Identification of a functional
Ca2+-sensing receptor in
normal human gastric mucous epithelial cells
Michael J.
Rutten1,
Kathy D.
Bacon1,
Katie L.
Marlink1,
Mark
Stoney1,
Camie L.
Meichsner1,
Fred P.
Lee2,
Susan A.
Hobson2,
Karin D.
Rodland2,
Brett C.
Sheppard1,3,
Donald D.
Trunkey1,
Karen E.
Deveney1, and
Clifford W.
Deveney1,3
1 Department of Surgery and
2 Department of Cell Biology,
Oregon Health Sciences University, and
3 Veterans Affairs Medical Center,
Portland, Oregon 97201
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ABSTRACT |
The purpose of the
present study was to determine whether human gastric mucous epithelial
cells express a functional
Ca2+-sensing receptor (CaR). Human
gastric mucous epithelial cells were isolated from surgical tissues and
cultured on glass coverslips, plastic dishes, or porous membrane
filters. Cell growth was assessed by the MTT assay, CaR localization
was detected by immunohistochemistry and confocal microscopy, CaR
protein expression was assessed by Western immunoblotting, and
intracellular Ca2+ concentration
([Ca2+]i)
was determined by fura 2 spectrofluorometry. In paraffin sections of
whole stomach, we found strong CaR immunohistochemical staining at the
basolateral membrane, with weak CaR-staining at the apical membrane in
mucous epithelial cells. Confocal microscopy of human gastric mucous
epithelial cell cultures showed abundant CaR immunofluorescence at the
basolateral membrane and little to no CaR immunoreactivity at the
apical membrane. Western immunoblot detection of CaR protein in cell
culture lysates showed two significant immunoreactive bands of 140 and
120 kDa. Addition of extracellular
Ca2+ to preconfluent cultures of
human gastric mucous epithelial cells produced a significant
proliferative response. Changes in
[Ca2+]i
were also observed in response to graded doses of extracellular Ca2+ and
Gd3+. The phospholipase C
inhibitor U-73122 specifically inhibited Gd3+-induced changes in
[Ca2+]i
in the gastric mucous epithelial cell cultures. In conclusion, we have
identified the localization of a functional CaR in human gastric mucous
epithelial cells.
gadolinium; intracellular calcium; signal transduction; stomach; fura 2; U-73122; confocal microscopy; Western immunoblotting; immunohistochemistry; cell culture; proliferation
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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).
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RESULTS |
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).

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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.
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In the next series of experiments, CaR immunohistochemical staining was
identified using a primary culture system of human gastric mucous
epithelial cells. As shown in Fig. 2,
cultures of human gastric mucous epithelial cells grown on Falcon
porous filters show polarity and have short stubby microvilli, apically located mucous granules, and tight junctions. Immunohistochemical staining for CaR in these cultures revealed positive CaR immunostaining in almost all the cells examined (Fig.
3B).
Depending on the amount of antibody used, the CaR immunostaining
pattern ranged from very intense cytoplasmic staining in each cell
(1:100 antibody dilution; data not shown) to a more defined pattern of
CaR cytoplasmic staining and peripheral cellular CaR fluorescence at
higher antibody dilutions (1:800 dilution) (Fig.
3B). The specific CaR staining in
the cultures could be eliminated by preabsorbing the CaR antibody with
blocking peptide (Fig. 3C).

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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.
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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.
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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).

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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.
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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).

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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.
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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).

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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.
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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).

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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).
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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.
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In the previous experiments, we found that extracellular
Ca2+ could produce changes in
[Ca2+]i.
In the next series of experiments, we wanted to use the CaR agonist
Gd3+ to determine whether it would
have similar effects on
[Ca2+]i
in cultures of human gastric mucous epithelial cells. As shown in the
representative tracing in Fig. 9, addition
of extracellular Gd3+ (0-400
µM) caused a dose-dependent increase in
[Ca2+]i.
Concentrations of Gd3+ >400 µM
did not produce any further change in
[Ca2+]i
but actually produced a decrease in
[Ca2+]i
(data not shown). Besides the changes in
[Ca2+]i,
other studies have shown that agonist activation of the CaR will also
produce changes in intracellular inositol trisphosphate (IP3) concentrations (13,
24-26, 30, 33), which can be attenuated or blocked using the
phospholipase C (PLC) inhibitor U-73122 (15). In the next series of
experiments, we pretreated gastric mucous epithelial cell cultures for
10 min with 1 µM of either U-73122 or its inactive analog U-73343,
and then Gd3+ was added and
[Ca2+]i
was measured. As shown in representative tracing in Fig.
10A, the
inactive PLC inhibitor U-73343 did not affect the
Gd3+-induced changes in
[Ca2+]i,
whereas the active analog U-73122 completely blocked
Gd3+-induced
[Ca2+]i
changes (Fig. 10B).

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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.
|
|

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|
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 |
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.
 |
ACKNOWLEDGEMENTS |
We thank Jodi Engstrom for excellent expertise on the use of the
confocal microscope and Charlie Meshul for generous help with electron microscopy.
 |
FOOTNOTES |
This study was supported in part by National Heart, Lung, and Blood
Institute Grant 1T35 HL-07890-01.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. J. Rutten, Oregon Health Sciences Univ., Dept. of Surgery/L223A, 3181 Sam
Jackson Park Rd., Portland, OR 97201 (E-mail:
ruttenm{at}ohsu.edu).
Received 16 March 1999; accepted in final form 16 June 1999.
 |
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