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Am J Physiol Cell Physiol 292: C1895-C1905, 2007. First published January 31, 2007; doi:10.1152/ajpcell.00404.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Ca2+ mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

Emmanuel M. Awumey,1 Allyn C. Howlett,2 James W. Putney, Jr.,3 Debra I. Diz,4 and Richard D. Bukoski1,{dagger}

1Cardiovascular Disease Research Program, 2Neuroscience of Drug Abuse Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham; and 3Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park; and 4Hypertension, Vascular Diseases Center and Department of Physiology, and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Submitted 25 July 2006 ; accepted in final form 22 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The rat dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca2+ concentration ([Ca2+]i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+ sensitivity with an EC50 of 1.75 ± 0.10 mM. NPS R-467 and Gd3+ activated the CaR. When [Ca2+]e was successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by ~50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C (PKC){alpha} levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]i transients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+ mobilization following CaR activation.

desensitization; protein kinase C

IN THE PARATHYROID GLAND, small changes in extracellular calcium (Cae2+) concentration ([Ca2+]e) modulate intracellular calcium (Cai2+) concentration ([Ca2+]i) by activating a calcium-sensing receptor (CaR) (7). The CaR belongs to a super family of G protein-coupled receptors (GPCRs), which include the metabotropic glutamate receptors (mGluR) (26, 33), {gamma}-aminobutyric acid type B receptors (GABABR) (29), putative pheromone receptors (50) and putative taste receptors (23). The receptor has a large NH2-terminal extracellular domain with multiple glycosylation sites, seven transmembrane-spanning domains, and a COOH-terminal intracellular tail with phosphorylation sites (14, 49). These domains have distinct properties that confer ligand binding and G protein-coupling specificity.

Molecular evidence for the existence of the CaR was initially provided by Brown et al. (6), who demonstrated the presence of a novel protein (with homology to the mGluR) in bovine parathyroid cells. Since then, the presence of similar receptors has been reported in kidney (1, 46), keratinocytes (4, 37), brain (49), intestinal epithelia (11, 16), and bone (40). It is well established that the CaR provides the primary mechanism for [Ca2+]e-mediated regulation of the release of parathyroid hormone as a result of parathyroid cells sensing small changes in serum [Ca2+]e (7), a process similar to the mechanism involved in catecholamine- and small peptide-induced G-protein coupled activation of cell surface receptors (22). However, the function of this receptor in other tissues has not been well characterized.

In earlier studies, we demonstrated that the dorsal root ganglion (DRG), which houses cell bodies of sensory nerves that send afferent processes to tissues, such as the perivascular adventitia, expresses the message encoding the CaR (8). We later showed that a protein immunoreactive with an antibody raised against the human parathyroid CaR is present in a subpopulation of periadventitial nerves (55). Furthermore, we determined that the 5' untranslated region of the mRNA that gives rise to the DRG CaR cDNA is significantly different from those of the parathyroid and kidney, suggesting that this protein is a product of the expression of alternate exons. These findings and physiological studies led us to hypothesize that activation of the perivascular CaR results in the release of a hyperpolarizing vasodilator compound, which relaxes adjacent smooth muscle cells (8, 9, 27). We have since cloned the rat DRG CaR cDNA into an EGFP vector (EGFP-N3) and functionally expressed it, in frame, as a fusion to the NH2-terminus (57). We also showed that the COOH-terminal amino acid sequence is considerably different from that of the CaR found in human parathyroid gland (57). This difference probably determines the behavior of the rat DRG CaR, including desensitization and coupling of G proteins in the signal transduction pathway. The present study was undertaken to further characterize this novel CaR because of its potential importance in the control of sensory nerve and vascular function (35, 55, 56) as well as its potential as a therapeutic target.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. G418 sulfate was from BioWhittaker (Walkersville, MD), 2-amino-ethyldiphenyl borate (2-APB), ionomycin and phorbol myristate acetate were from Calbiochem (La Jolla, CA), and gadolinium chloride was from Sigma (St. Louis, MO). The enhanced chemiluminescence (ECL) Western blotting system was from Amersham Biosciences (Piscataway, NJ), and DMEM, OPTI-MEM I reduced serum medium, Hanks' balanced salt solution (HBSS), fura 2-AM, Pluronic F-127, penicillin/streptomycin (100x), trypsin/EDTA (0.25% trypsin/1 mM EDTA-4Na), TRIzol reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). Phosphorylated protein kinase C (PKC) p-PKC{alpha}(Ser657) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), NPS R-467, NPS S-467, and CaR monoclonal antibody, raised against a synthetic peptide corresponding to residue 214–235 of the extracellular domain of the human parathyroid CaR, were gifts from NPS Pharmaceuticals (Salt Lake City, UT). This antibody is now available commercially as MA1–934 (Affinity Bioreagents, Golden, CO). All other chemicals used were of the purest grade available commercially.

Stable transfection of HEK293 cells expressing EGFP or CaR.EGFP. In-frame fusion of the cDNA encoding full-length DRG CaR with EGFP was carried out as previously described (57). HEK293 cells were grown as monolayer cultures in DMEM, supplemented with 10% FBS and penicillin/streptomycin (100 U ml–1·100 µg·ml–1) at 37°C in 5% CO2, and transfected with the CaR cDNA-EGFP fusion construct using Lipofectamine 2000. Briefly, a mixture of 4 µg plasmid DNA and Lipofectamine 2000 in OPTI-MEM I reduced serum medium was added to cells grown in DMEM (plus 10% FBS, but without penicillin/streptomycin) in 100 mm dishes for 24 h. After incubating at 37°C in 5% CO2 for 4 h, the medium was replaced with DMEM, supplemented with 10% FBS and penicillin/streptomycin (100 U ml–1/100 µg·ml–1) and cells grown for 48 h to allow expression of the CaR.EGFP fusion protein. Cells were then trypsinized and replated in fresh medium and the incubation continued overnight, followed by addition of G-418 sulfate (600 µg/ml). The medium was changed every 2–4 days for 3 wk to allow colonies of resistant cells to grow. Individual colonies were then selected and grown to generate cell lines (HEK-CaR.EGFP) stably expressing the CaR.EGFP fusion protein. HEK293 cells transfected with EGFP vector (HEK-EGFP) served as control. Cells were separated by fluorescence-activated cell sorting (FACS) to obtain populations uniformly showing green fluorescence and assayed for gene expression. A homogenous population of HEK293 cells, stably expressing the CaR.EGFP fusion protein were grown on 25 mm glass coverslips for 48 h and analyzed. For the present study, a population of cells in passages 1–5 (P1–P5) was used. Cells collected after sorting are designated as P0.

Microfluorimetric measurement of [Ca2+]i in HEK-CaR.EGFP cells. [Ca2+]i was measured in cells loaded with the fluorescent indicator, fura 2. Briefly, cells were washed twice with HBSS and loaded for 30 min with 5 µM fura 2-AM, dissolved in HBSS supplemented with Pluronic F-127 (0.02%). After excess dye was washed off, the coverslip was mounted in an Attofluor cell chamber in physiological salt solution composed of (in mM) 150 NaCl, 5.4 KCl, 1.2 MgSO4, 1.2 7H2O; 1.2 NaH2PO4, 6.0 NaHCO3, 0.25 CaCl2, 5.5 glucose, and 20 HEPES, pH 7.4, and placed on the stage of an Axiovert 100S inverted microscope equipped with a Zeiss Fluar x40 fluorescence oil-immersion objective. In experiments with gadolinium (Gd3+) chloride, NaH2PO4, and NaHCO3 were omitted from the solution. An imaging system (Dual-wavelength Fluorescence Imaging System; PTI, Birmingham, NJ) was used to measure [Ca2+]i. Cells, loaded with fura 2, were excited at 340 nm and 380 nm with a xenon light source (75 Watt Xe Compact Arc Lamp) and emission at 510 nm was captured by an IC-300 intensified charge-coupled device camera. The images were transmitted to a computer and processed using the ImageMaster Pro Ratio Fluorescence Imaging software, with a macro based on the concentration equation of Grynkiewicz et al. (20) to transform the data from ratio into Ca2+ concentrations. The [Ca2+]i data are reported as emission ratios (F340/F380) or nM [Ca2+]i. In all experiments, 20–30 cells from a single field were analyzed and the means calculated, and applications of 10 µM ionomycin and 20 mM EGTA followed Ca2+ response determinations to obtain the maximum possible level (Rmax) and the minimum level (Rmin) in Cai2+, respectively.

RT-PCR analysis of RNA extracted from control and transfected HEK293 cells. To demonstrate the presence of the CaR message in HEK-CaR.EGFP cells, total cellular RNA was extracted from HEK293, HEK-EGFP, and HEK-CaR.EGFP cells that had been grown in T75 flasks for 48 h. Total cellular RNA was amplified by RT-PCR with the same primers used to amplify the full-length CaR cDNA for cloning into the EGFP vector. The PCR products were subjected to restriction analysis using PstI and EcoRI endonucleases and the fragments visualized by ethidium bromide staining, after separation by electrophoresis on 1% agarose gel.

Western blot analysis of proteins extracted from transfected HEK293 cells. Cells were grown in 100 mm dishes for 48 h and total membrane protein extracted by homogenization in ice-cold solution of 10 mM Tris (pH 7.5), 0.25 M sucrose, and 3 mM MgCl2 with freshly added protease inhibitor cocktail (1 µM leupeptin, 1 mM dithiothreitol, 1 µM pepstatin, 5 mM pefabloc, 1 µg/ml bestatin, and 7 µg/ml calpain inhibitor II). The homogenate was then passed through a 22-gauge needle, centrifuged at 800 g for 10 min, and the supernatant from this step centrifuged at 100,000 g for 2 h. The resultant pellet was then dissolved in Tris buffer (pH 7.5), with 1% Triton X-100 and freshly added protease inhibitor cocktail as above, size separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was then blotted with the CaR antibody (19, 38, 39), incubated with horseradish peroxidase-conjugated anti-mouse IgG, and visualized using the ECL method. Membrane proteins were also prepared from HEK-CaR.EGFP cells stimulated with 1 mM Ca2+ for specified time periods and analyzed for phosphorylated-PKC (p-PKC) with anti-p-PKC antibody.

Statistical analysis. Data were analyzed using SigmaPlot 9.0 and SigmaStat 3.1 computer software programs (SYSTAT, Port Richmond, CA). Comparisons between treatment groups were made by ANOVA. Differences having P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RT-PCR analysis of total RNA and Western blot analysis of proteins. A population of HEK293 cells, stably expressing the CaR.EGFP fusion protein and showing uniform EGFP fluorescence as determined by FACS analysis (data not shown), was maintained in cell culture medium containing 600 µg/ml of G-418 sulfate and used in all experiments described in the present study. Figure 1A is a photograph of ethidium bromide-stained agarose gel analysis of RT-PCR products obtained with total RNA extracted from HEK293, HEK-EGFP, and HEK-CaR.EGFP cells and amplified with the CaR cDNA primers. Only RNA from the HEK-CaR.EGFP cells gave a product of similar size as the DRG CaR cDNA. The identity of the cDNA was established by restriction analysis using PstI and EcoRI, which cut the DNA at positions 1,369 bp and 1,567 bp, respectively in the full length DRG CaR cDNA sequence. Analysis of protein extracts from cells expressing EGFP protein alone or CaR.EGFP fusion protein is shown in Fig. 1B. The results show the expression of a protein doublet with molecular weights of 168–175 and 185 kDa in HEK-CaR.EGFP cells that was absent in HEK-EGFP cells. The size of the lower band corresponds to the fusion of EGFP (28 kDa) with the high-mannose, immature forms (140 kDa) of the CaR, and the upper band is probably a fusion of EGFP to a complex carbohydrate, mature form (157 kDa).


Figure 1
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  		Fig. 1. Expression of dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) cDNA in human embryonic kidney (HEK)293 cells stably transfected with enhanced green fluorescent protein (pEGFP)-CaR cDNA plasmid construct and Western blot analysis of cellular proteins. A: total RNA was extracted from 48-h cell cultures and used for RT-PCR with the same primers used in the cloning of the DRG CaR. Lane 1, 1 kb DNA ladder; lane 2, HEK293; lane 3, HEK-EGFP; lane 4, HEK-CaR.EGFP; lane 5, EcoRI digest of CaR cDNA (2.4 kB, 1.2-kB fragments); lane 6, PstI digest of CaR cDNA (2.5 kB, 1.03-kB fragments). Only RNA isolated from HEK-CaR.EGFP cells gave a product similar in size to the DRG CaR cDNA. The approximate sizes of the CaR cDNA restriction fragments are indicated. B: protein extracts from HEK-EGFP and HEK-CaR.EGFP cells were analyzed using a CaR monoclonal antibody and horseradish peroxidase (HRP)-conjugated IgG, and developed with the enhanced chemiluminescence (ECL) system. The HEK-CaR.EGFP cells expressed a doublet consistent with the expression of the CaR protein in HEK293 cells.

 
Intracellular Ca2+ transients. Responses of HEK-EGFP and HEK-CaR.EGFP cells, grown on glass coverslips, to Cae2+ are shown in Fig. 2A. The ratios of Rmax/Rmin, calculated from mean values were 6.37 and 6.33, respectively, for EGFP- and CaR.EGFP-expressing cells, indicating that cells expressing CaR.EGFP and control were identical in their ability to take up fura 2-AM and convert it to the active fluorophore. The addition of 10 µM ionomycin provided a mechanism for demonstrating maximum [Ca2+]i in response to an exogenous ionophore (Rmax), and 20 mM EGTA reduced the ionomycin-induced [Ca2+]i peak (Rmin). Increasing total [Ca2+]e from 0.5 to 1 mM in HEK-CaR.EGFP caused a transient increase in [Ca2+]i, which decayed to a plateau over time. The [Ca2+]i leveled off at a higher concentration than basal. No [Ca2+]i transients were observed in HEK-EGFP cells. Earlier studies indicated that the [Ca2+]i transient was not affected by the presence of 10 µM nifedipine, a Ca2+ channel blocker, in transiently transfected cells (57). The response was blocked by 30 µM 2-APB (Fig. 2B), which blocks store-operated Ca2+ entry (SOCE), inositol triphosphate (IP3) receptor and transient receptor potential (TRP) channels. Responses to Cae2+ were also obtained for several other clones with different levels of EGFP fluorescence intensity, four of which are shown in Fig. 2C. Figure 2D shows [Ca2+]e-dependent [Ca2+]i oscillations observed in some cells upon exposure to 2, 4, and 10 mM Ca2+e. The oscillation frequencies varied from 1/min at 2 mM Ca2+ to 2/min at 4 mM Ca2+, a twofold increase. At the higher [Ca2+]e of 10 mM, however, these oscillations disappeared.


Figure 2
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  		Fig. 2. Changes in intracellular Ca2+ concentration ([Ca2+]i) in HEK-EGFP and HEK-CaR.EGFP cells upon activation by extracellular Ca2+ (Cae2+) determined by microfluorimetric analysis. A: cells, grown on glass coverslips for 48 h, were loaded with 5 µM fura 2-AM and responses to 1 mM Cae2+ were determined in physiological salt solution (PSS). Maximum and minimum Ca2+ responses were determined by 10 µM ionomycin and 20 mM EGTA, respectively. B: responses of HEK-CaR.EGFP cells with (broken line) or without (solid line) 30 µM 2-APB to stimulation with 1 mM Cae2+. Cells were preincubated with 2-APB in PSS (containing 0.5 mM Ca2+) for 10 min before measurement of responses. The data are representative of five separate experiments carried out under similar conditions. C: I, responses of four different clones of HEK-CaR.EGFP cells to 1 mM Cae2+, determined in PSS with 0.25 mM Ca2+. II, bar chart indicating changes in [Ca2+]i. The data represent differences between peak and basal Cai2+ levels for each clone (*P < 0.001). D: [Ca2+]e-dependent [Ca2+]i oscillations in HEK-CaR.EGFP cells. Cells, grown on coverslips, were loaded with 5 µM fura 2-AM for 30 min and exposed to the indicated concentrations of Ca2+ for the specified time interval. Oscillations in cells were observed at 2 and 4 mM but disappeared at 10 mM. The data are representative of several experiments carried out under similar conditions.

 
NPS R-467 and Gd3+, activators of the human parathyroid CaR also stimulate the DRG CaR (Fig. 3). NPS R-467 (20 µM) and Gd3+ (50 µM) induced [Ca2+]i transients in these cells with a time course of intracellular Ca2+ rise and decay that resembled the response to 1 mM [Ca2+]e. No measurable responses were observed with the NPS S-467 isomer (Fig. 3C). As shown in Fig. 4A, stimulation of cell preparations on separate glass coverslips with single concentrations of Cae2+ indicated that changes in [Ca2+]i were related to [Ca2+]e, increasing in response to a concentration as low as 0.5 mM and reaching saturation at 10 to 20 mM Ca2+ (Fig. 4B). The EC50 for activation of the receptor, obtained from a nonlinear fit of data to a hyperbolic curve (R2 = 0.99), was 1.75 ± 0.10 mM with a Hill slope of 2.28 ± 0.26.


Figure 3
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  		Fig. 3. Effect of activators of the parathyroid CaR on Cai2+ transients in HEK-CaR.EGFP cells. Responses to 20 µM NPS R-467 (A) and 50 µM Gd3+ (B) are shown. Gd3+ responses were measured in PSS without NaH2PO4 and NaHCO3. Insets, data from 5 separate experiments, carried out under similar conditions, were analyzed for changes in Cai2+ and basal and peak responses are shown as means ± SE. C: comparative responses of cells to 10 µM NPS R-467 and its isomer, NPS S-467 under similar conditions. *P < 0.5, significantly different from basal.

 

Figure 4
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  		Fig. 4. Effect of additions of graded [Ca2+]e to HEK-CaR.EGFP cells on Cai2+ transients. A: [Ca2+]e-dependent changes in [Ca2+]i was measured in cells grown on glass coverslips and loaded with 5 µM fura 2-AM in Hanks' balanced salt solution (HBSS). The indicated concentrations of Cae2+ were added to cells on separate coverslips, in PSS containing 0.25 mM Ca2+, to obtain the responses shown. The tracings are representative of four experiments carried out under similar conditions. B: concentration-response relationship of [Ca2+]e-dependent mobilization of [Ca2+]i in HEK-CaR.EGFP cells. Differences between basal and peak [Ca2+]i were calculated from four separate experiments (see Fig. 4A) and plotted against [Ca2+]e. Values are means ± SE. The nonlinear regression curve was determined (R2 = 0.99) and the parameters of Hill slope (2.28 ± 0.26) and EC50 (1.75 ± 0.10) were extrapolated.

 
Desensitization. Cumulative increases in [Ca2+]e from 0.25 to 4 mM resulted in reduction of the peak [Ca2+]i (Fig. 5A). When responses were plotted against the [Ca2+]e, it was apparent that increasing the stimulus resulted in a desensitization of the response, which occurred at exposure to as little as 0.5 mM [Ca2+]e (Fig. 5B). The decrement in response was not overcome by increasing the stimuli to 2–4 mM [Ca2+]e. This desensitization response was also apparent in individual cells, as depicted in Fig. 5C, which shows the responses of six cells within a field to the serial addition of 1 mM increments in [Ca2+]e. Responses to repeated applications of 10 mM Ca2+ to the same cell preparation were determined between washes with physiological salt solution containing 0.25 mM Cae2+. As shown in Fig. 6, after the initial response, subsequent [Ca2+]i peaks were significantly lower (P < 0.05).


Figure 5
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  		Fig. 5. Effect of Cae2+ on Cai2+ mobilization in HEK-CaR.EGFP cells. A: cumulative [Ca2+]e-dependent changes in [Ca2+]i determined in HEK-CaR.EGFP cells. Responses of cells, loaded with fura 2, were determined in PSS containing 0.25 mM Ca2+. Basal [Ca2+]i at 0.25 mM [Ca2+]e was 96.7 ± 0.8 nM (n = 5). [Ca2+]e was increased successively from 0.25 to 4 mM over the indicated time period without being washed between additions. The [Ca2+]i peak was reduced as [Ca2+]e was raised to 4 mM. B: [Ca2+]e-dependent reduction in mobilization of [Ca2+]i resulting from cumulative addition of Cae2+. Differences between trough and peak [Ca2+]i were calculated from five individual experiments and plotted as means ± SE. C: responses of individual cells to cumulative applications of Ca2+. Tracings from six isolated cells are depicted as 1 mM increments of [Ca2+]e were added to the PSS solution.

 

Figure 6
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  		Fig. 6. Effect of high [Ca2+]e on [Ca2+]i levels in HEK-CaR.EGFP cells. A: cells, loaded with 5 µM fura 2-AM, were exposed alternatively to 10 and 0.25 mM Cae2+, followed by washes in PSS containing 0.25 mM [Ca2+]e to obtain changes in [Ca2+]i over the time course indicated. The data are typical of four separate experiments carried out under similar conditions. B: histogram showing the mobilization of [Ca2+]i resulting from repeated additions of 10 mM Cae2+. Differences between basal and peak [Ca2+]i were calculated from four individual experiments and plotted as means ± SE. *P < 0.05, significantly different from peak 1.

 
To determine whether Gd3+ and NPS R-467 also affect peak [Ca2+]i levels in cells expressing the CaR, responses to cumulative concentrations of these compounds were measured. Repeated applications of 1 or 10 µM NPS R-467 (Fig. 7) and 50 or 100 µM Gd3+ (Fig. 8) caused reductions from the initial peak [Ca2+]i. No measurable responses to NPS S-467 were observed (Fig. 7C).


Figure 7
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  		Fig. 7. Effect of cumulative additions of the calcimimetic, NPS R-467, and its inactive isomer, NPS S-467, on [Ca2+]i levels in HEK-CaR.EGFP cells. A: NPS R-467 (1 or 10 µM) was added repeatedly to cells, grown on glass coverslips and loaded with 5 µM fura 2-AM, in PSS medium containing 0.25 mM Ca2+. The data are representative of three to seven experiments carried out under similar conditions. B: histogram showing the mobilization of [Ca2+]i resulting from repeated additions of NPS R-467 to HEK-CaR.EGFP cells. Differences between basal and peak [Ca2+]i were calculated (see A) and plotted against the cumulative concentrations of NPS R-467. Values are means ± SE. *P < 0.05, significantly different from first application. C: NPS R-467 (10 µM) and NPS S-467 (10 µM) were added repeatedly to different cell preparations loaded with 5 µM fura 2-AM, in PSS medium containing 0.25 mM Ca2+. The data are representative of three experiments carried out under similar conditions. **P < 0.05, significantly different from third application; #P < 0.001, significantly different from 1 µM.

 

Figure 8
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  		Fig. 8. Effect of cumulative additions of Gd3+ on [Ca2+]i levels in HEK-CaR.EGFP cells. A: Gd3+ (50 or 100 µM) was cumulatively added to cells, grown on glass coverslips, and loaded with 5 µM fura 2-AM, and assayed for changes in [Ca2+]i in PSS medium containing 0.25 mM Ca2+ (without NaH2PO4 or NaHCO3). B: histogram showing mobilization of [Ca2+]i resulting from repeated additions of Gd3+ to HEK-CaR.EGFP cells. Differences between basal and peak [Ca2+]i were calculated from A and plotted against Gd3+ concentration. Values are means ± SE. The data is representative of several experiments carried out under similar conditions. *P < 0.05, significantly different from 100 µM; **significantly different from first application.

 
Effect of PKC on Ca2+i mobilization. A common mechanism for feedback regulation of GPCRs is through activation of PKC (32). GPCRs are regulated by phosphorylation-dependent and -independent mechanisms. To determine whether Cae2+-induced desensitization of Cai2+ mobilization involved PKC activation, we examined the disposition of PKC{alpha}, a conventional PKC isoform, following stimulation of the CaR. This isoform has been shown to co-localize with the CaR in lipid rafts in bovine parathyroid cells (30, 52), and is abundantly expressed, endogenously in HEK293 cells (51). We determined Cae2+-induced association of phosphorylated PKC{alpha} (pPKC{alpha}) with membrane proteins over time as well as the effects of pharmacological manipulations of PKC activity on Cai2+ mobilization. Increasing Cae2+ from 0.25 to 1.25 mM increased Cai2+ mobilization and resulted in increased pPKC{alpha} levels associated with cell membranes (Fig. 9). However, as shown in Fig. 10A, acute activation of PKC by 100 nM phorbol myristate acetate (PMA) had no effect on CaR-mediated [Ca2+]i signals. After chronic stimulation of cells with 100 nM PMA for 24 h, a procedure expected to cause downregulation of PKC activity, there was a partial reduction in Cae2+-activated [Ca2+]i signals (Fig. 10B). By contrast, [Ca2+]i signaling through endogenous muscarinic receptors was almost completely prevented by this concentration of PMA (data not shown).


Figure 9
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  		Fig. 9. Effect of Cae2+ on phosphorylated PKC levels in HEK-CaR.EGFP cells. Membrane proteins extracted from cells treated with 1.25 mM Cae2+ for the indicated time periods, were separated by SDS-PAGE (60 µg/lane), transferred onto nitrocellulose membranes, and blotted with pPKC{alpha}(Ser657) polyclonal antibody. Blots were developed and visualized by the ECL method. The data are representative of 3 experiments under similar conditions.

 

Figure 10
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  		Fig. 10. Effect of acute and chronic applications of phorbol myristate acetate (PMA) on CaR-mediated mobilization of Cai2+ in HEK-CaR.EGFP cells. A: cells grown on glass coverslips were loaded with 5 fura 2-AM and incubated with 100 nM PMA for 20 min. The response to cumulative addition of 1 mM Ca2+ was determined. B: cells grown on glass coverslips were treated with 100 nM PMA (broken line) or vehicle (solid line) for 24 h and loaded with 5 µM fura 2-AM before measurement of responses to cumulative addition of 1 mM Cae2+. The data are typical of five separate experiments carried out under similar conditions.

 
We next examined the effects of strategies expected to diminish PKC activity. We pretreated HEK-CaR.EGFP cells with 100 nM of several known PKC inhibitors (Bis-I, Ro 31–8220, Ro 32–0432, and Gö 6976). As shown in Table 1 and Fig. 11, in each case a partial inhibition of [Ca2+]i signaling by activation of the CaR was observed.


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  		Table 1. Effect of PKC inhibitors on peak Ca2+ in HEK-CaR.EGFP cells

 

Figure 11
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  		Fig. 11. Effect of PKC inhibitors on CaR-mediated mobilization of Cai2+ in HEK-CaR.EGFP cells. Cells, grown on glass coverslips, were loaded with 5 µM fura 2-AM for 30 min and pretreated with vehicle or 100 nM of each inhibitor (Bis-I, Gö 6976, Ro 31–8220, and Ro 32–0432) for 20 min before measurement of responses to cumulative addition of Cae2+. The data are typical of 4 separate experiments carried out under similar conditions.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In earlier studies from this laboratory, we showed that the sensory network of the perivascular adventitia express a CaR, which is a spliced variant of the parathyroid receptor and suggested that this receptor plays an important role in vasodilation (8). We have since cloned the CaR from DRG, which house the cell bodies of sensory nerves and transiently expressed it as an EGFP fusion protein in HEK293 cells (57). In the present study, we stably expressed this CaR as an EGFP fusion protein in HEK293 cells and characterized it with respect to Ca2+ signaling. The new findings indicate that the CaR can be stimulated by the same CaR agonists that stimulate the parathyroid receptor (Gd3+ and NPS R-467) and that the CaR showed Ca2+ sensitivity in the physiological range.

In the present study, the CaR doublet is consistent with the expression of this protein in HEK293 cells (25, 44), and probably indicates the fusion of EGFP with the high-mannose immature form of the CaR (57). The higher molecular weight band (185 kDa) is likely the fusion of the complex carbohydrate, mature form (157 kDa) of the receptor to EGFP. The HEK-CaR.EGFP cells responded to added Cae2+ with an increase in [Ca2+]i, indicating functional expression of the receptor. Binding of Cae2+ to the extracellular domain of the receptor initiated a signal linked to the release of Ca2+ from intracellular storage sites and transient elevation of [Ca2+]i. The data reported herein indicate stimulation of the CaR in HEK-CaR.EGFP cells by 1 mM Cae2+, which varied with different clones suggesting that response to stimulation depended on the expression levels of the CaR. The plateau above basal [Ca2+]i, suggests Ca2+ influx from the extracellular medium via SOCE channels. The CaR binds Ca2+ at the extracellular domain and transduces the signal through cAMP and phospholipases (PLC, PLA2, or PLD), depending on the cell type (7). Activation of PLC leads to IP3 production and release of stored intracellular Ca2+ following activation of IP3 receptors in the endoplasmic reticulum membrane. Reduction in [Ca2+]i is rapidly achieved by Ca2+ pumps (Ca2+-ATPase) located in the endoplasmic reticulum and plasma membranes. However, the depletion of stored Ca2+ also opens plasma membrane SOCE channels, that allow influx of Ca2+ resulting in a sustained plateau (41, 42) or periodic oscillations of [Ca2+ ]i that regulate cytosolic as well as nuclear functions of the cell (3, 12). Multiple signaling mechanisms are implicated in these events; however, the molecular mechanisms of these Ca2+ signaling pathways are not clear. Although multiple mechanisms have been described for Ca2+ influx (43), the nature of the entry channels is at present not known. Recent results implicate two new important players in this process, Stim1 (31, 47) and Orai1 (15, 54).

The [Ca2+]i oscillations, observed in this study, may be related to localized fluctuations in IP3 levels within a population of cells or recruitment of regulators of G protein signaling (RGS) protein to the membrane to promote GTP binding to G{alpha}q, thus uncoupling receptor-catalyzed loading of G{alpha}q-GTP from PLCbeta activation in the presence of persistent agonist stimulation (48, 32, 58). Other studies have shown that stimulation of the CaR leads to [Ca2+]i oscillations, which play a key role in signal transduction by regulating Ca2+/calmodulin-dependent protein kinase II (53), PKC (36), mitochondrial metabolism (21) and nuclear transcriptional activity that results in differential gene expression (13, 24). These oscillations can be organized at the subcellular level as propagating Ca2+ waves, and serve as an important mechanism in regulating cell function. For example, parathyroid cells exhibiting [Ca2+]i oscillations have been shown to secrete less parathyroid hormone (34) and therefore may contribute to CaR-mediated inhibition of the secretion of the hormone from parathyroid glands. The present findings conform to the earlier findings, which showed that cells heterogeneously expressing the CaR undergo [Ca2+]i oscillations at low [Ca2+]e (5). The concentration-dependent increase in oscillation frequencies is interesting and the mechanism and significance of this are being explored.

An important new finding of the present study is that cumulative addition of low concentrations of Cae2+ caused rapid reduction in peak [Ca2+]i, suggesting receptor desensitization. This finding is in contrast with that described for the parathyroid CaR, which showed increase in Cai2+ mobilization following cumulative additions of Cae2+ (2, 5, 17). Gd3+ and NPS R-467, activators of the parathyroid CaR, also desensitized the CaR. Gd3+ is an activator of the parathyroid receptor, but also blocks SOCE; therefore, the reduction in response observed after the second application could be attributable to blockade of receptor-mediated Cai2+ mobilization and entry. On the other hand, the reduction in the effect of NPS R-467, an allosteric activator of the CaR, could reflect receptor uncoupling from signal transduction mechanisms leading to endoplasmic reticulum Ca2+ release. We have also observed that Cae2+ cross-desensitized carbachol responses (data not shown), indicating depletion of the Ca2+ pool. This suggests that the reduction in Cai2+ mobilization by repeated stimulation of the CaR could be due, in part, to impaired store refilling. Gama and Breitwieser (17) have reported desensitization of the parathyroid CaR by 10 mM Cae2+; however, other studies have shown that this receptor is resistant to Ca2+-induced desensitization (18). The DRG CaR expressed in HEK293 cells shows higher Cae2+ sensitivity and is desensitized by physiological concentrations of Ca2+, unlike the parathyroid CaR expressed in HEK293 cells (5, 17). This is novel and suggests that the DRG receptor is functionally different. A possible explanation for this difference in behavior is the fact that the amino acid sequence of the cytoplasmic domain of the DRG CaR is different from the parathyroid receptor (57). The DRG CaR may therefore be coupled differently to G protein and the release of stored Ca2+. The possibility that the fusion of EGFP to the CaR may have changed the properties of the receptor is unlikely in view of the fact that some GPCR-EGFP fusions appear to retain normal receptor characteristics within the fusion protein (28). Furthermore, no differences in desensitization have been observed between the full-length parathyroid receptor and its EGFP fusion protein expressed in HEK293 cells (17).

The observation that PKC inhibition or down regulation reduced Cai2+ mobilization as a result of receptor stimulation, indicates a role for PKC in the signaling process. This finding suggests that phosphorylation plays a role. GPCR signal desensitization, following repeated agonist challenge, is thought to be initiated by second messenger-activated PKC or GPCR kinase-mediated phosphorylation of specific serine/threonine residues within the third intracellular loop and/or COOH-terminal tail of the receptor (22). In the present study, PKC{alpha} was translocated to the plasma membrane upon phosphorylation at Ser-657, and this event occurred following stimulation of the CaR, indicating PKC{alpha} activation. A similar finding was previously reported by Sakwe and colleagues (51), who showed that in both parathyroid and HEK293 cells expressing exogenous human kidney CaR, exposure to 2–5 mM Cae2+ resulted in the translocation of cytosolic PKC to the membrane fraction and p-PKC{alpha} remained associated with the membrane fraction for at least 6 h after treatment. Other studies have shown that PKC activators, such as PMA, substantially reduce Ca2+-evoked increase in IP3 and Cai2+ levels in bovine parathyroid cells (45). This effect was attributed to PKC phosphorylation of the receptor at specific phosphorylation sites in the intracellular domains of the CaR. Surprisingly, for the CaR, we observed just the opposite: PMA had little or no effect on [Ca2+]i, whereas inhibition of PKC reduced the responses. These findings clearly indicate that PKC plays an intriguing role in the regulation of the response mediated by the CaR. The mechanism of regulation, however, is quite complex and can involve multiple targets including the receptor as well as the various signal transduction proteins and Ca2+ channels and requires a rigorous examination of the role of other PKC isoforms.

Our data suggest that reduction in the amplitude of the [Ca2+]i transient following cumulative applications of Cae2+ was due to modulation of the coupling mechanism of the receptor to signaling proteins resulting from persistent stimulation. However, PKC does not seem to be involved in mediating the desensitization. Knowledge of the underlying mechanism will require additional investigation. Nonetheless, this finding is very important since the receptor can be a useful target for drugs that can modulate [Ca2+]i. The time-dependent reduction of the CaR responsiveness to [Ca2+]e constitutes an important physiological feedback control mechanism that probably protects the receptor from overstimulation. The parathyroid CaR exhibits persistent responsiveness to Ca2+ (6, 18), a property that probably allows some level of activation at all times unlike most GPCRs, which show prominent or complete loss of activation following repeated exposure to agonist (10). The DRG CaR appears to behave as the latter, and is therefore functionally different from the parathyroid receptor.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-64761 and 5UH1 HL-05968.


   ACKNOWLEDGMENTS
 
We thank Carl Bortner of the Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC, for performing the FACS analysis.

Present address for A. C. Howlett: Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. M. Awumey, Cardiovascular Disease Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central Univ., 700 George St., Durham, NC 27707 (e-mail: eawumey{at}nccu.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.

{dagger} Deceased March 2, 2006. Back


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