INTRODUCTION

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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.

	METHODS

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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.

	RESULTS

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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.

View larger version (7K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (38K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   View larger version (72K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   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.

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.

View larger version (44K): [in this window] [in a new window]   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.

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.

View larger version (18K): [in this window] [in a new window]   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).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (16K): [in this window] [in a new window]   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).

View larger version (14K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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+.

	DISCUSSION

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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 [K_0.5 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 [K_0.5 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.