aluminum (B) to the extracellular fluid. PI hydrolysis was enhanced, in a dose-dependent fashion, by additions of calcium between 3 and 20 mM. Aluminum also caused a modest increase in IP1 generation at the 0.5 and 1 mM concentrations. No significant increase in IP1 generation was detected at the 10, 25, or 100 M concentrations of aluminum. Values are the mean SEM of three separate determinations. The asterisk indicates a significant difference at P < 0.05 vs. 2 mM calcium (A) or vs. 10 M aluminum (B).

Full figure and legend (16K)

Because Al³⁺ has the potential to lower pH and possibly alter the ionized calcium concentrations, we performed additional studies to exclude indirect effects of Al³⁺ on the IP response. In this regard, we found that the addition of Al³⁺ resulted in a concentration-dependent reduction in pH from 7.24 at 25 M to a pH of 6.90 at 1 mM Al³⁺ chloride. Because the buffer lacked albumin and phosphate, there were no changes in ionized calcium after the addition of Al³⁺ to the media. The ionized calcium in the media was 0.5 mM before and after the addition of Al³⁺ chloride. Nevertheless, we examined the effects of pH reduction on IP generation in HEK cells expressing CaSR. We found that pH reductions from 7.4 to 6.8 had minimal effects on IP1 generation (data not shown). Moreover, we demonstrated the persistence of Al³⁺-stimulated IP1 generation after correcting the pH of the media to 7.4 (data not shown).

Figure 5 compares the effects of the trivalent cations Al³⁺ Figure 5a and gadolinium Figure 5b on [Ca²⁺]_i levels in cells transfected with rat CaSR. Al³⁺ concentrations of 1 mM caused a small increment in [Ca²⁺]_i levels. This response to Al³⁺ was dependent on the presence of CaSR, because nontransfected HEK control cells failed to respond to Al³⁺ at any of the tested doses (data not shown). Whereas millimolar concentrations of Al³⁺ activated the CaSR, we were unable to demonstrate Al³⁺-induced activation of rat CaSR at concentrations of Al³⁺ between 10 and 100 M, despite previous work that has demonstrated biological effects of Al³⁺ at low micromolar concentrations^16,26,27,28.

Figure 5.

Comparison of aluminum and gadolinium effects on [Ca²⁺]_i levels in HEK 293 cells transfected with rat CaSR. The ability of aluminum (A) and gadolinium (B) to increase [Ca²⁺]_i was assessed by monitoring [Ca²⁺]_i levels following the addition of the trivalent cations to the extracellular fluid using fura 2-loaded cells as described in the Methods section. Additions of 10 or 100 M aluminum did not cause an increase in [Ca²⁺]_i levels. At 1 mM aluminum, a small increment in [Ca²⁺]_i was observed that did not prevent the subsequent activation of CaSR by extracellular calcium. In contrast, the addition of 50 M gadolinium to the extracellular fluid caused an increase in [Ca²⁺]_i levels and blocked subsequent calcium-induced increases in [Ca²⁺]_i levels. Results are representative of four to six separate experiments per compound.

Full figure and legend (11K)

Pretreatment with Al³⁺ did not prevent the subsequent activation of CaSR by extracellular calcium Figure 5a. In contrast, pretreatment with gadolinium prevented subsequent calcium-induced increases in [Ca²⁺]_i in HEK 293 cells transfected with CaSR Figure 5b. Whether this apparent loss of receptor responsiveness induced by gadolinium represents gadolinium blockade of calcium binding to the receptor, down-regulation of CaSR receptor or other actions of gadolinium to modulate G-protein–coupled receptor function requires further study. Nevertheless, these results suggest that high-dose Al³⁺ effects on CaSR may be mediated by a mechanism distinct from gadolinium.

DISCUSSION

There is compelling evidence suggesting that extracellular Al³⁺ cation at micromolar concentrations activates a G-protein–coupled receptor-like signaling pathway in certain cells by a mechanism independent of its known effects to facilitate fluoride activation of G proteins^17,26. The overlapping cation specificity of the Al³⁺ response with that of CaSR agonists calcium, gadolinium, and neomycin suggests that CaSR may mediate the response to Al³⁺ in some cell systems. Therefore, we performed the current studies to determine if Al³⁺ is an agonist for the known CaSR. We found that Al³⁺ did not activate CaSR at bioactive micromolar concentrations Figure 4 and 5. With the possible exception of the effect of millimolar Al³⁺ suppressing PTH secretion in isolated parathyroid cell cultures⁹, the concentrations of Al³⁺ required to activate CaSR in HEK 293 cells greatly exceed those needed to stimulate a putative cation-sensing mechanism in other model systems^16,27,28. In this regard, micromolar concentrations of extracellular Al³⁺ stimulate a cell proliferative response in a variety of cell lines^16,27,28, as well as inhibit agonist-induced adenylyl cyclase activity and induce a PKC-dependent induction of the serum response promoter element in MC3T3-E1 osteoblasts^8,16. Our findings are consistent with other recent studies demonstrating no effect of Al³⁺ at concentrations between 1 and 100 M on IP production in CCL39 fibroblasts transfected with CaSR²⁵. These data suggest that the known CaSR does not transduce the biologically important actions of Al³⁺.

We did find, however, that pharmacological concentrations of Al³⁺ weakly activate CaSR transfected into HEK 293 cells as measured by PI-PLC activation. In this regard, demonstrable activation of CaSR occurred in the millimolar range Figure 4 and 5. Even at these high concentrations, the increments in [Ca²⁺]_i levels and PI hydrolysis in response to Al³⁺ were small in comparison to all other CaSR agonists tested Figure 4. We did not test to see if additional stimulation of CaSR occurred at even higher concentrations because the limited solubility of Al³⁺ makes such studies difficult to perform. Nevertheless, it is likely that Al³⁺ acts as a partial agonist for CaSR, although we cannot exclude the possibility that the observed effect of high-dose Al³⁺ may represent a nonspecific effect on this cation receptor. In spite of aluminum's ability to activate CaSR at high concentrations, it failed to reproduce the effect of gadolinium and calcium on CaSR Figure 5. Indeed, Al³⁺ failed to block subsequent CaSR activation by calcium, whereas pretreatment with the CaSR agonist gadolinium successfully blocked the ability of calcium to activate CaSR. This inability to mimic the effect of other agonists suggests that Al³⁺ has distinct effects on CaSR.

The current finding that Al³⁺ is a weak agonist for CaSR and the prior observations that Al³⁺ activates a G-protein–coupled receptor pathways by mechanisms independent of fluoroaluminate¹⁷ suggest that another cation-sensing mechanism exists that mediates the effects of Al³⁺ and possibly other cations. Indeed, there is growing evidence for the existence of a cation-sensing receptor that is distinct from CaSR. Several osteoblasts and fibroblast cell lines that exhibit responses to cations have not been shown to express CaSR^16,29,30. Although recent studies have identified CaSR in low abundance in certain osteoblast precursors and fibroblastic cell lines³¹, no studies have linked the functional response to Al³⁺ and other cations to the presence of known CaSR in these cells. Second, there are apparent differences in cation specificity, namely responsiveness to Al³⁺ but not magnesium, that suggest the presence of a functionally distinct extracellular cation-sensing mechanism¹⁶. Finally, the second-messenger coupling associated with Al³⁺ activation of the putative CaSR-like response differs from that of CaSR¹⁶. For example, in cation-responsive cells that lack known CaSR, Al³⁺, gadolinium, neomycin, and calcium stimulate proliferation but do not stimulate phosphoinositide turnover or [Ca²⁺]_i transients, as would be expected if CaSR were mediating the response. Therefore, it seems unlikely that activation of CaSR is sufficient to explained the cation-sensing response in osteoblasts, which may be mediated by a cation-sensing receptor distinct from the known CaSR.

In conclusion, prior studies indicate the presence of an extracellular cation-sensing receptor-like mechanism that is activated by micromolar concentrations of Al³⁺. Our studies of the transfected rat CaSR indicate that the concentration of Al³⁺ required to activate CaSR exceeded that required to activate G-protein–coupled receptor-like responses by extracellular Al³⁺ in other cells. These findings, taken together with the difficulty in demonstrating the presence of the CaSR in some cell culture systems that display a functional response to Al³⁺ and the differences in cation specificity and signal transduction pathways activated by Al³⁺, are inconsistent with the simple notion that Al³⁺ is activating CaSR in osteoblasts. Rather, our findings add further support for the existence of another extracellular cation-sensing mechanism that resembles CaSR but that is molecularly and functionally distinct. Given the apparent selectivity of Al³⁺ effects on this putative CaSR-like response, Al³⁺ might be used as a tool to identify the putative novel extracellular cation-sensing mechanism.

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Acknowledgments

This work was supported in part by grants RO1-AR37308 and RO1-AR43468 from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (L.D.Q.), and R29-DK47333 from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (R.F.S.). Dr. Spurney is also an established investigator of the American Heart Association. The authors thank Ms. Lauren L. Green for secretarial assistance in the preparation of this manuscript and Dr. Marc K. Drezner for his review of the manuscript.