Ca2+-sensing receptors in
intestinal epithelium
Lucio
Gama1,
Lynn M.
Baxendale-Cox2, and
Gerda E.
Breitwieser1
1 Department of Physiology,
School of Medicine and 2 School of
Nursing, Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
Expression of
Ca2+-sensing receptors (CaR) was
demonstrated in several human intestinal epithelial cell lines (T84,
HT-29, and Caco-2) and in rat intestinal epithelium by both reverse
transcriptase-polymerase chain reaction (PCR) and Northern blotting of
RNA. Restriction patterns of the PCR products were of the sizes
predicted by the human and rat sequences. CaR agonists
(Ca2+,
poly-L-arginine, protamine)
mediated an increase in intracellular Ca2+ in HT-29-18-C1 cells
(monitored by changes in fura 2 fluorescence), which was dependent on
release from thapsigargin-sensitive stores. U-73122, an inhibitor of
phosphatidylinositol-phospholipase C, eliminated the CaR
agonist-mediated rise in intracellular
Ca2+, whereas its inactive analog,
U-73343, had no effect. Pertussis toxin pretreatment had no effect on
CaR agonist-mediated modulation of intracellular
Ca2+. Taken together, these
studies demonstrate that CaR are expressed in intestinal epithelial
cells and couple to mobilization of intracellular Ca2+. The presence of CaR in
intestinal epithelial cells presents a new locus for investigations
into the role(s) of extracellular Ca2+ in modulating intestinal
epithelial cell differentiation and transepithelial
Ca2+ transport.
calcium-sensing receptor; polycation receptor; G protein-coupled
receptors; intracellular calcium
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INTRODUCTION |
CALCIUM-SENSING RECEPTORS (CaR), first identified and
cloned from bovine parathyroid cells (4), are expressed in tissues that
are involved in Ca2+ homeostasis,
including the parathyroid (4, 13), parafollicular cells (C cells) of
the thyroid (12, 14), and the kidney (29). Heterozygous and homozygous
CaR knockout mice display mild and severe derangements in
Ca2+ homeostasis, respectively,
suggesting a crucial role for CaR in organismal
Ca2+ homeostasis (18). Mutations
in human CaR cause several diseases of
Ca2+ handling (27). CaR are also
expressed in cell types that do not play a direct role in organismal
Ca2+ homeostasis but utilize
extracellular Ca2+ signals for a
variety of tasks, including triggering of differentiation [keratinocytes (1) and intestinal epithelial cells (22)] or
feedback regulation of cellular function in response to changes in
extracellular Ca2+ [as
suggested by the broad expression of CaR in neurons (11, 30, 35, 36,
38)].
Ca2+ plays several independent but
complementary roles in intestinal function. Intestinal epithelial cells
mediate Ca2+ absorption, and a
major role for 1,25-dihydroxyvitamin
D3 in modulating intestinal
Ca2+ absorption has been defined
(39). Ca2+ modulates intestinal
epithelial cell responsiveness to vitamin D (15) and thus may
contribute to overall organismal
Ca2+ availability (21), although a
well-defined mechanism for Ca2+ in
the regulation and/or modulation of
Ca2+ absorption has not been
established. In addition, high extracellular Ca2+ promotes intestinal
epithelial cell differentiation and decreases growth by as yet
undefined pathways (2, 7, 20), although a recent report suggests that
activation of CaR may be one step in the pathway (22).
CaR are G protein-coupled receptors that transduce extracellular
Ca2+ binding into a variety of
intracellular responses, including inhibition of adenylyl cyclase
(4-6), stimulation of
D-myo-inositol 1,4,5-trisphosphate (IP3)
production (11, 32), and release of intracellular
Ca2+ (11, 13, 34). In this report,
we test the hypothesis that the effects of
Ca2+ on intestinal epithelium are
mediated, in part, by CaR. Our results demonstrate that intestinal
epithelial cells express CaR and that alterations in extracellular
Ca2+ (and other CaR agonists)
activate CaR and induce changes in intracellular Ca2+ via activation of
phosphatidylinositol-phospholipase C and release from
thapsigargin-sensitive stores. These findings are consistent with a
potential role (or roles) for CaR in intestinal epithelial cell
function.
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METHODS |
Cell culture.
HT-29-18-C1 and -N2 [originally obtained by the Johns
Hopkins University Gastrointestinal Division from Dr. Daniel Louvard (19)] and T84 and Caco-2 cells (obtained from the American Type Culture Collection) were grown on plastic (Costar) in the appropriate media in humidified 95% air-5%
CO2 until confluent. T84 and
Caco-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; high glucose) with 10% heat-inactivated fetal bovine serum (Sigma), 50 U/ml penicillin, and 50 µg/ml streptomycin. HT-29 cells were grown in
DMEM containing 44 mM NaHCO3, 10 µg/ml human transferrin (Sigma), penicillin, streptomycin, 4 mM
glutamine, and 10% fetal bovine serum, supplemented with 25 mM
glucose.
RNA isolation.
Total RNA was isolated from cell lines (HT-29-18-C1, T84, and
Caco-2) and different sections of rat intestine with Trizol (Life
Technologies). Rat duodenum, jejunum, ileum, cecum, and colon were
isolated and rinsed with Hanks' solution, and the epithelial cells
were scraped from the underlying submucosa with a glass slide. To
determine subintestinal distribution, total RNA was extracted from the
ileum and separately from the ileal mucosa and submucosa. After RNA
preparation, samples were treated with deoxyribonuclease I for 30 min.
Reverse transcriptase-polymerase chain reaction
Total RNA samples (25 µg) were reverse transcribed into cDNA using
random hexamers (SuperScript II, Life Technologies) and then amplified
by 2 rounds of 35 cycles of polymerase chain reaction (PCR) using 2 sets of primers based on the published sequence of the human CaR gene
(13). The primer sequences were CaR 1, 5'-CAGACATCATCGAGTAT-3'; CaR 2, 5'-CACGTCGAAGTACTGAGG-3'; CaR 9A,
5'-ACCTGCTTACCTGGGAGAGG-3'; and CaR 10A,
5'-ACCTCCCTGGAGAACCCACT-3'. Primers 1 and 2 are
predicted to yield a 348-base pair (bp) product, and primers 9A and 10A
are predicted to yield a 305-bp product when amplifying either human or
rat CaR. PCR conditions were the same for both sets of primers:
denaturing at 94°C, primer annealing at 55°C for 1 min, and
primer extension at 72°C for 1 min. The positive control for PCR
was the full-length human cDNA for CaR. Negative controls included
primers but no cDNA or RNA that was not reverse transcribed.
Restriction analysis.
PCR products were concentrated using Microcon microconcentrators
(Amicon) and cut with specific restriction enzymes
(EcoR V or
Sau96 I) to confirm their identity as
CaR. Restriction fragments were analyzed on agarose gels (ethidium
bromide added to each sample). Direct sequencing of PCR products that
yielded the predicted restriction fragments was by the Sequenase method
(Amersham) with minor modifications.
Northern blots.
The probe for Northern blot analyses was constructed from the PCR
product amplified with CaR primers 1 and 2 from human intestinal cDNA.
The PCR product was subcloned into pCRScript and used as a template for
preparation of a biotinylated RNA probe (BIOTINscript, Ambion).
Northern blotting was carried out according to the protocols established in the Northern Max kit (Ambion). Total RNA (25 µg) from
each source was subjected to electrophoresis on 1% denaturing gels and
transferred to nylon membranes (Ambion) in 5× SSC buffer (1× SSC buffer is 0.15 M NaCl and 0.015 M sodium citrate, pH
7.0). After cross-linking with a Stratalinker ultraviolet cross-linker (Stratagene), the membranes were hybridized overnight with the biotinylated RNA probe at 65°C. Equivalent loading of lanes was assessed by ethidium bromide staining of gels to permit comparison of
ribosomal bands before transfer. High-stringency washes were then
performed at 68°C to reduce background interference. After nonisotopic detection, the membranes were exposed to Hyperfilm enhanced
chemiluminescence (Amersham) for 1-30 min. Films were scanned with
a Umax Vista-S6E scanner, using Adobe Photoshop software.
Fluorescence measurements.
Fluorescence measurements were made in HT-29-18-C1 cells, since
they are robust with respect to fura 2-acetoxymethyl ester (fura 2-AM)
uptake and deesterification. Neurotensin (100 nM) was used in each
experiment to assess the viability of the cells, i.e., the ability to
generate an intracellular Ca2+
transient via release from intracellular stores (25). HT-29-18-C1 cells were trypsinized (0.005% trypsin-EDTA) and resuspended for loading with fura 2-AM in a recovery medium containing (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.83 Na2HPO4,
0.17 NaH2PO4,
and 25 mannose, as well as 1 mg/ml bovine serum albumin, 1 mg/ml
trypsin inhibitor, and 0.5 µM fura 2-AM (Life Technologies or
Calbiochem), pH 7.4. Cells were allowed to recover and load for 60 min
at 37°C on a rocker. Aliquots of cells were gently pelleted and
resuspended in a minimal experimental solution (at 37°C) that
contained (in mM) 140 NaCl, 5.2 KCl, 0.55 MgCl2, and 10 HEPES, pH 7.4. Fluorescence was monitored at excitation wavelengths of 340 and 380 nm
(4-nm band pass) and an emission wavelength of 510 nm (2-nm band pass)
on an SLM/Aminco-Bowman Series 2 luminescence spectrometer. Results
were corrected for autofluorescence. Experiments were ended with
addition of 15 mM digitonin plus 5 mM
CaCl2, followed by 10 mM ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA; pH 7.4) to permit calibration. Ratios of 340-nm
fluorescence to 380-nm fluorescence were converted to concentrations
using a dissociation constant of 224 nM (17); minimum and maximum
fluorescence ratios were determined after digitonin and EGTA
treatments, respectively. Sequential additions of various
test agents were made to the cuvette during each experiment. For
additions that significantly altered the osmolality, control experiments were performed in which comparable changes in medium osmolality were produced by sucrose.
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RESULTS |
Reverse transcriptase-PCR analysis of CaR expression in intestinal
epithelium.
Reverse transcriptase (RT)-PCR was performed on RNA isolated both from
human intestinal epithelial cell lines (T84, several subclones of
HT-29, including HT-29-18-C1 and -N2, and Caco-2) and from mucosa
(epithelium) isolated from rat intestinal segments (duodenum, jejunum,
ileum, cecum, and colon). Figure
1A
illustrates the CaR exon structure and the location of the primer sets
that were used for the experiments.

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Fig. 1.
Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmation
of Ca2+-sensing receptor (CaR)
expression in human intestinal cell lines and rat mucosa.
A: exon-intron structure of human CaR
and location of primers. Exons are numbered, and vertical bars indicate
intron-exon boundaries. Arrows, locations of primers 1 and 2 (top) and primers 9A and 10A
(bottom). TM, transmembrane.
B: results of RT-PCR amplification with primers 9A and 10A applied to RNA isolated from various segments of rat intestinal mucosa. C:
restriction fragments resulting from Sau96 I digestion of 305-base pair
(bp) fragment derived from RT-PCR amplification of RNA derived from rat
ileal mucosa, utilizing primers 9A and 10A. Agarose gel was loaded with
uncut PCR product (uncut) and product of restriction enzyme reaction
(cut). D: Northern blot of rat ileal
fractions (mucosa, submucosa) and total ileum. Similar amounts of total
RNA were loaded for each fraction (25 µg), and blot was probed with a
biotinylated antisense RNA probe (see
METHODS); kb, kilobase.
E: RT-PCR with primers 1 and 2 applied to RNA isolated from 3 representative human intestinal epithelial cell
lines. F: RT-PCR with primers 1 and 2 applied to RNA isolated from rat intestinal mucosa.
G: restriction fragments resulting from EcoR V digestion of 348-bp
fragments derived from RT-PCR of RNA derived from either rat ileal
mucosa or HT-29-18-C1 cells, utilizing primers 1 and 2. Agarose
gel was run with uncut product from HT-29-18-C1 and cut products
from rat ileum and HT-29 cells.
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RT-PCR reactions were run utilizing primers 9A and 10A, which span from
exon 3 to exon 5. These primers were chosen to span intron-exon
boundaries (27) to eliminate the possibility that PCR amplification of
genomic DNA would lead to false positives. As illustrated in Fig.
1B, RT-PCR of rat intestinal RNA with
primers 9A and 10A yielded the expected 305-bp product. Restriction
fragment analysis of this PCR product was performed, and the predicted fragments from Sau96 I digestion were
obtained, as illustrated in Fig. 1C.
The sequence of this PCR fragment was identical to the published
sequence for the rat CaR cDNA (29, 31).
To determine which cell type(s) of the rat intestine expressed CaR, RNA
isolated from rat ileum (total), ileal mucosa (scraped epithelium), and
submucosa was subjected to Northern blotting. CaR expression is highly
enriched in the mucosa (Fig. 1D),
suggesting a role for CaR in intestinal epithelial cell function.
The predominantly epithelial nature of CaR expression was confirmed by
RT-PCR amplification of an appropriately sized product from all human
intestinal cell lines examined, including Caco-2, HT-29 (data shown is
for HT-29-18-C1; data not shown for -N2 and -19A subclones), and
T84 (Fig. 1E). For these
experiments, primers 1 and 2 were utilized, since they produced a
product in human and rat tissues that was not subject to potential
alternative splicing (12, 13). RT-PCR with primers 1 and 2, carried out on RNA isolated from epithelium scraped from segments of the rat intestine, also yielded the expected 348-bp product (Fig.
1F). The identity of the PCR
products was confirmed in two ways. First, as illustrated in Fig.
1G, the PCR products were isolated and subjected to restriction fragment analysis with an enzyme
(EcoR V) predicted from the nucleotide
sequence to yield a single asymmetric cut. The RT-PCR products from
both human (HT-29-18-C1) and rat (ileum) yielded the expected
restriction fragments. The 348-bp product of the PCR reaction from
HT-29-18-C1 cells was also sequenced and was identical to that
published for human CaR (13).
Northern blot analysis of rat intestine and human intestinal
epithelial cell lines.
To further confirm expression of CaR in intestinal epithelial cells, a
biotinylated, antisense RNA probe was generated from the 348-bp human
PCR product (Fig. 1E). Northern
blots of total RNA (25 µg) isolated from human intestinal epithelial
cell lines (Caco-2, HT-29-18-C1, and T84) and rat intestinal
epithelial cells (duodenum, jejunum, ileum, cecum, and colon) are
illustrated in Fig. 2,
A and
B, respectively. Both human and rat
intestinal epithelial cells exhibit multiple RNA transcripts, with the
major transcript in human being 4.7 kilobases (kb) (13) and that in rat
being slightly smaller, ~3.9 kb (29, 31). Expression varied among the
human epithelial cell lines, with HT-29 > T84 > Caco-2. Expression also varied along the rat intestine, with the greatest expression evident in early segments (duodenum, jejunum, and ileum) and greatly reduced expression in the cecum and colon. Probing of Northern blots
with a biotinylated probe produced from the full-length human cDNA of
CaR resulted in identification of the same array of transcripts from
human and rat RNA as were identified with the 348-bp probe (data not
shown), strongly suggesting that the transcripts were related to CaR
expression.

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Fig. 2.
Northern blotting of human intestinal cell lines and rat mucosa with
probes against human CaR. A: 25 µg
of Caco-2, HT-29-18-C1, and T84 cell total RNA.
B: 15 µg of total RNA derived from
rat mucosa scraped from duodenum, jejunum, ileum, cecum, and colon. Blots were prepared and probed as described in
METHODS.
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CaR agonists mobilize intracellular
Ca2+ in
HT-29-18-C1 cells.
The combined data of Figs. 1 and 2 demonstrate that the mRNA for CaR is
expressed both in the human intestinal cell lines examined (Caco-2,
HT-29, and T84) and in normal rat intestinal mucosa (epithelium).
Functional evidence for the presence of CaR requires examination of the
consequences of CaR activation. In many cell types [parathyroid
cells (13), C cells of the thyroid (12), pituitary cells (11, 35),
kidney cells (29), and cells transfected with cloned CaR (32)],
CaR activation mediates increases in intracellular
IP3 and subsequent release of
intracellular Ca2+ via activation
of IP3 receptors. HT-29-18-C1
cells were therefore loaded with fura 2-AM, and the effect(s) of bath
application of CaR agonists was examined by monitoring changes in fura
2 fluorescence at 340 and 380 nm. The viability of the cells and the
integrity of intracellular Ca2+
stores were assessed in each experiment by addition of 100 nM neurotensin, which activates neurotensin receptors in HT-29 cells (25,
37) and elicits an intracellular
Ca2+ transient primarily via
release from intracellular Ca2+
stores. Figure 3 illustrates the results of
such experiments. All experiments were performed in a minimal medium,
containing nominally zero Ca2+ and
0.55 mM Mg2+, to which the
illustrated serial additions were made. Figure 3A illustrates the response to an
increase in extracellular Ca2+
from nominally zero to 5 mM, followed by addition of 100 nM
neurotensin. Ca2+ elicited a
monotonic increase in intracellular
Ca2+ that decayed slowly, whereas
neurotensin elicited an intracellular Ca2+ transient that decayed to an
elevated steady state within 60 s. In representative experiments of
this type from two different cell preparations,
Ca2+ elicited a response that was
46.9 ± 7.4% of the peak response to 100 nM neurotensin
(n = 4). Because utilization of
Ca2+ as the CaR agonist alters the
driving force on Ca2+ and
potentially activates a variety of non-CaR-related
Ca2+ influx pathways, we also
examined the effects of two peptide agonists,
poly-L-arginine
(poly-L-Arg) (4, 5, 13) and protamine (5), under conditions in which extracellular
Ca2+ was nominally zero. Figure
3B illustrates the response of
HT-29-18-C1 cells to 2 µM
poly-L-Arg and the subsequent
response to 100 nM neurotensin. In experiments of this type in seven
cell preparations, 2 µM
poly-L-Arg elicited an increase
in intracellular Ca2+ that was
56.5 ± 6.3% of the peak response to 100 nM neurotensin (n = 16). Figure
3C illustrates the response to 200 and
400 µM protamine, followed by 100 nM neurotensin (representative of
experiments in 3 independent cell preparations, with an average
response that was 34.3 ± 6.3% of the peak neurotensin response;
n = 3). Both poly-L-Arg and protamine induce
increases in intracellular Ca2+
with an elevated plateau (even in the nominal absence of extracellular Ca2+), whereas neurotensin
induces a transient increase that rapidly decays to the plateau
established by the CaR agonist. These results demonstrate that a
variety of CaR agonists induce intracellular Ca2+ transients in
HT-29-18-C1 cells in the absence of extracellular Ca2+, indicative of CaR-mediated
release of Ca2+ from an
intracellular compartment.

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Fig. 3.
Intracellular Ca2+ changes in
response to application of various agonists. Intracellular
Ca2+ was monitored in
HT-29-18-C1 cells loaded with fura 2-acetoxymethyl ester (see
METHODS). Extracellular
Ca2+ at beginning of each
experiment was nominally 0. A:
intracellular Ca2+ responses to
bath addition of 5 mM CaCl2
followed by 100 nM neurotensin. B:
intracellular Ca2+ responses to 2 µM poly-L-arginine
(poly-L-Arg) followed by 100 nM
neurotensin (bath Ca2+ nominally 0 throughout experiment). C:
intracellular Ca2+ responses to
application of 200 and 400 µM protamine followed by addition of 100 nM neurotensin (bath Ca2+
nominally 0 throughout experiment).
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To further confirm that Ca2+ and
poly-L-Arg are inducing changes
in intracellular Ca2+ via CaR
activation, the dose-response relationships for the agonists were
determined in experiments such as those illustrated in Fig. 3.
Incremental additions in bath Ca2+
or poly-L-Arg were made until
the responses saturated, and the dose-response relationships were
determined by normalizing the data to the maximal response. Activation
of CaR by either Ca2+ or
poly-L-Arg was well fitted with
a single-site relation, with half-maximal stimulation at 1.15 mM for
Ca2+ (Fig.
4A) and
305 nM for poly-L-Arg (Fig.
4B).

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Fig. 4.
Dose-response relations for Ca2+
or poly-L-Arg-mediated
activation of intracellular Ca2+
transients. Experiments of type illustrated in Fig. 3 were performed with successive additions of either
Ca2+
(A) or
poly-L-Arg
(B) until no further changes were
elicited. Data for each experiment were normalized to maximal response
(10 mM Ca2+ for
A; 4,680 µM
poly-L-Arg for
B), and results from multiple experiments were combined (n = 3 in
A; n = 7 in B). Data were fitted with a
single-site model of the form Response = 100 × [(maximal response × [agonist]) / ([agonist] + K0.5)], where
[agonist] is the agonist concentration and
K0.5 is the half
maximal stimulatory concentration. A:
dose response for Ca2+-mediated
activation of intracellular Ca2+
increases. Ca2+o, extracellular
Ca2+ concentration.
K0.5 was 1.15 ± 0.34 mM. B: dose response for poly-L-Arg-mediated activation
of intracellular Ca2+ increases.
K0.5 was 305 ± 40 nM.
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The signal transduction pathway between CaR and an increase in
intracellular Ca2+ should include
activation of a GTP-binding protein, activation of
phosphatidylinositol-phospholipase C (PI-PLC), an increase in
intracellular IP3, and release of
Ca2+ from intracellular stores. We
utilized a combination of blockers and pertussis toxin to dissect this
pathway in HT-29-18-C1 cells.
To determine whether pertussis toxin-sensitive G proteins are involved
in CaR signaling in HT-29-18-C1 cells, we treated cells for 24 h
with 200 ng/ml pertussis toxin (List Biological Laboratories) (37).
Control cells were treated with vehicle (serum-free DMEM). Control and
pertussis toxin-treated cell populations responded to 2 µM
poly-L-Arg with increases in
intracellular Ca2+ of comparable
magnitude [control responses were 70.2 ± 12.5% (n = 6) and pertussis toxin-treated cell responses were 69.4 ± 14.6% (n = 5) of the peak neurotensin response].
These results suggest that pertussis toxin-sensitive GTP-binding
proteins are not involved in coupling CaR to intracellular
Ca2+ transients in
HT-29-18-C1 cells.
PI-PLC mediates increases in intracellular concentrations of
phosphoinositides. CaR activation by 2 µM
poly-L-Arg was assessed in the
presence of a blocker of PI-PLC (10), U-73122
{1-[6-((17
-3-methoxyestra-1,3,5(10)-trien-17yl)amino)hexyl]-1H-pyrrole-2,5-dione} and its relatively inactive analog, U-73343
{1-[6-((17
-3-methoxyestra-1,3,5(10)-trien-17yl)amino)hexyl]-2,5-pyrrolidinedione}. Figure 5 illustrates the results. Figure
5A depicts the control experiment, in
which poly-L-Arg (2 µM) was
added to cells in nominally zero extracellular
Ca2+. Figure
5B illustrates the response to
poly-L-Arg after a 3-min exposure to 1 µM U-73122. The response to
poly-L-Arg was completely blocked by U-73122. The inactive analog, U-73343, was not able to block
the response to poly-L-Arg (Fig.
5C). Figure
6 illustrates the tabulated responses from
experiments such as those in Fig. 5. We also tested the effect of
manoalide (a blocker of phospholipase A2) (16). Concentrations of
manoalide up to 2.5 µM had no effect on intracellular
Ca2+ (basal) or the cellular
responses to poly-L-Arg or
neurotensin.

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Fig. 5.
Effects of phosphatidylinositol-phospholipase C (PI-PLC) blockers on
poly-L-Arg-mediated activation
of intracellular Ca2+ transients.
All experiments were performed in nominally 0 bath Ca2+.
A: control experiment in which 2 µM
poly-L-Arg elicits an
intracellular Ca2+ transient.
B: response to 2 µM
poly-L-Arg after 2-min exposure to PI-PLC blocker U-73122 (1 µM). C:
response to 2 µM poly-L-Arg after 2-min exposure to 1 µM U-73343 (an inactive analog of
U-73122).
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Fig. 6.
Intracellular Ca2+ response to 2 µM poly-L-Arg in presence of
various PI-PLC blockers. Tabulated results from experiments of type
illustrated in Fig. 5. Intracellular
Ca2+ immediately before
poly-L-Arg addition (basal) is
compared with peak response in 2 µM
poly-L-Arg; 2 µM
poly-L-Arg induces an average increase in intracellular Ca2+ of
125 nM in control cells but no increase over that observed in presence
of U-73122. Although basal condition in U-73343 is slightly elevated, 2 µM poly-L-Arg induces an
increase in intracellular Ca2+ of
166 nM, which is greater than that observed in absence of blocker.
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Thapsigargin (1 µM) was used to deplete intracellular
Ca2+ stores (34), and Fig.
7 illustrates its effects on
Ca2+ signaling in HT-29-18-C1
cells. On application of 1 µM thapsigargin, intracellular
Ca2+ increases and then slowly
declines over the time course of the experiment (Fig.
7A). Figure
7B illustrates the effect(s) of
poly-L-Arg and neurotensin after
exposure of the cells to thapsigargin. The responses to both
poly-L-Arg and neurotensin are
eliminated by thapsigargin, indicating that both responses are mediated
by Ca2+ release from
thapsigargin-sensitive intracellular stores.

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Fig. 7.
Intracellular Ca2+ responses in
presence of thapsigargin. Cells were treated with 1 µM thapsigargin
to induce depletion of intracellular stores. Bath
Ca2+ was nominally 0. A: time-dependent response to 1 µM
thapsigargin. B: thapsigargin was
added at 30 s, and an intracellular
Ca2+ transient was observed.
Subsequent exposure to 2 µM
poly-L-Arg and 100 nM
neurotensin elicited minor decreases in intracellular Ca2+.
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DISCUSSION |
CaR is expressed in intestinal epithelial cells.
In this report, we present several lines of evidence to support the
hypothesis that intestinal epithelial cells express functional CaR.
First, RT-PCR with a primer set that spans several CaR intron-exon boundaries (from exon 3 to exon 5) yields an appropriately sized band
when applied to rat intestinal RNA. Identity with CaR was confirmed by
both restriction fragment analysis and sequencing. Similar results were
obtained with RT-PCR applied to several human colonic epithelial tumor
cell lines, including Caco-2, HT-29 (several subclones), and T84.
Second, Northern blotting with a probe generated from a segment of exon
3 of the human CaR reveals several bands in both human and rat total
RNA isolated from scraped mucosa (epithelium), consistent with
expression of CaR. Third, application of CaR agonists (Ca2+,
poly-L-Arg, protamine) results
in intracellular Ca2+ transients
in HT-29-18-C1 cells, consistent with agonist-mediated mobilization from thapsigargin-sensitive intracellular stores. The
affinity for extracellular Ca2+
observed in the present study (1.15 mM) is comparable to that observed
in isolated parathyroid cells
[K0.5 of
~1.5 mM (26, 28)], although it is lower than that observed for
CaR expressed in Xenopus oocytes or
human embryonic kidney (HEK)-293 cells (8, 13, 27, 32). These
differences may reflect tissue-specific alterations in
Ca2+ affinity, which has been
shown to be modulated by protein kinase C-mediated phosphorylation
(28). The affinity for
poly-L-Arg (340 nM) is higher
than that observed for
poly-L-Arg in parathyroid cells
[K0.5 of 40 nM for poly-L-Arg with average
molecular mass of 11,600 Da (5)]. The dose-response relations for
poly-L-Arg and
poly-L-lysine are dependent on
peptide length, with increasing length being reflected in a higher
affinity (5). The affinity for
poly-L-Arg in HT-29-18-C1
cells may reflect the differences in
poly-L-Arg preparations and
their relative degrees of polydispersity. Nevertheless, taken together,
these results strongly suggest the presence of functional CaR in
HT-29-18-C1 cells, with intracellular signaling consistent with
that observed in other cell types known to express CaR.
Minimal signal transduction pathway for CaR in intestinal epithelial
cells.
The functional studies presented in this report suggest that CaR in
intestinal epithelial cells couples via a pertussis toxin-insensitive G
protein to PI-PLC, presumably increasing intracellular
IP3, which ultimately results in
an intracellular Ca2+ transient
via release from thapsigargin-sensitive intracellular stores.
Neurotensin (100 nM) and carbachol (data not shown) elicit similar
results in HT-29-18-C1 cells, suggesting that a common signaling
pathway is utilized. The functional coupling of extracellular Ca2+ (or
poly-L-Arg or protamine) to
intracellular Ca2+ transients
suggests that intestinal cell function is modulated by CaR activation
under physiological conditions. Our studies do not address the question
of CaR localization, but this is clearly critical to understanding the
role(s) of CaR in intestinal epithelium. A recent study has suggested
that CaR is apically localized in rat kidney inner medullary collecting
duct (IMCD) (33), although functional studies in intact tubules have
failed to demonstrate intracellular
Ca2+ transients in response to
alterations in luminal Ca2+ in rat
IMCD (9). CaR may be apically localized in intestinal epithelium, since
reductions in apical (but not basolateral)
Ca2+ have been shown to induce
expression of c-myc in Caco-2 cells (22). Because alterations in extracellular
Ca2+ also affect tight junctional
permeability, a definitive conclusion about CaR localization in
intestinal epithelium awaits further studies. However,
Ca2+ has been postulated as a
controller of colonic epithelial growth and differentiation, and a
tentative apical localization of the intestinal CaR hints at
involvement in luminal Ca2+
sensing (40).
Possible roles for CaR in intestinal epithelial cells.
The intestinal mucosa is clearly an integral participant in organismal
Ca2+ homeostasis, mediating
Ca2+ absorption (21).
Ca2+ absorption occurs throughout
all segments of the intestine, although it is thought to be most
significant in the small intestine, due to longer residence times of
luminal contents. Both paracellular and transcellular pathways for
Ca2+ absorption have been
described, and the transcellular pathways are vitamin D dependent (39).
Recent evidence has suggested a synergistic effect between vitamin D
and Ca2+ in mediating
Ca2+ transport (15) as well as
vitamin D-mediated regulation of CaR expression (3), and thus
a role for CaR in modulation of intestinal
Ca2+ absorption is possible.
Functional studies of Ca2+
absorption in the homozygous CaR knockout mice (18) may be instructive
in this regard.
Ca2+ has been shown to mediate
intestinal epithelial cell differentiation in both patient studies (23,
24) and controlled studies in intestinal epithelial cell lines (2, 7,
20). Low Ca2+ stimulates cell
division and growth in Caco-2 and HT-29 cells (7, 18), and
extracellular Ca2+ in the range of
0.5-1 mM promotes differentiation via several criteria, including
thymidine incorporation and expression of differentiation markers (7,
20, 22). Although the mechanism(s) that mediates these effects of
extracellular Ca2+ is not known,
it is possible that CaR may transduce at least part of the
differentiation response. In summary, we have demonstrated intestinal
epithelial cell expression of CaR via several molecular biological and
functional criteria. These results provide the foundation for
investigations into the roles of CaR in modulating intestinal
Ca2+ absorption and epithelial
cell differentiation.
 |
ACKNOWLEDGEMENTS |
We thank Kelli Hettich for preliminary experiments, Dr. Laura Roman
for helpful discussions, and Dr. Marshall Montrose for fluorescence
advice.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-44484 (to G. E. Breitwieser), National
Institute of Nursing Research Grant 3800-02 (to L. M. Baxendale-Cox), and funds from the Johns Hopkins University School of
Nursing.
Address for reprint requests: G. E. Breitwieser, Johns Hopkins
University School of Medicine, Dept. of Physiology, 725 N. Wolfe St.,
Baltimore, MD 21205.
Received 9 October 1996; accepted in final form 19 June 1997.
 |
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