1 Endocrine-Hypertension
Division, The extracellular
calcium (Ca2+o)-sensing receptor (CaR)
plays vital roles in Ca2+o homeostasis, but no data are available on its expression in small and large intestine. Polymerase chain reaction products amplified from
reverse-transcribed duodenal RNA using CaR-specific primers showed
>99% homology with the rat kidney CaR. Northern analysis with a
CaR-specific cRNA probe demonstrated 4.1- and 7.5-kb transcripts in all
intestinal segments. Immunohistochemistry with CaR-specific antisera
showed clear basal staining of epithelial cells of small intestinal
villi and crypts and modest apical staining of the former, whereas
there was both basal and apical staining of colonic crypt epithelial cells. In situ hybridization and immunohistochemistry also demonstrated CaR expression in Auerbach's myenteric plexus of small and large intestines and in the submucosa in the region of Meissner's plexus. Our results reveal CaR expression in several cell types of small and
large intestine, in which it may modulate absorptive and/or secretomotor functions.
villi; crypts; nerve plexus; Northern analysis; immunohistochemistry; in situ hybridization histochemistry
MAINTENANCE OF A CONSTANT level of the extracellular
ionized calcium (Ca2+o) concentration is
a vital biological function in a wide variety of organisms
(4). The system regulating Ca2+ homeostasis involves several
organs and hormones. The former include the parathyroid glands, the
kidneys, the small and large intestines, and the skeleton, and the
latter comprise parathyroid hormone (PTH), calcitonin, and vitamin D
(for review, see Ref. 4). The parathyroid cell is extremely sensitive
to alterations in Ca2+o and responds
promptly with changes in PTH secretion designed to normalize
Ca2+o by acting on its target organs (4).
Acute PTH-mediated responses to hypocalcemia include increased
reabsorption of Ca2+ from the
kidney and resorption of Ca2+ from
bone. Protracted hypocalcemia leads to PTH-stimulated 1-hydroxylation and resultant activation of 25-hydroxyvitamin
D3 in the renal proximal tubule.
The 1,25-dihydroxyvitamin D3
produced by this reaction then acts through its receptor in the
duodenum to increase the absorption of
Ca2+ (4, 10).
Regulation of PTH secretion by Ca2+o is
dependent on a process of Ca2+o sensing
that is afforded by a recently identified
Ca2+o-sensing receptor (CaR), which is a
G protein-coupled heptahelical receptor (5). Initially cloned from
bovine parathyroid (5), CaR has subsequently been isolated from various
other organs that participate in Ca2+o homeostasis, such as rat (28) and rabbit kidney (7), as well as human
(20) and chicken parathyroid gland (15). It has also been cloned from
the brain (30) and is likewise expressed in several other tissues that
are not directly involved in the mineral ion homeostatic mechanism
[e.g., keratinocytes (1) and lens epithelial cells (11)].
The intestine is an important site for the maintenance of
Ca2+ homeostasis because of its
capacity for regulated absorption of dietary
Ca2+. The intestinal tract
exhibits functional heterogeneity along its length, and each intestinal
segment has specialized absorptive and/or secretory functions
(23), analogous to the nephron, which is also involved in the secretion
and absorption of solutes. The proximal portion of the small intestine
(i.e., the duodenum) absorbs large volumes of fluid against a
relatively small electrochemical gradient (31), similar to the proximal
tubule of the nephron. In addition, the duodenum is the major site for
1,25-dihydroxyvitamin D3
[1,25(OH)2D3]-dependent
Ca2+ absorption through increases
in transcellular, mucosal-to-serosal flux of
Ca2+, involving a process of
active transport that most likely includes the vitamin D-dependent
Ca2+-binding protein calbindin
(18, 26). Although the duodenum is the major site for absorption of
Ca2+, the jejunum and ileum, in
addition to absorbing lesser amounts of
Ca2+, are known to secrete
Ca2+ by a nonsaturable
paracellular mechanism (3, 23). Serosal-to-mucosal flux of
Ca2+ in these intestinal segments
may enable chelation of fatty acids and bile salts, thereby forming
insoluble "calcium soaps" that preclude potential damage to
colonic epithelial cells resulting from the actions of soluble,
unchelated fatty acids and bile salts. Although the duodenum, jejunum,
and ileum exhibit regional differences in their transport of various
nutrients, vitamins, minerals, and bile acids, there are less marked
regional differences in electrolyte transport (3). Even though the
major function of colon is to absorb water and sodium
(Na+), which is again
reminiscent of the function of certain portions of the nephron, it also
absorbs significant amounts of
Ca2+ by both vitamin D-dependent
and -independent mechanisms (17).
In addition to being absorbed and secreted by the intestine,
Ca2+o, along with vitamin D, exerts
several direct actions on intestinal function.
Ca2+o and vitamin D both increase the
level of expression of calbindin in duodenal explants (2). In addition,
there are several reports (6, 13, 14) that
Ca2+ and/or
1,25(OH)2 vitamin
D3 inhibit the proliferation of
colonic epithelial cells in vivo and in vitro. Similar effects have
been observed in cultured Caco-2 cells, a cell line derived from a human colon carcinoma (13, 14). Moreover, we have recently shown that
these cells express CaR, which may mediate the effects of
Ca2+o on the apical surface of the cells
to inhibit cellular proliferation and expression of
c-myc through activation of protein
kinase C (PKC) (22). These data led us to study the localization of CaR
in the small intestine and colon of the rat. We observed that it is
widely distributed and exhibits region-specific variations in
localization that are of potential physiological significance.
Animals.
Male Sprague-Dawley CD rats (weighing 225-250 g) were obtained
from Charles River Laboratories (Wilmington, MA). They were housed in a
controlled environment with a 12:12-h light-dark cycle and had free
access to standard rat chow and tap water. They were handled according
to the humane practices of animal care established by the Standing
Committee on Animals at Harvard University. Rats were killed after we
induced anesthesia with pentobarbital sodium.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C until further use. For immunohistochemistry, rats were
perfused transcardially with 4% buffered paraformaldehyde via the
descending aorta, followed by a solution containing phosphate-buffered saline (PBS) and sucrose having an osmolarity of 700 mosM. After perfusion, tissues were quickly removed, postfixed in buffered 4%
paraformaldehyde for 3 h on ice, and cryoprotected by incubation in
PBS-buffered 20% sucrose at 4°C overnight. The tissues were then
embedded in optimum-cutting compound (Miles, Elkhart, IN), snap frozen
in 2-methylbutane and liquid nitrogen, and stored at
70°C
until use.
Detection of CaR transcripts in isolated gastrointestinal segments by RT-PCR. Total RNA was isolated as previously described by Chirgwin et al. (12). We used 5 µg of total RNA for the synthesis of single-stranded cDNA. Briefly, RNA samples were reverse transcribed at 42°C for 1 h by incubation with 20 µl of an RT mixture containing the following constituents: 25 pmol random hexamer primers, 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 20 U RNasin, 0.5 mM deoxyribonucleotide triphosphates (dNTP), and 200 U superScript RT+ (Life Technologies, Gaithersburg, MD). The reverse transcriptase was inactivated by heating for 15 min at 70°C. The resultant first-strand cDNA was then used for the PCR procedure. PCR was performed in a total volume of 100 µl of a buffer solution containing the following: 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTP, 10 U of cloned Pfu polymerase (Stratagene, La Jolla, CA) as well as 0.25 µM of sense and antisense primers based on the sequence of the rat kidney Ca2+o-sensing receptor (RaKCaR) [forward primer, 5'-ACCTTTACCTGTCCCCTGAA-3' (RaKCaR bp 597-616) and reverse primer, 5'-GGGCAACAAAAACTCATG-3' (RaKCaR bp 964-980), respectively].
The primer pairs amplified a 383-bp fragment encoding a region located within the predicted NH2 terminus of RaKCaR. The optimum temperature cycling protocol was determined to be 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 30 cycles using a programmable thermal cycler (Omnigene, Hybaid Instruments, Holbrook, NY). Positive and negative control experiments were performed, respectively, by using 5 µg of RNA extracted from rat kidney and RNA samples from various gastrointestinal segments processed similarly but without RT. PCR products obtained from duodenal mucosa were subcloned into the pCR-Script SK(+) vector using the pCR-Script Amp SK(+) cloning kit (Stratagene). Bidirectional sequencing was performed using the dideoxy chain termination method with an Applied Biosystems model 373A automated sequencer (Department of Genetics, Children's Hospital, Boston, MA). Further nucleotide analyses were carried out using GeneWorks software (version 2.3.1, IntelliGenetics, Mountain View, CA).Detection of CaR transcripts by Northern blot analysis.
For the purpose of determining the size of the CaR transcripts in
various intestinal segments, we employed Northern blot analysis on
aliquots of 5 µg poly(A)+ RNA
obtained using oligo(dT) cellulose chromatography of total RNA.
Poly(A)+-enriched RNA samples were
denatured and electrophoresed in 2.2 M formaldehyde-1% agarose gels
along with a 0.24- to 9.5-kb RNA ladder (Life Technologies) and
transferred overnight to nylon membranes (Duralon, Stratagene). A
577-bp Bgl
II-Sac I fragment corresponding to
nucleotides 721-1,298 of the RaKCaR cDNA was subcloned into the
pBluescript(SK+) vector. The plasmid was then linearized with
Bgl II, and a
32P-labeled riboprobe was
synthesized with the MAXIscript T3
kit (Ambion, Austin, TX) using T3
polymerase and
[32P]UTP. Nylon
membranes were prehybridized for 2 h at 42°C in a solution
consisting of 50% formamide, 4× Denhardt's solution
[50× Denhardt's solution is 5 g Ficoll, 5 g
polyvinylpyrrolidone, and 5 g bovine serum albumin (BSA)],
5× SSPE (20× SSPE is 2.98 M NaCl and 0.02 M EDTA in 0.2 M
phosphate buffer, pH 7.0), 0.5% sodium dodecyl sulfate (SDS), 10%
dextran sulfate, 250 µg/ml yeast tRNA, and 200 µg/ml calf thymus
DNA. Labeled probe (2 × 106
cpm/ml) was then added, and the membranes were hybridized overnight at
the same temperature. Washing was carried out at high stringency [in 0.25× SSC (20× SSC is 3 M NaCl, 0.3 M
Na3-citrate · 2H2O), 0.5% SDS at 60°C] for 20 min. Membranes were then exposed to
X-ray film (Kodak XAR-5, Eastman Kodak, Rochester, NY) for 4 days at 70°C. Integrity of RNA samples was confirmed by reprobing
the blot with a
-actin cDNA probe.
In situ hybridization histochemistry.
In situ hybridization of transverse sections from rat duodenum and
proximal colon was performed as previously described (29). Briefly,
sense and antisense riboprobes corresponding to nucleotides 199
to +994 of the RaKCaR cDNA were transcribed using
[33P]UTP.
Ten-micrometer frozen sections were fixed in 4% paraformaldehyde-0.1 M
PBS, rinsed in PBS, and acetylated with acetic anhydride in 0.1 M
triethanolamine. Sense or antisense probes were mixed in hybridization
buffer (105 cpm/ml) [50%
deionized formamide, 250 µg/ml yeast tRNA, 100 µg/ml denatured salmon sperm DNA, 10 mM Tris, pH 7.5, 1 mM EDTA,
0.6 M NaCl and 1× Denhardt's solution] and applied to the
sections, which were then covered with Parafilm and incubated in a
humidified chamber at 55°C overnight. After hybridization, the
sections were washed in 1×SSC and treated with 20 µg/ml
ribonuclease A. The sections were subsequently washed in 0.1× SSC
at 55°C, dehydrated in a graded ethanol series, and air dried
overnight before being dipped in NTB-2 photographic emulsion (Eastman
Kodak) and stored in light-tight boxes at 4°C for 7 days. The
slides were then developed (D-19 developer, Eastman Kodak) and fixed
(Eastman Kodak fixative). The sections, covered by glass coverslips,
were photographed under dark-field microscopy.
Immunohistochemistry for CaR. Before immunohistochemistry, 2-µm-thick sections were subjected to "antigen rescue," according to the supplier's recommended procedure (9). Briefly, slides containing the sections were placed in a Tissue-TekR slide holder filled with 1× antigen-retrieval citra solution (concentrated format, 10×; BioGenex, San Ramon, CA) and microwaved at high power (800 W) until the solution came to a rapid boil, at which point the oven was reset at 500 W for 8 min. The slides were then allowed to cool for 30 min and were rinsed several times with distilled water, dried, and processed for immunohistochemistry as described below.
Endogenous peroxidases were inhibited by incubating the sections in peroxidase blocking reagent (DAKO, Carpenteria, CA) for 5 min followed by treatment with protein block serum-free solution (DAKO) for 15 min (7-9). The sections were then incubated overnight at 4°C with 10 µg/ml of affinity-purified anti-CaR antiserum (4637) raised against a peptide corresponding to amino acids 345-359, which resides within the predicted amino-terminal extracellular domain of the bovine parathyroid CaR. This antiserum has been characterized previously (9). Similar results (not shown) were obtained using another polyclonal antiserum raised against a peptide corresponding to residues 215-237 of the bovine CaR, which has likewise been characterized fully in our previous studies (8). Control sections were prepared by incubation with anti-CaR antiserum preabsorbed with the specific CaR peptide (10 µg/ml) against which it was raised. After washing the sections three times with 0.5% BSA in PBS for 10 min, we added peroxidase-coupled, goat anti-rabbit immunoglobulin G (1:100; Sigma Chemical, St. Louis, MO) for 1 h at room temperature. The slides were then washed in PBS three times for 10 min each, and the color reaction was developed using the DAKO AEC substrate system for ~5 min. The reaction was stopped by washing three times in water. ![]() |
RESULTS |
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Distribution of CaR mRNA in small and large intestine by RT-PCR. RT-PCR was performed separately on RNA isolated from the muscularis and mucosa of the duodenum as well as on RNA isolated from whole jejunum, ileum, and several regions of the colon to screen for the presence of CaR transcripts. Figure 1A shows that PCR products of the expected size, i.e., 383 bp, were amplified by CaR-specific primers from reverse-transcribed RNA isolated from all of the segments of small and large intestine studied. Moreover, these PCR products were of the same size as that obtained from the RaKCaR cDNA as a positive control (not shown). The primers that were employed spanned at least one intron, precluding amplification of products of the same size from contaminating genomic DNA; furthermore, no PCR products were amplified when the reverse transcriptase was omitted from the reaction (not shown). Nucleotide sequencing of a PCR product subcloned from duodenal mucosa revealed >99% nucleotide identity with the corresponding sequence of the RaKCaR cDNA, indicating that this PCR product was derived from bona fide CaR transcript(s) (Fig. 1B).
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Distribution of CaR mRNA in various intestinal segments by Northern blot analysis. Figure 2 shows Northern blot analysis on poly(A)+ RNA isolated from the same regions of small and large intestine utilized for RT-PCR, which revealed two transcripts with sizes of 7.5 and 4.1 kb in all of the intestinal segments studied (Fig. 2, lanes c-g). The duodenum showed an additional 3.0-kb transcript (Fig. 2, lanes c and d). The 4.1-kb transcript was the predominant CaR mRNA species in all of the intestinal segments studied (Fig. 2, lanes c-g), whereas in rat kidney and brain (Fig. 2, lanes a and b), the 7.5-kb transcript was expressed at the highest levels.
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Immunohistochemistry of CaR in small and large intestine. Immunohistochemistry using a specific anti-CaR antiserum (antiserum 4637, see MATERIALS AND METHODS) directed at a highly conserved epitope within the extracellular domain of the CaR (residues 345-359) identified CaR immunoreactivity in the duodenum that was localized predominantly on the basal aspects of the villus absorptive cells, the epithelial cells of the crypts and Brunner's glands, as well as in the region of the submucosa containing Meissner's plexus; there was also strong CaR immunostaining in the serosa (Fig. 3, A, B, and D-F). The specificity of the immunostaining was confirmed by its abolition after preabsorption of the anti-CaR antiserum with the peptide against which it was raised (Fig. 3C). No CaR immunoreactivity was observed in the smooth muscle layers. Staining of nerve fibers extending from the submucosa into Auerbach's plexus between the circular and longitudinal layers of the duodenal muscularis could be visualized in some sections (see also Fig. 5A). There was no change in the pattern of staining when the tissue sections were pretreated with collagenase before immunostaining; moreover, similar results were observed using another anti-CaR antiserum raised against a different epitope within the extracellular domain [antiserum 4641, raised against a peptide corresponding to amino acids 215-237 in the bovine CaR (8), which are identical to the corresponding residues in RaKCaR; not shown].
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In situ hybridization histochemistry of CaR mRNA in small and large intestine. In situ hybridization was performed using an antisense probe derived from a portion of the CaR cDNA corresponding to a region of the extracellular domain of the receptor, as described in MATERIALS AND METHODS. Cells expressing CaR mRNA were present in Auerbach's myenteric plexus between the circular and longitudinal muscle layers (Fig. 6, A and B). These cells appeared as intense clusters of silver grains (see arrows in Fig. 6A). There was additional, diffuse labeling of cells within the submucosa and in the crypt and villus cells of the duodenum. Strong labeling could also be seen in Auerbach's plexus of the colon (Fig. 6B). Specificity of the labeling was confirmed by incubating sections with a sense probe (data not shown).
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DISCUSSION |
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The CaR has been found in a variety of organs and cell types (10). It is highly abundant in the parathyroid gland and kidney, presumably reflecting the key roles of these tissues in the maintenance of Ca2+o homeostasis (10). Its presence in several additional organs or cell types, including numerous regions of the brain (30), AtT-20 cells (16), keratinocytes (1) and lens epithelial cells (11), which, however, are not directly involved in mineral ion homeostasis, suggests that CaR might have a role in regulating other types of processes as well. CaR has also been demonstrated to be present in several human intestinal epithelial cell lines in recent studies (19). Although the intestine plays an important role in Ca2+o homeostasis because of its capacity for regulated absorption of dietary Ca2+ and phosphate, the potential role(s) of the CaR in these processes has not been explored. In addition, various physiological processes in both the small and large intestine, such as exocrine and endocrine secretion, proliferation, and maturation of the mucosal epithelial cells as well as contraction of smooth muscle, are Ca2+ dependent. Therefore, we studied CaR localization in the small and large intestine as a first step toward investigating the potential physiological functions of CaR in the gastrointestinal tract.
Northern blot analysis demonstrated the presence of CaR transcripts with molecular sizes of 7.5 and 4.1 kb in all of the intestinal segments examined, whereas the duodenum alone expressed an additional, smaller transcript of 3.0 kb. CaR transcripts of 7.5 and 4.1 kb are also present in rat kidney (8) and brain (9); however, the relative ratios of the abundance of these two transcripts in kidney and brain varies from that of the various segments of intestine studied here. The ratio of the relative abundances of the 7.5- and 4.1-kb transcripts in kidney and brain is ~2.0, whereas it is ~0.3 throughout the intestinal tract. Because the 4.1-kb transcript encodes the entire functional CaR protein (28), the significance of the larger 7.5-kb transcript remains uncertain. However, the possibility of organ-specific, posttranscriptional regulation of CaR expression as a result of variations in the stabilities of the various CaR transcripts cannot be ruled out. Moreover, the presence of a distinct, lower molecular weight transcript of 3.0 kb might indicate expression of a different form of this or a related CaR in the duodenum. Even smaller (e.g., ~2 kb), CaR-related transcripts have also been described in the parathyroid (5), but their functional relevance is likewise unknown at present.
Sequencing of RT-PCR products from the duodenal muscularis revealed >99% sequence identity with RaKCaR cDNA. Specificity of the amplification of CaR transcripts by RT-PCR was confirmed by negative control experiments in which the reverse transcriptase was omitted from the RT-PCR reaction, since PCR products were not amplified under these conditions. In addition, the use of intron-spanning primers precluded amplification of a product of the size expected from bona fide CaR transcripts as a result of priming from contaminating genomic DNA. The low level of nonidentity between the sequences of the rat intestinal CaR transcripts and the RaKCaR cDNA most likely reflects PCR artifacts, since different nonidentities in nucleotide sequences were observed in RT-PCR products amplified from RNA isolated from the rat stomach (I. Cheng, N. Chattopadhyay, D. Soybel, and E. M. Brown, unpublished observations). Therefore, our data show that the CaR transcripts in the rat intestinal tract are essentially identical to those in the kidney and brain, at least in the 5' region that harbors the putative binding site for Ca2+o and other polycationic ligands. Because the probe used for Northern analysis was also derived from the 5' portion of the RaKCaR cDNA, the possibility that the intestinal tract expresses transcripts containing different transmembrane and/or cytosolic domains of the CaR cannot be ruled out without cloning the full-length transcript(s). The latter possibility appears unlikely in view of the similarity between the predominant CaR transcripts observed on Northern analysis and those observed previously from the rat kidney and brain.
In the duodenum, CaR immunoreactivity was principally localized on the basal aspects of the epithelial cells of the villi and crypts as well as in the submucosa, which consists of densely packed Brunner's glands and Meissner's plexus. The intensity of the CaR immunostaining was greater near the bases of the villi and within the crypts than at the tips of the villi. In the duodenum, epithelial proliferation occurs in crypt cells, but these latter cells also serve important secretory functions. Undifferentiated cells originating from proliferating stem cells within the crypt migrate up the villi and are eventually extruded into the gut lumen at the villus tips. Therefore, the abundance of the CaR in the crypts and at the bases of the villi and the lower levels of receptor expression at the tips of the villi suggest a potential role(s) for the receptor in the process of crypt-to-villus differentiation, wherein a signal for differentiation might also act as a signal for inhibiting CaR expression at the tip of the villus.
The duodenum manifests the greatest absorptive flux of Ca2+ per unit length. Therefore, the presence of CaR immunoreactivity at higher levels on the basal side of the epithelial cells of the villi relative to their apical surface might indicate some role for the CaR in absorptive processes involving sensing of Ca2+o on the systemic or blood side of the absorptive cells. In addition, further studies using costaining with specific markers will be needed to identify the additional CaR-expressing cells within the villi, which might potentially include enterochromaffin cells and/or lacteals. The presence of the receptor on the basal side of the crypts is also of interest in terms of a possible direct regulation of intestinal secretion by Ca2+o. Recent studies have shown that raising the level of Ca2+o perfusing the blood supply to the small intestine inhibits intestinal absorption and reduces secretion of Ca2+ (24), raising the possibility of a physiological role for CaR in mineral ion homeostasis via regulation of intestinal secretion. The localization of the CaR protein on the basal surface of the epithelial cells, without any apparent CaR immunoreactivity on the lateral cell surface, may indicate specific interactions of the receptor with molecular components present in the basal plasma membrane per se and/or the basement membrane. The presence of the CaR protein in Brunner's glands may suggest some role for the receptor in control of the secretion of the alkaline, mucus-rich solution that is produced by these glands. Additional studies using immunoelectron microscopy will be necessary to determine whether the CaR on the basal surface of Brunner's glands and/or the epithelial cells of the crypts and villi is localized solely on epithelial cells per se or is also present on CaR-expressing nerve endings that innervate these cells.
The jejunum and ileum are sites where Ca2+ secretion takes place, although a minor fraction of the total Ca2+ entering in the small intestine is also absorbed in these segments (23-25). Intense staining in Auerbach's and perhaps in Meissner's plexus in jejunum and ileum raises the possibility that the CaR has some function(s) in secretory or motor functions of these segments of the small intestine. Therefore, it is possible that the CaR could play diverse role(s) in both absorption of Ca2+ and in secretion of ions and mucus by various segments of the small intestine as well as in the motor functions of the intestine. It is a long-standing clinical observation that hypercalcemia reduces gastrointestinal motility, whereas hypocalcemia is associated with increased gastrointestinal motility. In view of the presence of the CaR within the enteric nervous system throughout the small and large intestine, it is possible that these effects of alterations in systemic levels of Ca2+o are mediated by the CaR. Direct investigation of the actions of specific CaR agonists on gastrointestinal motility, however, will be needed to address this latter point further. In addition, studies directed at colocalizing the CaR with specific neuronal markers, including specific neurotransmitters, will be important to address the specific type(s) of neurons expressing the receptor and its functional importance in the enteric nervous system.
Absorption of Ca2+ in the rat
colon is thought to contribute significantly to the overall intestinal
absorption of this mineral ion (17). Our results show the presence of
CaR on both the apical and basal aspects of the epithelial cells of the
colonic crypts as well as in the submucosa in the region of Meissner's
plexus by immunohistochemistry. CaR localization within the enteric
nervous system of the colon was confirmed by in situ hybridization
histochemistry, which revealed intense radioactive labeling of neuronal
cell bodies within Auerbach's myenteric plexus. A large body of data
has shown that Ca2+o inhibits
proliferation of colonic epithelial cells (6, 13, 14, 22), and recent
studies have suggested that this process is CaR mediated (22). We have
demonstrated that reduction in luminal
Ca2+o stimulates PKC activity and
c-myc expression in the Caco-2 cell
line, which are associated with increased cellular proliferation (22).
Moreover, expression of the CaR was apparent on the apical cell surface of Caco-2 cells. The difference in the apparent polarity of the receptor in normal rat colonic crypt cells vs. Caco-2 cells might be
contributed to by 1) species
variation, 2) neoplastic
transformation, and 3) changes
occurring in culture with the latter. It is likewise known that phorbol
esters inhibit Ca2+-dependent
Cl secretion by colonic
epithelial cells (21). It is also possible that the CaR could modulate
the activity of the
Na+-K+-2Cl
cotransporter in the colon, as has recently been shown to be the case
in the thick ascending limb of the nephron (32).
Our present results, therefore, show that the same CaR that is present in the parathyroid, kidney, and a variety of other cell types (10), both those involved and those uninvolved in systemic mineral ion homeostasis, is also expressed widely throughout the intestinal tract. CaR is located in epithelial cells, in which it could be involved in the control of intestinal absorptive and/or secretory functions, as well as in the enteric nervous system, where it could potentially regulate secretomotor functions. Additional studies are needed to define the functional implications of the CaR along the length of the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Olga Kifor's suggestions and expertise on immunohistochemistry.
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FOOTNOTES |
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We also gratefully acknowledge generous grant support from the National Institute of Diabetes and Digestive and Kidney Diseases (Grants DK-41415 and DK-48330 to E. M. Brown), the St. Giles Foundation (E. M. Brown and S. C. Hebert), NPS Pharmaceuticals, Inc. (E. M. Brown and S. C. Hebert), and the National Dairy Council (E. M. Brown).
Address for reprint requests: N. Chattopadhyay, Endocrine-Hypertension Division, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115.
Received 15 July 1997; accepted in final form 6 October 1997.
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