Mechanism of inhibition of Na+-glucose cotransport in the chronically inflamed rabbit ileum

U. Sundaram1, S. Wisel1, V. M. Rajendren2, and A. B. West3

1 Division of Gastroenterology, Departments of Medicine and Physiology, Ohio State University School of Medicine, Columbus, Ohio 43210; 2 Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06320; and 3 Department of Pathology, University of Texas, Galveston, Texas 77555

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

In a rabbit model of chronic ileal inflammation, we previously demonstrated that coupled NaCl absorption was reduced because of an inhibition of Cl-/HCO<SUP>−</SUP><SUB>3</SUB> but not Na+/H+ exchange on the brush-border membrane (BBM) of villus cells. In this study we determined the alterations in Na+-stimulated glucose [Na+-O-methyl-D-glucose (Na+-OMG)] absorption during chronic ileitis. Na+-OMG uptake was reduced in villus cells from the chronically inflamed ileum. Na+-K+-adenosinetriphosphatase (ATPase), which provides the favorable Na+ gradient for this cotransporter in intact cells, was found to be reduced also. However, in villus cell BBM vesicles from the inflamed ileum Na+-OMG uptake was reduced as well, suggesting an effect at the level of the cotransporter itself. Kinetic studies demonstrated that Na+-OMG uptake in the inflamed ileum was inhibited by a decrease in the maximal rate of uptake for OMG without a change in the affinity. Analysis of steady-state mRNA and immunoreactive protein levels of this cotransporter demonstrates reduced expression. Thus Na+-glucose cotransport was inhibited in the chronically inflamed ileum, and the inhibition was secondary to a decrease in the number of cotransporters and not solely secondary to an inhibition of Na+-K+-ATPase or altered affinity for glucose.

glucose absorption; sodium absorption; electrolyte transport; chronic intestinal inflammation; sodium-glucose cotransport; sodium-potassium-adenosinetriphosphatase; inflammatory bowel disease

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

INHIBITION OF Na+ and Cl- absorption and malnutrition have been well described in human inflammatory bowel diseases (1, 3, 5, 22). However, the cellular mechanisms of alterations of coupled NaCl and nutrient-dependent Na+ absorption during chronic ileal inflammation are not well understood. This is primarily because of the lack of good animal models of chronic ileitis and the inability to isolate viable villus and crypt cells from the inflamed intestine suitable for the study of electrolyte transport.

We recently reported a rabbit model of chronic ileal inflammation from which viable and relatively pure populations of villus and crypt cells can be isolated (27). In this model of chronic ileitis specific alterations in electrolyte transport pathways were demonstrated. For example, it was shown that the mechanism of inhibition of coupled NaCl absorption was due to an inhibition of Cl-/HCO<SUP>−</SUP><SUB>3</SUB> but not Na+/H+ exchange on the brush-border membrane (BBM) of villus cells (27).

Another important Na+-absorptive pathway in the normal ileum is Na+-nutrient cotransport (e.g., Na+-glucose cotransport). An alteration of this cotransport process in the chronically inflamed ileum will not only affect Na+ absorption but also the assimilation of important nutrients.

Na+-glucose cotransport is known to be present on the BBM of villus but not crypt cells in the normal ileum. In the intact cell, Na+-K+-adenosinetriphosphatase (ATPase) provides the favorable Na+ gradient for this cotransporter (7, 10, 15, 16, 25, 26, 28). Thus, during chronic ileal inflammation, cellular alterations in Na+-glucose cotransport may be at the level of the cotransporter and/or Na+-K+-ATPase. Therefore, the aims of this study were to test the hypothesis that chronic inflammation alters Na+-glucose cotransport and to determine the cellular mechanisms of this alteration.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Induction of chronic inflammation. Chronic ileal inflammation was produced in rabbits as previously reported (27). Pathogen-free rabbits were intragastrically inoculated with 10,000 oocytes of the coccidian protozoan Eimeria magna or sham inoculated with 0.9% NaCl (control animals). Oocytes were isolated from feces of infected rabbits by the method of Jackson (12). None of the sham inoculations and ~80% of inoculations with coccidia resulted in chronic ileal inflammation during days 13-15. Only enterocytes from those animals that had histologically confirmed chronic ileal inflammation were utilized for experiments.

Measurement of epithelial dynamics. Histological sections were analyzed to determine alterations in epithelial morphology during chronic ileal inflammation. The villus-to-crypt ratio was defined as total villus height from the base of the crypt divided by the total crypt height. Ten different villus/crypt units were measured in three control or inflamed intestines from different animals.

Cell isolation. Villus and crypt cells were isolated from the normal and inflamed ileum by a Ca2+-chelation technique as previously described (26, 27). Established criteria were utilized to validate good separation of villus and crypt cells. These criteria included 1) marker enzymes (e.g., thymidine kinase), 2) transporter specificity, 3) differences in intracellular pH, 4) morphological differences, and 5) differing rates of protein synthesis.

The following set of criteria was utilized to exclude cells that showed evidence of poor viability: 1) trypan blue exclusion, 2) the demonstration of Na+/H+ and Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange activities, and 3) the ability of the cells to maintain a baseline pH or imposed acid or alkaline gradient and return to baseline pH after perturbations. The cells were maintained in short-term culture for up to 6-8 h. For short-term culture, cells were resuspended at a final concentration of 0.1 g cells in 40 ml of Leibowitz-15 medium (GIBCO) with 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10% rabbit serum, 5,000 U/l penicillin, 5 mg/l streptomycin, and 10 mg/l gentamicin and gassed with 100% O2, pH 7.4 at 37°C and kept in sterile flasks until needed. Cells used for BBM vesicle (BBMV) preparation were frozen immediately in liquid nitrogen and stored at -70°C until required.

BBMV preparation. BBMV from rabbit ileal villus cells were prepared by CaCl2 precipitation and differential centrifugation (17). Frozen villus cells were thawed and suspended in 2 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.5) containing 50 mM mannitol. The suspension was homogenized, and 10 mM CaCl2 was added. The homogenate was centrifuged at 8,000 g for 15 min, and the supernatant was centrifuged at 20,000 g for 30 min. Then the pellet was resuspended in 10 mM Tris-HCl buffer (pH 7.5) containing 100 mM mannitol and homogenized. Vesicles were formed by adding MgCl2 (10 mM). The homogenate was centrifuged at 2,000 g for 15 min to remove debris, and the BBMV were precipitated by centrifugation at 27,000 g for 30 min. BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme (e.g., alkaline phosphatase) enrichment.

Uptake studies in villus and crypt cells. Villus or crypt cells (100 mg wet wt) were washed and resuspended in HEPES buffer containing (in mM) 1.25 3-O-methyl-D-glucose (3-OMG), 4.5 KCl, 1.2 KH2PO4, 1.0 MgSO4, 1.25 CaCl2, 20 HEPES, and either 130 mM NaCl or choline chloride and were gassed with 100% O2 (pH 7.4 at 37°C). Ten microcuries of [3H]OMG (Amersham) were added to 1-ml cell suspension in the HEPES buffer, and 100-µl aliquots were removed at desired time intervals. The uptake was arrested by mixing with 3 ml ice-cold stop solution (choline-HEPES buffer). The mixture was filtered on 0.65-µm Millipore (HAWP) filters. After two washes with ice-cold stop solution, the filter was dissolved in 4 ml Optifluor and the radioactivity was determined.

BBMV uptake studies. Uptake studies were performed by the rapid filtration technique as previously described (17). In brief, 10 µl of BBMV resuspended in (in mM) 100 choline chloride, 0.10 MgSO4, 50 HEPES-Tris (pH 7.5), 50 mannitol, and 50 KCl were incubated in 90 µl reaction medium that contained 50 mM HEPES-Tris buffer (pH 7.5), 1 mM OMG, 20 µM [3]OMG, 0.10 mM MgSO4, 50 mM KCl, 50 mM mannitol, 100 mM of either NaCl or choline chloride, 10 µM valinomycin, and 100 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. At desired times, uptake was arrested by mixing with ice-cold stop solution (in mM, 50 HEPES-Tris buffer, 0.10 MgSO4, 75 KCl, and 100 choline chloride, pH 7.5). The mixture was filtered on 0.45-µm Millipore (HAWP) filters and washed twice with 3 ml ice-cold stop solution. Filters with BBMV were dissolved in Optifluor, and radioactivity was determined. Amiloride (0.1 mM)-sensitive 22Na+ uptake in BBMV was also performed by rapid filtration as previously described (18).

Na+-K+-ATPase measurement. Na+-K+-ATPase was measured as Pi liberated by the method of Forbush (8) in cellular homogenates from the same amount of cells from normal or inflamed ileum as described below. The reaction was started by adding 20 mM ATP-Tris to 10 mg membrane protein in 50 mM Tris-HCl (pH 7.2), 5 mM MgCl2, and as appropriate 20 mM KCl and 100 mM NaCl. The reaction was stopped by the addition of ice-cold 2.8% ascorbic acid, 0.48% ammonium molybdate, and 2.8% sodium dodecyl sulfate (SDS)-0.48 M HCl solution and placed on ice for 10 min. The color was developed by incubating at 37°C for 10 min after the addition of 1.5 ml of 2% sodium citrate, 2% sodium arsenite, and 2% acetic acid. Readings were obtained at 705 nM, and K2HPO4 was used as Pi standard. Enzyme specific activity was expressed as nanomoles of Pi released per milligram protein per minute.

Northern blot studies. Total RNA was extracted from rabbit ileal villus cells by the guanidinium isothiocyanate-cesium chloride method. After denaturation total RNA was electrophoresed on 1.8% agarose-formaldehyde gel, transferred to nylon membrane (Schleicher and Schuell, Keene, NH), and incubated with prehybridization solution. Membranes were hybridized with 32P-labeled SGLT1 cDNA. The cDNA was random labeled with [32P]CTP with Klenow polymerase. beta -Actin was used to ensure equal loading of total RNA onto the electrophoresis gels. Hybridized membrane was exposed to autoradiography film (NEN, Boston, MA). The SGLT1 cDNA was generously provided by Dr. E. M. Wright.

Western blot studies. BBMV (4 mg) were diluted in SDS-reducing buffer, boiled, and electrophoresed on a 12% SDS-polyacrylamide gel electrophoresis. The gel was electroblotted onto polyvinylidene difluoride membrane (NEN) and blocked for 2 h in 5% bovine serum albumin at room temperature. The membrane was incubated at room temperature with 1:3,000 anti-rabbit SGLT1 anti-serum followed by goat anti-rabbit immunoglobulin G coupled to horseradish peroxidase (1:10,000, Pierce, Rockford, IL). After each incubation the membrane was washed extensively with phosphate-buffered saline-0.2% Tween 20. The signal was developed with the chemiluminescence Western blot kit (NEN). The SGLT1 antibody was generously provided by Dr. E. M. Wright.

Data presentation. When data are averaged, means ± SE are shown except when error bars are inclusive within the symbol. All uptakes were done in triplicate. The number (n) for any set of experiments refers to vesicle or isolated cell preparations from different animals. Preparations in which cell viability was <85% were excluded from analysis. Student's t-test was used for statistical analysis.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The histological observation of villus blunting in the inflamed ileum was confirmed by measurements of crypt and villus height. Villus-to-crypt ratio was noted to be diminished from 7.97 ± 0.7 to 3.98 ± 0.2 (P < 0.0001) from the normal to the chronically inflamed ileum. The morphological changes have been observed in detail previously (27).

Crypt-villus cell separation is an important concern in intestine in which the normal maturation process is altered. Thus the following criteria were used to ensure good cell separation (27): 1) marker enzymes (alkaline phosphatase for villus cells and thymidine kinase for crypt cells), 2) higher intracellular baseline pH in crypt compared with villus cells, 3) presence of Na+/H+ exchange in the BBM of villus but not crypt cells, 4) a better developed BBM on villus cells, 5) higher rate of protein synthesis in crypt cells compared with villus cells, and 6) the presence of Na+-nutrient cotransport on the BBM of villus but not crypt cells.

Cell viability by trypan blue exclusion was observed in 94 ± 4% of villus cells from the normal rabbit ileum and in 93 ± 5% of villus cells from the inflamed ileum (n = 11).

We have previously demonstrated that Na+-dependent glucose uptake is present in villus but not crypt cells of the normal rabbit ileum (26). The initial study was performed to confirm this observation. In villus cells from the normal ileum 3-OMG uptake was significantly stimulated by extracellular Na+ (e.g., 8.56 ± 0.90 nmol/mg protein at 15 min in the presence of Na+ and 1.03 ± 0.13 nmol/mg protein in the absence of Na+, n = 6, P < 0.0001). However, Na+-stimulated 3-OMG uptake was not observed in crypt cells from the normal ileum (e.g., 1.10 ± 0.36 nmol/mg protein at 15 min in the presence of Na+ and 1.20 ± 0.11 nmol/mg protein in the absence of Na+, n = 6, P not significant).

Na+-stimulated 3-OMG uptake in villus and crypt cells from the inflamed ileum was next determined (Fig. 1). In villus cells from the inflamed ileum 3-OMG uptake was also significantly stimulated by extracellular Na+ (Fig. 1A). Similar to the normal ileum, Na+-stimulated 3-OMG uptake was also not present in crypt cells from the inflamed ileum (Fig. 1B). Thus these data demonstrate that Na+-stimulated glucose uptake is present in villus but not crypt cells from the normal and inflamed ileum.


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Fig. 1.   Effect of extracellular Na+ on 3-O-methyl-D-glucose (3-OMG) uptake as a function of time in intact villus and crypt cells from chronically inflamed rabbit ileum. A: villus cells, n = 6, * P < 0.005. Extracellular Na+ significantly stimulated 3-OMG uptake at all time points in villus cells from inflamed ileum. B: crypt cells, n = 6. Similar to the normal ileum, Na+-stimulated 3-OMG uptake was also not present in crypt cells from inflamed ileum. bullet , Na+ present; open circle , Na+ absent.

Figure 2 compares the Na+-dependent uptake of 3-OMG in villus cells from the normal and inflamed ileum. Na+-dependent 3-OMG uptake was significantly diminished in villus cells from the inflamed ileum. These data indicate that Na+-glucose cotransport was reduced in intact villus cells from the inflamed ileum.


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Fig. 2.   Effect of chronic inflammation on Na+-dependent uptake of 3-OMG in villus cells. Na+-dependent 3-OMG uptake as a function of time is shown and is significantly inhibited in villus cells from inflamed ileum. Six inflamed (open circle ) and six control (bullet ) animals were studied. * P < 0.01.

Inhibition of Na+-glucose cotransport at the cellular level may represent a direct effect on the cotransporter located on the BBM and/or may be secondary to an inhibition of Na+-K+-ATPase on the basolateral membrane (BLM), which provides the favorable Na+-electrochemical gradient for this cotransport process. Thus Na+-K+-ATPase activity was measured as previously described (14) in homogenates of villus cells from normal and inflamed ileum. Na+-K+-ATPase activity was reduced ~50% in villus cells from the inflamed ileum compared with the normal ileum (Fig. 3). These data suggest that the reduction in Na+-glucose cotransport in inflamed ileal villus cells may, at least in part, be due to reduced electrochemical gradients of Na+ across the BBM resulting from an alteration in Na+ extrusion capacity by the cells.


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Fig. 3.   Effect of chronic inflammation on Na+-K+-ATPase in villus cells. Na+-K+-ATPase activity was decreased ~50% in villus cells from inflamed ileum (10.8 ± 2.0 nmol · mg protein-1 · min-1 in normal and 5.8 ± 1.2 nmol · mg protein-1 · min-1 in inflamed, n = 6, * P < 0.05).

To determine whether chronic inflammation has a direct effect on the Na+-glucose cotransporter itself, 3-OMG uptake was determined in BBMV prepared from villus cells from the normal and inflamed ileum. Na+-dependent 3-OMG uptake was significantly reduced in villus cell BBMV from the inflamed ileum (Fig. 4). These data suggest that the cotransporter itself was directly inhibited during chronic ileal inflammation.


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Fig. 4.   Na+-dependent uptake of 3-OMG in villus cell brush-border membrane vesicles (BBMV) as a function of time from normal (bullet ) and chronically inflamed (open circle ) ileum. Na+-dependent glucose uptake was significantly reduced in villus cell BBMV from inflamed ileum (n = 3, * P < 0.05).

Altered Na+ permeability would be expected to diminish the Na+ gradient across the membrane and may explain the inhibition of 3-OMG uptake seen in BBMV from the inflamed ileum. Therefore, 22Na+ uptake in villus cell BBMV from the normal and inflamed ileum was determined and was found not to be different (0.10 nmol · mg protein-1 · 15 s-1 in normal and 0.11 nmol · mg protein-1 · 15 s-1 in inflamed, n = 3, P not significant). Unaltered amiloride- sensitive 22Na+ uptake (i.e., Na+/H+ exchange) also indicates that the villus cell BBM from the inflamed ileum is not significantly contaminated by crypt cell BBM, because it is known that Na+/H+ exchange is only present on the BBM of villus but not crypt cells in the rabbit ileum (16).

BBM purity was also ensured by a similar degree of enrichment of the villus cell BBM marker enzyme, alkaline phosphatase (11.0 ± 1.0-fold enrichment in normal villus cell BBM and 10.1 ± 1.1-fold enrichment in inflamed villus cell BBM, n = 3).

To determine whether the inhibition of Na+-glucose cotransport during chronic ileal inflammation was due to an alteration in the affinity for glucose and/or in the maximal rate of uptake (Vmax) of glucose, kinetic studies were performed. Uptake for all the concentrations was carried out at 6 s because in initial uptake studies Na+-dependent glucose uptake in BBMV was linear for at least 10 s (data not shown) in the normal and the inflamed ileum. Figure 5 demonstrates the kinetics of glucose uptake in villus cell BBMV from the normal and inflamed ileum. Figure 5A shows the uptake of Na+-dependent 3-OMG as a function of varying concentrations of extravesicular glucose. As the concentration of extravesicular glucose was increased, the uptake of Na+-dependent 3-OMG was stimulated and subsequently became saturated in the normal as well as in the inflamed ileum. With use of Enzfitter, a Lineweaver-Burk plot of these data was generated and is shown in Fig. 5B. Kinetic parameters derived from these data demonstrate that the affinity [1/Michaelis constant (1/Km)] for 3-OMG uptake was not different between the normal and inflamed ileum (Km for 3-OMG uptake in BBMV was 6.6 ± 1.5 mM in normal and 7.0 ± 2.0 in inflamed ileum, n = 3, P not significant). However, the Vmax of 3-OMG was reduced severalfold in the inflamed ileum (Vmax for 3-OMG uptake in BBMV was 5.0 ± 0.5 nmol · mg protein-1 · 6 s-1 in normal and 1.2 ± 0.4 nmol · mg protein-1 · 6 s-1 in inflamed ileum, n = 3, P < 0.05). These data suggested that Na+-glucose cotransport was inhibited in the chronically inflamed ileum secondary to a decrease in the number of cotransporters rather than altered affinity for glucose. To confirm these findings we next looked at steady-state levels of mRNA for SGLT1 in villus cells.


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Fig. 5.   Kinetics of glucose uptake in villus cell BBMV from normal (bullet ) and chronically inflamed (open circle ) ileum. A: representative of 3 experiments. Na+-dependent uptake of [3H]OMG is shown as a function of varying concentration of extravesicular D-glucose. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 6 s. As concentration of extravesicular glucose was increased, uptake of glucose was stimulated and subsequently became saturated in villus cell BBMV from both normal and inflamed ileum. B: analysis of these data with Lineweaver-Burk plot yielded kinetic parameters. Affinity for 3-OMG uptake is not affected during chronic ileal inflammation. However, maximal rate of uptake of 3-OMG is reduced severalfold in inflamed ileum.

Steady-state levels of mRNA transcripts for SGLT1 were markedly reduced in villus cells from the chronically inflamed ileum (Fig. 6). Because steady-state mRNA levels may not directly correlate with functional protein levels on the BBM, immunoreactive SGLT1 levels on the BBM were also determined. Western blot analysis of BBMV showed that the anti-SGLT1 antibody recognized one major immunoreactive protein band at the expected size of 70 kDa, which was reduced in intensity in the chronically inflamed ileum (Fig. 7).


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Fig. 6.   Northern blot analysis demonstrates that steady-state levels of SGLT1 mRNA are reduced in villus cells from chronically inflamed ileum. Representative of 4 experiments each with different animals.


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Fig. 7.   Western blot analysis demonstrates reduction in amount of immunoreactive SGLT1 in BBMV from chronically inflamed ileum.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

This study demonstrates that Na+-glucose cotransport is inhibited in the chronically inflamed ileum. This inhibition is not entirely a consequence of a reduction in the capacity of the villus cells to extrude Na+. In the chronically inflamed ileum there is an inhibition at the level of the Na+-glucose cotransporter itself. Kinetic parameters indicate that while the affinity for glucose is not affected, the Vmax of glucose is reduced. Diminished levels of steady-state mRNA for SGLT1 and SGLT1 immunoreactive protein are observed in the chronically inflamed ileum. These data indicate that the mechanism of Na+-glucose cotransport inhibition is due to a decrease in the number of Na+-glucose cotransporters in the chronically inflamed ileum.

Our laboratory had previously demonstrated the presence of Na+-glucose cotransport in isolated villus but not crypt cells from the normal rabbit ileum (16, 26). This distribution was shown to be preserved in the chronically inflamed ileum as well (Fig. 1). However, one previous study has indicated the presence of Na+-glucose cotransport in both villus and crypt cells from the normal rabbit ileum (19). Inadequate cell separation criteria resulting in the contamination of crypt cells with villus cells may account for this finding. Indeed, immunocytochemistry and in situ hybridization studies have demonstrated that the Na+-glucose cotransporter (SGLT1) is present only in the mature villus cells in the normal rabbit ileum (11, 28). Thus most of the currently available evidence supports the observation that Na+-glucose cotransport is limited to villus cells in the normal ileum.

Although the location of Na+-glucose cotransport along the crypt-villus axis in the ileum is fairly clear, how it is altered during chronic ileal inflammation is not known. Previous studies have looked at the effect of acute inflammation on Na+-glucose cotransport in several animal models. Differing observations about the effect of acute inflammation on Na+-glucose cotransport have been reported; Na+-glucose cotransport was found to be unaltered in acute enteritis produced by Giardia in gerbils (4), whereas inhibition of Na+-glucose cotransport was observed in acute enteritis produced by Cryptosporidium in pigs (2) and Yersinia enterocolitica in rabbits (21). It was not possible to address the mechanism of alteration in Na+-glucose cotransport at the cellular level based on these intact tissue studies because in acute enteritis there is near complete loss of mature villus cells that contain the Na+-glucose cotransporter. In acute enteritis produced by transmissible gastroenteritis virus in piglets (9, 13, 14) the inhibition of Na+-glucose cotransport has been postulated to be secondary to the loss of high affinity D-glucose carrier. However, multiple Na+-glucose cotransport systems have not been described in other animals, including rabbits (19). Thus the mechanism of alteration of Na+-glucose cotransport during acute intestinal inflammation is not completely understood.

The alterations that occur in Na+-glucose cotransport during chronic ileal inflammation have not previously been investigated. Undoubtedly, this is a result of a lack of good animal models of chronic ileal inflammation. Two other models of chronic small intestinal inflammation, peptidoglycan polysaccharide-induced enterocolitis in rats (23) and alloimmunization-induced enterocolitis in guinea pigs (20), have not yet been utilized for electrolyte transport studies. At present there are no perfect animal models of chronic ileitis comparable to the human disease. Although this rabbit model of chronic intestinal inflammation should not be considered as an example of the human disease, it does possess many of the same features; the ileum is thickened with a cobblestone appearance, the villi are blunted, the crypts are hypertrophied, and the immune response is characterized by chronic rather than acute inflammatory cells. Thus it may be a suitable animal model to study the effect of chronic ileal inflammation on electrolyte transport at the cellular level.

Inhibition of Na+-glucose cotransport in villus cells from the chronically inflamed ileum may occur at the level of the cotransporter and/or secondary to an alteration in Na+ extrusion from the cell facilitated by Na+-K+-ATPase. This study indicates that during chronic ileal inflammation the mechanism of inhibition of Na+-glucose cotransport is at the level of the cotransporter and is not exclusively secondary to an alteration in the Na+ extrusion capacity of the villus cell. Kinetic studies and Northern and Western blot studies indicate that the number of Na+-glucose cotransporter is reduced in villus cells from the chronically inflamed ileum.

One possible explanation for the reduction in the number of Na+-glucose cotransporters in the chronically inflamed ileum may be a significant contamination of the isolated villus cell population with crypt cells or immunocytes. Fortunately, in the chronically inflamed ileum many of the characteristics of mature villus cells are clearly preserved, which allowed us to separate villus and crypt cells consistently (27). Furthermore, the cell isolation process did not result in any significant contamination of epithelial cells with immunocytes (27). Thus it is unlikely that the inhibition of Na+-glucose cotransport during chronic ileal inflammation demonstrated in this study is a result of the contamination of villus with crypt cells or immunocytes.

Alterations in absorption and secretion by ileal villus and crypt cells undoubtedly occur as a result of the numerous immune-inflammatory mediators endogenously produced in the chronically inflamed intestine (5, 6, 22, 24). In this model of chronic inflammation we have previously demonstrated that coupled NaCl absorption, which occurs by the dual operation of Na+/H+ and Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange on the BBM of villus cells, is inhibited as a result of impairment of Cl-/HCO<SUP>−</SUP><SUB>3</SUB> but not Na+/H+ exchange. Unlike the villus cells, in the crypt cells Na+/H+ exchange, known to be present only on the BLM of crypt cells, was stimulated in the chronically inflamed ileum. The BLM Na+/H+ exchange stimulation alkalinizes the crypt cells, which may subsequently stimulate the BBM Cl-/HCO<SUP>−</SUP><SUB>3</SUB> exchange resulting in HCO<SUP>−</SUP><SUB>3</SUB> secretion by these cells (27).

The previous findings (27) and this study demonstrate that specific transport pathways are altered in villus and crypt cells during chronic ileal inflammation to inhibit coupled NaCl and glucose-stimulated Na+ absorption and promote HCO<SUP>−</SUP><SUB>3</SUB> secretion. Given the numerous immune-inflammatory mediators known to be released in the chronically inflamed intestine and that at least some of them are capable of affecting electrolyte transport pathways, it is hypothesized that different immune-inflammatory mediators released in the chronically inflamed ileum may have unique effects on transport pathways in villus and crypt cells. Which of these agents are responsible for the transport abnormalities observed in this model of chronic ileal inflammation has yet to be elucidated.

In conclusion, Na+-glucose cotransport is inhibited during chronic ileal inflammation. The mechanism of inhibition is not solely secondary to a diminution in the Na+ extrusion capacity of the villus cell. In fact, our studies suggest that the number of Na+-glucose cotransporters is reduced in villus cells in the chronically inflamed ileum.

    ACKNOWLEDGEMENTS

We thank K. M. Sundaram and V. Annapurna for technical assistance. We thank Dr. E. M. Wright for generously providing SGLT1 cDNA and antibodies used in the Northern and Western blot studies.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R29-DK-45062 and the American Gastroenterological Association Research Industry Scholar Award to U. Sundaram.

Address for reprint requests: U. Sundaram, Div. of Gastroenterology, Ohio State Univ. School of Medicine, N-214 Doan Hall, 410 W. Tenth Ave., Columbus, OH 43210.

Received 24 September 1996; accepted in final form 30 June 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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