Role of actin in EGF-induced alterations in enterocyte SGLT1 expression

Brian M. Chung, Jason K. Wong, James A. Hardin, and D. Grant Gall

Gastrointestinal Research Group, Health Sciences Centre, University of Calgary, Calgary, Alberta, Canada T2N 4N1


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Na+-glucose cotransporter (SGLT1) expression and the role of actin in epidermal growth factor (EGF)-induced alterations in glucose transport and brush-border surface area were examined in New Zealand White rabbit jejunal loops. In separate experiments, EGF or EGF concurrent with cytochalasin D, an inhibitor of actin polymerization, was administered to the experimental loop and compared with its vehicle control. SGLT1 expression was measured by Western blot in brush-border membrane vesicles (BBMV) after 5-min and 1-h exposure. Glucose kinetics were determined by a rapid filtration technique, and brush-border surface area was examined by electron microscopy after 1-h exposure. The effect of cytochalasin D alone on BBMV glucose kinetics and brush-border surface area was also assessed. EGF resulted in a significant increase in BBMV SGLT1 expression (P < 0.05), glucose maximal uptake (Vmax; P < 0.001), and absorptive brush-border surface area (P < 0.001). These effects were abolished with concurrent cytochalasin D treatment. Cytochalasin D alone had no effect on glucose transport or brush-border surface area. The findings suggest that EGF acutely upregulates jejunal brush-border surface area and the Vmax for jejunal glucose uptake via the recruitment and insertion of SGLT1 from an internal pool into the brush border by a mechanism that is dependent on actin polymerization.

epidermal growth factor; sodium-glucose cotransporter; glucose transport


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

EPIDERMAL GROWTH FACTOR (EGF) is a 53-amino acid peptide derived from numerous sources in the gastrointestinal tract, including saliva, bile, Paneth cells (34), and Brunner's glands of the duodenum (26). EGF is a potent mitogen and has been shown to promote DNA synthesis (6, 26) and transcription of RNA leading to protein synthesis (6). EGF has also been shown to regulate small intestinal transport function. EGF increases intestinal nutrient and ion absorption both in vivo and in vitro (32). This effect is associated with increases in both brush-border surface area and total absorptive surface area, which appears to be due to the recruitment of a pool of preformed microvillus membrane (13). In addition, our laboratory as well as others (14, 16) have demonstrated an increase in maximal uptake (Vmax) for glucose transport in jejunal brush-border membranes after EGF treatment. These findings suggest EGF stimulates jejunal glucose transport via the insertion of new membrane and transport proteins into the brush border.

For EGF to exert its biological effects it must first bind to a specific transmembrane receptor. This receptor has three main regions: an extracellular ligand binding domain, a transmembrane segment, and an intracellular domain possessing intrinsic tyrosine kinase activity (2, 26). The binding of EGF to its receptor induces dimerization of bound receptors, resulting in activation of the EGF receptor complexes by autophosphorylation on tyrosine residues (38, 42). The activated EGF receptor complex has been shown to be associated with the actin cytoskeleton (10, 35, 43), as well as to act on many intracellular substrates, including four enzymes linked to the cytoskeleton: diacylglycerol kinase, phosphoinositol kinase, phosphoinositol 4-phosphate kinase, and phospholipase Cgamma (33).

Activation of the EGF receptor has been reported to lead to increased F-actin content in cultured cells (35), and stimulation of cells with EGF has been shown to induce serine phosphorylation on actin filaments (44). The brush border of intestinal epithelial cells, an evolutionary adaptation designed to increase surface area for digestion and absorption of ions and nutrients, appears to be a primary site of action of EGF in the small intestine. A major component of the brush border is actin, which comprises the core of each microvillus. Actin is also known to be involved in the regulated cycling and insertion of membrane proteins, including transport proteins (11, 28, 41). Thus we hypothesized that EGF increases glucose absorption by increasing the insertion of membrane and Na+-glucose cotransporter protein (SGLT1) into the brush border through a mechanism involving the polymerization of actin.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal model. New Zealand White rabbits (800-1,000 g) were used. Experimental procedures followed standards set by the Canadian Council of Animal Care. Animals were anesthetized with halothane, a laparotomy was performed, jejunum was isolated, and two blind 10- to 15-cm loops, separated by a 1-cm segment, were tied off 5 cm distal to the ligament of Treitz. Three separate experimental conditions were examined: either EGF (60 ng/ml; Sigma, St. Louis, MO) in 1.8-2.0 ml of Krebs buffer [(in mM) 140 Na+, 127.5 Cl-, 25 HCO-3, 10 K+, 1.25 Ca2+, 1.1 Mg2+, 2 H2PO4, pH 7.4], EGF (60 ng/ml) and cytochalasin D (2 µM; Sigma) in 1.8-2.0 ml of Krebs buffer, or cytochalasin D (2 µM) alone in 1.8-2.0 ml of Krebs buffer. Experimental solutions were added alternately to either the proximal or distal loop, and vehicle alone (0.1% DMSO in 1.8-2.0 ml Krebs buffer) was added to the remaining loop. Cytochalasins have previously been shown to effectively inhibit actin polymerization (4, 7, 31). After a 1-h incubation, loops were removed and tissue was prepared for glucose transport measurements and electron microscopy.

Brush-border membrane vesicle glucose transport. For the glucose transport studies the mucosa was scraped and brush-border membrane vesicles (BBMV) prepared by a calcium chloride precipitation method as previously described (20). BBMV were stored in liquid nitrogen until day of assay. All measurements were normalized to membrane protein, as determined by the method of Lowry et al. (23). Purity of brush-border membranes was evaluated by measuring sucrase activity in both the initial mucosal homogenate and the microvillus membrane preparation. Sucrase activity was determined by the method of Dalquist (9). Basolateral contamination was assessed by comparing Na+-K+-ATPase activity in the initial homogenate and the microvillus preparation by the method of Kelly et al. (19).

BBMV uptake of D-glucose was measured using a rapid filtration technique and a 5-s time course as previously described (18). All chemicals were obtained from Sigma except for the D-[3H]glucose, which was obtained from DuPont NEN Research products (Mississauga, Ontario). Vesicles were resuspended to a final concentration of 8-15 mg protein/ml in (in mM) 100 KCl, 300 mannitol, and 10 Tris-HEPES (pH 7.5). Valinomycin (4 µM) was added immediately before transport measurements to voltage clamp the vesicle preparations.

Glucose transport was measured by mixing 10 µl membrane vesicles with 50 µl reaction buffer containing a concentration of mannitol ranging from 0 to 100 mM, 100 mM NaSCN, 10 mM Tris-HEPES, 100 mM KCl (pH 7.5), 4 µM D-[3H]glucose, and varying concentrations of unlabeled D-glucose ranging from 0 to 100 mM. Experiments were performed at 22°C. Glucose transport was stopped by addition of 5 ml ice-cold stop solution [(in mM) 100 NaCl, 100 mannitol, 10 Tris-HEPES, 100 KCl, pH 7.5]. The solution was then rapidly filtered through a 0.45-µm filter (Millipore/Continental Water Systems, Bedford, MA) and washed with 5 ml of stop solution. Filters were placed in 10 ml scintillation fluid and counted in a liquid scintillation counter. Brush-border membrane kinetic analysis was performed by nonlinear regression techniques as previously described (25). Data are expressed as nanomoles of D-[3H]glucose taken up per minute per milligram protein.

Brush-border surface area. Jejunal brush-border surface area was determined from three animals for each of the three treatment groups. After 1 h, EGF, EGF-cytochalasin D, cytochalasin D alone, or control loops were removed and fixed in 5% glutaraldehyde in phosphate buffer [(in M) 0.2 NaH2PO4, 0.2 Na2HPO4, pH 7.3], osmolality of 300 mosmol/kgH2O at 20°C. Tissue was postfixed in 1% OsO4 for 2 h, dehydrated in graded alcohols, cleared with propylene oxide, and infiltrated with and embedded in Spurr's low-viscosity medium (J. B. EM Services, Dorval, Quebec). Ultrathin sections were obtained and double-stained with uranyl acetate in 50% ethanol and 0.4% lead citrate. Micrographs were obtained from the midvillus region of the sections as determined by a low-magnification observation of complete villi. Duplicate measurements from each micrograph were obtained, and jejunal midvillus brush-border area was calculated as previously described (5). To avoid observer bias, the study was performed under blinded conditions.

Brush-border membrane SGLT1 expression. Animals were anesthetized, and two blind jejunal loops were prepared as described above. Two separate experimental conditions were examined: either EGF (60 ng/ml) in 1.8-2.0 ml of Krebs buffer was administered into one loop and 1.8-2.0 ml Krebs buffer were added to the paired control loop or EGF (60 ng/ml) and cytochalasin D (2 µM) in 1.8-2.0 ml of Krebs buffer were added to one loop and 1.8-2.0 ml of Krebs buffer were added to the other loop. Experimental solutions were added alternately to either the proximal or distal loop, and vehicle (0.1% DMSO) alone was added to the remaining loop. In separate experiments, loops were removed after 5 min or 1 h, and the mucosa was harvested. BBMV were prepared as described above.

Western blot. BBMV proteins were separated on denaturing 8% SDS-PAGE minigels according to the method of Laemmli (22). Briefly, BBMV were diluted 1:1 in 2× Laemmli SDS protein sample buffer (S-2401, Sigma) and boiled for 5 min at 100°C. BBMV were then loaded onto 8% SDS minigels (25 µg protein/lane) and separated using a constant current. High-range molecular weight markers (V-5251, Promega, Madison, WI) were concurrently run to determine molecular weights. Separated proteins were transferred overnight onto nitrocellulose paper (0.45 mM, Trans-Blot transfer medium, Bio-Rad, Mississauga, Ontario) with a low-ionic transfer buffer at constant current. The finished blots were rinsed in 0.05% Tween 20 Tris-buffered saline (TTBS), blotted dry on filter paper, and stored at -20°C until staining.

Blots were probed with a polyclonal antibody raised against a proposed extracellular loop of the rabbit SGLT1 sequence (8327-1109, Cedarlane Laboratories, Hornby, Ontario) (15). In preliminary experiments, a densitometric analysis of serial dilutions of the peptide against which the antibody was raised was performed. Optical density was linear for concentrations of the peptide ranging from 1 to 2.25 pmol. All samples measured in this study fell within the linear portion of the curve. Immunoreactive bands were developed using a modification of the immunogold-silver process described by Fowler (12). Briefly, blots were thawed and rehydrated with TTBS. Blots were blocked for 1 h with 5% wt/vol BSA (A-3803, Sigma). Blots were then incubated for 4 h with a 1:40 vol/vol dilution of the antibody in 1% BSA-TTBS and subsequently washed three times for 10 min. Blots were then probed with a secondary antibody (G-7402, Sigma) diluted 1:300 vol/vol in gelatin buffer (0.1% BSA, 0.4% gelatin, TTBS) for 1.5 h. Finally, blots were washed three times with TTBS and once in distilled water and then incubated with silver enhancer (RPN 492, Cedarlane Laboratories, Hornby, Ontario) for 20-30 min to amplify the gold signal. Immunoreactive bands were assessed by two-dimensional scanning densitometry with incident illumination on a Scanalytics SCPI Masterscan densitometer, using Camscan (Scanalytics, Fairfax, VA). The image was analyzed by RFLPScan 1.01 and expressed as units of integrated optical density.

Statistical analysis. Data are expressed as means ± SE, and statistical analyses were performed by Student's t-test or ANOVA with repeated measurements. Western blots were analyzed by paired t-test. Statistical comparison of kinetic curves was performed as previously described (27). Significance levels were set at 0.05.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Glucose kinetics. Jejunal BBMV prepared from EGF-, EGF-cytochalasin D-, and cytochalasin D-treated loops were compared with control vesicle preparations. All BBMV preparations used in this study demonstrated at least a 10-fold increase in sucrase activity compared with their respective mucosal homogenates and <3% basolateral contamination as determined by Na+-K+-ATPase. Kinetic parameters for the uptake of glucose into brush-border vesicles were calculated and are presented in Table 1. Luminal exposure of tissue to 60 ng/ml EGF for 1 h resulted in a significant (P < 0.001) increase in the Vmax (53%) for glucose transport in BBMV. To determine the role of actin in the EGF-induced increase in glucose transport, an inhibitor of actin polymerization, cytochalasin D (2 µM), was concurrently administered with EGF. As shown in Table 1, glucose transport kinetics in vesicles from EGF-cytochalasin D-treated tissue did not significantly differ from vesicles obtained from control tissue. To determine if cytochalasin D alone had any effect on brush-border glucose transport kinetics, a further series of experiments was performed. When cytochalasin D alone was administered to loops for 1 h, glucose transport kinetic values did not significantly differ compared with vesicles from control loops. No significant effect on the Michaelis-Menten constant (Km) was observed compared with control in any of the three treatment groups. Kinetic parameters did not differ between control BBMV preparations, and thus all control kinetic data were pooled.

                              
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Table 1.   Jejunal BBMV glucose transport kinetics

Brush-border surface area. Representative electron micrographs of the apical brush border from the midvillus region of EGF-, EGF-cytochalasin D-, and control-treated loops after 1 h luminal exposure are shown in Fig. 1. Brush-border surface area and enterocyte ultrastructure in tissue exposed to cytochalasin D alone did not differ from control; therefore, only control tissue is shown. No ultrastructural abnormalities were seen in any of the treatment groups. Villus and crypt architecture was unaltered, and epithelial cells appeared normal, showing no evidence of damage. The striking difference observed was a notable increase in microvillus height in the EGF group. As shown in Fig. 2, when brush-border surface area was calculated, EGF (60 ng/ml) significantly (P < 0.001) increased jejunal brush-border surface area [41%; 49.9 ± 2.1 vs. 35.5 ± 1.8 µm2 for EGF (n = 26) and control (n = 36), respectively]. The control values for the various treatment groups examined did not significantly differ; thus all control data were pooled. The increase in brush-border surface area was mostly due to a significant increase in microvillus height. Microvillus width and density did not significantly differ between treatment groups (Fig. 2). Concurrent addition of the inhibitor of actin polymerization, cytochalasin D, abolished the increase in brush-border surface area seen with EGF treatment (36.9 ± 2.2 µm2, n = 22). When the effect of cytochalasin D alone was assessed, cytochalasin D exhibited no significant effect on any brush-border parameter, including brush-border surface area (35.2 ± 1.7 µm2, n = 26). Consistent with the findings of Madara et al. (24), treatment with cytochalasin D (either EGF-cytochalasin D or cytochalasin D alone) appeared to result in the slight opening of tight junctions compared with control (data not shown).


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Fig. 1.   Electron micrographs of representative brush border from midvillus of control (A), epidermal growth factor- (EGF; B), and EGF-cytochalasin D-treated tissues (C), after loops were exposed to 1 h of experimental solution. Tissue treated with cytochalasin D alone did not differ from control (micrograph not shown). Bars = 1 µm.


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Fig. 2.   Microvillus height, width, density, and surface area of jejunal brush border from midvillus region in EGF-, EGF-cytochalasin D-, and cytochalasin D-treated tissue compared with control. Solid bars represent experimental group and open bars respective control values. Data are expressed as means ± SE. * P < 0.001. CYTO, cytochalasin D. Control data did not differ between any experimental groups and were therefore pooled.

Brush-border SGLT1 expression. All BBMV preparations utilized for Western blots had a >10-fold increase in sucrase activity compared with initial homogenates and <3% basolateral contamination as assessed by Na+-K+-ATPase activity. BBMV protein, sucrase, and Na+-K+-ATPase activity did not differ between any of the groups after either 5 min or 1 h as shown in Table 2.

                              
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Table 2.   Jejunal BBMV protein, sucrase activity, and Na+-K+-ATPase activity

Probing with the polyclonal antibody showed a single immunoreactive band at 70-75 kDa. This is in agreement with previous reports for the molecular mass of the rabbit intestinal SGLT1 (15). Immunoreactive bands are shown in Figs. 3 and 4. Densitometric analysis demonstrated that 5-min (Fig. 3, P < 0.01) or 1-h (Fig. 4, P < 0.05) luminal exposure to EGF (60 ng/ml) significantly increased BBMV SGLT1 band density over saline-exposed controls. Concurrent administration of cytochalasin D with EGF for 1 h completely abolished the increase in BBMV SGLT1 band density (Fig. 4).


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Fig. 3.   Na+-glucose cotransporter (SGLT1) expression in EGF and control brush-border membrane vesicle (BBMV) preparations exposed to EGF or saline for 5 min. Top: representative immunoblots of BBMV protein separated on 8% SDS gel, depicting single immunoreactive band corresponding to SGLT1 from EGF and vehicle control membrane preparations. Bottom: bar graph of integrated optical density expressed as means ± SE of BBMV harvested from EGF- and vehicle control-treated loops (n = 5). Integrated optical density was significantly increased in BBMV harvested from EGF-treated loops compared with controls. * P < 0.01 compared with respective paired control BBMV preparations.


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Fig. 4.   SGLT1 expression in EGF, EGF-cytochalasin D, and control BBMV preparations. Top: representative immunoblots of BBMV protein separated on 8% SDS gel, depicting single immunoreactive band corresponding to SGLT1 from EGF, EGF-cytochalasin D, and vehicle control membrane preparations. Bottom: bar graph of integrated optical density expressed as means ± SE of BBMV harvested from EGF-, EGF-cytochalasin D-, and vehicle control-treated loops (n = 5). Integrated optical density was significantly increased in BBMV harvested from EGF-treated loops compared with controls. Concurrent EGF + cytochalasin D administration blocked increase in SGLT1 expression. * P < 0.05 compared with respective paired control BBMV preparations.


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The results suggest that EGF acutely upregulates jejunal brush-border surface area and the Vmax for jejunal glucose uptake via the recruitment and insertion of membrane rich in SGLT1 from an internal pool into the brush border. This process is dependent on actin filaments. EGF treatment resulted in a significant increase in the Vmax for brush-border glucose transport, brush-border surface area, and brush-border SGLT1 content. Concurrent cytochalasin D administration inhibited EGF-induced increases in brush-border glucose transport, surface area, and SGLT1 content, suggesting that these are linked phenomena and that actin polymerization is required for EGF-induced effects. Furthermore, previous studies have demonstrated that EGF-stimulated elevations in Vmax are not associated with any alteration in brush-border membrane lipid composition, physical characteristics, or sucrase content (13), and no alteration in either affinity of the Na+-coupled cotransporter for glucose or brush-border membrane sodium permeability was observed after EGF treatment (14). As demonstrated, the response to EGF is very rapid (<5 min); EGF stimulated a significant increase in brush-border SGLT1 content. This is in keeping with our previous results demonstrating a rapid upregulation of transport (32) and absorptive surface area (13). Thus it seems likely that the observed elevation in glucose Vmax after EGF treatment is due to the insertion of additional membrane containing transport proteins into the brush-border membrane. This effect appears to represent a general increase in membrane nutrient transport, inasmuch as previous studies have demonstrated a similar increase in brush-border proline uptake after EGF treatment (14). The inhibition of EGF-induced effects by cytochalasin D may reflect either an inhibition of actin-dependent membrane cycling and insertion or a combination of reduced vesicle trafficking and alterations in microvillus and terminal web actin polymerization.

Evidence from a variety of sources suggests that cells may contain a pool of preformed membrane-bound transporters available for recruitment to the plasma membrane. Insulin stimulates increased glucose transporter isoform-4 expression on the surface of CHO cells due to the translocation of an intracellular pool of membrane-bound transporters (17) and increases glucose uptake in isolated rat adipose cells via the incorporation of intracellular glucose transporters into the plasma membrane (8). Similar to the effect of EGF on glucose transport with its associated increase in brush-border surface area, these effects of insulin are also associated with an increase in cell surface area (8, 17). Insulin has also been shown to increase the plasma membrane peptide transporter content of Caco-2 cells by a mechanism that is microtubule dependent but does not require an increase in gene expression (40). cAMP-stimulated insertion of the cystic fibrosis transmembrane conductance regulator into the membrane of Xenopus laevis oocytes is also associated with an increase in membrane surface area (39).

The cytoskeletal protein actin exists both as actin monomers (G-actin) and long actin filaments (F-actin) composed of many polymerized monomer subunits. After nucleation of an actin filament, subsequent elongation of the filament occurs by addition of G-actin monomers. Stability of actin filaments and their rate of growth is closely regulated and is determined by a number of factors including the free monomer concentration and a variety of actin-binding proteins (1). Cytochalasin D is a fungal product that binds to and caps the ends of actin filaments, inhibiting further addition of monomer subunits (1). There is some evidence that cytochalasins may also interact with G-actin monomers (37). Various roles have been characterized for actin in cellular function. Aside from its well-recognized structural role as a major cytoskeletal component, actin is thought to both spatially organize cellular organelles and proteins and provide a pathway for the movement of intracellular vesicles bound to motor proteins.

As previously shown, EGF acutely upregulates brush-border surface area primarily due to an increase in microvillus height (13). Inhibition of actin polymerization by cytochalasin D abolished the effect of EGF on brush-border surface area. The microvillus core is mainly composed of polymerized actin filaments, and, depending on the species, 20-30 actin filaments are bundled per microvillus (3, 29). There is evidence that the EGF receptor is associated with actin filaments (10, 35, 36, 43), and the EGF receptor itself has been shown to bind to F-actin (10, 43). Furthermore, in cell culture, EGF induces serine phosphorylation on actin, causing a rapid polymerization of actin in the cytoskeleton (44), and stimulates a 30% rise in cellular filamentous actin levels (36). Of particular interest are experiments by Mooseker et al. (30), in which addition of excess G-actin induced an acute increase in microvillus length in isolated brush-border preparations. Thus alterations in actin polymerization may lead to increases in the length of the microvillus actin core, resulting in increased brush-border surface area. On the other hand, in the current study brush-border cytoskeletal morphology was not disrupted, and there was no evidence of a direct effect of cytochalasin D on brush-border actin. In rabbit ileum, basolateral EGF stimulation has been shown to result in a signal transduction cascade terminating at the brush border (21). Therefore, although our findings indicate that the EGF receptor can associate with actin filaments and suggest that EGF may exert some of its biological effects via alterations in intracellular actin pools, the mechanism by which EGF may alter cellular actin remains to be determined.

In summary, the findings in the current study suggest that luminal EGF acutely increases the expression of brush-border SGLT1 by a mechanism dependent on actin polymerization. Concurrent treatment with cytochalasin D, a specific inhibitor of actin polymerization, abolishes EGF-induced increases in apical surface area, glucose transport, and brush-border SGLT1 expression, indicating that the observed increases in microvillus height and recruitment of absorptive membrane and transport proteins are linked phenomena.


    ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council of Canada.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: D. G. Gall, Dept. of Pediatrics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1.

Received 16 July 1998; accepted in final form 29 October 1998.


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Abstract
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Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(2):G463-G469
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