Department of Medicine, Christian-Albrechts University, 24105 Kiel, Germany
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ABSTRACT |
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Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport.
jejunal absorption; brush-border membrane vesicles
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INTRODUCTION |
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CONJUGATED BILE ACIDS are secreted into the intestine during digestion. Bile acid absorption from the small and large intestine is efficient, with >90% of the bile acids secreted with a meal being conserved (9). As a consequence of this efficient absorption, a large pool of bile acids accumulates and circulates multiple times each day.
Conjugated bile acid uptake by the small intestine is mainly by carrier-mediated transport. Separate transporters appear to be present in the jejunum and ileum. In addition, a small fraction of the more lipophilic conjugates may be absorbed passively in protonated form if the pH of duodenal contents is sufficiently acidic (2). Conjugated bile acids may undergo deconjugation by bacterial enzymes in the small intestine before absorption (9). The liberated unconjugated bile acids, being weak acids, can be absorbed from the small intestine by passive mechanisms.
Active absorption of conjugated bile acids has long been known to involve a sodium-dependent transporter in the apical membrane of the ileal enterocyte. This transporter has been cloned from hamster (32), rat (25), and human ileum (33); has been shown to be present in the renal proximal tubule in the rat (5); and has been found to have homology with the sodium-dependent transporter for conjugated bile acids that is present in the basolateral membrane of the hepatocyte (7). Because resection of the ileum causes severe bile acid malabsorption, the ileal transporter was considered to be the major transporter responsible for the efficient intestinal absorption of conjugated bile acids.
Nonetheless, there is a large body of evidence indicating that conjugated bile acids are absorbed from the jejunum despite convincing evidence that the ileal transporter is absent in the jejunum, at least in several species. In humans, the evidence for jejunal absorption includes perfusion studies that have shown uptake of conjugated bile acids (4, 8, 30, 31), as well as measurements of bile acids in jejunal aspirates (3) and plasma (21) that suggested a more proximal and therefore earlier absorption of dihydroxy-conjugated bile acids. In the pig, diversion of intestinal content around the jejunum greatly decreases bile acid absorption, suggesting that the majority of conjugated bile acids occurs in the jejunum (12). In rats, estimates of jejunal absorption range from 12 to 60% of total bile acid absorption (17, 20, 27, 29). Evidence for proximal absorption of conjugated bile acids in nonmammals such as hens (10), pigeons (28), and turkeys (26) has been reported.
In an attempt to define the mechanism for jejunal absorption of conjugated bile acids, we performed jejunal perfusion studies in the guinea pig and found evidence for carrier-mediated transport based on saturability and competitive inhibition (2). The transport rate was greater for dihydroxy-conjugated bile acids than trihydroxy-conjugated bile acids in contrast to ileal transport, where trihydroxy-conjugates are transported more rapidly (18).
Perfusion studies do not permit assessment of ion dependency of transport and also do not clearly distinguish transcellular from paracellular absorption, even though paracellular absorption seems unlikely for large, charged molecules such as conjugated bile acid anions. In the present study, we have prepared isolated brush-border membranes from the rat to 1) gain further insight into the mechanism of jejunal transport of conjugated bile acid anions, 2) test whether a transport mechanism for conjugated bile acid anions is also present in the apical membrane of the rat jejunal enterocyte, and 3) determine whether the apical transporter of the rat enterocyte had a substrate affinity profile that was similar to that observed in the perfused guinea pig jejunum.
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MATERIALS AND METHODS |
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Male Sprague-Dawley rats (Harlan Winkelmann, Borchen, Germany) weighing 250-275 g were fed ad libitum and kept on a 12:12-h light-dark cycle for at least 1 wk before use. Food, but not water, was removed from the cage 24 h before conducting an experiment.
D-[U-14C]glucose (9.5-13.3 GBq/mmol) and cholyltaurine (C-tau), [G-3H]taurocholate (74-185 GBq/mmol), were purchased from DuPont NEN (Bad Homburg, Germany). Chenodeoxycholyltaurine (CDC-tau), 22-23-[3H]taurochenodeoxycholate, was a generous gift from Dr. Alan F. Hofmann (University of California, San Diego, CA). Adenosine triphosphate, lactate dehydrogenase, nicotine adenine nucleotide, phosphoenolpyruvate, and pyruvate kinase were obtained from Boehringer Mannheim. Nonradioactive bile acids [C-tau, CDC-tau, and deoxycholyltaurine (DC-tau)], ouabain, dithiothreitol, D-glucose, D-mannitol, DMEM, EDTA, and EGTA were bought from Sigma-Aldrich (Deisenhof, Germany). All other reagents were of analytic grade and were obtained from Merck (Darmstadt, Germany). Rapid filtration experiments were done with cellulose nitrate filters (25-mm diameter, 0.2-µm pore size) and a manifold (DN 025/0 Schleicher & Schuell, Dassel, Germany).
Brush-border membrane vesicles were prepared from isolated intestinal epithelial cells in contrast to a whole mucosal homogenate. Epithelial cells were isolated according to the method of Schwenk et al. (24). Briefly, rats were killed by neck fracture. The abdomen was opened, and either a segment of jejunum or distal ileum was removed. Jejunum, defined as the 30 cm distal to the ligament of Treitz, and ileum, defined as the 30 cm proximal to the ileocecal valve, were obtained. The intestine was rinsed and filled with solution A (in mM: 96 NaCl, 27 sodium citrate, 5.6 KH2PO4-K2HPO4, 1.5 KCl). Solution A was then replaced by solution B (in mM: 140 NaCl, 16 KH2PO4-Na2HPO4, 1.5 EDTA, 0.5 dithiothreitol) and palpated gently for 2 min. The intestinal content, containing detached cells, was then drained. Cells and mucus were separated by filtration. The cells were suspended in DMEM at 4°C. Cell viability was >95% as checked regularly by the trypan blue exclusion method.
Preparation of brush-border membrane vesicles was done according to the
method of Schmitz et al. (23), as modified by Kessler et al. (13). The
following steps were done at 4°C. Cells were centrifuged (2 min,
500 g), and the pellet was suspended
in buffer A (in mM: 50 D-mannitol, 12 Tris, pH 7.5).
This suspension was homogenized for 30 s at 1,400 rpm (Homogenisator,
B. Braun, Melsungen, Germany). Calcium chloride was added to give a
final concentration of 10 mM. The suspension was incubated for 20 min
before centrifugation at 3,000 g for
15 min (Kontron centrifuge Z 364-K, Berthold, Gosheim, Germany). The
supernatant was centrifuged at 27,000 g for 30 min (Beckman L7-55,
Munich, Germany). The pellet was suspended in buffer
B (in mM: 50 D-mannitol, 10 HEPES, 10 Tris,
pH 7.5) and homogenized again. The final pellet was suspended in
buffer C (in mM: 0.2 CaCl, 290 D-mannitol, 10 HEPES-KOH, 1 MgCl, pH 7.5) after centrifugation at 27,000 g for 30 min. For anion exchange experiments, buffer C was substituted
for buffer D (in mM: 230 D-mannitol, 10 HEPES-Tris, 25 NaHCO3, pH 7.5). Vesicles were
visualized with electron microscopy. The vesicles were either used
immediately or after short storage at 70°C.
The protein concentration was measured by the Lowry method. Saccharase (6) and leucine aminopeptidase (19) were used as marker enzymes for brush-border membranes. Na+-K+-ATPase was measured spectrophotometrically (22) as a basolateral marker enzyme.
To define the time course of substrate uptake, 60 µl of vesicle suspension were added to 3 ml of the reaction assay at room temperature. Aliquots of 0.5 ml were taken at defined time intervals and transferred immediately to cellulose nitrate filters; the retentate was washed with stop solution (in mM: 130 NaCl, 10 HEPES-Tris, pH 7.5). To measure the initial rate of uptake, 11 µl of vesicle suspension were added to 550 µl of the reaction assay. After 10 s the reaction was stopped by immediate filtration of 500 µl vesicle suspension followed by rinsing with stop solution. Filters were placed in scintillation vials, and scintillation fluid (Quickszint 200, Zinsser Analytic, Frankfurt, Germany) was added. Vials were stored overnight (to eliminate chemiluminescence), and radioactivity was determined using a liquid scintillation counter with automatic external standard for quench correction (Beckman LS 2800).
Data are given as means ± SD. The Student's t-test for unpaired samples was used.
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RESULTS |
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Validation studies.
Marker enzymes for brush-border and basolateral poles of the enterocyte
were measured to ensure that the isolated brush-border membrane vesicle
preparations were of satisfactory purity. Saccharase was enriched seven
times (P < 0.001) based on
measurement of activity of the brush-border membrane fraction compared
with that of the homogenate (Table 1).
Leucine aminopeptidase, a second brush-border membrane enzyme, was
increased 6.1 times (P < 0.001).
Activity of
Na+-K+-ATPase,
a basolateral marker enzyme, was diminished by a factor of 10 (P < 0.001).
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Bile acid transport by jejunal brush-border membranes.
The uptake of C-tau was not stimulated by an inwardly directed sodium
gradient but trans-stimulated by the presence of an outwardly directed
bicarbonate gradient (Fig. 1). By
trans-stimulation, absorption rate increased severalfold and showed a
typical overshoot phenomenon. Maximum absorption was reached after 1 min with a transport rate of 2.2 ± 0.1 nmol/mg protein.
Steady-state levels were 0.6 ± 0.2 nmol/mg protein for the inwardly
directed sodium gradient and 0.4 ± 0.2 nmol/mg protein for the
outwardly directed bicarbonate gradient.
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DISCUSSION |
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These experiments indicate that two natural taurine-conjugated bile acids that are representative of endogenous bile acids in many vertebrate species are transported across rat jejunal brush-border membrane vesicles by an anion-dependent process. Based on its stimulation by an outwardly directed bicarbonate concentration gradient and its inhibition by DIDS, the process is likely to be an antiport process. Additional lines of evidence that the uptake of conjugated bile acids by these jejunal brush-border membrane vesicles was carrier mediated are 1) saturability, 2) inhibition of uptake by other conjugated bile acids and an organic anion, and 3) temperature dependency of uptake. Transport of CDC-tau was more rapid than C-tau, in contrast to transport by brush-border membrane vesicles from the ileum where the opposite is true. The results thus confirm and extend our previous perfusion studies performed using the guinea pig (2).
Brush-border membrane vesicles were prepared from isolated enterocytes rather than from the total epithelial cell population. The steps required for enterocyte isolation are time consuming, but the final preparation of brush-border membranes was of high purity as evidenced by the enrichment in marker enzymes. Preparation of brush-border vesicles increased the activity of the brush-border marker enzymes by approximately six to seven times compared with the homogenate (Table 1). This increase of marker enzymes of the brush-border membrane is somewhat smaller than in published studies (13), most likely because of the high purity of epithelial cells at the beginning of the vesicle preparation. The marker enzyme for the basolateral membrane, Na+-K+-ATPase, was almost not measurable in the preparation of brush-border membrane vesicles, suggesting that the preparation of brush-border membrane vesicles had negligible contamination by basolateral membranes.
Before the experiments were started, vesicles were kept in a small volume of bicarbonate-rich buffer to preload the vesicles with bicarbonate. At the beginning of the experiment the outside solution was isosmotically diluted (~40 times), leading to a steep outwardly directed concentration gradient of bicarbonate. This dilution step led to a rather high volume of incubation buffer compared with previous studies (13). A drawback of this method is the requirement for a substantial amount of radioactively labeled bile acids.
The nature of the brush-border membrane transport system mediating the uptake of conjugated bile acids in the jejunum is not known. By photoaffinity labeling, an 87-kDa protein that is involved in bile acid transport based on binding of photoaffinity-labeled conjugated bile acids is present throughout the small intestine in the rabbit (15). A similar protein with a molecular mass of 94 kDa is present in the jejunum of the rat (14). The sodium ion-independent organic anion transport protein present in the basolateral membranes of the hepatocyte has been cloned (11, 16) and shown to transport conjugated bile acids. Whether it is present in the brush-border membrane of the jejunal enterocyte has not been reported. It is not present in the proximal and distal colon (11).
The present work indicates that anion exchange transport together with passive absorption of protonated glycine-conjugated bile acids provides an explanation for jejunal uptake of conjugated bile acids in mammals. Such jejunal absorption may not only conserve conjugated bile acids but could modulate intracellular events, as has been claimed for conjugated bile acids in cholangiocytes (1).
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ACKNOWLEDGEMENTS |
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We thank Dr. Alan F. Hofmann for continuous support.
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FOOTNOTES |
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This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft; Am 84/3-1).
Present address and address for reprint requests and other correspondence: A. Amelsberg, Boehringer Ingelheim Pharma KG, Birkendorfer Str. 65, 88397 Biberach, Germany (E-mail: andree.amelsberg{at}bc.boehringer-ingelheim.com).
Received 15 December 1997; accepted in final form 3 December 1998.
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