INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE CALCIUM-SENSING RECEPTOR (CaR), although originally identified and cloned from bovine parathyroid cells (7, 17), is now found widely expressed in both epithelial (5, 7-9, 11, 12, 14, 15, 25, 30, 31) and nonepithelial (24, 26, 32, 33, 38, 39) cells. All of the isolated CaR mRNA transcripts identified so far are related to a class of G protein-coupled receptors with a size of ~120 kDa (7, 17, 19). One of the first functions given to the CaR was the regulation of Ca²⁺ homeostasis by parathyroid cells (5, 7). However, because the CaR is now found in many cell types, its biological function is likely to involve several physiological responses. Besides extracellular Ca²⁺, the reported number of divalent and trivalent agonists that activate the CaR now includes strontium (Sr²⁺), magnesium (Mg²⁺), gadolinium (Gd³⁺), barium (Ba²⁺), and aluminum (Al³⁺) (14, 23, 24, 28, 29, 33).

In the gastrointestinal tract, the CaR has been identified in rat intestine (8, 15), amphibian stomach (11), rat stomach (9), human Caco-2 and HT-29 cancerous intestinal cell lines (15, 20), and the human gastrin-secreting G cells in the gastric antrum (31). In the stomach, contradictory data exist on the localization of CaR to gastric mucous epithelial cells. That is, CaR immunolocalization was not found in human antral mucous epithelial cells (31), whereas Cheng et al. (9) reported CaR immunolocalization to rat gastric mucous epithelial cells. Although a specific role for the CaR in human antral G cells has been described, the characterization and a role for the CaR in gastric mucous epithelial cells have yet to be defined. It has been proposed for intestinal epithelial cells that the CaR may be involved in cell growth and differentiation (8, 15). In this regard, the mucous epithelial cells in the stomach have a high proliferative rate, which contributes to the "barrier" function and protection of stomach against noxious agents (27). Therefore, in the present study, we wanted to examine both the expression and functionality of the CaR in human gastric mucous epithelial cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and peptides. Fura 2-AM (special packaging) was obtained from Molecular Probes (Eugene, OR), stored at 20°C, and dissolved as a 5 mM stock in cultured grade DMSO (Sigma). U-73122 and U-73343 were purchased from Calbiochem (San Diego, CA) and dissolved in DMSO. Gadolinium chloride, type I collagenase, RIA-grade BSA, light green stain, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit were purchased from Sigma. All cell culture media were purchased from GIBCO Life Technologies; fetal bovine serum was purchased from Hyclone (Logan, UT).

Cell culture. Human gastric mucous cells were isolated and cultured as previously described (34). Briefly, gastric tissue was obtained from Helicobacter pylori-free patients undergoing routine surgical gastrectomy. All procedures and handling of human tissue were approved by the Oregon Health Sciences University Human Studies Subcommittee. The surgical specimens were washed twice in serum-free medium and pinned down on polymerized Sylgard, and the epithelium was removed by scraping the surface with a glass slide. The scraped tissue pieces were minced using razor blades and then washed three times at 100 g for 3 min in serum-free medium. The pellets were then transferred to siliconized 125-ml screw-cap Erlenmeyer flasks containing 20 ml of serum-free culture medium with 20 mg/ml of type I collagenase and 0.1% BSA. The flasks were then gassed with 95% O₂-5% CO₂, put into a 37°C shaking water bath, and gyrated at 120 oscillations/min for 45 min. At the end of the incubation period, the collagenase-digested mixture was put into a 50-ml syringe with an attached 15-gauge Luer-stub adapter, and the contents were pushed out over a 200-µm nylon mesh screen. The mesh-filtered suspension was washed twice in serum-free medium and centrifuged at 100 g for 3 min, then the pellet was resuspended in 15 ml of serum-free culture medium, and a 200-µl aliquot was taken for cell counts in a Coulter counter. The 15-ml suspension was divided into three 5-ml aliquots in 16 × 125-cm Falcon round-bottom tubes; 5 ml of isosmotic Percoll (34) were then added to each tube. The tubes were centrifuged for 15 min at 100 g at 24°C, and the bottom pellet containing the gastric mucous epithelial cells was removed. The pellet was washed three times and centrifuged at 20 g for 3 min in serum-free cell culture medium, and then the cells were plated on 0.45-µm Falcon porous filters, 16 × 125-mm rectangular glass slides, or 96-multiwell plastic dishes. Cultures of Rat-1 fibroblasts (gift from Dr. Karin Rodland) were also cultured and grown in the same medium as the gastric mucous cells.

Immunohistochemical detection of CaR in gastric tissue. Human gastric tissue samples obtained during surgery were immediately placed into zinc-formalin fixative and then processed for paraffin embedding. From the paraffin-embedded blocks, 4- to 5-µm sections were cut, deparaffinized, rinsed in PBS, and incubated for 10 min in 0.1% hydrogen peroxide-PBS, pH 7.4, to quench endogenous peroxidase activity. The sections were then incubated overnight with the primary anti-CaR antibody (polyclonal anti-CaR, Affinity BioReagents, Golden, CO) at 4°C and then washed in PBS and incubated with a secondary peroxidase-labeled goat anti-rabbit antibody for 1 h at room temperature. Controls for nonspecific staining included incubation of the primary anti-CaR antibody with an excess of CaR peptide (50 µg/ml) or replacement of the primary anti-CaR antibody with a nonspecific IgG. Sections were then rinsed for 10 min in PBS and lightly counterstained with 0.01% light green; a coverglass with mounting medium was then added for microscopic visualization. The slides were then visualized using a Nikon Diaphot inverted microscope, and pictures were taken with an attached 35-mm camera using Kodak color TechPan film (Rochester, NY).

Immunohistochemical detection of CaR in cell monolayers. Gastric mucous epithelial cells were grown to confluence on Falcon porous filters, washed with PBS, and then fixed in 3.5% zinc-formaldehyde solution for 10 min at 24°C. The fixative was removed, the cell monolayers were washed five times with PBS at 24°C, and then the cells were permeabilized with the addition of 10°C methanol for 2 min. After 2 min, the methanol was removed and the cultures were air dried and washed three times with PBS. Nonspecific binding sites were blocked by incubation in 10% normal goat serum for 30 min. The goat serum was then removed, the monolayers were washed 3 times with PBS, and then the cells were incubated overnight at 4°C with anti-CaR antibody (1:800 dilution in PBS). The anti-CaR antibody was removed, and the cultures were washed twice with PBS at 24°C and then treated with rhodamine-labeled goat anti-rabbit IgG at a 1:100 dilution in PBS for 1 h at 24°C. The secondary antibody was then removed, and the cells were washed twice with PBS at 24°C. Controls for nonspecific staining included incubation of the primary anti-CaR antibody with an excess of CaR peptide (25 µg/ml) or omission of the first primary antibody. The cultures were then visualized using a Nikon Diaphot inverted microscope, and pictures were taken with an attached 35-mm camera using Kodak color TechPan film.

Confocal microscopy. The processed immunofluorescent cell culture monolayers were removed from their plastic inserts by rimming the "outside" of the filter with a no. 11 scalpel blade. The detached filters were then placed on top of a few drops of FluorSave on a no. 1 round coverglass (0.13-17 mm thickness, 25 mm diameter) with the monolayer side facing up. Additional drops of FluorSave were added to the top of the monolayer, and another coverglass was placed on top. The edges of the coverslips were sealed and placed on the stage of a Nikon Diaphot 2000 microscope with the monolayer side facing down. The microscope was attached to a Bio-Rad MRC-1000 confocal laser scanning system (Bio-Rad Laboratories, Richmond, CA). All images were visualized through a Nikon Neofluor ×60, numerical aperture 1.25 oil objective using a rhodamine filter set, and the images were downloaded into an IBM computer containing COMOS analysis software (Bio-Rad).

Western immunoblotting of CaR. Western blotting for CaR was done as described by Rodland and colleagues (25, 26). Cell cultures were grown to preconfluency in 10-cm Falcon plastic dishes; at the appropriate times, the medium was removed and the cells were scraped in 1 ml of homogenization buffer. The homogenization buffer contained 50 mM Tris, pH 7.5, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The cell scrapings were then homogenized with 15 strokes of a 1.5-ml Dounce homogenizer, and nuclei were removed by centrifugation at 800 g for 10 min. The supernatant was subjected to centrifugation at 43,000 g in a Beckman TLA 100.3 rotor for 1 h to pellet the plasma membrane fragments, and the resulting pellet was resolubilized in homogenization buffer with 1% Triton X-100. Lysate protein was quantitated using the Bradford method with a commercially available kit (Bio-Rad), and 5 µg of protein were added to each lane of 8% SDS polyacrylamide gels. Western immunoblotting for CaR proteins was done by size fractionation with SDS-gel electrophoresis, and then the proteins were transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore) by electroblotting. Membranes were blocked in 3% BSA-0.05% sodium azide for 1 h at room temperature, followed by overnight incubation with primary CaR-antibody at 4°C in TTBS (0.05% Tween 20, 20 mM Tris, pH 7.5, 150 mM NaCl). Membranes were then washed three times in TTBS, incubated with a secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, and washed extensively in TTBS. The protein bands were then visualized by chemiluminescence (Renaissance, DuPont NEN, Boston, MA) and exposed to Kodak X-Omat AR film. The film was then photographed, and densitometric analysis was performed on the bands using SigmaGel software (SPSS, San Rafael, CA).

MTT growth assay. Proliferation of human gastric mucous epithelial cells was assayed using the MTT growth assay. Freshly isolated human gastric epithelial cells were first cultured in DMEM-10% serum medium in 96-multiwell plates (Falcon). When the cultures became ~30% confluent, they were switched to a serum-free, low-Ca²⁺ (0.1 mM) DMEM medium for 24 h. At the end of this time, the medium was replaced with fresh serum-free (phosphate-free) DMEM medium containing varying concentrations of extracellular Ca²⁺ (0.250-8.00 mM) and growth was measured 1, 2, and 3 days later. At the end of each time period, the medium was removed and 50 µl of a 0.4 mg/ml MTT solution in RPMI medium were added to each well. The cultures were then incubated at 37°C in a CO₂ incubator for 2.5 h. After this time, the MTT solution was aspirated and 50 µl of a 0.1 N HCl-isopropanol solution were added. The cultures were then incubated for 30 min at 24°C on a gyratory platform shaker. After 30 min, the 96-multiwell plate was then placed in an ELISA 96-multiwell plate reader (Molecular Devices) using wavelengths of 570 and 690 nm. The final MTT absorbance was calculated by subtracting the 690-nm background absorbance from the 570-nm measurement readings.

Fura 2 and intracellular Ca²⁺ measurements. The day before the experiments, the cell cultures were switched to growth factor-free, serum-free medium for 24 h. Fura 2 loading of cells was achieved by removing the serum-free medium and incubating the cultured cells on glass coverslips with 2.5 µM fura 2-AM in fresh serum-free medium for 30 min at 37°C. After the 30-min loading period, the coverslips were washed two times with fresh serum-free medium, washed two times with mammalian Ringer, and then placed in fresh mammalian Ringer for 30 min. After this time, the coverslips were placed into a sealed cuvette of a temperature-controlled fluorometer (SLM-AMINCO) with an inlet and outlet tube at the top of the cuvette for solution perfusion. The mammalian Ringer solution consisted of (in mM) 137 NaCl, 4 KCl, 25 NaHCO₃, 2 KH₂PO₄, 15 HEPES, 1 MgSO₄, 2 CaCl₂, and 25 glucose, pH 7.4. Ca²⁺-free Ringer solutions consisted of Ringer solution without CaCl₂ and the addition of 2.0 mM EGTA-EDTA, pH 7.4. When high concentrations of Ca²⁺ or Gd³⁺ were needed for experiments, the KH₂PO₄ and MgSO₄ in the Ringer were replaced with KCl and MgCl₂, respectively, to avoid cation-phosphate precipitations. All solutions were oxygenated with 5% CO₂ and 95% O₂, and corrections were made for changes in pH before being perfused into the cuvette containing the cultured cells. Intracellular Ca²⁺ measurements were obtained at excitation wavelengths of 340 and 380 nm (10-nm bandwidth) at an emission wavelength of 500 nm, and the signals were analyzed using software provided by the SLM-AMINCO fluorometer. The actual intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was calculated using the method of Grynkiewicz et al. (18). The entire volume of the cuvette (2.25 ml) could be replaced in <5 s when the Millipore pump was set at a high speed without any disruption of the cell monolayer.

Statistics. All data points are expressed as means ± SE. The differences between means were considered significant when the P value calculated from Student's t-test for paired cultures was <0.05. Multiple cell culture comparisons were analyzed using ANOVA and Duncan's multiple range tests. In this study, n represents the total number of different individual cell preparations isolated from different surgical specimens. All statistical calculations were made using Sigma-Stat statistical software (Jandel Scientific, San Rafael, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Immunohistochemical detection of CaR in human gastric mucous epithelial cells. Immunohistochemical staining using a specific affinity-purified antibody to the human CaR was used to detect CaR in paraffin sections of the human gastric mucosa. As shown in Fig. 1, we found intense basolateral staining of CaR in the surface mucous epithelial cells (Fig. 1A). Preabsorbing the CaR antibody with CaR-blocking peptide eliminated the specific CaR staining (Fig. 1B). There was also lighter positive CaR immunostaining in the cytoplasm of both parietal and chief cells (data not shown).

View larger version (97K): [in this window] [in a new window]   Fig. 1.   Photographs showing the immunohistochemical detection of Ca2+-sensing receptor (CaR) protein in paraffin sections of human gastric mucosa. Samples of human gastric mucosa were fixed in 4% zinc-paraformaldehyde, paraffin embedded, and sectioned for light microscopy immunostaining. Note the intense positive CaR immunostaining in the basolateral region of the surface mucous epithelial cells with an occasional intermittent light staining for CaR at the apical membrane of the cells (A). B: control section in which the anti-CaR antibody was preincubated with blocking CaR peptide. Bars = 100 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 2.   An electron micrograph showing the morphology of normal human gastric epithelial cells grown on a Falcon porous filter. Note that the gastric cells display polarity with apically located mucous granules and short microvilli, tight junctions (arrows), and basally located nuclei. Bar = 5 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Immunohistochemical detection of CaR in cultures of human gastric mucous epithelial cells grown on Falcon porous filters. A: light microscopy photograph looking down on the luminal surface of confluent cultures of gastric mucous epithelial cells. When cultures were immunostained with the anti-CaR antibody (1:800 dilution), we found both a diffuse cellular fluorescence and a more intense peripheral fluorescence around the periphery of the cells (B). C: control culture in which the cells were incubated with the anti-CaR antibody that was pretreated with the blocking peptide. Bars = 30 µm.

Confocal microscopy detection of CaR in human gastric mucous epithelial cells. In the previous experiments, we found that cultures of gastric mucous epithelial cells contained specific CaR immunoreactivity. However, the exact cellular location of the CaR immunoreactivity in the cultured cells was difficult to detect given the one-dimensional picture of the cells. Using three-dimensional confocal microscopy, we were better able to identify the cellular distribution of the CaR in cultures of human gastric mucous epithelial cells grown on porous filters. As shown in Fig. 4A, we typically found strong CaR immunofluorescence within the basal portion of the cell and along the entire basolateral membrane. There was also some weak intermittent immunofluorescence at the apical membrane of the cells (Fig. 4B). We found little to no specific immunostaining at the apical cytoplasmic portion of the cell (Fig. 4B). Control cultures treated with the CaR antibody preabsorbed with the CaR-blocking peptide eliminated the specific fluorescence (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Confocal microscopy immunofluorescence detection of CaR in primary cultures of human gastric mucous epithelial cells. A: positive immunofluorescence for CaR with a specific anti-CaR antibody on cultures of human gastric mucous epithelial cells grown on porous filters viewed in the vertical plane of the cell. Note the abundant positive CaR fluorescence at the basal portion of the cells (double arrow) as well as along the lateral membrane and light intermittent CaR-immunoreactive staining at the apical membrane of the cells (single arrow). B: control culture in which the anti-CaR antibody was preincubated with blocking CaR peptide. Bars = 10 µm.

Western immunoblot of CaR in gastric mucous epithelial cells. Western immunoblotting of cultures of gastric mucous epithelial cell lysates was performed using the specific CaR antibody. Cultures of Rat-1 fibroblast lysates were also used as a control, since previous studies have shown that they contain a specific and functional CaR (26). Immunoblotting of both cell culture lysates revealed strong and moderate CaR staining in gastric and Rat-1 cultures, respectively (Fig. 5). As shown in Fig. 5A, the major band detected in the gastric lysates had a molecular mass of ~140 kDa with a second lighter band of ~120 kDa. Parallel analysis of Rat-1 fibroblast lysates with the anti-CaR antibody also detected a major protein band of ~120 kDa, with a much smaller band at ~140 kDa (Fig. 5A). Both major and minor bands from both lysates were eliminated when the primary anti-CaR antibody was preabsorbed with CaR blocking peptide (Fig. 5B).

View larger version (90K): [in this window] [in a new window]   Fig. 5.   Western immunoblot detection of CaR protein in cultures of human gastric mucous epithelial cells (left) and in cultures of Rat-1 fibroblasts (right). In 2 lanes designated A, equal amounts of protein (5 µg) from each cell culture lysate were subjected to 8% SDS-PAGE, blotted, and incubated with an anti-CaR antibody as described in MATERIALS AND METHODS. In the gastric lysates, arrows indicate positions of 2 immunoreactive bands at ~140 and ~120 kDa and a major band and minor bands at ~120 and ~140 kDa, respectively, in the Rat-1 fibroblast lysates. In the 2 lanes designated B, gels were incubated with the primary anti-CaR antibody that was preabsorbed with the blocking CaR peptide.

Effect of extracellular Ca²⁺ on gastric mucous epithelial cell proliferation. A role for extracellular Ca²⁺ in modulating the growth of epithelial cells has now been postulated. However, the role of extracellular Ca²⁺ in the regulation of gastric mucous epithelial proliferation has not been thoroughly examined. In the next series of experiments, preconfluent gastric mucous epithelial cells were pretreated with 0.100 mM extracellular Ca²⁺ for 24 h, and then varying concentrations of extracellular Ca²⁺ were added and changes in cell proliferation were measured using the MTT assay. As shown in Fig. 6, changing the extracellular Ca²⁺ concentration from 0.250 to 0.500 mM produced stimulated growth rates over a 3-day period. Concentrations of extracellular Ca²⁺ >2 mM produced no further increase in cell proliferation but actually produced a slow decrease in cell growth compared with the lower extracellular Ca²⁺ concentrations (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Graph showing the proliferation of gastric mucous epithelial cells in response to various concentrations of extracellular Ca2+. Cultures of human gastric cells were grown to ~35% confluency and then switched to serum-free DMEM medium (0.3 mM Ca2+) for 24 h. After 24 h in serum-free, low-Ca2+ medium, cultures were switched to fresh DMEM containing various concentrations of extracellular Ca2+. Cultures were then assayed for changes in cell proliferation using the MTT assay at 1, 2, and 3 days after the addition of the Ca2+-containing medium. Each data point represents mean ± SE of 32 individual data points done in quadruplicate.

Effects of extracellular Ca²⁺ and Gd³⁺ on changes in [Ca²⁺]_i. The addition of extracellular Ca²⁺ or Gd³⁺ has been shown to increase [Ca²⁺]_i, which has been used to prove the functionality of the CaR (7, 21, 24-26, 28). In the next series of experiments, gastric mucous epithelial cell cultures were pretreated with nominally Ca²⁺-free Ringer and then exposed to pulsatile or continuous changes in extracellular Ca²⁺ according to modifications of previously described techniques (31). As shown in the representative tracing in Fig. 7A, the initial exposure of the cultures to 0.250 mM Ca²⁺ resulted in a small but a significant increase in [Ca²⁺]_i. Subsequent exposure to consecutive pulses of extracellular Ca²⁺ up to 2 mM increased [Ca²⁺]_i with little change in [Ca²⁺]_i beyond 2 mM extracellular Ca²⁺. The calculated extracellular Ca²⁺ KD₅₀ for producing a half-maximal change in [Ca²⁺]_i was found to be 0.66 mM. In the next series of experiments, changes in [Ca²⁺]_i were made using rapid consecutive changes in extracellular Ca²⁺ concentrations from 0.250 to 8.0 mM. As shown in the representative tracing in Fig. 7B, a significant increase in [Ca²⁺]_i was observed again at 0.250 mM extracellular Ca²⁺, but there was little significant change in [Ca²⁺]_i beyond that shown with 2.0 mM extracellular Ca²⁺ (P < 0.05, n = 7). The switch to nominally Ca²⁺-free Ringer at the end of the experiment returned [Ca²⁺]_i to near baseline levels (Fig. 7B). Compared with the sequential method of extracellular Ca²⁺ exposure, the pulsatile method of extracellular Ca²⁺ exposure produced quantitatively higher changes in [Ca²⁺]_i (Fig. 8). However, the relative qualitative changes between the two methods produced nearly similar results in gastric mucous epithelial cell [Ca²⁺]_i. Any difference between the two methods is likely to reflect an altered intracellular Ca²⁺ baseline level, most likely due to the constant exposure of the cells to the extracellular Ca²⁺ dose (4).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Representative tracings showing changes in intracellular Ca2+ concentration ([Ca2+]i) in response to changes in extracellular Ca2+ ranging in concentration from 0.250 to 8.0 mM in a consecutive "pulsatile" (A) or "sequential" (B) exposure protocol. In the pulsatile procedure (A), there was a 4- to 5-min washout period between each Ca2+ pulse using nominally Ca2+-free Ringer. The initial exposure of the cultures to 0.250 mM Ca2+ resulted in a small but significant increase in [Ca2+]i. Subsequent exposure to sequential pulses of extracellular Ca2+ up to 2 mM increased [Ca2+]i with little change in [Ca2+]i beyond that shown with 2 mM extracellular Ca2+. In B, changes in [Ca2+]i were next made using a rapid sequential protocol of varying extracellular Ca2+ concentrations from 0.250 to 8.0 mM. Note that there was a significant increase in [Ca2+]i at 0.250 mM extracellular Ca2+ with little change in [Ca2+]i beyond 2.0 mM extracellular Ca2+. Switch to Ca2+-free Ringer at the end of the experiment returned [Ca2+]i to near baseline levels (B).

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Graph summarizing the effects of increasing concentrations of extracellular Ca2+ concentration on [Ca2+]i comparing the consecutive pulsatile or sequential protocols (see MATERIALS AND METHODS). Note that the greatest increase in [Ca2+]i by both procedures occurred within the range of 0.250 and 2.0 mM extracellular Ca2+. Each data point represents the summation of 3 individual measurements from 5 (n = 7) separate cell culture isolations. * Significantly different (P < 0.050) values between pulsatile and sequential methods.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Representative tracings showing the effects of varying extracellular Gd3+ concentrations on [Ca2+]i in cultures of human gastric mucous epithelial cells. Cultures were initially incubated in nominally Ca2+-free Ringer for 30 min, and then Gd3+ was added (thick arrow) and [Ca2+]i was measured. Addition of various doses of Gd3+ (0-400 µM; long arrows) caused dose-dependent transient increases in [Ca2+]i that eventually returned to baseline levels.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Representative tracings showing the effects of the phospholipase C (PLC) inhibitor U-73122 on Gd3+-induced changes in [Ca2+]i in cultures of human gastric mucous epithelial cells. Cultures were pretreated for 10 min with either 1 µM of the inactive PLC analog U-73343 (A) or 1 µM of active PLC analog U-73122 (B). Gd3+ (400 µM) added to the cultures (arrows) produced the typical transient change in [Ca2+]i in the presence of the inactive PLC analog U-73343 (A), whereas the Gd3+-induced change in [Ca2+]i was eliminated in cultures pretreated with the active PLC analog U-73122 (B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we have identified the localization of a functional CaR in human gastric mucous epithelial cells. Immunohistochemical detection confirmed CaR expression in both paraffin sections of full thickness gastric tissues and in primary cultures of human gastric mucous epithelial cells. In addition, confocal microscopy immunofluorescence specifically localized the CaR in human gastric mucous epithelial cells to the basal portion and basolateral membrane of the cells. We also found weak CaR immunofluorescence at the apical membrane of the gastric mucous epithelial cells in both cell culture and in paraffin sections of intact stomach. These results differ from those of Ray et al. (31) in which CaR immunoreactivity was not detected in the mucous epithelial cells of the human antrum. These contradictions in the data could be due to differences in methodology, antibodies used, or the possibility that human antral G cells react more strongly to the CaR-antibody than antral mucous epithelial cells. Our findings do agree with other reports in the stomach in which CaR immunoreactivity was found in Necturus gastric surface cells (11) and in rat gastric surface cells (9).

In general, the predominant localization of the CaR to the basolateral membrane in human gastric mucous epithelial cells would suggest that the CaR would be primarily involved in "sensing" changes to serum Ca²⁺ concentrations that would regulate growth or secretion. However, another possibility is that damage to the luminal surface mucous cells could produce an influx of luminal Ca²⁺ across the damaged gastric epithelia to activate the basolateral CaR of underlying surface mucous cells to help aid in cell proliferation and gastric mucosal repair. Also, one of the most well-established physiological responses in the stomach is the increase in gastric acid secretion in response to luminal extracellular Ca²⁺ (e.g., calcium carbonate) (22). This biological effect most likely starts with luminal Ca²⁺ activating a CaR-mediated increase in [Ca²⁺]_i in antral G cells (31), which then triggers gastrin granule release through a cytoskeletal-dependent mechanism (35). The release of gastrin eventually stimulates gastric acid secretion through both direct and indirect mechanisms (10). However, CaR activation is likely not the sole mediator for changes in [Ca²⁺]_i, since other compounds such as bombesin have also been shown to increase [Ca²⁺]_i within antral G cells (35, 36). In this regard, gastric peptides along with the CaR might also play a role in mucin granule release and secretion in gastric mucous epithelial cells. On a comparative basis, CaR localization has been reported in the HT-29 human intestinal mucous-secreting goblet cell line (15).

Additional identification of the CaR in human gastric mucous epithelial cells was done using Western immunoblot analysis. We found in lysates of these cultures a predominant protein band at ~140 kDa and a smaller band at ~120 kDa. As reported by others, the higher-molecular-mass band is likely to represent a glycosylated form of the CaR, whereas the smaller 120-kDa band may represent the unglycosylated form of the CaR protein (1). Additional confirmation that we had CaR protein in the human gastric epithelial cell culture lysates was done with parallel Western blot analysis on Rat-1 fibroblast lysates in which a functional CaR protein has been well characterized (26). The specificity of CaR protein localization in the immunohistochemical, immunofluorescent, and Western blot assays was also confirmed using CaR antibody preabsorbed with CaR protein that eliminated all CaR detection.

In determining the functionality of the CaR in our cells, we tested the effects of various concentrations of extracellular Ca²⁺ on [Ca²⁺]_i. We found that dose dependently increasing extracellular Ca²⁺ up to 2 mM caused rapid rises in [Ca²⁺]_i in human gastric mucous epithelial cells. Concentrations of extracellular Ca²⁺ >2 mM did not produce any further change in [Ca²⁺]_i whether the extracellular Ca²⁺ was given in rapidly consecutive or single pulsatile doses. However, decreasing the extracellular Ca²⁺ level to nominally Ca²⁺-free Ringer rapidly returned the [Ca²⁺]_i to baseline levels. These rapid Ca²⁺ responses suggest that human gastric mucous epithelial cells possess a mechanism for adjusting to fluctuations in extracellular Ca²⁺ in the control intracellular Ca²⁺ homeostasis. It is also important to note that we obtained these results from multiple samples from seven different culture preparations compared with multiple samples from a single cell culture preparation. There is always the possibility that a mutated CaR may exist in a single patient, but a range of sampled tissues and cultured cells is likely to be a better depiction of the normal range of CaR activity. The sensitivity of our cells to extracellular Ca²⁺ was also consistent with those values reported for other cell types such as parathyroid cells (5, 7), CaR-positive HEK-293 cells (2), the AtT-20 pituitary cell line (13), Rat-1 fibroblasts (26), human antral G cells (31), and the intestinal HT-2918-C1 cell line (15), in which extracellular Ca²⁺ produced specific changes in [Ca²⁺]_i. Also, the addition of extracellular Gd³⁺ (a specific CaR agonist; Refs. 1, 5, 24, 25) dose dependently increased intracellular Ca²⁺ through a PLC-dependent pathway. That is, we found that the PLC antagonist U-73122 blocked Gd³⁺-induced [Ca²⁺]_i changes in cultures of human gastric mucous epithelial cells. We also found that U-73122 could reduce the Gd³⁺-induced change in [Ca²⁺]_i. These data suggest that the CaR in gastric mucous epithelial cells is at least partially regulated by activation of PLC, which agrees with reports in intestinal epithelial cells (15). However, although the U-73122 data suggest the involvement of IP₃ on CaR-mediated changes in [Ca²⁺]_i, the specific details of this mechanism are not known. It is also important to note that changes in the extracellular Ca²⁺ concentration to study changes in [Ca²⁺]_i is an experimental maneuver in itself that may modify existing Ca²⁺ feedback loops or second messenger systems producing nonspecific reactions unrelated to any specific CaR investigation (4, 6).

In summary, we have identified the localization of a functional CaR in human gastric mucous epithelial cells. For the future, it will be important to determine how the expression or activation of the CaR is integrated with other components involved in gastric mucosal growth and repair such as growth factors (16), polyamines (3), or aging (37). It is of interest that polyamines can act as agonists for the CaR (30), and polyamines are essential in gastric epithelial repair by influencing microtubule-dependent polymerization that is a Ca²⁺-dependent process (3). Additional studies will be needed to further define the role(s) of the CaR in gastric mucous cell proliferation, differentiation, or mucin secretion.