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The ontogenic regulation of intestinal gene expression in the rat is characterized by the abrupt onset of a number of genes near the time of weaning(1). One example of this is the appearance of sodium-dependent bile acid transport activity in the terminal ileum(2, 3). It is not known if this is a preprogrammed system or one that is designed to respond to an expanding circulating bile salt pool(4, 5). Sodium-dependent bile acid transport in the ileum is mediated at least in part by a 48-kD protein, ASBT(gene name, SLC10A2)(6). Descriptive molecular analysis of the ontogeny of ASBT in the rat intestine reveals that it undergoes a transcriptionally mediated increase in expression at the time of weaning, which is consistent with previous functional studies of sodium-dependent bile acid transport activity(7). Interestingly, the expression of the same gene in the kidney is constitutive during this same time period(8).

Once bile salts traverse the apical membrane of ileal enterocytes, a 14-kD bile acid-binding protein, ILBP, appears to be involved in intracellular trafficking(9). ILBP undergoes a similar abrupt increase in expression at the time of weaning in the mouse and in the rat(9–11). The initial activation of ILBP expression in the mouse is much earlier, near the time of cytodifferentiation of the intestine (i.e. late fetal). Therefore ILBP expression is initially activated prenatally, repressed in the postnatal period, and then reinduced at weaning(10). It is not known whether these events are associated with a similar pattern of expression of ASBT and whether there is functional ileal bile salt reclamation in the fetal/neonatal period in the rat. Therefore ASBT and ILBP expression and sodium-dependent bile acid transport activity were investigated during this time period in the rat.

METHODS

Animals. Sprague-Dawley rat pups were obtained from Charles River (Raleigh, NC). Animals were housed, fed, and handled according to the protocol approved by the NIH Guide for the Care and Use of Laboratory Animals and under a protocol approved by the Yale Animal Care and Use Committee. Sprague-Dawley rat pups were housed with their mother and were exposed to 12-h day/night cycles. Fetal rats were obtained by cesarean section.

Northern blot analysis. Total cellular RNA was prepared by the guanidine isothiocyanate-cesium chloride method(12) in three separate preparations from the terminal ileum (defined as the distal 25% of the small intestine) and kidneys of embryonic d 22 (E22), and postnatal d 1,3,7,and 21 (P1,P3,and so forth) rats. Two to three litters (natural litters ranging from 8 to 14 animals per litter) of E22 and P1, one litter of P3 and P7, and six P21 rats were used for each total RNA preparation. Polyadenylated RNA was isolated and purified from aliquots of total RNA using oligo(dT) linked to magnetic beads as per the manufacturer's instruction (Promega, Madison, WI). Poly(A+) RNA was electrophoresed through a 1.2% agarose gel containing 1.6 M formaldehyde. RNA was transferred to an uncharged GeneScreen membrane (NEN Research, Boston, MA) through capillary action and UV cross-linked using a Stratalinker (Stratagene, La Jolla, CA). Blots were sequentially probed for ASBT, ILBP (mouse ILBP cDNA probe generously provided by Jeffrey Gordon, Washington University, St. Louis, MO)(10), and for cyclophilin(13). The 1.2-kb insert of the rat ileal ASBT clone BS37C1(7), which was excised by digestion with EcoRI and XhoI, and full-length ILBP and cyclophilin probes were radiolabeled with[32P]dCTP by random primer labeling (Life Technologies, Inc., Gaithersburg, MD). Blots were washed twice for 5 min at room temperature in 6× SSC, then twice at 60 °C in 2 × SSC for 30 min followed by two 30-min washes at room temperature in 0.1 × SSC. Signal intensity was determined using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). All signals are reported relative to the signal intensity for E22, which was arbitrarily assigned 1.00.

Western blot analysis. Western blotting for ASBT was performed using a previously characterized carboxy-terminal antipeptide antibody and BBMV, which were prepared by divalent cation precipitation as previously described(7, 8). Western blotting for ILBP was performed using a polyclonal ILBP antibody(10) and homogenates of terminal ileum. Three litters of fetal rats were required for each BBMV preparation and yielded between 350 and 500 μg of BBMV. Apical membrane marker enrichments were not performed primarily because of the lack of information of appropriate apical membrane markers for these developmental stages in the terminal ileum. An enhanced chemiluminescent detection system was used according to the manufacturer's instructions (Amersham Corp., Arlington Heights, IL) for visualization of the ASBT protein. ASBT protein abundance was approximated by volume densitometry of the 48-kD ASBT band(Personal Densitometer, Molecular Dynamics, Sunnyvale, CA). Because of the large number of animals required for these analyses, BBMV preparations and blotting were performed in duplicate and a representative blot is shown.

Taurocholate transport. Given the low yield of BBMV proteins from the fetal/neonatal rat ileum and kidney the method of preparation of BBMV in this investigation was modified slightly from previous investigations(8, 14). For a single preparation of crude ileal or renal BBMV, whole nonflushed terminal ileum (operationally defined as the last 25% of the small intestine) or intact kidneys were removed from two litters of pups and were homogenized in an appropriate solution (ileum, 50 mM mannitol, 2 mM Tris, pH 7.1; kidney, 200 mM mannitol, 80 mM HEPES/41 mM KOH, pH 7.5). All solutions used in membrane preparation contained a mixture of protease inhibitors consisting of 25 μg/mL leupeptin, 5 μg/mL aprotinin, 40 μg/mL phenylmethylsulfonyl fluoride, 50 μg/mL benzamide, 0.5 μg/mL pepstatin. Ileal and renal homogenates were incubated for 15 min at 4 °C with 10 mM CaCl2 or 10 mM MgCl2, respectively. Cellular debris and nuclei were removed with a 10-min 2987 × g spin, and then crude apical membranes were sedimented at 26 890 × g for 20 min. The resulting pellet was resuspended in 300 mM mannitol, 10 mM HEPES/Tris, pH 7.5, homogenized with 10 low speed strokes of a tight Teflon homogenizer, and then sedimented at 43 140 × g for 30 min. The resulting pellet was resuspended in 300 mM mannitol, 10 mM HEPES/Tris, pH 7.5, stored on ice, and used within 6 h for transport assays. Protein concentrations were determined using the Bradford(15) method (Bio-Rad, Richmond, CA), with BSA as the standard. Sodium-dependent uptake of 4 μM [3H]taurocholate was measured at 60 s in triplicate by rapid filtration using previously published methods(14).

To further validate these transport studies temperature sensitivity and transport into an osmotically sensitive space was assessed with a single preparation of vesicles from fetal d 22, postnatal d 7, and postnatal d 21 animals. Temperature sensitivity of the transport process was assessed by incubating the vesicles with 4 μM [3H]taurocholate at 4 °C instead of the standard 37 °C. Transport into an osmotically sensitive space was measured by preincubating the vesicles for 30 min at 4 °C with a solution that yielded a final osmotic concentration that was 560 mosmol as opposed to the standard 310 mosmol solution. The incubation solution was also altered to yield a 560 mosmol solution (125 mM NaCl, 300 mM mannitol, 10 mM HEPES versus 125 mM NaCl, 50 mM mannitol, 10 mM HEPES).

Bile acid analysis of ileal contents. Bile acids were extracted from samples of ileal homogenates (E22, P7, and P21) after addition of the internal standard nordeoxycholic acid (1-2 μg). All samples were sonicated in 80% methanol (50 mL) for 30 min, refluxed for 2 h, and taken to dryness on a rotary evaporator(16). The samples were diluted with 0.01 M acetic acid and extracted on Lipidex 1000 (Packard Instrument Co., Groningen, The Netherlands) and reverse-phase octadecylsilane-bonded silica cartridges (Bond Elut C18 Analytichem Inc., Harbor City, CA) by the methods described by Setchell and Worthington(17). Bile acids were recovered by elution of both columns with methanol, and the combined extracts were taken to dryness. Solvolysis was carried out in a mixture of methanol, distilled tetrahydrofuran, and 1 M trifluoracetic acid in dioxane (1:9:0.1 by volume), heated to 45 °C for 2 h(18). After evaporation of the reagents, the residue was enzymatically hydrolyzed with cholylglycine hydrolase (Sigma Chemical Co., St. Louis, MO) in 0.2 M phosphate buffer at 37 °C overnight(19). Unconjugated bile acids were again extracted by passage of the sample through a Bond Elut C18 cartridge and recovered by elution with methanol. The bile acids were isolated by lipohilic anion exchange chromatography(20) on a column of diethylaminohydroxypropyl Sephadex LH-20 (Lipidex DEAP, Packard Instrument Co.) and recovered by elution with 0.1 M acetic acid in 72% ethanol. Volatile methyl estertrimethylsilyl ether derivatives were prepared(21) by reaction with freshly distilled ethereal diazomethane followed by TriSil reagent (Pierce Chemicals, Rockford, IL) and purified(22) on Lipidex 5000 (Packard Instruments Co.) in hexane. The bile acid methyl ester-trimethylsilyl ethers were separated by gas chromatography on a 30 m × 0.25-mm DB-1 fused silica capillary column (J & W Scientific, Folson, CA) using a temperature-programmed operation from 225 to 295 °C. Identification of individual peaks was confirmed by GC-MS analysis performed on a VG Autospec Q magnetic sector mass spectrometer. Electron ionization (70 eV) mass spectra obtained by repetitive scanning of eluting components were recorded over the mass range 50-800 Da. Quantification of individual bile acids was achieved by comparison of the peak height of each bile acid derivative with the peak height response of the added internal standard.

Statistical analysis. Unless otherwise noted all values are the mean ± SD, and means are compared using a t test. Error bars on all graphs are standard deviations.

RESULTS

Northern blot analysis. Northern blotting of ileal poly(A+) RNA revealed that the ASBT transcript could be detected on E22, its level of expression was reduced by approximately 50% on P7, and it was reinduced on P21 to a level that was approximately 15 times that observed prenatally (Fig. 1, A and B). In contrast, analyses of renal poly(A+) RNA demonstrated that expression in the kidney was relatively constitutive during the same time period (Fig. 1, C and D). ILBP transcript could be detected in ileum but not kidney (data not shown). ILBP mRNA could not be detected in fetal or neonatal RNA samples(E22, P1, P3, P7), but was easily detected at P21 (data not shown). Results were similar whether the ASBT or ILBP signals were corrected for cyclophilin or 260 nm UV light absorbance.

Figure 1
figure 1

Northern blot analysis. (A and C) Representative Northern blot analyses of the fetal/neonatal expression of ASBT in the rat ileum (A) and kidney (C). Five micrograms of poly(A+) RNA have been probed for ASBT (top panel) and cyclophilin (bottom panel) using coding region cDNA probes. The 5.0-kb ASBT transcript can be easily detected in fetal tissues, shows reduced expression on postnatal d 1, 3, and 7 in ileum but not in the kidney and then undergoes marked up-regulation at weaning on postnatal d.21 in the ileum but not in the kidney. (B and D). Cumulative Northern blot analysis of ileal (B) and renal (D) ASBT mRNA expression. Three separate preparations of poly(A+) RNA were probed for ASBT and cyclophilin by Northern blotting. Signal intensity was measured using a PhosphorImager, and the intensity of the ASBT signal was corrected for loading using the cyclophilin signal from the same blot. The unitless value was arbitrarily set at 100% for E22 on each of the three blots, and the remaining values are relative to the signal intensity at E22 for each organ. Error bars represent SDs of three separate measurements relative to E22. In ileum there is a modest reduction in ASBT signal in P1, 3, and 7 relative to E22 and at least a 17-fold increase in signal intensity on P21 (note bar is off scale). In kidney, the ASBT signal is relatively constant throughout this same time period. Given the limited number of samples and the variability in the measurements, statistically significant changes were not observed.

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Western blot analysis. Qualitatively similar results were obtained after Western blotting of fetal and neonatal ileal and renal BBMV using a carboxy-terminal antipeptide antibody. The 48-kD ASBT protein was found to follow the same biphasic pattern of expression in the ileum and was constitutively expressed in the kidney (Fig. 2A). Semiquantitative volume densitometric analysis of the level of expression of the ASBT protein was similar to that observed for the ASBT mRNA (Fig. 2B). The 14-kD ILBP protein could not be detected in fetal or neonatal ileal homogenates, but was readily detected from weaning(P21) and adult rat ileum (Fig. 2C).

Figure 2
figure 2

Western blot analysis. (A) Representative Western blot of fetal/neonatal ileal and renal BBMV using a carboxy-terminal pentadecapeptide ASBT antibody. Fifty micrograms of BBMV were loaded in all lanes except the P21 ileum (10 μg). The 48-and 92-kD ASBT bands can be seen in each of the samples (arrows), although the signal is quite faint in the ileal P7 sample. (B) Volume densitometry of this blot is plotted with signal corrected for the amount of protein that was loaded. ASBT protein expression is down-regulated in the immediate postnatal period in the ileum and is then markedly up-regulated at weaning. ASBT protein expression in the kidney is relatively constitutive during this time period. (C) Representative Western blot of ileal homogenates using a polyclonal antimouse ILBP antibody. The 14-kD ILBP protein cannot be detected at E22 or P3, is faintly detected at P7 and is much more abundant at P21 and in adult rat ileum[loading E22, P3, P7, P21, 40 μg; adult (P60), 5 μg].

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Taurocholate transport. Crude BBMV were used for the transport studies because the yields of highly purified BBMV from fetal animals were too low for the measurement of sodium-dependent taurocholate transport. Sodium-dependent taurocholate uptake was measured at 60 s using 4 μM[3H]taurocholate (2.1 Ci/mmol; Amersham Corp., Northbrook, IL) to maximize signal intensity. Sodium-dependent bile acid transport activity was easily measured in fetal ileal membranes and was significantly reduced by P7(E22, 17.0 ± 5.7 pmol/mg/60 s; P7, 3.9 ± 2.1, p = 0.02, n = 3) (Fig. 3). This reduction in transport is specific as previous investigations have shown that P7 ileal membranes are able to support sodium-dependent uptake of taurine(8). The relatively constant association of[3H]taurocholate with the ileal vesicles in the presence of potassium indicates that the vesicles have relatively similar physical properties(i.e. nonspecific binding and/or sodium-independent transport). The transport in E22 ileal membranes was both temperature-dependent (84% reduction at 4 °C) and occurred into an osmotically sensitive space (51% reduction in hyperosmolar conditions) (Fig. 4). These characteristics were similar to that observed in crude membranes prepared from P21 ileum. Interestingly, the sodium-dependent bile acid transport activity in the E22 and P21 membranes was quantitatively similar despite significant differences in the abundance of the ASBT protein (6-fold greater in P21 relative to E22) (Fig. 2B). However, these measurements were performed at the time of maximal uptake, which optimized detection of low levels of transport activity, but is not necessarily a good indication of protein abundance. The limited yield of membrane vesicles did not permit kinetic analysis of the transprot activity, which would have generated maximal velocity rates for direct and more valid comparison with protein expression. The transport properties of the renal vesicles were less satisfactory. The sodium independent fraction was relatively high (Fig. 3) and this transport was not into an osmotically sensitive space (percent reduction in hyperosmolar media E22, 20; P7, 0; P21, 0). Therefore the functional consequences of the renal ASBT expression require further investigation in a modified transport system.

Figure 3
figure 3

[3H]Taurocholate uptake into fetal/neonatal ileal and renal membrane vesicles. The result of three separate sets of taurocholate transport experiments are depicted. The uptake in the presence of a Na or K gradient is depicted. The error bars represent standard deviations. In the ileum a clear reduction in the sodium-dependent (Na-K) taurocholate transport can be seen between E22 and P7.

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Figure 4
figure 4

Temperature and osmotic sensitivity of[3H]taurocholate uptake into fetal/neonatal ileal membrane vesicles. The results of the mean of three measurements from a single preparation of E22, P7, and P21 vesicles are depicted. The first bar in each set represents uptake in the presence of a sodium gradient, the second in the presence of potassium, the third in the presence of sodium at 4 °C, and the fourth in the presence of a hyperosmolar sodium solution. Significant sodium dependent(Na-K) transport is seen at E22 and P21. Uptake in the presence of sodium is markedly reduced at E22 and P21 by temperature reduction or osmotic shrinkage of the vesicles.

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Bile acid analysis of ileal contents. Ileal homogenates that were generated in the preparation of the crude membranes in these experiments were analyzed for bile acid content. The ileal segements were not flushed before homogenization so that this value represents the sum of bile salts found in both the intestinal lumen and mucosa. The volume of material in the intestinal lumen was not measured so that the quantity of bile salts is expressed relative to the amount of protein in the homogenate rather than as a concentration. By this analysis the total bile salt concentration in the E22 terminal ileum was apparently physiologically significant especially when compared with that found at P21 (E22, 1.6; P7, 3.9; P21, 2.6 ng/μg of protein).

DISCUSSION

These investigations demonstrate a biphasic pattern of expression of sodium-dependent bile acid transport activity and of ASBT mRNA and protein in the rat ileum. In contrast, ASBT mRNA and protein expression in the kidney are relatively constitutive. This biphasic pattern of expression in the ileum is reminiscent of the expression of the ileal lipid binding protein in the mouse(10), although in the rat ileum ILBP's expression is monophasic. This monophasic induction of gene expression around the 2nd to 3rd wk of development is similar to that described by Henning(1) for several other intestinal genes. The observed discordance in the developmental activation patterns of rat ASBT and ILBP gene expression suggests that they may be controlled by different regulatory signals.

The discordance in expression of ASBT and ILBP in the fetal rat ileum raises important questions about the physiologic relationship of these two proteins. ILBP has clearly been shown to have a bile acid binding capacity in vitro and along with its predominantly ileal-specific expression suggests a role in intracellular trafficking of bile salts(9, 23). Although the physiologic role of ILBP in ileal reclamation of bile salts remains undefined. The demonstration of sodium-dependent bile acid transport, in isolated BBMVs from fetal and neonatal rats, indicates that the transcriptional, translational, and posttranslational mechanisms are present to process a functional bile acid transporter (i.e. ASBT). The lack of ILBP expression in the fetal rat ileum suggests that fetal handling of bile salts is different from mature rats or that ILBP is not essential for bile acid reclamation. Alternatively the fetal expression of ASBT is not physiologically significant. To address this last issue fetal ileal bile salt levels were assessed.

GC/MS analysis of fetal ileal homogenates revealed significant concentrations of bile salts, findings which are similar to previous observations using RIAs for taurocholic acid(24). The presence of significant concentrations of fetal ileal bile salts indicates that passive jejunal absorption is incomplete and that a role for active ileal transport exists(25). The primary sources of fetal bile salts is hepatic synthesis, which begins before birth in the rat(26). In addition, there is evidence for a contribution to the fetal pool of bile salts by maternal to fetal transfer, despite the primary role of the placenta for bile acid excretion(27). Canalicular excretion of endogenously synthesized bile salts in the fetus presumably occurs by an ATP-dependent transporter, which is expressed before the potential driven transporter(24, 28). This canalicular excretion of bile salts may be crucial for the normal development of the biliary tract and appears to produce a significant intestinal load of bile salts, whose ileal reclamation is most likely to be mediated by the expression of the ASBT gene. The absence of fetal hepatic basolateral sodium-dependent bile acid(29, 30) transport and bypassing of the liver via the ductus venosus necessitates placental excretion to avoid potentially injurious fetal hypercholanemia. It is possible that the repression of the ileal reclamation of bile salts in the postnatal period serves to allow ileal malabsorption of bile salts and excretion, until such time as the hepatic excretory process become fully mature. In addition malabsorption of bile salts may lead to depression of bile acid synthetic pathways allowing expansion of the bile salt pool. Increased bile flow may be a signal that this process has matured and necessitates the reexpression of ileal bile salt reclamation(14).

Regardless of the physiologic role of the fetal expression of the ASBT gene, the repression of its expression in the neonatal period is an important phenomenon to understand. Promoter mapping studies of several intestinal genes[ILBP(10), sucrase isomaltase(31–33), intestinal fatty acid binding protein(34), and liver fatty acid binding protein(35)] suggests that suppression appears to be a relatively common mechanism involved in the regulation of intestinal gene expression. In transgenic mice, 8.5 kb of the sucrase-isomaltase gene flanking region directs reporter expression to all four intestinal cell lineages, whereas shorter constructs are restricted to enterocytes and enteroendocrine cells(29–31). In contrast, ILBP/human GH fusion gene expression is appropriately directed to enterocytes alone by a 913-nucleotide construct, whereas shorter constructs direct expression appropriately to enterocytes but also aberrantly to goblet cells, implying the presence of a goblet cell suppressor element between nucleotides 417 and 913(10). In addition a relatively short ILBP promoter construct is able to recapitulate its normal postnatal down-regulation in transgenic mice(10). This implies that the mechanism of this suppression is primarily regulated at the level of transcription. Nuclear run on studies with E22 and P7 ileum might suggest that a similar mode of transcriptional regulation occurs with the ASBT gene. Unfortunately size limitations preclude a nuclear run on analysis of fetal rat ileum. Interestingly, nuclear run on assays of ASBT transcription on postnatal d 7 in rat kidney and ileum seem to indicate that the decreased expression in the ileum is related to relative instability of the ASBT transcript(8). Elucidation of the mechanisms involved in the postnatal down-regulation of ASBT expression may lead to the identification of endogenous factors that might be useful for pharmacologic reduction of ASBT expression leading to bile salt wasting. It has been clearly demonstrated that the induction of bile salt wasting by partial ileal bypass is an effective means of reducing cholesterol levels and long-term morbidity and mortality from coronary artery disease(36).

In summary, these studies have demonstrated that the ontogeny of the ASBT gene in the rat ileum is characterized by a biphasic pattern of expression. ASBT function and gene products can be detected in near term fetal rats, are repressed in the early neonatal period, and then abruptly reexpressed at the time of weaning. In contrast, the pattern of expression in the kidney is relatively constitutive. The level of regulation of the postnatal ileal repression of ASBT expression is not known but may be related to the steady-state level of ASBT mRNA (i.e. transcription or mRNA stability) and/or translation of that mRNA.