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Am J Physiol Gastrointest Liver Physiol 290: G439-G450, 2006. First published September 15, 2005; doi:10.1152/ajpgi.00163.2005
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MUCOSAL BIOLOGY

Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters

Dirk Weihrauch,1 Jainuch Kanchanapoo,1,2,* Mei Ao,1,* Roli Prasad,1 Pawinee Piyachaturawat,2 and Mrinalini C. Rao1

1Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois; and 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand

Submitted 8 April 2005 ; accepted in final form 10 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intestinal handling of bile acids is age dependent; adult, but not newborn, ileum absorbs bile acids, and adult, but not weanling or newborn, distal colon secretes Cl– in response to bile acids. Bile acid transport involving the apical Na+-dependent bile acid transporter (Asbt) and lipid-binding protein (LBP) is well characterized in the ileum, but little is known about colonic bile acid transport. We investigated colonic bile acid transport and the nature of the underlying transporters and receptors. Colon from adult, weanling, and newborn rabbits was screened by semiquantitative RT-PCR for Asbt, its truncated variant t-Asbt, LBP, multidrug resistance-associated protein 3, organic solute transporter-{alpha}, and farnesoid X receptor. Asbt and LBP showed maximal expression in weanling and significantly less expression in adult and newborn rabbits. The ileum, but not the colon, expressed t-Asbt. Asbt, LBP, and farnesoid X receptor mRNA expression in weanling colon parallel the profile in adult ileum, a tissue designed for high bile acid absorption. To examine their functional role, transepithelial [3H]taurocholate transport was measured in weanling and adult colon and ileum. Under short-circuit conditions, weanling colon and ileum and adult ileum showed net bile acid absorption: 1.23 ± 0.62, 5.53 ± 1.20, and 11.41 ± 3.45 nmol·cm–2·h–1, respectively. However, adult colon secreted bile acids (–1.39 ± 0.47 nmol·cm–2·h–1). We demonstrate for the first time that weanling, but not adult, distal colon shows net bile acid absorption. Thus increased expression of Asbt and LBP in weanling colon, which is associated with parallel increases in taurocholate absorption, has relevance in enterohepatic conservation of bile acids when ileal bile acid recycling is not fully developed.

taurocholate transport; apical sodium-dependent bile acid transporter; multidrug resistance-associated protein 3; lipid-binding protein; ileal bile acid-binding protein

BILE ACIDS ARE AMPHIPATHIC steroid molecules that are essential for the solubilization and absorption of lipids and fat-soluble vitamins. In humans, ≥95% of the bile acids are recycled to the liver via the enterohepatic circulation, chiefly by active reabsorption in the ileum (52). Thus, under physiological conditions, only ~5% of bile acids enter the colon. Active bile acid transport in the ileum is developmentally regulated: it is absent in the newborn and increases in the weanling and adult animal (13, 21–23, 37). However, the colon plays an important role in bile acid metabolism. Intestinal bacteria, especially in the colon, deconjugate bile acids and dehydroxylate primary bile acids at position 7 to form secondary bile acids (25). In certain pathophysiological states, such as irritable bowel syndrome, Crohn's disease, and surgical ileal resection, active bile acid absorption in the ileum is compromised (26, 52). Under these conditions, more bile acids enter the colon, where dihydroxy bile acids, such as chenodeoxycholic acid and deoxycholic acid, stimulate net electrolyte and fluid secretion, resulting in diarrhea (8). In vitro studies have demonstrated that, in human colonic cell lines (15, 16) and in rabbit (44) or rat (41) colonic mucosa mounted in Ussing chambers, taurodeoxycholate (TDC) activates Cl– secretion. Interestingly, this action of TDC is age specific in rabbits: it affects only the adult distal colon and has no effect in newborns (44). These findings were extended by our previous investigations showing TDC- and taurochenodeoxycholate-activated Cl– flux in isolated adult colonocytes but not in colonocytes from newborn (7–10 day old) or weanling (~4 wk old) rabbits (14, 56). Although bile acid transport and the underlying transporters and receptors in the small intestine and liver are well characterized, there are only a few studies in the mammalian colon. For example, by sampling portal blood, Fujii et al. (17) showed a small, but measurable, absorption of taurocholate (TC) from the colon of adult mongrel dogs.

In the ileum, the secondary active Na+-dependent bile acid transporter (Asbt/ASBT)1 is localized to the apical membrane and transports conjugated primary and secondary bile acids with high affinity (10). Asbt is also expressed in the kidney and in cholangiocytes (10, 33, 59). Although Na+-dependent organic anion transporter 3 (Oatp3) is not as prominent as ileal Asbt, transport of bile acids by Oatp3 in the jejunum has been reported (7, 19, 57). Bile acids also interact with cytosolic binding proteins and members of the nuclear receptor superfamily in liver and small intestine. In enterocytes, a 14-kDa cytosolic protein, termed ileal bile acid-binding protein (iBABP) in most species but named lipid-binding protein (LBP) in rabbits, has been identified (31, 51); here, the term LBP is used for rabbits and iBABP for other species. Pre- and postnatal expression of Asbt and iBABP/LBP has been examined in a few species. In the rabbit, uptake of radioactive bile acids into the ileum increases from weanlings (6 wk old) to adults (1 yr old) (53). Postnatally, expression of Asbt and iBABP/LBP shows dramatic increases at around the time of weaning. For example, Asbt mRNA first appears at postnatal day 16 in rat ileum, followed by protein expression by day 21 (47), with a parallel, steep increase of iBABP mRNA expression from day 16 to day 22 (27). On the basis of photoaffinity labeling and target size analysis of rabbit ileal proteins, it has been suggested that a complex of four iBABP molecules and two Asbt dimers play a critical role in transcelluar bile salt movements (29–32). The coexpression of Asbt and iBABP during postnatal development, as well as their parallel expression in large cholangiocytes (4), supports the hypothesis that Asbt and iBABP are essential for ileal bile acid uptake. In contrast to its absence in the early postnatal stage, Asbt expression is high in the rat and mouse in the late fetal stage (47). A similar biphasic developmental pattern is seen with respect to iBABP in the mouse; however, in the rat, iBABP expression is not seen until the postnatal stage, i.e., it is monophasic. Because iBABP is absent in the rat during late fetal development when Asbt is present (47) and iBABP is absent in other tissues possessing Asbt, such as renal tissues (18), the hypothesis that iBABP is always essential for transcellular bile acid transport is questionable.

Once transported into the cell, bile acids elicit their regulatory effects via their interaction with members of the nuclear receptor superfamily. In the ileum, bile acids act via the farnesoid X receptor (FXR) to increase expression of iBABP/LBP (55) and decrease expression of Asbt in mice, rabbits, and the human colonic cell line Caco-2 (8, 36). The influence of FXR on expression of the basolateral membrane (BLM) transporters is unknown. Four isoforms of FXR, FXR-{alpha}1, FXR-{alpha}2, FXR-beta1, and FXR-beta2, have been described in mice, with the intestines showing very low expression of FXR-{alpha} (62). FXR-beta1 and FXR-beta2 differ by an insertion of four amino acids (MYTG) between the DNA- and ligand-binding domains of FXR-beta2, and it has been postulated that activation of FXR target genes may depend on the ratio of FXR-beta2 to FXR-beta1 (62). Bile acids also bind to and activate the pregnane X receptor and the vitamin D3 receptor (VDR). When activated, VDR and pregnane X receptor have largely been shown to upregulate pathways that detoxify bile acids (38, 50). Preliminary studies imply a role for VDR in regulation of Asbt and LBP (9).

In contrast to the well-characterized Asbt mechanism on the apical membrane, the mechanism by which bile acids exit the BLM of the ileal enterocytes is not fully understood. In addition, whether expression of the putative basolateral transporters is age dependent is not known. Functionally, exit via an anion exchanger has been shown in rat small intestinal BLM vesicles (58). In 2000, Lazaridis et al. (34) provided evidence to suggest that basolateral extrusion of bile acids occurs via a 154-amino acid protein, termed truncated Asbt (t-Asbt). This protein is encoded by an alternatively spliced, truncated variant of Asbt mRNA and has a distinct COOH terminus. Oocytes expressing t-Asbt exhibit Na+-independent TC efflux. Another candidate for basolateral bile acid efflux is the multidrug resistance-associated protein 3 (Mrp3/MRP3), expressed in the BLM of rat and human small and large intestines (28, 45). Membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 showed ATP-dependent transport of several bile acid species (24, 61), leading to the suggestion that Mrp3 plays a role in intestinal BLM bile acid extrusion (39, 49). Sinusoidal efflux of TC has also been correlated with hepatic expression of Mrp3 (1). Very recently, Dawson et al. (12) provided evidence showing that the heteromeric organic solute transporter-{alpha} (Ost-{alpha})-Ost-beta may be the basolateral transporter in mouse ileum.

Although age-related changes in bile acid absorption and Asbt and iBABP/LBP expression have been well documented in the small intestine (5, 13, 27, 35, 37, 42, 47), little is known about these processes in the developing distal colon, despite the relevance of bile acids to colon physiology. In the present study, we used adult (>6 mo old), weanling (~4 wk old), and newborn (5–8 day old) rabbits as a model to examine age-related differences in bile acid transport and expression of iBABP/LBP in mammalian colon. In rabbits, weaning occurs at around day 21 postpartum, and the animals were used 4–7 days later. We report significant age-related differences in mRNA expression of bile acid transporters/receptors and demonstrate parallel changes in function determined by transepithelial fluxes. In particular, mRNA expression of Asbt, LBP, and FXR in weanling colon parallels the profile found in adult ileum, a tissue designed for high bile acid flow. We demonstrate for the first time that weanling, but not adult, distal colon shows net bile acid absorption. We postulate that weanling colon has a salvage system to preserve the loss of valuable bile acids during this critical stage of intestinal development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. TRIzol reagent, SuperScript II RNase H– reverse transcriptase, oligo(dT)12–18 primer, RNaseOUT, and recombinant RNase inhibitor were obtained from Invitrogen (Carlsbad, CA); First Choice RLM-RACE (rapid amplification of cDNA ends) kit and RNA-Later from Ambion (Austin, TX); RedTaq polymerase, 10 mM dNTP mix, DNase kits, and protease inhibitor cocktail from Sigma (St. Louis, MO); oligonucleotide primers from Sigma Genosys (St. Louis, MO); goat polyclonal anti-Mrp3 antibody from Santa Cruz Biotechnology (Santa Cruz, CA); donkey anti-goat secondary antibody from Chemicon (Temecula, CA); SuperSignal West Pico Chemiluminescent Substrate Kit from Pierce (Rockford, IL); 3H-labeled TC (2 µCi/µg) from Perkin-Elmer (Boston, MA); and scintillation cocktail from JT Baker (Phillipsburg, NJ). All other reagents were of analytic grade and were purchased from Sigma Chemical.

Animals and tissue procurement. New Zealand White adult (>6 mo old), weanling (25–28 day old; weaned at day 21), and newborn (5–8 day old) rabbits of either sex were purchased from New Franken Rabbitry (New Franken, WI) and housed at the institutional Biologic Resources Laboratory according to guidelines of the American Association for Accreditation of Laboratory Animal Care. Animal protocols were approved by the Institutional Animal Care Committee.

Rabbits were anesthetized with ketamine (10 mg/kg) and xylazine (2.5 mg/kg) given intramuscularly and then euthanized by pentobarbital sodium overdose (75 mg/kg iv). Distal colon from the anal verge to the splenic flexure and distal ileum were excised and flushed with ice-cold (4°C) lactated Ringer solution containing 5% glucose (LRG) and opened along the mesenteric border. Tissues were rinsed in LRG, and the mucosal surface was gently blotted to remove mucus and maintained in oxygenated LRG. For RNA isolation and Western blot analyses, mucosae were separated from underlying muscles by mechanical scraping. Tissues for RNA isolation were placed in RNA-Later and stored at –80°C until use. For flux studies, muscle layers of the ileum and distal colon were stripped using curved forceps to avoid mechanical damage to the epithelium.

Semiquantitative RT-PCR. Total RNA was isolated using TRIzol reagent, and contaminating DNA was eliminated using a DNase kit according to the manufacturer's instructions. Total RNA was quantitated photometrically at 260 nm. Before the RT reaction, DNase-purified RNA was checked for purity in a high (i.e., 36) cycle PCR using a primer pair designed to amplify GAPDH (see Table 1 in supplemental data for this article at http://ajpgi.physiology.org.cgi/content/full/00163.2005/DC1). Only RNA samples that showed no amplification products were used in subsequent RT assays, which employed 2 µg of total RNA and Superscript II reverse transcriptase.

For each primer pair (see Table 1 in supplemental online data), optimal PCR amplification conditions were determined employing a hot-start protocol and RedTaq polymerase. In all PCR assays comparing mRNA expression levels of different age groups or tissues, equal amounts of template (RT product) were employed. To verify amplification of the desired target gene, PCR products were sequenced at the Marine DNA Sequencing Center (Mount Desert Island Biological Laboratory, Salisbury Cove, ME). For semiquantitative RT-PCR, the sampled PCR product strictly depended on the abundance of the target cDNA, and cycle numbers were chosen in which the PCR ran in the exponential phase. Thus, for each set of primers, the PCR using RT products from the three age groups were run for different cycles, and the optimal cycle number in the exponential phase was determined. For GAPDH, this was 24 cycles (cycle numbers for the other target genes are provided in Figs. 1–3 and 5; see representative diagram in Fig. 1 addendum in supplemental online data). PCR products (12 µl) were separated on 1–2% agarose gels and visualized by ethidium bromide staining. The band densities were determined under UV light by digital analysis using a Kodak Digital Science DC 120 Zoom digital camera and Kodak Digital Science 1D 2.0.2 software (Eastman Kodak, Rochester, NY). For each PCR assay, a parallel PCR was performed employing GAPDH primers (see Table 1 in supplemental online data) at an annealing temperature of 55°C and 24 PCR cycles. GAPDH is equally expressed in rabbit intestinal tissues of all age groups (see Figs. 1–5) and served as an internal control.


Figure 1
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  		Fig. 1. Semiquantitative mRNA expression of the apical Na+-dependent bile acid transporter (Asbt) in developing rabbit intestine. Total RNA was extracted from adult (AD), weanling (WN), and newborn (NB) distal colon (dC; A) and from adult and weanling ileum (Ile; B). Top: representative agarose gels showing ethidium bromide-stained PCR products from exponential phase of Asbt (36 and 28 PCR cycles for colonic and ileal tissues, respectively) and internal standard GAPDH (24 PCR cycles). Bottom: quantitation of expression, calculated as ratio (mean ± SE; n = 4 and 3 in A and B, respectively) of pixels, obtained by digital analysis, of Asbt to GAPDH mRNA expression. ND, not detectable. *P < 0.05.

 

Figure 3
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  		Fig. 3. Semiquantitative mRNA expression of nuclear farnesoid X receptor (FXR) in developing rabbit intestine. FXR expression was assessed using pan FXR primers. Total RNA was extracted from adult, weanling, and newborn distal colon and adult ileum. Top: representative agarose gels showing ethidium bromide-stained PCR products from exponential phase of FXR (30 PCR cycles) and GAPDH (24 PCR cycles). Bottom: quantitation of FXR-to-GAPDH mRNA expression ratio calculated as described in Fig. 1. Values are means ± SE; n = 3–4. *P < 0.05.

 

Figure 5
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  		Fig. 5. Semiquantitative mRNA expression of multidrug resistance-associated protein 3 (Mrp3) in developing rabbit distal colon and adult ileum. Total RNA was extracted from adult, weanling, and newborn distal colon and adult ileum. A: colonic samples. B: comparison of expression in adult distal colon and adult ileum. Top: representative agarose gels of ethidium bromide-stained PCR products from exponential phase of Mrp3 (32 PCR cycles) and GAPDH (24 PCR cycles). Bottom: quantitation of Mrp3-to-GAPDH mRNA expression (means ± SE; n = 3) calculated as described in Fig. 1. *P < 0.05.

 
Cloning of rabbit Mrp3, Ost-{alpha}, t-Asbt, and FXR. Rabbit Mrp3, Ost-{alpha}, t-Asbt, and FXR have not been cloned; therefore, we used the following strategy to identify these rabbit transcripts. Degenerate primers, based on conserved regions of published sequences of the cDNA of interest (see Table 1 in supplemental online data), were designed and used to amplify the rabbit transcript by PCR. Products of the predicted size were gel purified, sequenced, and subjected to Basic Local Alignment Search Tool (BLAST) searches to determine homology with known transcripts. This sequence was then used to design specific primers for use in semiquantitative PCR to compare different age groups and to obtain the entire open reading frame (ORF) of t-Asbt and FXR with use of the RACE technique (Ambion).

Protein preparation and Western blotting. With the use of previously published methods (56), ileum from adult animals and distal colonic mucosal epithelia from adult, weanling, and newborn animals were homogenized in a buffer containing (in mM) 1 EDTA, 2 MgCl2, 5 beta-mercaptoethanol, 1 DTT, and 25 Tris·HCl, pH 7.4, and protease inhibitor cocktail. The homogenates were further processed by sonication on ice (3 pulses for 5 s each, output 4, 40% of duty cycle; Branson Sonifier Cell Disruptor model 350). The homogenate was centrifuged (2,000 g for 5 min) to pellet out the nuclei and unbroken cells, and the supernatant was referred to as total homogenate and stored at –80°C until use. Protein was determined by the Bradford method with a kit from Bio-Rad (Hercules, CA).

As described earlier (56), a modification of the method of Towbin et al. (54) was used to perform Western blotting after SDS-PAGE on 7.5% gels. After SDS-PAGE, the homogenate samples were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Bedford, MA) at 250 mA for 2 h in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS). The blots were washed in Tris-buffered saline (TBST: 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 10 min and then blocked with Blotto (5% Carnation nonfat dry milk in TBST) for 1 h at room temperature. The blots were incubated with primary polyclonal goat anti-human MRP3 antibodies generated against a peptide corresponding to the COOH-terminal region of human MRP3 (Santa Cruz Biotechnology). The antibodies were used at a dilution of 1:200 in Blotto overnight at 4°C on a shaker. After incubation with the primary antibody, the blots were washed three times for 10 min each in TBST and then incubated with donkey anti-goat secondary antibodies conjugated to horseradish peroxidase in Blotto at room temperature for 1 h. The blots were washed three times for 15 min each in TBST, and horseradish peroxidase was visualized using a SuperSignal West Pico Chemiluminescent Substrate Kit.

Measurement of transepithelial TC fluxes. Distal colonic and distal ileal epithelia were dissected from the underlying muscle layers and mounted in 0.33-cm2 Ussing chambers (Physiologic Instruments, San Diego, CA). The tissues were bathed in HCO3–-Ringer buffer (buffer A; 4 ml/chamber) containing (in mM) 141.8 Na+, 125.6 Cl–, 3.0 HPO42– + H2PO4–, 1.2 Ca2+, 1.2 Mg2+, 5.4 K+, and 21.0 HCO3– (pH 7.4). The tissues were oxygenated with 95% O2-5% CO2 and maintained at 37°C. The mucosal bath solution contained 10 mM mannitol and the serosal bath contained 10 mM glucose to circumvent the compounding effects of glucose-stimulated short-circuit current (Isc) in the ileum. Transmural Isc (µA/cm2) was measured using an automatic voltage-clamp apparatus (model VCC-MC6, Physiologic Instruments). The electrode offset and the fluid resistance were compensated between the potential-monitoring electrode bridges. The potential difference (mV) was measured via a pair of calomel half-cells connected to agar-salt bridges (3% agarose and 3 M KCl), whereas the tissue was short-circuited via a pair of Ag-AgCl electrodes that connected to separate agar-salt bridges. All experiments were conducted under short-circuit conditions. Total tissue conductance (Gt, mS/cm2) and tissue resistance (Rt, {Omega}·cm2) were obtained by introduction of a 5-mV bipolar pulse at 10-s intervals and calculated by Ohm's law.

3H-labeled TC was used to measure unidirectional and net transepithelial bile acid fluxes in paired tissues from the same animal with comparable Rt (±25%). From each rabbit, two pairs of distal colonic tissue were studied, and the values were averaged to yield n = 1. A single pair of distal ileal tissue from the same animal was studied in parallel. After stabilization of electrical parameters in each tissue pair (~30 min after mounting), 100 µM 3H-labeled TC (1 µCi) was added to the mucosal or serosal side for measurement of mucosal-to-serosal (Jm -> s) or serosal-to-mucosal (Js -> m) flux. Immediately after radioisotope addition, 10 µl were sampled from the radioactive side to determine the specific activity. TC fluxes were determined from a 400-µl sample obtained from the nonradioactive side every 30 min beginning with TC addition (time 0) and continuing for 3 h. The samples were replaced with another 400 µl of fresh nonradioactive buffer A, and this dilution was factored into the calculations. At the end of the experiment, viability of the tissues was tested by determination of the Isc response to mucosal addition of 10 mM glucose to the ileum or serosal addition of 10 µM forskolin to the distal colon. Scintillation fluid (10 ml) was added to all samples, and radioactivity was assessed in a Tri-Carb liquid scintillation analyzer (Perkin-Elmer). TC fluxes are expressed as nanomoles per square centimeter per hour, and net flux (Jnet) is the difference between Jm -> s and Js -> m (see Figs. 8–10).


Figure 8
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  		Fig. 8. Transepithelial unidirectional 3H-labeled taurocholate (TC) fluxes and electrical parameters in developing ileal epithelia stripped of underlying muscles. A1 and B1: mucosal-to-serosal and serosal-to-mucosal flux (Jm -> s and Js -> m) of TC over 30-min periods from addition of TC (time 0) in adult (A1; n = 4 or 5) and weanling distal ileum (B1; n = 6 or 7). A2 and B2: transmural short-circuit current (Isc) and total conductance (Gt) measured in parallel with fluxes in adult (A2; n = 5) and weanling distal ileum (B2; n = 7). Arrows, addition of 10 mM glucose to mucosal bath to assess tissue viability. *Significantly different from time 0 (P < 0.05).

 

Figure 10
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  		Fig. 10. Unidirectional and net transepithelial TC fluxes in adult and weanling ileum (A) and adult and weanling distal colon (B) epithelia stripped of underlying muscles. Jnet, net flux. Values were calculated from steady-state Jnet values in Fig. 9 from 90 to 180 min. *Significantly different from each other. Jm -> s and Jnet were significantly lower (P < 0.05) in weanling and adult distal colon than in weanling or adult ileum.

 
Statistics. Each n value depicts a separate animal. If more than one sample was tested from one animal, the values were averaged to represent n = 1. Values are means ± SE. Independent t-test and one-way ANOVA (for >2 means) followed by the least-significant difference were used to evaluate statistical significance. For Isc and Gt, significance was determined by least-squares regression analysis and a one-sample t-test. P ≤ 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Asbt. To examine Asbt expression in rabbit colon, specific primers (RbAsbtF/RbAsbtR; see Table 1 in supplemental online data) were employed in semiquantitative RT-PCR. The antisense primer corresponded to exon 2, a region unique to the Asbt sequence and not found in t-Asbt. Expression of colonic Asbt mRNA varied with age: it was highest in the weanling, lower in the newborn, and below detection in the adult colon (Fig. 1A). Although mean values for the newborn appeared to be >0, these values, as assessed by a one-sample t-test, were not different from 0 (see DISCUSSION). In contrast, adult ileum showed significantly higher Asbt mRNA abundance than weanling ileum, and Asbt was not detectable in the newborn at 28 cycles (Fig. 1B). At all ages, 36 cycles were needed for amplification in the linear phase for colonic samples, whereas only 28 cycles were needed for ileal samples.

LBP. Because expression of iBABP/LBP closely corresponds with that of Asbt in developing rat ileum (27, 47), we evaluated mRNA expression of LBP in developing rabbit distal colon. Although weanling distal colon showed a robust expression of LBP at 28 cycles, equivalent amounts of RNA in adult and newborn distal colon showed no detectable expression of LBP (Fig. 2A). At 32 cycles, LBP was detectable in the adult, but not in the newborn (data not shown). Expression of LBP mRNA was greater in the ileum than in the colon, with only 18 cycles needed to obtain a strong amplification signal for adult and weanling animals. Differences in expression between adult and weanling ileum were not statistically significant. LBP was not detectable in newborn ileum at 18 cycles (Fig. 2B).


Figure 2
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  		Fig. 2. Semiquantitative mRNA expression of lipid-binding protein (LBP) in developing rabbit colon (A) and ileum (B). Total RNA was extracted from adult, weanling, and newborn distal colon and adult and weanling ileum. Top: representative agarose gels showing ethidium bromide-stained PCR products from exponential phase of LBP (28 and 18 PCR cycles for colon and ileum, respectively) and GAPDH (24 PCR cycles). Bottom: quantitation of LBP-to-GAPDH mRNA expression ratio calculated as described in Fig. 1. Values are means ± SE; n = 3 in A and B.

 
FXR. Because FXR has been shown to regulate expression of mouse and rabbit ileal Asbt and iBABP/LBP mRNA, we examined mRNA expression of FXR in newborn, weanling, and adult rabbit distal colon and adult ileum. Rabbit FXR has not been cloned, so we used the RACE technique (see MATERIALS AND METHODS) to obtain the full-length cDNA rabbit FXR sequence. Amplification of the 3'-untranslated region (UTR) employing the primer combination RbFXRF2 (see Table 1 in supplemental online data) and the inner 3'-RACE primer (provided by the kit) gave two distinct cDNA fragments (data not shown). The cDNA sequences of these forms contained identical ORFs coding for proteins of 482 amino acids and differed only with respect to position of the poly(A) tail at nucleotide 1824 in the longer isoform, 3'-UTR isoform L (GenBank Accession No. AF497630), and at nucleotide 1717 in the shorter isoform, 3'-UTR isoform S (GenBank Accession No. AF497631). The deduced amino acid sequence for rabbit FXR was 94% identical with human FXR-beta1 (GenBank Accession No. AAM53550) and 90% identical to golden hamster FXR-beta1 (GenBank Accession No. AAM53548).

Semiquantitative RT-PCR employing the primer pair RbFXRFA/RbFXRRA (pan FXR primers; see Table 1 in supplemental online data), designed to amplify a 267-bp sequence of all FXR isoforms, showed an age-dependent decline in FXR mRNA expression in distal colon, with maximal expression in the newborn (Fig. 3). In addition, expression was 2.55-fold greater in adult ileum than in adult distal colon (1.18 ± 0.11 vs. 0.46 ± 0.17 relative units) but less than in weanling and newborn colon (Fig. 3).

Recent evidence (62) suggests that higher expression of the ratio of FXR-beta2 to FXR-beta1 may be important in regulating the activation of target genes, such as LBP, and may parallel LBP expression. Using specific FXR-beta2 and FXR-beta1 primers, we determined that FXR-beta1 is the dominant intestinal isoform; however, the ratio of FXR-beta2 to FXR-beta1 expression did not parallel LBP expression. Thus ratios of FXR-beta2 to FXR-beta1 were lower in adult ileum than in adult distal colon (data not shown).

t-Asbt. To determine whether rabbit ileum and colon possess the putative basolateral bile acid transporter t-Asbt, we designed primers based on the rat sequence (34). Exon 2 was missing in t-Asbt, and there was a frame-shift event at the splicing site. Primer pairs (Rbt-AsbtF/Rbt-AsbtR; see Table 1 in supplemental online data) specific for the rabbit were designed to span the putative splicing site for t-Asbt, such that, if present, Asbt (302 bp) and t-Asbt (183 bp) could be detected in the same PCR. A band corresponding to the predicted size of t-Asbt product (183 bp) was found in adult rabbit ileum but not adult or weanling distal colon. As reported for the rat (34), high (i.e., 40) cycles of PCR amplification (Fig. 4A) were needed to detect t-Asbt in the rabbit. No product was seen when RNA was used as the template, confirming that our samples were free of DNA contamination. In contrast, with use of the same primers, a band corresponding to Asbt (302 bp), as would be predicted from Fig. 1, was prominent in adult ileum and weanling distal colon and less prominent in adult distal colon. The relative expression of Asbt to t-Asbt in adult ileum was 13.5:1.


Figure 4
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  		Fig. 4. Ethidium bromide-stained agarose gel of PCR products of truncated apical Na+-dependent bile acid transporter (t-Asbt) and Asbt. A: cDNA and total RNA of adult ileum and adult and weanling distal colon were amplified (40 cycles) using the primer pair Rbt-AsbtF/Rbt-AsbtR. Ratio of Asbt to t-Asbt in adult ileum was 13.5:1. GAPDH (24 cycles) served as internal control. B: nucleotide sequence of predicted open reading frame and corresponding deduced amino acid sequence. *Potential sites of N-glycosylation. Putative transmembrane domains are underlined.

 
The adult ileal RT-PCR sample product was further gel purified and sequenced. Specific primers were designed from this sequence to determine the entire ORF (see MATERIALS AND METHODS). As in the rat (34), this rabbit t-Asbt sequence (GenBank Accession No. AY292653) represented a splice variant of the ileal Asbt (GenBank Accession No. Z54357) with the omission of exon 2 (119 bp) and a frame-shift event. However, the deduced ORF of rabbit t-Asbt lacked the COOH-terminal 12 amino acids found in rat t-Asbt (34). The coding region of the deduced rabbit sequence consisted of 143 amino acids with 3 predicted transmembrane domains and 2 N-glycosylation sites at the extracellular NH2 terminus (Fig. 4B; see TMHMM online at http://workbench.sdsc.edu and Prosite at http://www.expasy.org).

Mrp3. We next examined whether Mrp3, another putative basolateral transporter, was present in the colon. Using the strategy described in MATERIALS AND METHODS, we identified and cloned a partial rabbit Mrp3 sequence (GenBank Accession No. AY289920; see Table 1 in supplemental online data for primers). A GenBank BLAST search revealed that the predicted partial amino acid sequence of rabbit Mrp3 had 91% identity to the published human MRP3 sequence (GenBank Accession No. NP_003777) and 84% identity to rat Mrp3 (GenBank Accession No. NP_542148). With use of specific rabbit primers (RbMrp3F1/RbMrp3 R1, see Table 1 in supplemental online data) and semiquantitative RT-PCR, Mrp3 expression was found to be significantly higher in adult than in newborn colon, with intermediate values in weanling colon (Fig. 5A); however, values in weanling and newborn colon were not significantly different. Although semiquantitative analyses were not described in an earlier study, the rat ileum appeared to express less Mrp3 than the rat colon as determined by RT-PCR (43). Therefore, we compared Mrp3 expression in adult rabbit ileum and colon by semiquantitative RT-PCR. Significantly more Mrp3 was expressed in the distal colon than in the ileum (Fig. 5B).

Ost-{alpha}. Finally, we examined whether the most recently described putative basolateral transporter, Ost-{alpha}, was present in the colon. Using the strategy described in MATERIALS AND METHODS, we identified and cloned a partial rabbit Ost-{alpha} sequence (GenBank Accession No. DQ122755; see Table 1 in supplemental online data for primers). A GenBank BLAST search revealed that the predicted partial amino acid sequence of rabbit Ost-{alpha} had 86% identity to the published human Ost-{alpha} sequence (GenBank Accession No. AAP23993.1) and 80% identity to rat Ost-{alpha} (GenBank Accession No. XP221376.2). With use of specific rabbit primers (RbOst{alpha}F1/RbOst{alpha}R1, see Table 1 in supplemental online data) and semiquantitative RT-PCR, Ost-{alpha} expression in the colon was found to be low in all age groups, and, equally important, there was no significant difference in expression among age groups (Fig. 6A). Ost-{alpha} mRNA expression was greater in the ileum than in the colon, inasmuch as 36 cycles were required for exponential-phase PCR amplification of colonic cDNA at all ages, whereas only 28 cycles were required for exponential-phase PCR amplification of ileal cDNA (Fig. 6B). Expression in adult and weanling ileum was significantly different from that in newborn ileum but were not significantly different from each other (Fig. 6B).


Figure 6
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  		Fig. 6. Semiquantitative mRNA expression of organic solute transporter-{alpha} (Ost-{alpha}) in developing rabbit colon (A) and ileum (B). Total RNA was extracted from adult, weanling, and newborn distal colon and adult and weanling ileum. Top: representative agarose gels showing ethidium bromide-stained PCR products from exponential phase of Ost-{alpha} (36 and 28 PCR cycles for colon and ileum, respectively) and GAPDH (24 PCR cycles). Bottom: quantitation of ratio of Ost-{alpha} to GAPDH mRNA expression calculated as described in Fig. 1. Values are means ± SE; n = 3. *P < 0.05.

 
Mrp3 protein expression. The results of the semiquantitative RT-PCR of the putative basolateral bile acid transporters (Figs. 4–6) suggested that Mrp3 may be the candidate transporter in the colon, whereas Ost-{alpha} may be the candidate transporter in the ileum. To determine whether Mrp3 protein expression paralleled the RT-PCR observations, Mrp3-specific polyclonal antibodies were used to probe ileal and colonic tissues from different age groups by immunoblotting. As shown in Fig. 7, adult and weanling distal colon expressed Mrp3, a 190-kDa protein, in high abundance. In marked contrast, Mrp3 protein expression in newborn distal colon (Fig. 7A) and adult ileum (Fig. 7B) was very low. In contrast, in the rat, although mRNA expression was lower, as determined by RT-PCR, Mrp3 protein expression was equivalent in the ileum and colon (45).


Figure 7
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  		Fig. 7. Protein expression of Mrp3 in developing rabbit distal colon and adult ileum. Protein was isolated from adult, weanling, and newborn distal colon and adult ileum, and Western blotting was performed. A: representative Western blots of protein from adult, weanling, and newborn distal colon probed with anti-Mrp3 antibody (left) and quantitation of Mrp3 protein as determined by densitometric scans of each blot and expressed in pixels (means ± SE; n = 3; right). B: Western blots of adult distal colon and ileum probed with anti-Mrp3 antibody. *P < 0.05.

 
Bile acid flux. The increased mRNA expression in weanling distal colon of proteins such as Asbt, LBP, and FXR characteristically involved in ileal bile acid reabsorption suggested that developing distal colon may also be designed for transepithelial bile acid uptake. To test this hypothesis, bile acid transport was measured across stripped adult and weanling distal ileal and colonic mucosa mounted in Ussing chambers. Jm -> s and Js -> m of 3H-labeled TC (100 µM) were measured under short-circuit conditions (n = 4–7), and Isc and Gt were monitored. TC was selected because it is readily available commercially and has been utilized in a number of studies to examine intestinal absorption in the rabbit (3); however, the major bile acids reported in the rabbit circulation are secondary bile acids, especially glycodeoxycholic acid (20). Another reason 3H-labeled TC is an appropriate marker is that, in a perfused rabbit model, ileal and hepatic rates of saturable uptake were much greater for TC than for TDC (2). TC caused a slight, but significant, increase in Gt (14%) in weanling ileum, but not in any of the other tissues examined (Figs. 8 and 9). TC also did not affect Isc in adult (Fig. 9A2) or weanling (Fig. 9B2) colon. In adult (Fig. 8A2) and weanling (Fig. 8B2) ileum, TC caused a gradual increase in Isc over time, whether it was added to the mucosal or serosal side. By least-squares regression analysis and one-sample t-test, this increase was found to be significant (P < 0.02) in adult and weanling ileum but not in adult (P = 0.13) or weanling (P = 0.06) distal colon.


Figure 9
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  		Fig. 9. Transepithelial unidirectional TC fluxes and electrical parameters in developing distal colonic epithelia stripped of underlying muscles. A1 and B1: Jm -> s and Js -> m of TC over 30-min periods from addition of TC (time 0) in adult (A1; n = 3 or 4) and weanling distal colon (B1; n = 6 or 7). A2 and B2: transmural Isc and Gt measured in parallel with fluxes in adult (A2; n = 4) and weanling distal colon (B2; n = 7). Arrows, addition of 10 µM forskolin to serosal bath to assess tissue viability.

 
Viability was assessed at 180 min as the Isc response to addition of mucosal glucose in the ileum (arrow in Fig. 8, A2 and B2) or serosal forskolin in the colon (arrow in Fig. 9, A2 and B2). TC uptake increased with time and was linear, beginning anywhere from 30 min (weanling ileum and adult colon) to 60–90 min (adult ileum and weanling colon) and continuing for ≥3 h. If we consider the differences in expression of transporters in these tissues, it is not surprising that different times were needed to achieve steady state. On the basis of these flux rates and to achieve uniformity for comparison purposes, Jm -> s and Js -> m were calculated from the slope between 90 and 180 min for all four tissues, and Jnet values were calculated as the difference (Jnet = Jm -> s – Js -> m). Net uptake of TC was observed in adult and weanling ileum: 11.41 ± 3.45 (n = 4) and 5.53 ± 1.20 nmol·cm–2·h–1 (n = 6; Fig. 10), respectively. When calculated as the mean of the steady-state fluxes between 90 and 180 min, Jm -> s and Jnet in adult and weanling ileum were statistically significantly different. On the other hand, the adult distal colon exhibited a small, yet significant, net TC secretion (–1.39 ± 0.47 nmol·cm–2·h–1, n = 3; Fig. 10). In marked contrast to the adult colon and as predicted by the expression of bile acid transporters, weanling distal colon showed a significant, although much smaller, net TC absorption than the ileum (1.23 ± 0.62 nmol·cm–2·h–1, n = 6; Fig. 10). Jm -> s and Jnet of colonic tissues were significantly different from those of ileal tissues.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
Asbt and LBP. The present study demonstrates that the array of bile acid transporters and receptors expressed in weanling distal colon is very different from that expressed in adult or newborn rabbit. The mRNA expression of LBP and Asbt in distal colon appears to be biphasic with respect to age, with no (LBP) or moderately low (Asbt) expression in newborns, followed by a clear expression in weanlings and disappearance in adults (Figs. 1 and 5). Asbt and LBP are key proteins in the enterohepatic circulation of bile acids. Mutations or specific inhibition of Asbt lead to malabsorption of primary bile acids, a steep decrease of the bile acid pool, and an increase in fecal bile acid secretion (11, 43, 46). Studies by Kramer et al. (32) using photoaffinity labeling suggested that LBP may be the only physiologically relevant bile acid-binding protein in the rabbit ileal cytosol. Whether this holds true for all species and during different stages in development remains to be established, because, when Asbt is high in the fetal rat, there is no expression of iBABP (46), and iBABP is not found in all tissues expressing Asbt (18). Although the overall mRNA expression of Asbt and LBP was lower in weanling distal colon than in adult ileum (Figs. 1 and 4), the presence of these two transcripts suggests that weanling distal colon is designed for bile acid absorption.

This study demonstrates for the first time that the distal colon of weanling mammals transports bile acids from lumen to serosa at a net rate that is ~11% that of adult ileum and 22% that of weanling ileum (Figs. 8 and 9). In sharp contrast to these findings, the distal colon of adults, where neither mRNA of Asbt nor mRNA of LBP can be detected by PCR, promotes a small, but significant, net secretory bile acid flux. Perhaps other members of the Mrp superfamily underlie this secretion (Fig. 11). The concentrations of bile acids at the serosal surface under normal physiological conditions may be low because of efficient clearance by capillary flow; therefore, these secretory mechanisms of bile acids may come into play under conditions of low serosal clearance.


Figure 11
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  		Fig. 11. Summary of TC net transport and mRNA expression of bile acid transporters and bile acid-binding proteins in developing distal colon and adult ileum of rabbits. Dashed arrows, activation of expression. Widths of open arrows indicate relative net transepithelial transport of TC.

 
Although the total bile acid pool increases steeply during weaning, as shown for rats and guinea pigs (40, 60), the ileum has not yet reached the adult morphology. In weanling rabbits, the mucosal surface area is ~9 mm2/mm2 serosa compared with ~25 mm2/mm2 serosa in adults, with a villus surface area of 88 and 278 µm2/villus, respectively (53). As shown in the present study, Asbt and LBP mRNA expression in weanling ileum is also lower than adult levels (Figs. 1 and 2). Furthermore, this is borne out by the functional studies which demonstrate that Jm -> s and Jnet are significantly lower in weanling than in adult ileum (Fig. 10). A lower bile acid uptake into ileal enterocytes in weanling rabbits was also shown earlier (53).

FXR. An increased interaction between weanling distal colon and bile acids is also indicated by higher abundance of the mRNA of the nuclear bile acid sensor FXR in weanling than in adult distal colon (Fig. 3). FXR cloned from adult rabbit colon represents FXR-beta1, which is also the predominant isoform in mouse small intestine (62). All rabbit intestinal tissues also express FXR-beta2 mRNA, although at much lower levels than FXR-beta1 (data not shown). FXR is involved in the regulation of LBP and also, indirectly, Asbt (9). The high expression of Asbt and LBP in distal ileum and the distinctive increase in their expression in weanling colon (Figs. 1A and 2A), together with the similar expression levels of FXR mRNA (Fig. 3) in these two tissues, corroborate the speculation that, as in the ileum, FXR may play a role in ensuring that bile acid absorption occurs in weanling distal colon.

t-Asbt, Mrp3, and Ost-{alpha}. Transcellular bile acid transport also requires basolateral exit mechanisms, and candidate transporters are anion exchangers (58): t-Asbt (34) and Mrp3 (45) in the rat and Ost-{alpha}-Ost-beta (12) in the mouse. The evidence is largely based on immunohistochemical and functional studies in heterologous cell systems. The physiological role of these transporters remains to be established. In the present study, we demonstrate that rabbit ileum expresses mRNA for t-Asbt, and, as in the case of the rat (34), t-Asbt mRNA abundance is rather low. However, critical to this study is that t-Asbt mRNA was not detected in adult or weanling distal colon (Fig. 4).

Ost-{alpha}-Ost-beta has recently been described as the putative basolateral bile acid transporter in the mouse, with strong expression in the ileum but not the distal colon (12). Similarly, our results show that, in the rabbit, Ost-{alpha} is strongly expressed in adult ileum (Fig. 6A), with much lower expression in adult distal colon. Equally important, we demonstrate an age-dependent expression of Ost-{alpha} (adult > weanling >> newborn) in the ileum but uniformly low expression at all ages in the colon. We postulate that although Ost-{alpha} may play an important role in ileal bile acid absorption in rabbit ileum, it may have a small, if any, role in weanling colon.

In contrast, in terms of mRNA and protein, Mrp3 exhibits an age-dependent (adult > weanling > newborn) expression in distal colon, whereas it exhibits very low expression in adult distal ileum compared with adult colon (Figs. 5B and 7). These results also suggest parallel expressions of Mrp3 protein and mRNA in rabbit tissues. Earlier studies in the rat (45) also showed a greater expression of colonic than ileal Mrp3 mRNA by RT-PCR but a similar Mrp3 protein expression in the two tissues. However, the PCR data were not quantitated in that study. Furthermore, immunohistochemistry in rat small and large intestine showed that Mrp3 is localized at the BLM of the villar and surface cells, respectively (45). Preliminary results from our laboratory also show a similar distribution in rabbit colon (unpublished observations). All the above observations lead us to suggest that Mrp3 may be the basolateral exit mechanism in weanling rabbit colon, whereas Ost-{alpha} or t-Asbt is the putative basolateral transporter in the ileum.

Bile acid transporters in distal colon of newborn rabbits. The lack of detectable LBP mRNA in distal colon of newborn rabbits is consistent with studies in rat small intestine, where iBABP mRNA was not detectable in postnatal week 1 (48). In contrast, mRNA expression of Asbt in newborn distal colon was variable (see standard error in Fig. 1), although by one-sample t-test the mean values were not different from 0. A biphasic expression pattern of ileal Asbt was also noted during prenatal/early postnatal development of the rat (48). Thus Asbt mRNA was expressed in rats on embryonic day 22, decreased within postnatal week 1, and then increased steeply at weaning. It is possible that the variances in expression in the colon of newborn rabbits are due to the time span (postnatal days 5–8) in which the animals were killed. Together, these observations indicate that newborn distal colon is not designed for transcellular bile acid uptake. Although the physiological function of high FXR expression in the newborn remains unclear, a preadaptive high abundance might be advantageous in initiation of LBP expression in distal colon as the animal is weaned.

Transport of bile acids. Bile acid transport has been reported to be regulated at the transcriptional level. This is the first report of a detailed examination of age-related changes in unidirectional and net transepithelial fluxes of bile acids in the small and large intestine. The gradual increase in Isc in adult and weanling rabbit ileum is intriguing and may be due to the electrogenic TC flux; because the flux is of much smaller magnitude in weanling distal colon, a similar trend is not seen in that tissue. The changes we report during development might implicate a regulatory adaptation to balance the changing milieu of the gut in the transition from newborn to weanling to adult. The alterations in mRNA expression in ileum and distal colon with age in the present study occur in concert with the changes in bile acid flux. The great absorptive capability for bile acid in adult ileum may be balanced by the potential for secretion in the distal colon, whereas the lower absorption in weanling ileum may be supported by a secondary salvage absorptive process in the distal colon. It is conceivable that such a salvage mechanism may be present in a variety of mammalian species, inasmuch as most species exhibit age-dependent expression of ileal Asbt. To put this in the context of rabbit physiology, it is very useful to the animal to have a mechanism for conserving bile acids in the colon during development. The adult rabbit is a coprophagic animal and ingests soft feces with a high content of fibrous material; therefore, unconjugated bile acids can be found in its proximal intestinal lumen. However, with the increase of cereal grains in standard rabbit chow, the contribution of soft feces to total protein intake may be <10% (6). On the other hand, in the developing animal fed rabbit milk, the diet is high in fat and low in fiber. Under these circumstances, especially when ileal absorption has not reached adult levels, a colonic salvage pathway allowing for bile acid reabsorption to optimize nutrient (fat) absorption would be highly advantageous to the animal.

In summary, the present study provides new insights into bile acid transport and expression of transporter/receptor proteins in developing mammalian distal colon. We demonstrate for the first time that weanling distal colon possesses the necessary bile acid transporter machinery and exhibits net bile acid absorption. We postulate that, in weanling distal colon, active bile acid absorption occurs via Asbt and increases intracellular bile acid concentrations, because the ability of weanling ileum to absorb bile acids is below that of the adult. This in turn might activate FXR, which then leads to an increased LBP expression. We propose that bile acids exit the BLM via Mrp3. In contrast, adult colon has no Asbt and significantly lower FXR expression than weanling colon and, therefore, cannot absorb bile acids. Physiologically, this may be relevant in the enterohepatic conservation and recycling of bile acids at a stage when the ileal capacity for bile acid absorption is not maximal. The mechanisms regulating these changes remain to be elucidated.


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 MATERIALS AND METHODS
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58135 (to M. C. Rao) and the Royal Golden Jubilee Program, Thailand (to P. Piyachaturawat and J. Kanchanapoo).


   ACKNOWLEDGMENTS
 
The authors are deeply indebted to Cynthia Kay for editorial assistance and Dr. John Walker (Dept. of Physiology and Biophysics, University of Illinois, Chicago, IL) for help with the statistical analyses. The authors thank Dr. Alan Hoffmann (University of California, San Diego, CA) for his input when these data were presented at a meeting.

Present address of D. Weihrauch: Dept. of Animal Physiology, University of Osnabrück, Osnabrück, Germany.

Present address of J. Kanchanapoo: Div. of Biopharmacy, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, Thailand.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Rao, Dept. of Physiology and Biophysics, Univ. of Illinois, College of Medicine, 835 S. Wolcott Ave., Chicago, IL 60612 (e-mail: meenarao{at}uic.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.

* These authors also made major contributions to this work. Back

1 Alternative teminology includes sodium-dependent bile acid transporter or ileal sodium-dependent bile acid transporter. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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