Mechanism of glucocorticoid-mediated reversal of inhibition of Clminus /HCOminus 3 exchange during chronic ileitis

Steve Coon and Uma Sundaram

Division of Digestive Diseases, Departments of Medicine and Physiology, Ohio State University School of Medicine, Columbus, Ohio 43210


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na+/H+ and Cl-/HCO-3 exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl-/HCO-3 but not Na+/H+ exchange was reduced in the BBM of villus cells during chronic ileitis. Cl-/HCO-3 exchange was inhibited secondary to a decrease in the affinity for Cl- rather than an alteration in the maximal rate of uptake of Cl- (Vmax). Methylprednisolone (MP) stimulated Cl-/HCO-3 exchange in the normal ileum by increasing the Vmax of Cl- uptake rather than altering affinity for Cl-. MP reversed the inhibition of Cl-/HCO-3 exchange in rabbits with chronic ileitis. However, MP alleviated the Cl-/HCO-3 exchange inhibition by restoring the affinity for Cl- rather than altering the Vmax of Cl- uptake. These data suggest that glucocorticoids mediate the alleviation of Cl-/HCO-3 exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.

glucocorticoids; inflammatory bowel disease; chloride/bicarbonate exchange; sodium/hydrogen exchange; coupled sodium chloride absorption; immune regulation of electrolyte transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

COUPLED NaCl absorption occurs via the dual operation of Na+/H+ and Cl-/HCO-3 exchange on the brush border membrane (BBM) of villus cells in the rabbit ileum (4). Many agents have been demonstrated to regulate coupled NaCl absorption by directly affecting these two exchangers in the normal intestine (3, 7, 9). For example, serotonin, an intestinal secretagogue that is thought to mediate its effects via intracellular Ca2+, has been demonstrated to inhibit coupled NaCl absorption by inhibiting Cl-/HCO-3 but not Na+/H+ exchange on the BBM of villus cells in the rabbit ileum (16). In contrast, forskolin, which mediates its effect via intracellular cAMP, has been demonstrated to inhibit coupled NaCl absorption by inhibiting Na+/H+ but not Cl-/HCO-3 exchange also on the BBM of villus cells (17).

Unlike these two secretagogues, an absorptagogue such as clonidine has been demonstrated to stimulate coupled NaCl absorption by stimulating Na+/H+ but not Cl-/HCO-3 exchange on the BBM of villus cells in the rabbit ileum (13). Also, in intact tissue studies other intestinal absorptagogues such as methylprednisolone have been demonstrated to stimulate Na+ and Cl- absorption in the rabbit ileum (11, 12) and Na+ absorption in rat ileum (22). Furthermore, glucocorticoids have been demonstrated to increase the message for the putative absorptive isoform of Na+/H+ exchange, specifically NHE-3 (23). These studies illustrate that Na+/H+ and Cl-/HCO-3 exchange are uniquely affected by absorptagogues and secretagogues to alter absorption and secretion in the normal ileum.

In diarrheal diseases characterized by chronic inflammation of the intestine, malabsorption of NaCl has been well described (1-3, 7). However, the exact mechanism of alteration of coupled NaCl absorption in inflammatory bowel disease (IBD) is unknown. In a rabbit model of chronic ileal inflammation, it was previously demonstrated that inhibition of coupled NaCl absorption by the villus cells occurs as a result of diminished Cl-/HCO-3 but not Na+/H+ exchange activity (18). The specific mechanisms of alteration of Cl-/HCO-3 exchange was not further delineated in that study. The effect of chronic intestinal inflammation directly on the BBM Na+/H+ exchange was also not addressed in that study.

Malabsorption of electrolytes and fluid that is known to occur in IBD may, at least partially, be caused by the effects of a wide variety of immune-inflammatory mediators known to be produced in the chronically inflamed intestine (3, 7). Furthermore, broad-spectrum immune modulation with glucocorticoids is known to, at least partially, alleviate electrolyte and fluid malabsorption in IBD (2, 8). However, the mechanism of glucocorticoid-mediated reversal of inhibition of coupled NaCl absorption during chronic intestinal inflammation is unknown.

Given this background, one of the aims of this study was to determine the mechanism of inhibition of Cl-/HCO-3 exchange that resulted in diminished coupled NaCl absorption in the chronically inflamed ileum. Another aim was to test the hypothesis that glucocorticoids would alleviate the inhibition of Cl-/HCO-3 exchange, thereby alleviating the inhibition of coupled NaCl absorption during chronic ileitis. The final aim was to determine the mechanism of glucocorticoid-mediated reversal of inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Induction of chronic inflammation and drug treatment. Chronic ileal inflammation was produced in rabbits as previously reported (18). Pathogen-free 1.8- to 2.0-kg New Zealand White male rabbits (Prince's Rabbitry, Oakmill, KY) were intragastrically inoculated with Eimeria magna oocytes or sham inoculated with 0.9% NaCl (control animals). 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 used for experiments. Control or inoculated rabbits were treated intramuscularly with saline or 40 mg of water-soluble methylprednisolone sodium succinate (MP) per day on days 12 and 13 after inoculation and killed on day 14 after inoculation.

Cell isolation. Villus and crypt cells were isolated from the ileum by a calcium chelation technique as previously described (15, 18). Previously established criteria were used to validate good separation of villus and crypt cells (15, 18). Some of these criteria included 1) marker enzymes (e.g., thymidine kinase, alkaline phosphatase), 2) transporter specificity (e.g., Na+/H+ on the BBM of villus but not crypt cells), 3) differences in intracellular pH (e.g., intracellular pH is higher in crypt cells compared with villus), 4) morphological differences (e.g., villus cells are larger and with better-developed BBM compared with crypt cells), and 5) differing rates of protein synthesis (e.g., higher synthesis rate in crypt cells compared with villus).

Previously established criteria were also used to study cells with good viability and to exclude cells that showed evidence of poor viability (15, 18). Some of these criteria included 1) trypan blue exclusion, 2) the demonstration of Na+/H+ and Cl-/HCO-3 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.

BBM vesicle preparation. BBM vesicles (BBMV) from rabbit ileal villus cells were prepared by CaCl2 precipitation and differential centrifugation as previously reported (20). BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (e.g., alkaline phosphatase).

Uptake studies in BBMV. BBMV uptake studies were performed by a rapid filtration technique as previously described (20). For Cl-/HCO-3 exchange experiments, BBMV were resuspended and incubated for 2 h in 105 mM K gluconate, 50 mM HEPES-Tris, and either 50 mM KHCO3 gassed with 5% CO2-95% N2 or 50 mM K gluconate gassed with 100% N2 with a final pH of 7.7. The reaction was started by adding 5 µl of vesicles to 95 µl of reaction mix containing 5 mM N-methyl-D-glucamine (NMG)36Cl, 149.7 mM K gluconate, 10 mM valinomycin, 50 mM MES-Tris, and 0.3 mM KHCO3, pH 5.5 gassed with 5% CO2-95% N2, or 5 mM NMG36Cl, 10 mM valinomycin, 50 mM MES-Tris, and 150 mM K gluconate, pH 5.5 gassed with 100% N2. One millimolar DIDS was added to the reaction mix when relevant. For Na+/H+ exchange experiments BBMV were resuspended in 150 mM mannitol, 100 mM tetramethylammonium (TMA) gluconate, and 50 mM MES-Tris (pH 5.5) or 50 mM HEPES-Tris (pH 7.5). The reaction was started by adding 5 µl of vesicles to 95 µl of reaction mix containing 150 mM mannitol, 99 mM TMA gluconate, 50 mM HEPES-Tris (pH 7.5), and 1 mM 22Na gluconate. One millimolar amiloride was added to the reaction mix when relevant. At desired times, uptake was arrested by mixing with ice-cold stop solution. The mixture was filtered on 0.45-µm Millipore (HAWP) filters and washed twice with 5 ml of ice-cold stop solution. Filters with BBMV were dissolved in Liquiscint, and radioactivity was determined.

Data presentation. When data are averaged, means ± SE are shown in figures except when error bars are inclusive within the symbol. All uptake studies were done in triplicate. The n number 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cl-/HCO-3 exchange. Initially, the villus-crypt distribution of Cl-/HCO-3 exchange in the normal rabbit ileum was determined. Cl-/HCO-3 exchange was defined as HCO3-dependent and DIDS-sensitive 36Cl uptake. HCO-3-dependent 36Cl uptake was present in BBMV prepared from villus cells (Fig. 1A). Furthermore, this HCO-3-dependent 36Cl uptake was DIDS sensitive (Fig. 1A). Similarly, HCO-3-dependent 36Cl uptake was present in BBMV prepared from crypt cells (Fig. 1B). This uptake was also DIDS sensitive (Fig. 1B). Thus Cl-/HCO-3 exchange is present in both villus and crypt cell BBM from the normal rabbit ileum.


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Fig. 1.   Villus-crypt distribution of Cl-/HCO-3 exchange in normal rabbit ileum. A: villus cells. HCO-3-dependent 36Cl uptake was present in brush-border membrane (BBM) vesicles (BBMV). This uptake was DIDS inhibitable. B: crypt cells. HCO-3-dependent 36Cl uptake was also present in BBMV. This uptake was also DIDS inhibitable. Thus Cl-/HCO-3 exchange is present in both villus and crypt cell BBM from normal rabbit ileum.

We next determined the effect of chronic ileal inflammation on Cl-/HCO-3 exchange in villus and crypt cells. HCO-3-dependent and DIDS-sensitive 36Cl uptake is shown in Fig. 2. In villus cell BBMV from the chronically inflamed ileum HCO-3-dependent and DIDS-sensitive 36Cl uptake was significantly reduced (Fig. 2A). However, in crypt cell BBMV from the chronically inflamed ileum HCO-3-dependent and DIDS-sensitive 36Cl uptake was unaffected. Thus Cl-/HCO-3 exchange is inhibited in villus but not crypt cell BBM during chronic ileitis.


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Fig. 2.   Effect of chronic ileal inflammation on Cl-/HCO-3 exchange in villus and crypt cells. HCO-3-dependent, DIDS-sensitive uptake of 36Cl in BBMV as a function of time is shown. A: villus cells. HCO-x-dependent, DIDS-sensitive 36Cl uptake was significantly diminished in villus cell BBMV from chronically inflamed ileum. B: crypt cells. HCO-3-dependent, DIDS-sensitive 36Cl uptake was unaffected in crypt cell BBMV from chronically inflamed ileum. Thus Cl-/HCO-3 exchange is inhibited in villus but not crypt cells during chronic ileitis.

Na+/H+ exchange. Because coupled NaCl absorption is known to occur via the dual operation of Cl-/HCO-3 and Na+/H+ exchange in the intestine, we next studied Na+/H+ exchange. First the villus-crypt distribution of Na+/H+ exchange in the normal ileum was determined. Na+/H+ exchange was defined as H+ gradient-stimulated and amiloride-sensitive 22Na uptake. H+ gradient-dependent 22Na uptake was present in BBMV prepared from villus cells (Fig. 3A). Furthermore, this H+ gradient-stimulated 22Na uptake was amiloride sensitive (Fig. 3A). However, H+ gradient-stimulated 22Na uptake was not present in BBMV prepared from crypt cells (Fig. 3B). This nonspecific 22Na uptake was also not affected by amiloride (Fig. 3B). Thus Na+/H+ exchange activity is present in villus but not in crypt cell BBM from the normal rabbit ileum.


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Fig. 3.   Villus-crypt distribution of Na+/H+ exchange in normal rabbit ileum. A: villus cells. An in > out H+ gradient-stimulated 22Na uptake was present in BBMV. This 22Na uptake was amiloride inhibitable. B: crypt cells. H+ gradient-stimulated 22Na uptake was not present in BBMV. This nonspecific 22Na uptake was also not amiloride sensitive. Thus Na+/H+ exchange is present in villus but not crypt cell BBM from normal rabbit ileum.

We next determined the effect of chronic ileal inflammation on Na+/H+ exchange in villus and crypt cells. H+ gradient-stimulated and amiloride-sensitive 22Na uptake is shown in Fig. 4. In villus cell BBMV from the chronically inflamed ileum H+ gradient-stimulated and amiloride-sensitive 22Na uptake was unaltered (Fig. 4A). Similar to that in the normal ileum, Na+/H+ exchange was also not present in crypt cell BBMV from the chronically inflamed ileum (Fig. 4B). Thus Na+/H+ exchange is unaffected in villus cell BBM, and it is not present in crypt cell BBM during chronic ileitis.


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Fig. 4.   Effect of chronic ileal inflammation on Na+/H+ exchange in villus and crypt cells. H+ gradient-stimulated and amiloride-sensitive 22Na uptake in BBMV as a function of time is shown. A: villus cells. H+ gradient-stimulated and amiloride-sensitive 22Na uptake was unaffected in villus cell BBMV from chronically inflamed ileum. B: crypt cells. H+ gradient-stimulated and amiloride-sensitive 22Na uptake was also not present, and nonspecific 22Na uptake was unaffected in crypt cell BBMV from chronically inflamed ileum. Thus Na+/H+ exchange is unaffected in villus cells and is not present in crypt cells from chronically inflamed ileum.

Kinetic studies. To decipher the mechanism of inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum, kinetic studies were performed. Uptake studies for various concentrations were carried out at 3 s because initial uptake studies showed that HCO-3-dependent and DIDS-sensitive 36Cl uptake in BBMV was linear for at least 8 s (data not shown). Figure 5A demonstrates the kinetics of Cl- uptake in villus cell BBMV from normal and chronically inflamed ileum. This figure shows the HCO-3-dependent and DIDS-sensitive 36Cl uptake in BBMV as a function of varying concentrations of extravesicular Cl-. As the concentration of extravesicular Cl- was increased, HCO-3-dependent and DIDS-sensitive 36Cl uptake was stimulated and subsequently became saturated in all conditions (Fig. 5A, left). With the use of Enzfitter, kinetic parameters derived from this data (Table 1) demonstrated that the maximal rate of uptake (Vmax) of Cl- was not altered in the chronically inflamed ileum (Fig. 5A, right; Vmax for Cl- uptake in villus cell BBMV was 45.8 ± 1.6 and 48.4 ± 1.7 nmol · mg protein-1 · min-1 in normal and inflamed, respectively; n = 6, P = not significant). However, the affinity [1/Michaelis constant (Km)] for Cl- was significantly reduced in the chronically inflamed ileum (Km for Cl- uptake in villus cell BBMV was 21.0 ± 1.2 and 50.1 ± 1.7 mM in normal and inflamed, respectively; n = 6, P < 0.001). These data indicated that Cl-/HCO-3 exchange was inhibited in the chronically inflamed ileum secondary to a decrease in the affinity for Cl- rather than a change in the number of transporters.




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Fig. 5.   Kinetic studies of Cl- uptake in villus cell BBMV. Uptake for all concentrations was determined at 3 s. All figures are representative of 4-6 such experiments. A: normal and chronically inflamed ileum. Left, HCO-3-dependent, DIDS-sensitive 36Cl uptake as a function of varying concentrations of extravesicular Cl-. As concentration of extravesicular Cl- was increased, Cl- uptake was stimulated and subsequently became saturated in villus cell BBMV from normal and chronically inflamed ileum. Right, analysis of these data with Lineweaver-Burk plot yielded kinetic parameters. Maximal rate of uptake (Vmax) of 36Cl was not affected during chronic ileal inflammation (Vmax for 36Cl uptake in BBMV was 45.8 ± 1.6 and 48.4 ± 1.7 nmol · mg protein-1 · min-1 in normal and inflamed, respectively; n = 6, P = not significant). However, affinity [1/Michaelis constant (Km)] for Cl- was reduced significantly in chronically inflamed ileum (Km for 36Cl uptake in BBMV was 21.0 ± 1.2 and 50.1 ± 1.7 mM in normal and inflamed, respectively; n = 6, P < 0.001). B: kinetics of methylprednisolone (MP)-mediated stimulation of Cl-/HCO-3 exchange in villus cell BBMV from normal rabbit ileum. Left, HCO-3-dependent, DIDS-sensitive uptake of 36Cl as a function of varying concentrations of extravesicular Cl-. As concentration of extravesicular Cl- was increased, uptake of Cl- was stimulated and subsequently became saturated in all conditions. Right, analysis of these data with Lineweaver-Burk plot yielded kinetic parameters. Affinity for Cl- was unaffected by MP treatment (Km for 36Cl uptake in BBMV was 21.8 ± 1.0 and 25.5 ± 2.0 mM in normal and normal + MP, respectively; n = 4). However, Vmax for 36Cl uptake was significantly increased by MP treatment (Vmax for 36Cl uptake in BBMV was 43.5 ± 1.9 and 85.5 ± 2.1 nmol · mg protein-1 · min-1 in normal and normal + MP, respectively; n = 4, P < 0.05). C: kinetics of MP-mediated reversal of inhibition of Cl-/HCO-3 exchange in villus cell BBMV from chronically inflamed rabbit ileum. Left, HCO-3-dependent, DIDS-sensitive uptake of 36Cl as a function of varying concentrations of extravesicular Cl-. As concentration of extravesicular Cl- was increased, Cl- uptake was stimulated and subsequently became saturated in all conditions. Right, analysis of these data with Lineweaver-Burk plot yielded kinetic parameters. Vmax for 36Cl uptake was not affected by treatment with MP during chronic ileal inflammation (Vmax for 36Cl uptake in BBMV was 43.5 ± 1.9, 50.9 ± 1.8, and 45.6 ± 2.8 nmol · mg protein-1 · min-1 in normal, inflamed, and inflamed + MP, respectively; n = 4). However, affinity for Cl-, which was reduced in chronically inflamed ileum, was completely reversed by treatment with MP (Km for 36Cl uptake in BBMV was 21.8 ± 1.0 , 49.9 ± 4.0, and 21.3 ± 1.0 mM in normal, inflamed, and inflamed + MP, respectively; n = 4, P < 0.01).


                              
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Table 1.   Kinetic parameters for Cl-/HCO-3 exchangers

Effect of glucocorticoids on Cl-/HCO-3 exchange. First we characterized the effect of MP on Cl-/HCO-3 exchange in villus cell BBMV from the normal rabbit ileum. HCO-3-dependent and DIDS-sensitive 36Cl uptake is shown in Fig. 6A. In villus cell BBMV from MP-treated normal rabbits HCO-3-dependent and DIDS-sensitive 36Cl uptake was significantly enhanced (Fig. 6A). Thus MP treatment stimulates Cl-/HCO-3 exchange in villus cells from the normal rabbit ileum.



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Fig. 6.   Effect of MP on Cl-/HCO-3 exchange. HCO-3-dependent, DIDS-sensitive uptake of 36Cl in villus cell BBMV as a function of time is shown. Statistical comparisons are made of uptakes of different conditions for each time point. A: effect of MP treatment on Cl-/HCO-3 exchange in villus cell BBMV from normal ileum. HCO-3-dependent, DIDS-sensitive uptake of 36Cl into villus cell BBMV was significantly stimulated by treatment with MP of normal ileum. Thus MP stimulates Cl-/HCO-3 exchange in villus cells from normal rabbit ileum. B: effect of MP treatment on Cl-/HCO-3 exchange inhibition in villus cell BBMV from chronically inflamed ileum. Diminished HCO-3-dependent, DIDS-sensitive uptake of 36Cl into chronically inflamed ileal villus cell BBMV was significantly stimulated by treatment with MP. Thus MP alleviates inhibition of Cl-/HCO-3 exchange in villus cell BBM from chronically inflamed rabbit ileum.

To determine the mechanisms of MP-mediated stimulation of Cl-/HCO-3 exchange in the normal rabbit ileum, kinetic studies were performed. Again, as the concentration of extravesicular Cl- was increased, HCO-3-dependent and DIDS-sensitive 36Cl uptake was stimulated and subsequently became saturated in all conditions (Fig. 5B, left). With the use of Enzfitter, kinetic parameters derived from this data (Table 1) demonstrated that the affinity for Cl- was unaffected in the ileum treated with MP (Fig. 5B, right; Km for Cl- uptake in villus cell BBMV was 21.8 ± 1.0 and 25.5 ± 2.0 mM in normal and normal + MP, respectively; n = 4). However, the Vmax for Cl- uptake was significantly increased in the MP-treated ileum (Vmax for Cl- uptake in villus cell BBMV was 43.5 ± 1.9 and 85.5 ± 2.1 nmol · mg protein-1 · min-1 in normal and normal + MP, respectively; n = 4, P < 0.05). These data indicated that Cl-/HCO-3 exchange was stimulated by MP in villus cells from the normal ileum secondary to an increase in the number of transporters rather than a change in the affinity for Cl-.

Next, to determine whether MP treatment could alleviate the inhibition of Cl-/HCO-3 exchange seen in the chronically inflamed ileum, HCO-3-dependent and DIDS-sensitive 36Cl uptake was determined in BBMV prepared from villus cells from MP-treated and sham-treated chronically inflamed ileum. MP treatment of rabbits with chronic ileitis resulted in a significant reversal of reduced HCO-3-dependent and DIDS-sensitive 36Cl uptake in villus cell BBMV (Fig. 6B). Thus MP treatment alleviates the Cl-/HCO-3 exchange inhibition seen in the chronically inflamed ileum.

To delineate the mechanism of glucocorticoid-mediated reversal of inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum, kinetic studies were performed. Figure 5C demonstrates the kinetics of Cl- uptake in villus cell BBMV from chronically inflamed ileum and MP-treated inflamed ileum. As before, as the concentration of extravesicular Cl- was increased, the uptake of Cl- was stimulated and subsequently became saturated in all conditions (Fig. 5C, left). Kinetic parameters derived from this data (Table 1 and Fig. 5C, right) demonstrated that, unlike the normal ileum, in which MP increased the Vmax for Cl- uptake, MP did not alter the Vmax for Cl- uptake in the chronically inflamed ileum (Vmax for Cl- uptake in villus cell BBMV was 43.5 ± 1.9, 50.9 ± 1.8, and 45.6 ± 2.8 nmol · mg protein-1 · min-1 in normal, inflamed, and inflamed + MP, respectively; n = 4). However, the affinity (1/Km) for Cl- uptake, which was reduced in the chronically inflamed ileum, was almost completely reversed by treatment with MP (Km for Cl- uptake in villus cell BBMV was 21.8 ± 1.0, 49.9 ± 4.0, and 21.3 ± 1.0 mM in normal, inflamed, and inflamed + MP, respectively; n = 4, P < 0.05). These data indicated that the mechanism of reversal of inhibition of Cl-/HCO-3 exchange by MP during chronic ileitis was secondary to a restoration in the affinity for Cl- rather than an increase in transporter numbers. Thus the findings of the kinetic studies demonstrated the following mechanisms. 1) MP stimulates Cl-/HCO-3 exchange in the normal ileum by increasing transporter numbers without altering affinity for Cl-. 2) During chronic ileitis Cl-/HCO-3 exchange is inhibited secondary to a decrease in affinity for Cl- without an alteration in transporter numbers. 3) MP-mediated reversal of inhibition of Cl-/HCO-3 exchange during chronic ileitis is via restoration of affinity for Cl- and not via upregulation of transporter numbers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that coupled NaCl absorption is inhibited in the chronically inflamed ileum secondary to an inhibition of villus cell BBM Cl-/HCO-3 exchange. At the level of the transporter, the mechanism of inhibition of Cl-/HCO-3 exchange during chronic ileitis is a decrease in affinity for Cl-. Treatment with glucocorticoids stimulates Cl-/HCO-3 exchange in the normal ileum. At the level of the transporter, the MP-mediated stimulation of Cl-/HCO-3 exchange in the normal ileum is secondary to an increase in transporter numbers without an alteration in affinity for Cl-. In the chronically inflamed ileum, treatment with MP reverses the inhibition of Cl-/HCO-3 exchange. However, the MP-mediated reversal of inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum is secondary to a restoration of the affinity for Cl- without an alteration in transporter numbers.

Although malabsorption of electrolytes and fluid has been well documented in human IBD (1-3, 7, 8), the mechanism of alteration of coupled NaCl absorption during chronic ileal inflammation had not been investigated. Undoubtedly, this is a result of a lack of good animal models of chronic ileal inflammation. Two other models of chronic enterocolitis in rats (10) and guinea pigs (6) have not yet been used for electrolyte transport studies. This rabbit model of chronic ileal inflammation possesses many of the same features as human IBD (18). Thus it has been used previously to describe alterations in electrolyte and nutrient transport properties and now to determine the mechanisms of inhibition of Cl-/HCO-3 exchange and the effect of glucocorticoids on this inhibition during chronic ileitis.

Although the intention is not to suggest that this model of chronic intestinal inflammation is the same as human IBD, this rabbit model of chronic enteritis shares many similarities with IBD in 1) trigger (an offending agent in a susceptible host triggers the immune system, which remains upregulated even after the loss of the agent), 2) clinical sequelae (malabsorption and diarrhea), 3) gross morphology (the intestine is thickened and erythematous with a cobblestone appearance), and 4) histology (microscopically no parasites are seen, the villi are blunted, the crypts are hypertrophied, and there is an increase in intraepithelial and lamina propria lymphocytes, plasma cells, and mast cells). Despite these similarities it is not suggested that this model is representative of IBD, merely that in the absence of other models of chronic small intestinal inflammation from which viable villus and crypt cells can be isolated, this is an important model for understanding the effects of inflammation. Furthermore, most transport pathways have been well characterized in the normal rabbit ileum; thus comparison in pathophysiological states is possible.

In previous intact cell studies (18) this laboratory demonstrated that during chronic ileal inflammation Cl-/HCO-3 but not Na+/H+ exchange activity was reduced in villus cells. The inhibition of Cl-/HCO-3 exchange can be expected to inhibit coupled NaCl absorption by the ileum because this occurs by the dual operation of Cl-/HCO-3 and Na+/H+ exchange on the BBM of villus cells (4). A potential concern with these observations was that when intact cells were used an alteration in the intracellular buffering capacity in the chronically inflamed ileum may have been responsible for the perceived changes in transporter activity. Because buffering capacity was not altered in cells from the normal and inflamed ileum, an alteration in buffering capacity was not felt to explain the alteration in exchanger activity. Another concern is that the perceived unaltered Na+/H+ exchange activity may be a combination of stimulation of the basolateral membrane Na+/H+ exchanger and inhibition of the BBM Na+/H+ exchanger. The use of BBMV will alleviate these concerns. Indeed, studies using BBMV demonstrated that Na+/H+ exchange is unaffected whereas Cl-/HCO-3 exchange is inhibited in villus cells from the chronically inflamed ileum.

The previous studies also did not describe the mechanism of inhibition of villus cell Cl-/HCO-3 exchange during chronic ileitis. This study demonstrates that Cl-/HCO-3 exchange was inhibited in the chronically inflamed ileum secondary to diminished affinity for Cl- rather than altered transporter numbers. This suggests that Cl-/HCO-3 exchange may be altered at the level of glycosylation and/or phosphorylation during chronic enteritis.

Glucocorticoids were chosen for study because they are a mainstay of therapy of IBD. Furthermore, although the mechanism of the glucocorticoid action on electrolyte transport in IBD is not known, this class of drugs has been demonstrated to, at least partially, alleviate the impairment of electrolyte malabsorption in IBD (2, 9). This study demonstrates that glucocorticoids alleviate the inhibition of Cl-/HCO-3 exchange, which can result in the alleviation of NaCl absorption during chronic ileitis. Furthermore, glucocorticoids reverse the same mechanism that caused the inhibition of Cl-/HCO-3 exchange during chronic ileitis, specifically, altered affinity for Cl-.

It is of note that glucocorticoids had different effects on Cl-/HCO-3 exchange in the normal and the chronically inflamed ileum. Although glucocorticoids increased the relative activity of Cl-/HCO-3 exchange in both instances, in the normal ileum the stimulation was secondary to an increase in transporter numbers. However, in the chronically inflamed ileum, the diminished affinity for Cl- was restored to increase Cl-/HCO-3 exchange activity. The unique effect of glucocorticoids on Cl-/HCO-3 exchange in the normal and chronically inflamed ileum led us to hypothesize that glucocorticoids differentially regulate the Cl-/HCO-3 exchanger in the normal and chronically inflamed ileum. In the normal ileum the data are consistent with a positive effect of MP at the level of the Cl-/HCO-3 exchanger. In the chronically inflamed ileum this pressor effect of MP is lost. However, MP reverses the Cl-/HCO-3 exchanger inhibition during chronic enteritis by the same mechanism by which it was inhibited in the chronically inflamed ileum. Thus we speculate that glucocorticoids may have inhibited the release of an immune-inflammatory mediator that was responsible for the inhibition of Cl-/HCO-3 exchange during chronic enteritis.

It is known that a variety of immune-inflammatory mediators are released in the chronically inflamed intestine (3, 7). Furthermore, many of these agents are known to alter electrolyte and nutrient transport processes (3, 7). On the basis of the unique alterations in electrolyte transport processes, it was hypothesized that different immune-inflammatory mediators may regulate different transport pathways in the chronically inflamed ileum (14, 18-21). For example, as demonstrated in this study, coupled NaCl absorption is inhibited during chronic ileitis by an inhibition of Cl-/HCO-3 but not Na+/H+ exchange on the BBM of villus cells. However, Cl-/HCO-3 exchange in the BBM of crypt cells is unaffected during chronic ileitis. Furthermore, this laboratory previously demonstrated (5) that a different anion exchange process, specifically short-chain fatty acid (SCFA)/HCO-3 exchange, which is only found in the BBM of villus cells, was also inhibited during chronic ileitis. However, unlike villus cell BBM Cl-/HCO-3 exchange inhibition, which is secondary to a decrease in the affinity for Cl-, SCFA/HCO-3 exchange was inhibited secondary to a decrease in transporter numbers and not altered affinity for the SCFA (5). Thus these three anion exchange processes in villus and crypt cells are uniquely affected in the chronically inflamed intestine. The unique responses of these three anion exchanges may be secondary to their intrinsic differences and/or unique effects of different immune-inflammatory mediators released in the chronically inflamed ileum.

The anion exchanger family of transporters is not the only one to be uniquely affected during chronic ileitis. In fact, the Na+-solute cotransporter family of transporters is also uniquely affected during chronic ileitis. At the cellular level, Na+-glucose, Na+-amino acid, and Na+-bile acid cotransport were inhibited secondary to an effect at the level of the cotransporter as well as a reduction in Na+-K+-ATPase in the chronically inflamed ileum. However, at the cotransporter level each pathway was uniquely altered during chronic ileitis: Na+-glucose cotransport was inhibited by a decrease in the number of cotransporters without a change in affinity for glucose (20). In contrast, Na+-amino acid cotransport was inhibited by a reduction in affinity for the amino acid without a change in the number of cotransporters (19). Unlike these two Na+-solute cotransport processes, Na+-bile acid cotransport was inhibited by a decrease both in affinity for the bile acid and in cotransporter numbers (21). Therefore, these three types of Na+-dependent solute cotransport processes are inhibited by different mechanisms during chronic ileitis. As previously stated, because a variety of immune-inflammatory mediators are known to be released in the chronically inflamed ileum, it is hypothesized that different immune-inflammatory mediators may regulate different transport pathways in the chronically inflamed ileum.

This hypothesis might be further supported if a broad-spectrum immune modulator such as glucocorticoids were to reverse the malabsorption during chronic ileitis by reversing the same unique mechanism that resulted in the inhibition of each of the transport pathways. Indeed, we previously demonstrated (14) that Na+-glucose cotransport that was inhibited by a decrease in cotransporter numbers but not altered affinity for glucose during chronic ileitis was reversed by MP by restoring the transporter numbers without affecting the affinity for glucose (14). Furthermore, as this study demonstrates, glucocorticoids reverse the inhibition of Cl-/HCO-3 exchange by reversing the same mechanism that resulted in the inhibition of this transporter during chronic ileitis. Thus we postulate that MP may act as a broad-spectrum immune modulator by inhibiting the release of different immune-inflammatory mediators that cause specific alterations in various transport processes in the chronically inflamed ileum.

MP as an immune modulator may exert its effect at many levels. One possibility is that it normalizes the morphological alterations seen in the chronically inflamed ileum (e.g., villus blunting, crypt hyperplasia). However, we previously demonstrated (14) that this brief duration of treatment with MP is not sufficient to restore the morphological alterations seen during chronic ileitis. Another possibility is that MP inhibits the formation of immune-inflammatory mediators that are responsible for the specific transport alterations during chronic ileitis. This allows for the restoration of transporter activity. This hypothesis of glucocorticoids having an effect on circulating immune-inflammatory mediators that can inhibit electrolyte transport in the intestine is supported by studies in which glucocorticoids reversed the inhibition in electrolyte and fluid absorption in patients with IBD (2, 8). Specifically which immune-inflammatory mediators are responsible for the inhibition of various transport processes in the chronically inflamed rabbit ileum will need to be delineated in future studies using specific pathway inhibitors, agonists, and antagonists.

In conclusion, this study demonstrates that in the normal ileum glucocorticoids stimulate Cl-/HCO-3 exchange by increasing cotransporter numbers without altering affinity for Cl-. It also demonstrates that the mechanism of inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum is secondary to a decrease in affinity for Cl-. Furthermore, it demonstrates that the inhibition of Cl-/HCO-3 exchange in the chronically inflamed ileum can be reversed by treatment with glucocorticoids. The mechanism of reversal at the cotransporter level is secondary to restoration of affinity for Cl- rather than altered transporter numbers. Thus the effects of MP on Cl-/HCO-3 exchange are profoundly different in the normal and chronically inflamed intestine.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45062 to U. Sundaram.


    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 and other correspondence: U. Sundaram, Division of Digestive Diseases, Ohio State Univ. School of Medicine, N-214 Doan Hall, 410 W. Tenth Ave., Columbus OH 43210 (E-mail: sundaram-1{at}medctr.osu.edu).

Received 29 January 1999; accepted in final form 5 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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