1 Department of Cellular and Molecular Physiology and 2 Department of Surgery, Yale University, New Haven, Connecticut 06520-8026
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ABSTRACT |
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The calcium-sensing receptor
(CaSR) is activated by extracellular calcium (Ca
calcium-sensing receptor; intracellular calcium; net fluid movement; isolated perfused colonic crypt
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INTRODUCTION |
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THE EXTRACELLULAR
CALCIUM (Ca
The CaSR is expressed in gastrointestinal tract, including colon
(7, 14), suggesting multiple potential roles for this receptor in gastrointestinal biology. In the intestine, and colon in
particular, previous studies have indicated that high Ca2+
dietary intake promotes colonic mucosal epithelial cell
differentiation, decreases cell growth, and reduces risks for
development of colorectal cancer [see recent summary by Bresalier
(2) and also Refs. 17, 18, and
31]. There is also evidence that, as in the small
intestine, the colon can absorb and secrete Ca2+ in
response to changes in Ca
To date, however, there has been no documentation of the functioning of this receptor in colon nor its involvement in the regulation of colonic function. In addition, despite the presence of CaSR transcripts and protein in the colon mucosal epithelium, there has been uncertainty with regard to the receptor distribution and localization within this tissue. We previously (7) suggested that the CaSR is present in the apical and basolateral membranes of rat colon crypt cells, although the CaSR distribution in the surface epithelium was unclear. Sheinin et al. (32) recently examined the CaSR immunolocalization in human large intestine using a monoclonal anti-CaSR antibody and found that the CaSR immunoreactivity was only present in certain enteroendocrine cells of the crypts.
In the present study, we further investigated the expression and
localization of the CaSR in both rat and human colonic epithelial cells, the functioning of this receptor in rat colon, and its modulation of fluid movement in isolated perfused rat colonic crypts.
The expression of CaSR in colon surface and crypt epithelial cells was
verified by RT-PCR and Western blot analyses. In addition, immunofluorescence and immunohistochemistry studies demonstrated CaSR
protein at both the apical and basolateral surfaces of rat colonic
surface and crypt cells. Moreover, by measuring the CaSR agonist-induced changes in Ca
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MATERIALS AND METHODS |
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Animals. Experiments were performed using adult male Sprague-Dawley rats (weighing 150-300 g) obtained from Charles River Laboratories and Taconic Farms. The use of rats as well as the protocol for isolating colon tissues and cells was approved by the Institutional Animal Care and Use Committee (IACUC # 2000-10253) at Yale.
Animals were fed and maintained on regular chow (PMI Nutrition International) with free access to water before the investigation. Rats were anesthetized and euthanized with isoflurane or pentobarbital and killed by cervical dislocation before colonectomy.Isolation of surface and crypt cells from colon.
The procedure for the isolation of surface and crypt cells from rat
colon is based on the method previously described (21) with minor modifications. Colons were removed from rats and were cut
open longitudinally to eliminate fecal pellets. After being dissected
and washed, the tissues were everted to expose the mucosal surface. To
obtain surface cells, the everted colons were either scraped gently
over the mucosa with a glass slide or incubated in Na-citrate buffer
containing (in mM): 96 NaCl, 27 Na citrate, 0.8 KH2PO4, 5.6 Na2HPO4,
and 1.5 D-glucose, pH 7.4. To obtain isolated crypts, the
colonic segments were further incubated in Na-EDTA buffer containing
(in mM): 96 NaCl, 1.5 KCl, 21 Na EDTA, 55 sorbitol, 22 sucrose, and 10 HEPES, pH 7.4. At the end of each incubation period, colonic segments
were agitated for 30 s to release surface cells or individual
crypts. Released cells or crypts were immediately mixed with 2 volumes
of standard Ringer solution containing (in mM): 125 NaCl, 5.0 KCl, 1.0 CaCl2, 1.2 MgSO2, 2.0 NaH2PO4, 5.0 D-glucose, and 32 HEPES, pH 7.4. Cells were collected by centrifugation (2,000 rpm for 5 min in a Beckman Coulter Allegra 6R centrifuge), washed in initial
Ringer solution, and resuspended in initial Ringer solution unless
otherwise specified. Initial Ringer solution contains the same
composition as standard Ringer except that Ca2+ was reduced
to 0.1 mM and Mg2+ was either 0 mM or 0.5 mM. These
solutions with reduced divalent cation concentrations were used to
minimize stimulation of CaSR by Ca
Detection of CaSR transcripts in isolated surface and crypt cells by RT-PCR. Total RNA was isolated, separately, from surface and crypt cells as well as from rat kidneys (RNeasy Mini Kit; QIAGEN, Valencia, CA) and reverse transcribed into cDNA using oligo(dT)12-18 primers (SuperScript preamplification system, Life Technologies), and aliquots of the reverse transcripts were amplified by 30 cycles of PCR using a set of primers based on the published sequence of the rat CaSR gene. The primer sequences were 5'-ACC TTT ACC TGT CCC CTG AA-3' and 5'-GGG CAA CAA AAC TCA AGG TG-3'. This primer pair spans an intron, is predicted to yield a 383-bp fragment when amplifying rat CaSR transcripts, and corresponds to a region within the predicted NH2 terminus of the CaSR. The PCR reactions were performed in a Robocycler 40 (Stratagene, La Jolla, CA) under the following conditions: 94°C for 1 min, 50°C for 2 min, and 72°C for 1.5 min for 1 cycle, followed by 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min for 30 cycles. Positive control for PCR experiments was performed by using 1 pg of the full-length rat cDNA for CaSR, whereas negative controls included primers but no templates or RNA not reverse transcribed.
For nucleotide sequencing studies, PCR products of the expected size were extracted from the agarose gel and were sequenced bidirectionally by using the primer pairs described above. Nucleotide sequencing was performed by the dideoxy chain termination method with an Applied Biosystems automated sequencer at the Yale Keck facility. Further nucleotide sequence analyses were carried out using VectorNTi software (version 6).Detection of CaSR protein expression in isolated colonic surface and crypt cells by Western blot analysis. Cells were lysed for 15 min on ice in a buffer containing: 62.5 mM Tris, pH 6.8, 10% sucrose, 2% SDS, 5% 2-mercaptoethanol, 100 µM phenylmethylsulfonyl fluoride, and Mini Complete protease inhibitors (Roche). The lysates were briefly sonicated and were incubated at room temperature for 15 min before they were loaded onto a 6% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad) by electroblotting. The membranes were rinsed in Tris-buffered saline (TBS) and quenched with 5% nonfat milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. Expression of CaSR was detected with an affinity-purified polyclonal antibody raised against a 22-amino acid region of the NH2 terminus of the receptor (29). Incubation with primary antibody was made overnight in 5% nonfat milk containing TBS-T (1:100 dilution). After three 20-min washes at room temperature in TBS-T, membranes were blocked with milk, avidin, and biotin solutions and were incubated with anti-rabbit IgG secondary antibody conjugated to biotin (1:20,000 dilution) and with avidin/biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA). Each treatment was followed by three 20-min washes at room temperature in TBS-T. Immunoreactive signals were visualized by chemiluminescence (Amersham Pharmacia Biotech UK).
Immunofluorescence and immunohistochemistry. The colons from male Sprague-Dawley rats (weighing 200-250 g) were perfused via the descending aorta with PBS, followed by 4% paraformaldehyde, and then by 12.5% sucrose in PBS solution. The same fixative was also used for a human colon sample taken via biopsy (graciously donated by Mary Kay Washington, Associate Professor of Pathology, Vanderbilt University Medical Center, in conjunction with the Vanderbilt-Ingram Cancer Center, Human Tissue Acquisition and Pathology Shared Resource Core). Tissues were kept overnight at 4°C in 30% sucrose in PBS. Tissues were then embedded in Tissue Tek optimum cutting temperature (Sakura Finetek USA, Torrence, CA) and frozen in isopentane cooled on dry ice. Five-micrometer frozen sections were cut with a Leica CM3050 cryostat and thaw mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA).
Cryosections were antigen retrieved by using Citra Microwave Solution (Biogenex, San Ramon, CA) followed by treatment with 1% SDS in PBS for 5 min to expose antigenic sites. After SDS treatment, slides were rinsed three times with PBS and incubated 30 min with 1% BSA/PBS followed by three 5-min washes in PBS. Slides were then incubated with 4% Seablock for 1 h at room temperature followed by overnight incubation with primary affinity-purified anti-CaSR polyclonal antibody (1:100 dilution). For immunohistochemistry, slides were preincubated in peroxidase blocking reagent (DAKO, Carpenteria, CA) for 5 min and in protein blocking serum-free solution (DAKO) for 15 min before overnight incubation with the primary antibody. To assess nonspecific staining, control experiments were performed by incubating the slides without primary antibody or with primary antigen absorbed antibody for 1 h at room temperature. Secondary antibody was diluted with 1% BSA/PBS and applied to sections for 1 h at room temperature at the following dilutions: Alexa 594 anti-rabbit IgG (Molecular Probes, Eugene, OR) 1:5,000 diluted in 1% BSA/PBS, and peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories) 1:100 diluted in PBS, pH 7.4. The slides were then washed in two 5-min washes in high salt PBS + 2.8% NaCl then two 5-min washes in PBS. For immunohistochemistry, the color reaction was developed at room temperature for 5 min by using the DAKO AEC substrate system. The reaction was stopped by three rinses in water. Slides were then mounted using Vectashield (Vector Laboratories) and were examined with a Nikon Eclipse 800 Research microscope equipped with a charge-coupled device camera. Fluorescence and histochemical photomicrographs were stored in a computer and were processed using Adobe Photoshop 5.0 software.Fluo 3 fluorescence imaging. Crypts were adhered to glass coverslips precoated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA) and were loaded with fluo 3-AM (Molecular Probes). Following dye loading, the crypts were washed for 5 min and the coverslips were transferred to a perfusion chamber where the isolated crypt cells were imaged with a confocal laser scanning microscope (LSM 410, Carl Zeiss, Thornwood, NY) using a 40 × 1.4 oil immersion lens with infinity corrected optics. Dye molecules were excited with a multiline argon laser at a wavelength of 488 nm, and emission was detected in the wavelength range of 515-565 nm. Images of crypt cells (512 × 512 × 12 bit deep) were recorded before and after addition of stimulant, and each image was an average of eight sequential frames acquired at 2-s intervals. Neither solution changes per se nor addition of vehicle affected the fluorescent signals.
Measurement of Ca
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Measurement of Ca
Measurement of Jv in isolated perfused colonic
crypts.
Determination of fluid movement in the isolated perfused crypt followed
a method that we have previously developed (33). Briefly,
following isolation of the crypt by either a hand dissection or by the
same EDTA dissection technique outlined above, the crypt was
transferred to the stage of an inverted microscope and mounted on a
series of concentric glass micropipettes. Following cannulation of both
ends of the crypt, perfusion was established on both the luminal and
basolateral membranes. For the luminal perfusion, rates were maintained
at ~4-8 nl/min, whereas the basolateral perfusion was at 3 ml/min (which equates to 3 bath exchanges/s). The length, diameter, and
contact time for particles were recorded, and the time to fill a
calibrated collection pipette was used to determine the luminal
perfusion. The luminal perfusates contained 10 µCi/ml
methoxy-[3H]inulin (New England Nuclear, Boston, MA).
Jv was calculated as previously described
(33) as the rate at which the effluent accumulated in the
collection pipette and the concentrations of methoxy-[3H]inulin in the perfusate and effluent.
Positive Jv values
(nl · mm1 · min
1) indicate
net fluid movement from lumen to bath (absorption), and negative values
indicate fluid movement from bath to lumen (secretion). The bath was
routinely assayed for methoxy-[3H]inulin, and experiments
were discarded if the bath concentration exceeded background.
Determinations of Ins(1,4,5)P3 levels. Measurements of Ins(1,4,5)P3 were performed by using an Ins(1,4,5)P3 binding protein kit (Amersham Pharmacia Biotech). Following drug treatment, cells were immediately lysed by exposure to perchloric acid and cell lysates were centrifuged at 2,000 g at 4°C for 15 min. The supernatant was transferred to a siliconized glass tube and neutralized with ice-cold 1.5 M KOH containing 60 mM HEPES buffer to pH 7.5, and the concentration of Ins(1,4,5)P3 was then measured according to the manufacturer's instructions. Samples were normalized on the basis of total protein by using a Pierce protein assay.
Chemicals and solutions. CaCl2, GdCl3, neomycin, U-73122, and forskolin were purchased from Sigma (St. Louis, MO), thapsigargin from Molecular Probes (Eugene, OR), and nonfat milk from Nestle (Solon, OH). All physiological solutions were prepared freshly and were equilibrated with 100% oxygen before use.
Statistics. Values are given as means ± SE of n experiments. Statistical comparisons between two means were performed by Student's t-test, whereas comparisons among multiple means were by ANOVA. Both tests were performed using Microsoft Excel 97 for Windows. P < 0.05 was considered significant. Curve fitting and calculations for kinetic index of CaSR activation were performed using GraphPad Prism version 3 for Windows (GraphPad Software, San Diego, CA).
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RESULTS |
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Although previous studies have identified CaSR transcripts and
protein in the colon (7, 32), there remains uncertainty as
to the specific colonic epithelial cells expressing the CaSR as well as
the sidedness (apical or basolateral) of expression in these cells.
Thus we further investigated the expression and localization of the
CaSR in rat colonic surface and crypt cells. Figure
1A shows that PCR products of
the expected size, i.e., 383 bp, were amplified by CaSR-specific
primers from transcripts isolated from both surface and crypt cells.
Nucleotide sequencing of the PCR products from colon crypts revealed
>99% identity with the corresponding sequence of the full-length CaSR
cDNA from rat kidney. Thus both surface and crypt cells express CaSR
transcripts.
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An affinity-purified polyclonal antibody that is specific for the CaSR
(29) identified a specific band of ~135 kDa in lysates of both surface and crypt cells (Fig. 1B), verifying CaSR
protein expression in both of these colonic epithelial regions. The
Western blot was performed under reducing conditions, and the band at ~135 kDa represents the monomer form of CaSR. However, when blots were performed under nonreducing conditions (i.e., absence of 2-mercaptoethanol), bands at both 135 and 200-235 kDa were
observed, representing, respectively, the monomer and dimer forms of
CaSR (data not shown). The CaSR signals detected in surface and crypt cells of colon were similar to that from the kidney (Fig.
1B), which is known to express high levels of CaSR (4,
30). To assess which specific cells, and which membranes in
these cells, express CaSR protein, we examined the receptor
immunolocalization (29). CaSR antigenic sites were made
accessible in frozen sections by "antigen retrieval" treatment
before immunodetection. Figure 2,
A-D show the immunofluorescence and
immunohistochemistry of CaSR in the rat distal colon epithelial cells.
Both apical and basolateral aspects of surface and crypt cells
exhibited intense CaSR staining (Fig. 2,
B-D). A similar pattern of CaSR
immunostaining was observed in proximal colon epithelial cells (not
shown). To assess if CaSR is expressed in human colonic crypts, we
stained a colon biopsy specimen with the CaSR-specific antibody. Figure 2, E and F show representative differential
interference contrast and immunofluorescence images of the same human
colonic crypt and demonstrate that the CaSR staining pattern in human
colonic crypt is essentially identical to that in rat.
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After verifying that CaSR transcripts and protein were present in both
surface and crypt cells, we next determined if this receptor is
functionally active. We assessed Ca
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We next examined Ca
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CaSR-mediated Ca
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The dose-response relationships for Ca
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To further assess the functional relevance of the CaSR expression in
the colon epithelial cells, particularly its potential role in
modulating intestinal fluid movement in certain diarrheal states, the
effect of either luminal or basolateral CaSR activation on
Jv was determined in isolated rat distal colonic
crypts in both basal and forskolin-stimulated states. Figure
7 summarizes the changes in
Jv in perfused colonic crypts in the absence and presence of 100 µM forskolin and before and after raising
Ca1 · min
1, indicating
net fluid absorption. Exposure to forskolin induced net fluid
secretion; Jv averaged between
0.195 and
0.202 nl · mm
1 · min
1.
Raising Ca
1 · min
1 for
basolateral application and 0.302 ± 0.017 and 0.300 ± 0.024 nl · mm
1 · min
1 for luminal
application.
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DISCUSSION |
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Our data demonstrate that CaSR is both expressed and functionally
active in the colon epithelium and that this colonic CaSR is a
modulator of intestinal fluid transport. We and others (7, 32) have shown the presence of CaSR transcripts and protein in
colon. In the present study, we extend these observations by demonstrating that CaSR transcripts and protein are expressed in
isolated surface and crypt epithelial cells from rat distal colon.
Immunohistochemistry and immunofluorescence showed the presence of CaSR
on both apical and basolateral surfaces of these epithelial cells. This
receptor protein was also found in a similar pattern in crypts from a
human colon biopsy specimen, providing support for potential roles of
the CaSR in human colon. The increases in Ca
The CaSR localization in rat colonic epithelium shown here is
consistent with the distribution pattern in crypts previously reported
in rat by Chattopadhyay et al. (7) but additionally demonstrates distinct apical and basolateral receptor expression in
surface cells. In contrast, Sheinin et al. (32) recently examined the CaSR immunolocalization in the large intestine of humans
and showed receptor immunoreactivity only in epithelial cells at the
base of crypts. Since the CaSR protein was found in the present study
in a similar pattern in colonic crypts from both rat and human,
differences in CaSR localization between our study and that of Sheinin
et al. are unlikely to be due to species differences. The most likely
explanation is differences in experimental conditions, including use of
polyclonal vs. monoclonal antibody, cryosections vs. paraffin sections,
and with vs. without "antigen retrieval" pretreatment. In previous
studies (29, 30), antigen retrieval was critical to
identifying CaSR expression in rat kidney using our well-studied
polyclonal antibody against the CaSR. Further support for our CaSR
expression pattern in both surface and crypt cells in rat colon is
given both by the receptor-mediated responses in Ca
The EC50 value for Cas) in isolated rat colon mucosa. It is
likely that the influence of extracellular levels of Ca2+
on these two different aspects of colon function is mediated by the
same mechanism for Ca
The major function of the colon is to absorb fluid. In kidney,
modulation of salt and fluid absorption by CaSR has been demonstrated in various tubular segments where the receptor is localized (5, 29). To assess if the colonic CaSR is a modulator of intestinal fluid movement, in the present study we measured changes in
Jv in isolated perfused colonic crypts using the
in vitro microperfusion technique (33). We found that
activation of either the apical or basolateral CaSR reversed net fluid
secretion elicited by forskolin stimulation to net absorption (see Fig.
7). This reversal of forskolin-stimulated net fluid secretion may be
due to either a reduction in secretory flux or a stimulation of
absorptive flux. The mechanism for modulation of forskolin-stimulated
fluid secretion may be due to CaSR-mediated reduction in intracellular
cAMP accumulation as observed in cortical thick ascending limb
(10) and in several other cell types [see review
(6)]. In thick ascending limb, Ca
Even though the present study has identified the colonic CaSR as
a potential modulator of regulated intestinal fluid transport, the high
expression of CaSR in both luminal and basolateral surfaces of colon
epithelium may also indicate physiological roles and functions in
addition to regulating fluid absorption/secretion. The CaSR is
abundantly expressed in the parathyroid gland, intestines, and kidney.
The expression of the receptor in these tissues presumably reflects the
key roles of CaSR in the maintenance of Ca
The CaSR in colon may also be regulating epithelial cell proliferation and differentiation. The epithelium of the colon as well as the small intestine is in a state of constant renewal. Cells proliferate and become differentiated as they migrate out of the base of the crypt to the surface. Thus cells at the base of the crypt are highly proliferative but less differentiated, and cells at the surface of the colon epithelium, on the other hand, are highly differentiated with little or no proliferation. In this regard, the CaSR in kerotinocytes and certain other cells has been shown to modulate proliferation/differentiation and to alter the activities of mitogen-activated protein kinases and tyrosine kinases associated with cell proliferation (19, 22, 34, 36). Thus it is possible that the CaSR may be mediating the high dietary Ca2+ responses of colonic mucosal epithelium in promoting cell differentiation, decreasing cell growth, and reducing the risk for development of colorectal cancer (2, 17, 18, 31).
Of particular interest in the colon is the intense CaSR
immunoreactivity localized on the apical aspects of cells (see Fig. 2,
B-D). The apical localization is consistent
with potential roles for this receptor in sensing and responding to
changes in luminal Ca
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ACKNOWLEDGEMENTS |
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Support for this work was from local funds from Yale Medical School and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50230 and DK-17433.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. C. Hebert, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St. SHM B147, P.O. Box 208026, New Haven, CT 06520-8026 (E-mail: steven.hebert{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 20, 2002;10.1152/ajpgi.00500.2001
Received 20 November 2001; accepted in final form 17 March 2002.
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