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Chloride conductance and sodium-dependent glucose transport in rat and human enterocytes
GASTROENTEROLOGY 1997;112:1213–1220
Chloride Conductance and Sodium-Dependent Glucose Transport in Rat and Human Enterocytes ALEX H. BEESLEY,* JACQUELINE HARDCASTLE,* PETER T. HARDCASTLE,* and CHRISTOPHER J. TAYLOR‡ Departments of *Biomedical Science and ‡Paediatrics, Sheffield University, Sheffield, England
Background & Aims: In cystic fibrosis intestine, there is an increase in the rate of Na/-dependent glucose absorption. This may result from enterocyte hyperpolarization after defective Cl0 channel function, but only if Cl0 secretion and Na//glucose cotransport occur in the same membrane. This study examined the effects of Cl0 gradients on Na//glucose uptake in brush border membrane vesicles from rat and human small intestine. Methods: Vesicles were prepared by Mg2/-precipitation, and the active uptake of tritiated glucose was measured using a filtration-stop protocol. Results: An outwardly directed Cl0 gradient inhibited active glucose uptake in rat vesicles, whereas an inward Cl0 gradient stimulated uptake. These effects were sensitive to blockers of the cystic fibrosis transmembrane regulator but not to inhibitors of other Cl0 channels. Active glucose uptake into vesicles prepared from normal human intestine was also inhibited by an outward Cl0 gradient, whereas uptake into vesicles prepared from a single sample of human cystic fibrosis intestine was not. Conclusions: A Cl0 conductance resembling the cystic fibrosis transmembrane regulator is colocalized with Na//glucose cotransport in rat and human small intestine, supporting the possibility that abnormalities in glucose absorption in cystic fibrosis may be a secondary effect of defects in Cl0 channel function.
C
ystic fibrosis (CF) is characterized by defects in epithelial Cl0 secretion attributable to abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR), which normally acts as a Cl0 channel.1 However, there is also an increase in the rate of Na/ 2,3 and Na/linked nutrient absorption4 in this disease, which contributes to CF pathology by exacerbating the luminal dehydration accompanying defective secretion. One possible explanation for abnormalities in Na/ transport is that a decrease in Cl0 conductance causes a cellular hyperpolarization, increasing the electrochemical driving force for Na/ entry at the luminal membrane. This does not seem to be the case in the airways because microelectrode studies have shown potential differences across the apical membrane to be lower in CF than non-CF cells.2 Similar measurements have never been made in the intestinal / 5e1b$$0056
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tract; such a mechanism could therefore be responsible for the enhanced rate of Na/-linked glucose absorption observed in CF enterocytes. However, if defects in Cl0 secretion can influence Na//glucose absorption in this fashion, these processes must be either simultaneously present within the same cells or, at the very least, exist within neighboring cells that are electrically coupled. The debate regarding the site of secretory and absorptive processes in the intestine remains unresolved. There may be considerable overlap in the distribution of such mechanisms along the crypt/villus axis, but there is little evidence that the proteins responsible for Cl0 secretion and active glucose absorption specifically colocalize at the cellular level. CFTR is the most clearly identified Cl0 channel of intestinal epithelia (see Anderson et al.5), yet, until recently, the apical membrane expression of this protein has been reported in õ3% of villus enterocytes,6 the location of the Na//glucose cotransporter, SGLT1.7,8 The purpose of the present study was to investigate whether Na//glucose cotransport is colocalized with a Cl0 conductance in the apical membrane of small intestinal enterocytes and, thus, whether defects in Cl0 channel function could feasibly be expected to directly alter electrogenic glucose absorption in CF. This was achieved by studying the effect of Cl0 gradients on active glucose absorption in brush border membrane vesicles (BBMVs) prepared from rat and human small intestine.
Materials and Methods Experimental Tissues Male Wistar rats weighing 230–250 g were obtained from the Sheffield Field Laboratories and allowed free access to food and water. They were anesthetized with sodium pentobarbitone (Sagatal; 60 mg/kg intraperitoneally; Rhoˆne Me´rieux Abbreviations used in this paper: BBMV, brush border membrane vesicle; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; D-FSK, 1,9-dideoxyforskolin; DIDS, 4,4*-diisothiocyanato-stilbene-2,2*-disulfonic acid; DPC, N-phenylanthrancilic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; ORCC, outwardly rectifying chloride channel. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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Ltd., Harlow, Essex, England), and mucosal scrapes were taken from the first 25 cm of the jejunum. These were frozen at 0807C for later use. Histological examination showed that mucosal scraping removed the top half of the villi and left crypt tissue intact. Control human bowel was obtained by surgical resection from patients with intestinal obstruction. The single CF tissue used in this study was an elective surgical section removed as the result of recurrent obstruction. These tissues were collected freshly whenever possible and cleaned with saline before being snap-frozen with liquid nitrogen. They were then stored at 0807C until the day of use. All human tissues used in this study were morphologically normal. Approval for this research was granted by the South Sheffield Research Ethics Committee, and animal experiments were conducted under Home Office licence.
Preparation of BBMVs From Rat and Human Intestine BBMVs were prepared from rat jejunum using an Mg2/-precipitation protocol that has been described previously.9 The techniques for the preparation of BBMVs from human small intestine were similar to those for rat tissue and were based on those of Shirazi-Beechey et al.10 with the following modifications. A frozen piece of human jejunum or distal ileum, typically Ç1.5 cm2 in size, was removed from the 0807C freezer and pinned out onto a cork bed to thaw. The mucosa was then removed from the underlying muscle layers by blunt dissection. This tissue was placed into an ice-cold tube with 10–20 mL of homogenization buffer (50 mmol/L mannitol, 2 mmol/L sodium-HEPES, and 0.02% NaN3 , pH 7.4 at 47C) and homogenized for short bursts at 8000 rpm with a T25 Ultra-Turrex homogenizer (Janke & Kunkel, Staufen, Germany) using a precooled S20-25N-18G blade. Between each burst, the blades of the homogenizer tool were checked for large pieces of muscle or connective tissue, which were found to block effective homogenization. When all such pieces had been removed, the sample was homogenized at 8000 rpm for another minute. The probe and tube were rinsed with 21 10–20 mL homogenization buffer, which was then added to the sample. The sample was incubated with 10 mmol/L MgCl2 and centrifuged as previously described for the preparation of rat vesicles.9 Final rat and human BBMV pellets were resuspended in the buffers as described below.
Assay of Marker Enzymes The activities of brush border, basolateral, and mitochondrial marker enzymes were determined in the initial homogenate and in the BBMV suspension to evaluate the degree of purification of the final preparation. Alkaline phosphatase activity was measured using a commercial kit (Sigma 104; Sigma Chemical Co., Poole, England) with p-nitrophenyl phosphate as substrate. Sucrase activity was determined using a method based on that of Dahlqvist,11 and basolateral Na/,K/ – adenosine triphosphatase (ATPase) activity was assayed as described by Hardcastle et al.12 Succinate dehydrogenase activity
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was measured as described by Pennington.13 All enzyme activities were calculated per milligram of protein, determined with the Bio-Rad assay technique using bovine serum albumin as standard.
Measurement of BBMV Na//Glucose Uptake Glucose uptakes into both rat and human BBMVs were determined using a filtration-stop technique as described previously.9 Briefly, 3 mL vesicle suspension was added to 60 mL incubation buffer, and the reaction was terminated after set time points by the addition of 2.5 mL ice-cold stop solution consisting of 165 mmol/L NaCl, 20 mmol/L sodium-HEPES, 0.25 mmol/L phlorhizin, and 0.02% NaN3 , pH 7.4 at 47C. The exact composition of the incubation medium in each experiment is described later. After the addition of stop solution, the reaction mixture was rapidly filtered through a 0.45-mm (rat BBMVs) or 0.2-mm (human BBMVs) nitrocellulose membrane filter (Whatman Ltd., Maidstone, England) under vacuum. The filters and incubation tubes were then washed with an additional 5 mL of stop solution, and the radioactivity retained on the filter paper was measured using a scintillation counter. In all experiments, passive uptake of glucose was determined in the presence of 0.25 mmol/L phlorhizin and was subtracted from total uptakes to provide a measure of active Na/-dependent glucose uptake. Glucose uptakes were expressed as picomoles per milligram of protein in the BBMV sample, and each experimental value was taken as the mean of duplicate experiments made on a single sample.
Effect of Cl0 Gradients on BBMV Active Glucose Uptake BBMVs were preloaded by resuspending pellets in buffer of the appropriate composition (Table 1). Active glucose uptakes were driven with sodium gluconate, and Cl0 gradients (outwardly or inwardly directed) were established by substituting potassium gluconate or mannitol for KCl in the internal and external solutions, using concentrations that were calculated to maintain constant solution osmolality. When the channel blocking effects of HEPES were studied, the biological buffer sodium–N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid was directly substituted for sodium-HEPES in all experimental solutions. 4,4*-Diisothiocyanato-stilbene2,2*-disulfonic acid (DIDS) was dissolved in a solution of 20 mmol/L sodium-HEPES (pH 7.4) and added directly to the external incubation buffer. 5-Nitro-2-(3-phenylpropylamino)benzoate (NPPB), N-phenylanthrancilic acid (DPC), and 1,9dideoxyforskolin (D-FSK) were all initially dissolved in 100% dimethylsulfoxide and subsequently diluted 1:10 with 100% ethanol to give stocks of the required strength. When these stocks were added to the incubation buffer, the final concentrations of dimethylsulfoxide and ethanol were 0.05% and 0.5% (vol/vol), respectively. None of the above solvents had any significant effect on BBMV glucose transport. In all experiments involving the use of drugs, control data therefore represent uptakes in the presence of the appropriate vehicle.
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Table 1. Experimental Conditions for BBMV Glucose Uptakes Experiment condition
Additions to internal solution
Mi/Mo KGi/Mo KCli/Mo KCli/KGo KCli/KClo Mi/KClo
178 105 100 100 100 178
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
mannitol KG KCl KCl KCl mannitol
Additions to external solution 178 178 178 105 100 100
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
mannitol mannitol mannitol KG KCl KCl
Chloride gradient
Potassium gradient
None None 100 mmol/L in ú out 100 mmol/L in ú out None 100 mmol/L out ú in
None 100 mmol/L in ú out 100 mmol/L in ú out None None 100 mmol/L out ú in
NOTE. Experiment condition refers to the inside(i)/outside(o) presence of mannitol (M), potassium gluconate (KG) or KCl. BBMVs were loaded with an internal solution containing 200 mmol/L mannitol, 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus the additional compounds indicated. The external solution contained (final concentrations) 100 mmol/L sodium gluconate, 100 mmol/L [3H]D-glucose (4 GBq mmol01), 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus the additional compounds indicated.
Effect of Internal Anion Composition on Na//Glucose Uptake
was a generous gift from Prof. R. Greger, Freiburg University, Germany. All other reagents were of analytical grade.
The above methods were extended to study the effect of outwardly directed gradients for iodide and bromide as well as chloride. This was achieved by loading vesicles with 200 mmol/L mannitol, 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus 100 mmol/L of either KI, KBr, or KCl, or the osmotically equivalent amount of potassium gluconate (105 mmol/L). Glucose uptakes were then measured from an external solution containing (final concentrations) 100 mmol/L sodium-gluconate, 200 mmol/L mannitol, 100 mmmol/L [3H]D-glucose (4 GBq/mmol), 20 mmol/ L sodium-HEPES, 0.1 mmol/L MgSO4 , and 0.02% NaN3 (pH 7.4 at 207C) to create outwardly directed gradients for I0, Br0, and Cl0 (KIi/Mo , KBri/Mo , and KCli/Mo vs. KGi/Mo).
Effect of External Anion Composition on Na//Glucose Uptake This was assessed by studying the ability of different sodium salts to drive active glucose uptake into rat BBMVs. Vesicles were loaded with 300 mmol/L mannitol, 20 mmol/L sodium HEPES, 0.1 mmol/L MgSO4 , and 0.02% NaN3 (pH 7.4 at 207C), and glucose uptakes were measured from an external solution containing (final concentrations) 120 mmol/ L mannitol, 100 mmol/L [3H]D-glucose (4 GBq/mmol), 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus 100 mmol/L of NaSCN, NaI, NaCl, NaBr, NaF, or the osmotically equivalent amount of sodium gluconate (105 mmol/L).
Materials DIDS, D-FSK, and diagnostic kits for the determination of alkaline phosphatase activity were obtained from Sigma Chemical Co. DPC was purchased from Fluka Chemika (Buchs, Switzerland), [3H]D-glucose from Amersham International plc. (Amersham, England), and phlorhizin dihydrate from Phase Separations Ltd. (Queensferry, England). The scintillation fluid used was Emulsifier Safe from Canberra Packard Ltd. (Pangbourne, England). Bio-Rad protein assay reagents were obtained from Bio-Rad (Hemel Hempstead, England). NPPB
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Analysis of Data Results are expressed as the mean { SEM with the number of observations (n). In the case of human tissue, data derived from multiple tissue specimens taken from the same patient were averaged to deliver a single result for that subject (n), which was later used for statistical analysis. Statistical significance was assessed using the Student’s t test (paired or unpaired) or by one-way analysis of variance (ANOVA) for multiple comparisons (parametric or nonparametric as appropriate), with P values of õ0.05 considered significant.
Results Purification of Rat and Human BBMVs Rat vesicles showed enrichment of brush border enzyme markers (sucrase and alkaline phosphatase) compared with the original homogenate (Table 2). In contrast, markers for basolateral membrane (Na/,K/ATPase) and mitochondrial enzyme activity (succinate dehydrogenase) were impoverished in these vesicles. Enrichments for BBMVs prepared from non-CF human tissue compared favorably with those of rat vesicles (Table 2), showing the purification of the brush border membrane over other cellular membrane types in both vesicle preparations. Succinate dehydrogenase activity was not assayed for human BBMVs to conserve limited tissue stocks. Effect of Cl0 Gradients on Na//Glucose Uptake Into Rat BBMVs The effect of an outwardly directed Cl0 gradient on sodium gluconate–dependent glucose uptake can be seen in Figure 1. In the presence of bilateral KCl (100 mmol/L), total glucose uptake reached a peak by 60 seconds and decreased to equilibrium levels after 15 minutes. Substitution of external KCl with potassium gluconate significantly reduced 60-second total uptake by 65% WBS-Gastro
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Table 2. Enzyme Enrichments for Rat and Human BBMVs
Rat BBMVs Human BBMVs
Sucrase
Alkaline phosphatase
Succinate dehydrogenase
Na/,K/-ATPase
9.8 { 0.4 (8) 23.7 { 4.4 (4)
22.9 { 1.3 (18) 21.3 { 6.3 (5)
0.1 { 0.0 (6) Not assayed
0.7 { 0.2 (14) 1.9 { 0.6 (4)
NOTE. Values represent mean enrichment factors { SEM (n) for enzyme activities in vesicles compared with the original homogenate.
(P õ 0.001, Student’s t test) but did not affect passive uptake in the presence of 0.25 mmol/L phlorhizin (Figure 1). Equilibrium levels were not significantly altered by the presence of a Cl0 gradient (P ú 0.05, Student’s t test). This phenomenon is unlikely to be attributable to an inhibitory effect of additional gluconate because active Na//glucose uptake was also reduced when the Cl0 gradient was established without the use of potassium gluconate, i.e., in the presence of internal KCl alone (Figure 2A), indicating that another mechanism must be responsible. Because there was no gradient for K/ movement in KCli/KGo experiments and the direction of the K/ gradient in KCli/Mo experiments was the reverse of that required to inhibit Na//glucose uptake electrically (Table 1 and Figure 2A), it is possible that the electrogenic movement of Cl0 ions may have been responsible for these changes. Further evidence for this was provided by experiments in which the direction of the Cl0 gradient was reversed, a condition that stimulated BBMV active glucose uptake despite the presence of an inwardly directed K/ gradient (Figure 2B).
nificantly greater than uptake occurring in the presence of impermeant sodium gluconate (Figure 3A). The permeability for these anions (relative to Cl0) was SCN0 (2.4) ú I0 (1.8) ú Br0 (1.1) É Cl0 (1.0) É F0 (0.8) ú gluconate (0.4). To confirm that this order of halide selectivity was not direction-dependent, the effects of outwardly directed gradients of different anions on active Na//glucose uptake were compared (Figure 3B). The inhibition of active uptake by intravesicular KI compared with potassium gluconate controls (77%) was significantly greater than that caused by internal KCl (46%), confirming that I0 is more permeable in this system than
BBMV Cl0 Conductance Properties After 60-second incubations, active uptakes driven by NaSCN, NaI, NaBr, and NaCl were all sig-
Figure 1. Time course for sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs (n Å 10) in the absence (h, KCli/KClo) or presence (s, KCli/KGo) of an outwardly directed 100-mmol/L Cl0 gradient: open symbols indicate total uptake; filled symbols indicate passive uptake in the presence of phlorhizin.
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Figure 2. Effect of Cl0 gradients (100 mmol/L) on Na/-dependent glucose uptake ({SEM) into rat BBMVs at 60 seconds. Vesicles were loaded with internal/external KCl (100 mmol/L), potassium gluconate (KG, 105 mmol/L), or mannitol (M) as indicated. (A ) Effect of outwardly directed Cl0 gradient. *P õ 0.05; one-way ANOVA. (B ) Effect of inwardly directed Cl0 gradient (n Å 4). *P õ 0.05; paired Student’s t test.
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Figure 4. Time course for active sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs with (s, KCli/KGo) or without (h, KCli/ KClo) an outwardly directed 100-mmol/L Cl0 gradient in the absence (open symbols) or presence (filled symbols) of 100 mmol/L DIDS (n Å 4).
NPPB (500 mmol/L) and DPC (1 mmol/L) did reduce this inhibition (by 62% and 32%, respectively), but lower concentrations of DPC (300 mmol/L) had no effect (P ú 0.05). When the gradient for Cl0 was reversed, a stimulation of glucose uptake that was inhibited by 84% by 1 mmol/L DPC (Figure 5) was observed. Figure 3. Effect of anion gradients (100 mmol/L) on active glucose uptake ({SEM) into rat BBMVs. (A ) Uptake driven by external sodium anion salts or sodium gluconate (NaG) after 60 seconds. *P õ 0.05 vs. all other groups; one-way ANOVA, n Å 8. (B ) Sodium gluconate– driven uptake (60 seconds) with internal K/ anion salts or potassium gluconate (KG) and external mannitol (M). P õ 0.05 for groups noted on the graph; one-way ANOVA.
Glucose Uptake Into Human BBMVs After studies of the effect of Cl0 gradients on active glucose uptake in rat BBMVs, similar measurements were made in vesicles prepared from human small intestine. There was no significant difference (P ú 0.05) in active glucose uptake between jejunal and ileal vesicles prepared from control tissues under non-Cl0 gradient
Cl0. The selectivity sequence of anion permeation from the intravesicular compartment (relative to Cl0) was I0 (1.7) ¢ Br0 (1.5) ¢ Cl0 (1.0). Effects of Cl0 Channel Blockers To determine whether intrinsic BBMV Cl0 conductance represented the presence of a specific ion channel, the effect of the Cl0 channel blocker DIDS on the time course for rat BBMV glucose uptake was examined (Figure 4). No significant effect of 100 mmol/L DIDS on active uptake was observed at any time point in either the presence or absence of a Cl0 gradient (P ú 0.05 for all, paired Student’s t tests; n Å 4). To investigate further, the effects of other Cl0 channel blockers on peak (60 seconds) changes in uptake associated with outwardly or inwardly directed Cl0 gradients were studied (Figure 5). Substitution of HEPES buffer with TES did not alter the decrease in uptake associated with an outward Cl0 gradient (P ú 0.05), neither did the addition of 100 mmol/L D-FSK compared with its controls (P ú 0.05). / 5e1b$$0056
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Figure 5. Effect of 20 mmol/L TES, 100 mmol/L D-FSK, 500 mmol/ L NPPB, and 300 mmol/L or 1 mmol/L DPC2 on the change in sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs caused by outward (replacement of KCli/KClo with KCli/KGo) or inward (replacement of Mi/Mo with Mi/KClo) 100-mmol/L Cl0 gradients at 60 seconds. *P õ 0.05 vs. controls ( ); one-way ANOVA or Student’s t test as appropriate.
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Figure 6. Sodium gluconate–driven glucose uptake into human BBMVs at 60 seconds in the absence (KCli/KClo: h, non-CF; j, CF) or presence (KCli/KGo : ) of an outwardly directed 100-mmol/L Cl0 gradient. (A ) Individual data for BBMVs prepared from jejunum (J) or distal ileum (DI). (B ) Mean uptakes ({SEM) for non-CF (jejunum and ileum combined) and CF BBMVs expressed as percentage of value obtained in the absence of a Cl0 gradient. *P õ 0.05, paired Student’s t test.
(KCli/KClo) conditions (Figure 6A). However, in each case, a decrease in active uptake was observed when a 100-mmol/L outwardly directed Cl0 gradient was applied (Figure 6A), with the mean value decreasing from 339 { 76 to 55 { 71 pmol glucose/mg protein (Figure 6B; P õ 0.05, n Å 5). In contrast, active glucose uptake into BBMVs prepared from the one CF tissue used in this study was unaffected by an outwardly directed Cl0 gradient (Figure 6A and B), although the basal value was within the range observed in non-CF tissues. The patient from whom this tissue was obtained was pancreatic insufficient and had the genotype DF508/R553X.
Discussion Because Na//glucose absorption is an electrogenic process, the presence of a Cl0 gradient in vesicles that also express a Cl0 channel would be expected to affect the rate of active glucose absorption through changes in membrane potential. Such an approach has been used to show the existence of an apical membrane Cl0 conduc/ 5e1b$$0056
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tance in proximal tubules from rat kidney cortex.14 The observations that glucose uptake into intestinal BBMVs was inhibited by the imposition of an outwardly directed Cl0 gradient and that a gradient in the opposite direction stimulated active glucose uptake are consistent with the simultaneous existence of a Cl0 conductance and the Na//glucose transporter in the same vesicles. Theoretically, this apparent colocalization could result from the fusion of apical membrane fragments from different cell types; however, there is no direct evidence that this occurs in such vesicle preparations. Endosomal vesicles can be induced to fuse together for the purposes of patch clamping by a process of dehydration and subsequent rehydration, but the same technique has been unsuccessful in fusing together apical membrane vesicles,15 suggesting that BBMVs do not readily fuse with other membranes. The most clearly identified Cl0 channel in the intestinal epithelium is CFTR. The importance of this protein in intestinal secretion is shown by the fact that secretory responses to a multitude of agonists are abolished in CF intestine (for review see Anderson et al.5). In contrast, airway epithelium, which is known to possess additional Cl0 channels, retains the facility for Ca2/-mediated secretion in CF.5 In the present study, I0 consistently had a larger permeability than Cl0 (a ratio of nearly 2:1) regardless of the direction of the imposed gradient, a sequence more typically quoted for the outwardly rectifying Cl0 channel (ORCC) than CFTR.16,17 However, the permeability sequence quoted for CFTR (Cl0 ú I0) has been shown to be dependent on both experimental conditions and the direction of imposed electrochemical gradients.18 Other electrophysiological properties of CFTR have also been shown to vary in response to experimental conditions19; more evidence is therefore required to establish channel identity. DIDS, a well-recognized ORCC blocker, had no significant effect on the inhibition of Na//glucose uptake caused by an outwardly directed Cl0 gradient in the present investigation. Similarly, substitution of HEPES buffer with TES in all experimental solutions had no effect on the size of this inhibition. HEPES has been reported to inhibit ORCC channel activity,20 and the fact that substitution with TES (a similar buffer that is not believed to inhibit ORCC activity) had no effect supports the conclusion that these channels are not present in rat BBMVs. D-FSK (100 mmol/L), which has been shown to inhibit Cl0 channel activity associated with cell volume regulation in response to hypotonic shock,21 similarly had no effect in this system. DPC and NPPB are also inhibitors of the ORCC at low concentrations22,23 but are known to block both WBS-Gastro
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ORCC and CFTR channel activity at higher concentrations.17,23,24 No effect of DPC on the Cl0-dependent inhibition of Na//glucose uptake occurred at 300 mmol/L, but a significant reduction was observed at 1 mmol/L, suggesting that CFTR may be present in rat BBMV membranes. That a high concentration of NPPB (500 mmol/L) similarly diminished the size of this response and that 1 mmol/L DPC reduced the stimulation of glucose uptake observed with the presence of a Cl0 gradient in the opposite direction support this conclusion. In BBMVs prepared from non-CF human small intestine, an outwardly directed Cl0 gradient inhibited active Na//glucose uptake in an analogous manner to vesicles prepared from rat small intestine. In contrast, glucose uptake in the one section of CF tissue obtained during this period of investigation was not affected by the imposition of a Cl0 gradient. The patient involved had a severe phenotype, was pancreatic insufficient, and had the genotype DF508/R553X. The latter mutation results in the production of an unstable messenger RNA that is not successfully translated,25 whereas DF508 CFTR is incorrectly processed within intracellular compartments.26 Because both mutations result in the absence of functional CFTR in the apical membrane, the most likely explanation for the inhibitory effect of a Cl0 gradient on electrogenic glucose uptake in non-CF BBMVs is that the channel responsible is CFTR, although this requires substantiation. It should be emphasized that control data were derived from paired experiments with Cl0 gradient–dependent decreases being observed in every sample. Moreover, a substantial (43%) inhibition was shown even in control vesicles with a basal rate of active glucose uptake lower than that of CF vesicles, indicating that a low level of transporter activity probably does not account for the failure to observe such an effect in the CF sample. We have previously shown that the basal rate of Na//glucose cotransport in vesicles prepared from CF intestinal biopsy specimens is not significantly different from those prepared from non-CF tissue, which suggests that the intrinsic activity of the transporter is not altered in this disease.27 Colocalization of a Cl0 channel with the Na//glucose cotransporter is a novel finding, especially because these proteins have traditionally been believed to be localized to different regions of the crypt/villus axis. The highest levels of the CFTR Cl0 channel within the small intestine have previously been shown to exist in crypt enterocytes, with only 3% expressed in non–crypt cells.6 However, it is unlikely that the results of the present study represent the colocalization of Cl0 channel activity and Na// glucose transport in crypt cells because the preparation of rat vesicles began with mucosal scrapes that did not / 5e1b$$0056
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seem to encompass crypt tissue. Although the finding that rigorous mucosal scraping does not remove crypt tissues is at first surprising, it is a consistent observation in our laboratory; similar results have been obtained using mouse small intestine. Other investigators have reported similar findings.28 Studies of the distribution of the brush border glucose transporter all report the highest levels of expression to be in the villi.7,8 Although CFTR expression has been reported to be ‘‘switched off’’ as enterocytes migrate past the crypt/villus boundary,29 in situ hybridization in human intestine has shown a gradation of CFTR messenger RNA expression from crypt to villus, with low levels still detected in the very tips.30 A more recent study using human jejunum has concluded that the majority of villus cells may in fact express CFTR.31 The same report also described changes in intracellular Cl0 levels in non-CF villus enterocytes consistent with a normal role for these cells in intestinal secretion. In addition to these observations, it has been reported that wild-type CFTR expression is necessary for the adenosine 3*,5*cyclic monophosphate–mediated inhibition of electroneutral NaCl absorption in mouse small intestine.32 This process is thought to involve the parallel activity of Na// H/ and Cl0/HCO30 exchangers33,34; because Na//H/ antiport has been reported to be localized only within villus enterocytes,35 this again indicates that at least some CFTR must reside in these cells. The concept that CFTR may be involved with the processes of both Cl0 secretion and NaCl absorption is also consistent with the effects of cholera toxin on intestinal transport. In normal small intestine, cholera toxin causes diarrhea not only by stimulating Cl0 secretion but also by inhibiting electroneutral NaCl absorption.36 In contrast, it has been reported that there is no change in net fluid transport after cholera toxin exposure in CF intestine,37 clearly illustrating the importance of wild-type CFTR in both of these processes. In summary, a Cl0 conductance seems to be colocalized with Na//glucose cotransport in both rat and human BBMVs prepared from small intestinal villus enterocytes. These data therefore lend weight to the hypothesis that part of the increase in Na//glucose cotransport in CF may be an indirect secondary effect of defects in Cl0 secretion. Moreover, the possibility that this channel represents CFTR raises interesting questions regarding the potential cellular relationship of these two proteins.
References 1. Riordan JR. The cystic fibrosis transmembrane regulator. Annu Rev Physiol 1993;55:609–630. 2. Boucher RC, Stutts MJ, Knowles MR, Cantley L, Gatzy JT. Na/ transport in cystic fibrosis respiratory epithelia. J Clin Invest 1986;78:1245–1252.
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3. Boucher RC, Cotton CU, Gatzy JT, Knowles MR, Yankaskas JR. Evidence for reduced Cl0 and increased Na/ permeability in cystic fibrosis human primary cell cultures. J Physiol 1988;405:77– 103. 4. Baxter P, Goldhill J, Hardcastle J, Hardcastle PT, Taylor CJ. Enhanced intestinal glucose and alanine transport in cystic fibrosis. Gut 1990;31:817–820. 5. Anderson MP, Sheppard DN, Berger HA, Welsh MJ. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am J Physiol 1992;263:L1–L14. 6. Ameen NA, Ardito T, Kashgarian M, Marino CR. A unique subset of rat and human intestinal villus cells express the cystic fibrosis transmembrane conductance regulator. Gastroenterology 1995; 108:1016–1023. 7. Kinter WB, Wilson TH. Autoradiographic study of sugar and amino acid absorption by everted sacs of hamster intestine. J Cell Biol 1965;25:19–39. 8. Hwang ES, Hirayama BA, Wright EM. Distribution of the SGLT1 Na//glucose co-transporter and mRNA along the crypt-villus axis of rabbit small intestine. Biochem Biophys Res Commun 1991; 181:1208–1217. 9. Beesley A, Hardcastle J, Hardcastle PT, Taylor CJ. Influence of peppermint oil on absorptive and secretory processes in rat small intestine. Gut 1996;39:214–219. 10. Shirazi-Beechey SP, Davies AG, Tebbutt K, Dyer J, Ellis A, Taylor CJ, Fairclough P, Beechey RB. Preparation and properties of brush border membrane vesicles from human small intestine. Gastroenterology 1990;98:676–685. 11. Dahlqvist A. Assay of intestinal disaccharides. Anal Biochem 1968;22:99–107. 12. Hardcastle J, Hardcastle PT, Cookson J. Inhibitory actions of loperamide on absorptive processes in rat small intestine. Gut 1986;27:686–694. 13. Pennington RJ. Biochemistry of dystrophic muscle. Biochem J 1961;80:649–654. 14. Brown CDA, King N, Simmons NL. Co-expression of an anion conductance pathway with Na/-glucose co-transport in rat renal brush-border membrane vesicles. Pflu¨gers Arch 1993;423:406– 410. 15. Schmid A, Burckhardt G, Gogelein H. Single chloride channels in endosomal vesicle preparations from rat kidney cortex. J Membr Biol 1989;111:265–275. 16. Cliff WH, Frizzell RA. Separate Cl0 conductances activated by cAMP and Ca2/ in Cl0 secreting epithelial cells. Proc Natl Acad Sci USA 1990;87:4956–4960. 17. Schwiebert EM, Flotte T, Cutting GR, Guggino WB. Both CFTR and outwardly rectifying chloride channels contribute to cAMPstimulated whole cell chloride currents. Am J Physiol 1994;266: C1464–C1477. 18. Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW. The cystic fibrosis transmembrane conductance regulator chloride channel: iodide block and permeation. Biophys J 1992;62:1–4. 19. Overholt JL, Saulino A, Drumm ML, Harvey RD. Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current. Am J Physiol 1995;268:C636–C646. 20. Hanrahan JW, Tabcharani JA. Inhibition of an outwardly rectifying anion channel by HEPES and related buffers. J Membr Biol 1990; 116:65–77. 21. Diaz M, Valverde MA, Higgins CF, Rucareanu C, Sepulveda FV. Volume activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin. Pflu¨gers Arch 1993;422:347– 353. 22. Wangemann P, Wittner M, Englert HC, Lang HJ, Schlatter E, Greger R. Cl channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship. Pflu¨gers Arch 1986; 407:S128–S141.
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23. Fischer H, Kreusel KM, Illek B, Machen TE, Hegel U, Clauss W. The outwardly rectifying Cl0 channel is not involved in cAMPmediated Cl0 secretion in Ht29 cells: evidence for a very low conductance Cl0 channel. Pflu¨gers Arch 1992;422:159–167. 24. Diener M, Rummel W. Actions of the Cl0 channel blocker NPPB on absorptive and secretory transport processes of Na/ and Cl0 in rat descending colon. Acta Physiol Scand 1989;137:215– 222. 25. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993;73:1251– 1254. 26. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O’Riordan CR, Smith AE. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 1990;63:827–834. 27. Beesley AH, Hardcastle J, Hardcastle PT, Taylor CJ. Na//glucose co-transporter activity in cystic fibrosis. Arch Dis Child 1996;75: 170. 28. Murray D, Wild GE. Effect of fasting on Na-K-ATPase activity in rat small intestinal mucosa. Can J Physiol Pharmacol 1980;58: 643–649. 29. Trezise AEO, Romano PR, Gill DR, Hyde SC, Sepulveda FV, Buchwald M, Higgins CF. The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression. Embo J 1992;11:4291–4303. 30. Strong TV, Boehm K, Collins FS. Localisation of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridisation. J Clin Invest 1994;93: 347–354. 31. O’Loughlin EV, Hunt DM, Bostrom TE, Hunter D, Gaskin KJ, Gyory A, Cockayne DHJ. X-ray microanalysis of cell elements in normal and cystic fibrosis jejunum: evidence for chloride secretion in villi. Gastroenterology 1996;110:411–418. 32. Clarke LL, Harline MC. CFTR is required for cAMP inhibition of intestinal Na/ absorption in a cystic fibrosis mouse model. Am J Physiol 1996;270:G259–G267. 33. Nellans HN, Frizzell RA, Schultz SG. Coupled sodium-chloride influx across the brush border of rabbit ileum. Am J Physiol 1973; 225:467–475. 34. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol 1985;249:G236–G245. 35. Knickelbein RG, Aronson PS, Dobbins JW. Membrane distribution of sodium-hydrogen and chloride bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum. J Clin Invest 1988; 82:2158–2163. 36. De Jonge HR. The response of small intestinal villous and crypt epithelium to choleratoxin in rat and guinea pig: evidence against a specific role of the crypt cells in chlorine-induced secretion. Biochim Biophys Acta 1975;381:128–143. 37. Gabriel SE, Brigman KN, Koller BH, Boucher RC, Stutts MJ. Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 1994;266:107–109.
Received June 19, 1996. Accepted December 12, 1996. Address requests for reprints to: Jacqueline Hardcastle, Ph.D., Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, England. Fax: (44) 114-2765413. e-mail: [email protected]. Supported by the Special Trustees of the Former United Sheffield hospitals and the University of Sheffield. The authors thank Dr. E. Debnam and Dr. S. P. Shirazi-Beechey for their assistance in developing the techniques for the preparation of brush border membrane vesicles.
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Chloride Conductance and Sodium-Dependent Glucose Transport in Rat and Human Enterocytes ALEX H. BEESLEY,* JACQUELINE HARDCASTLE,* PETER T. HARDCASTLE,* and CHRISTOPHER J. TAYLOR‡ Departments of *Biomedical Science and ‡Paediatrics, Sheffield University, Sheffield, England
Background & Aims: In cystic fibrosis intestine, there is an increase in the rate of Na/-dependent glucose absorption. This may result from enterocyte hyperpolarization after defective Cl0 channel function, but only if Cl0 secretion and Na//glucose cotransport occur in the same membrane. This study examined the effects of Cl0 gradients on Na//glucose uptake in brush border membrane vesicles from rat and human small intestine. Methods: Vesicles were prepared by Mg2/-precipitation, and the active uptake of tritiated glucose was measured using a filtration-stop protocol. Results: An outwardly directed Cl0 gradient inhibited active glucose uptake in rat vesicles, whereas an inward Cl0 gradient stimulated uptake. These effects were sensitive to blockers of the cystic fibrosis transmembrane regulator but not to inhibitors of other Cl0 channels. Active glucose uptake into vesicles prepared from normal human intestine was also inhibited by an outward Cl0 gradient, whereas uptake into vesicles prepared from a single sample of human cystic fibrosis intestine was not. Conclusions: A Cl0 conductance resembling the cystic fibrosis transmembrane regulator is colocalized with Na//glucose cotransport in rat and human small intestine, supporting the possibility that abnormalities in glucose absorption in cystic fibrosis may be a secondary effect of defects in Cl0 channel function.
C
ystic fibrosis (CF) is characterized by defects in epithelial Cl0 secretion attributable to abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR), which normally acts as a Cl0 channel.1 However, there is also an increase in the rate of Na/ 2,3 and Na/linked nutrient absorption4 in this disease, which contributes to CF pathology by exacerbating the luminal dehydration accompanying defective secretion. One possible explanation for abnormalities in Na/ transport is that a decrease in Cl0 conductance causes a cellular hyperpolarization, increasing the electrochemical driving force for Na/ entry at the luminal membrane. This does not seem to be the case in the airways because microelectrode studies have shown potential differences across the apical membrane to be lower in CF than non-CF cells.2 Similar measurements have never been made in the intestinal / 5e1b$$0056
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tract; such a mechanism could therefore be responsible for the enhanced rate of Na/-linked glucose absorption observed in CF enterocytes. However, if defects in Cl0 secretion can influence Na//glucose absorption in this fashion, these processes must be either simultaneously present within the same cells or, at the very least, exist within neighboring cells that are electrically coupled. The debate regarding the site of secretory and absorptive processes in the intestine remains unresolved. There may be considerable overlap in the distribution of such mechanisms along the crypt/villus axis, but there is little evidence that the proteins responsible for Cl0 secretion and active glucose absorption specifically colocalize at the cellular level. CFTR is the most clearly identified Cl0 channel of intestinal epithelia (see Anderson et al.5), yet, until recently, the apical membrane expression of this protein has been reported in õ3% of villus enterocytes,6 the location of the Na//glucose cotransporter, SGLT1.7,8 The purpose of the present study was to investigate whether Na//glucose cotransport is colocalized with a Cl0 conductance in the apical membrane of small intestinal enterocytes and, thus, whether defects in Cl0 channel function could feasibly be expected to directly alter electrogenic glucose absorption in CF. This was achieved by studying the effect of Cl0 gradients on active glucose absorption in brush border membrane vesicles (BBMVs) prepared from rat and human small intestine.
Materials and Methods Experimental Tissues Male Wistar rats weighing 230–250 g were obtained from the Sheffield Field Laboratories and allowed free access to food and water. They were anesthetized with sodium pentobarbitone (Sagatal; 60 mg/kg intraperitoneally; Rhoˆne Me´rieux Abbreviations used in this paper: BBMV, brush border membrane vesicle; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; D-FSK, 1,9-dideoxyforskolin; DIDS, 4,4*-diisothiocyanato-stilbene-2,2*-disulfonic acid; DPC, N-phenylanthrancilic acid; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoate; ORCC, outwardly rectifying chloride channel. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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Ltd., Harlow, Essex, England), and mucosal scrapes were taken from the first 25 cm of the jejunum. These were frozen at 0807C for later use. Histological examination showed that mucosal scraping removed the top half of the villi and left crypt tissue intact. Control human bowel was obtained by surgical resection from patients with intestinal obstruction. The single CF tissue used in this study was an elective surgical section removed as the result of recurrent obstruction. These tissues were collected freshly whenever possible and cleaned with saline before being snap-frozen with liquid nitrogen. They were then stored at 0807C until the day of use. All human tissues used in this study were morphologically normal. Approval for this research was granted by the South Sheffield Research Ethics Committee, and animal experiments were conducted under Home Office licence.
Preparation of BBMVs From Rat and Human Intestine BBMVs were prepared from rat jejunum using an Mg2/-precipitation protocol that has been described previously.9 The techniques for the preparation of BBMVs from human small intestine were similar to those for rat tissue and were based on those of Shirazi-Beechey et al.10 with the following modifications. A frozen piece of human jejunum or distal ileum, typically Ç1.5 cm2 in size, was removed from the 0807C freezer and pinned out onto a cork bed to thaw. The mucosa was then removed from the underlying muscle layers by blunt dissection. This tissue was placed into an ice-cold tube with 10–20 mL of homogenization buffer (50 mmol/L mannitol, 2 mmol/L sodium-HEPES, and 0.02% NaN3 , pH 7.4 at 47C) and homogenized for short bursts at 8000 rpm with a T25 Ultra-Turrex homogenizer (Janke & Kunkel, Staufen, Germany) using a precooled S20-25N-18G blade. Between each burst, the blades of the homogenizer tool were checked for large pieces of muscle or connective tissue, which were found to block effective homogenization. When all such pieces had been removed, the sample was homogenized at 8000 rpm for another minute. The probe and tube were rinsed with 21 10–20 mL homogenization buffer, which was then added to the sample. The sample was incubated with 10 mmol/L MgCl2 and centrifuged as previously described for the preparation of rat vesicles.9 Final rat and human BBMV pellets were resuspended in the buffers as described below.
Assay of Marker Enzymes The activities of brush border, basolateral, and mitochondrial marker enzymes were determined in the initial homogenate and in the BBMV suspension to evaluate the degree of purification of the final preparation. Alkaline phosphatase activity was measured using a commercial kit (Sigma 104; Sigma Chemical Co., Poole, England) with p-nitrophenyl phosphate as substrate. Sucrase activity was determined using a method based on that of Dahlqvist,11 and basolateral Na/,K/ – adenosine triphosphatase (ATPase) activity was assayed as described by Hardcastle et al.12 Succinate dehydrogenase activity
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was measured as described by Pennington.13 All enzyme activities were calculated per milligram of protein, determined with the Bio-Rad assay technique using bovine serum albumin as standard.
Measurement of BBMV Na//Glucose Uptake Glucose uptakes into both rat and human BBMVs were determined using a filtration-stop technique as described previously.9 Briefly, 3 mL vesicle suspension was added to 60 mL incubation buffer, and the reaction was terminated after set time points by the addition of 2.5 mL ice-cold stop solution consisting of 165 mmol/L NaCl, 20 mmol/L sodium-HEPES, 0.25 mmol/L phlorhizin, and 0.02% NaN3 , pH 7.4 at 47C. The exact composition of the incubation medium in each experiment is described later. After the addition of stop solution, the reaction mixture was rapidly filtered through a 0.45-mm (rat BBMVs) or 0.2-mm (human BBMVs) nitrocellulose membrane filter (Whatman Ltd., Maidstone, England) under vacuum. The filters and incubation tubes were then washed with an additional 5 mL of stop solution, and the radioactivity retained on the filter paper was measured using a scintillation counter. In all experiments, passive uptake of glucose was determined in the presence of 0.25 mmol/L phlorhizin and was subtracted from total uptakes to provide a measure of active Na/-dependent glucose uptake. Glucose uptakes were expressed as picomoles per milligram of protein in the BBMV sample, and each experimental value was taken as the mean of duplicate experiments made on a single sample.
Effect of Cl0 Gradients on BBMV Active Glucose Uptake BBMVs were preloaded by resuspending pellets in buffer of the appropriate composition (Table 1). Active glucose uptakes were driven with sodium gluconate, and Cl0 gradients (outwardly or inwardly directed) were established by substituting potassium gluconate or mannitol for KCl in the internal and external solutions, using concentrations that were calculated to maintain constant solution osmolality. When the channel blocking effects of HEPES were studied, the biological buffer sodium–N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid was directly substituted for sodium-HEPES in all experimental solutions. 4,4*-Diisothiocyanato-stilbene2,2*-disulfonic acid (DIDS) was dissolved in a solution of 20 mmol/L sodium-HEPES (pH 7.4) and added directly to the external incubation buffer. 5-Nitro-2-(3-phenylpropylamino)benzoate (NPPB), N-phenylanthrancilic acid (DPC), and 1,9dideoxyforskolin (D-FSK) were all initially dissolved in 100% dimethylsulfoxide and subsequently diluted 1:10 with 100% ethanol to give stocks of the required strength. When these stocks were added to the incubation buffer, the final concentrations of dimethylsulfoxide and ethanol were 0.05% and 0.5% (vol/vol), respectively. None of the above solvents had any significant effect on BBMV glucose transport. In all experiments involving the use of drugs, control data therefore represent uptakes in the presence of the appropriate vehicle.
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Table 1. Experimental Conditions for BBMV Glucose Uptakes Experiment condition
Additions to internal solution
Mi/Mo KGi/Mo KCli/Mo KCli/KGo KCli/KClo Mi/KClo
178 105 100 100 100 178
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
mannitol KG KCl KCl KCl mannitol
Additions to external solution 178 178 178 105 100 100
mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L
mannitol mannitol mannitol KG KCl KCl
Chloride gradient
Potassium gradient
None None 100 mmol/L in ú out 100 mmol/L in ú out None 100 mmol/L out ú in
None 100 mmol/L in ú out 100 mmol/L in ú out None None 100 mmol/L out ú in
NOTE. Experiment condition refers to the inside(i)/outside(o) presence of mannitol (M), potassium gluconate (KG) or KCl. BBMVs were loaded with an internal solution containing 200 mmol/L mannitol, 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus the additional compounds indicated. The external solution contained (final concentrations) 100 mmol/L sodium gluconate, 100 mmol/L [3H]D-glucose (4 GBq mmol01), 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus the additional compounds indicated.
Effect of Internal Anion Composition on Na//Glucose Uptake
was a generous gift from Prof. R. Greger, Freiburg University, Germany. All other reagents were of analytical grade.
The above methods were extended to study the effect of outwardly directed gradients for iodide and bromide as well as chloride. This was achieved by loading vesicles with 200 mmol/L mannitol, 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus 100 mmol/L of either KI, KBr, or KCl, or the osmotically equivalent amount of potassium gluconate (105 mmol/L). Glucose uptakes were then measured from an external solution containing (final concentrations) 100 mmol/L sodium-gluconate, 200 mmol/L mannitol, 100 mmmol/L [3H]D-glucose (4 GBq/mmol), 20 mmol/ L sodium-HEPES, 0.1 mmol/L MgSO4 , and 0.02% NaN3 (pH 7.4 at 207C) to create outwardly directed gradients for I0, Br0, and Cl0 (KIi/Mo , KBri/Mo , and KCli/Mo vs. KGi/Mo).
Effect of External Anion Composition on Na//Glucose Uptake This was assessed by studying the ability of different sodium salts to drive active glucose uptake into rat BBMVs. Vesicles were loaded with 300 mmol/L mannitol, 20 mmol/L sodium HEPES, 0.1 mmol/L MgSO4 , and 0.02% NaN3 (pH 7.4 at 207C), and glucose uptakes were measured from an external solution containing (final concentrations) 120 mmol/ L mannitol, 100 mmol/L [3H]D-glucose (4 GBq/mmol), 20 mmol/L sodium-HEPES, 0.1 mmol/L MgSO4 , 0.02% NaN3 (pH 7.4 at 207C), plus 100 mmol/L of NaSCN, NaI, NaCl, NaBr, NaF, or the osmotically equivalent amount of sodium gluconate (105 mmol/L).
Materials DIDS, D-FSK, and diagnostic kits for the determination of alkaline phosphatase activity were obtained from Sigma Chemical Co. DPC was purchased from Fluka Chemika (Buchs, Switzerland), [3H]D-glucose from Amersham International plc. (Amersham, England), and phlorhizin dihydrate from Phase Separations Ltd. (Queensferry, England). The scintillation fluid used was Emulsifier Safe from Canberra Packard Ltd. (Pangbourne, England). Bio-Rad protein assay reagents were obtained from Bio-Rad (Hemel Hempstead, England). NPPB
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Analysis of Data Results are expressed as the mean { SEM with the number of observations (n). In the case of human tissue, data derived from multiple tissue specimens taken from the same patient were averaged to deliver a single result for that subject (n), which was later used for statistical analysis. Statistical significance was assessed using the Student’s t test (paired or unpaired) or by one-way analysis of variance (ANOVA) for multiple comparisons (parametric or nonparametric as appropriate), with P values of õ0.05 considered significant.
Results Purification of Rat and Human BBMVs Rat vesicles showed enrichment of brush border enzyme markers (sucrase and alkaline phosphatase) compared with the original homogenate (Table 2). In contrast, markers for basolateral membrane (Na/,K/ATPase) and mitochondrial enzyme activity (succinate dehydrogenase) were impoverished in these vesicles. Enrichments for BBMVs prepared from non-CF human tissue compared favorably with those of rat vesicles (Table 2), showing the purification of the brush border membrane over other cellular membrane types in both vesicle preparations. Succinate dehydrogenase activity was not assayed for human BBMVs to conserve limited tissue stocks. Effect of Cl0 Gradients on Na//Glucose Uptake Into Rat BBMVs The effect of an outwardly directed Cl0 gradient on sodium gluconate–dependent glucose uptake can be seen in Figure 1. In the presence of bilateral KCl (100 mmol/L), total glucose uptake reached a peak by 60 seconds and decreased to equilibrium levels after 15 minutes. Substitution of external KCl with potassium gluconate significantly reduced 60-second total uptake by 65% WBS-Gastro
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Table 2. Enzyme Enrichments for Rat and Human BBMVs
Rat BBMVs Human BBMVs
Sucrase
Alkaline phosphatase
Succinate dehydrogenase
Na/,K/-ATPase
9.8 { 0.4 (8) 23.7 { 4.4 (4)
22.9 { 1.3 (18) 21.3 { 6.3 (5)
0.1 { 0.0 (6) Not assayed
0.7 { 0.2 (14) 1.9 { 0.6 (4)
NOTE. Values represent mean enrichment factors { SEM (n) for enzyme activities in vesicles compared with the original homogenate.
(P õ 0.001, Student’s t test) but did not affect passive uptake in the presence of 0.25 mmol/L phlorhizin (Figure 1). Equilibrium levels were not significantly altered by the presence of a Cl0 gradient (P ú 0.05, Student’s t test). This phenomenon is unlikely to be attributable to an inhibitory effect of additional gluconate because active Na//glucose uptake was also reduced when the Cl0 gradient was established without the use of potassium gluconate, i.e., in the presence of internal KCl alone (Figure 2A), indicating that another mechanism must be responsible. Because there was no gradient for K/ movement in KCli/KGo experiments and the direction of the K/ gradient in KCli/Mo experiments was the reverse of that required to inhibit Na//glucose uptake electrically (Table 1 and Figure 2A), it is possible that the electrogenic movement of Cl0 ions may have been responsible for these changes. Further evidence for this was provided by experiments in which the direction of the Cl0 gradient was reversed, a condition that stimulated BBMV active glucose uptake despite the presence of an inwardly directed K/ gradient (Figure 2B).
nificantly greater than uptake occurring in the presence of impermeant sodium gluconate (Figure 3A). The permeability for these anions (relative to Cl0) was SCN0 (2.4) ú I0 (1.8) ú Br0 (1.1) É Cl0 (1.0) É F0 (0.8) ú gluconate (0.4). To confirm that this order of halide selectivity was not direction-dependent, the effects of outwardly directed gradients of different anions on active Na//glucose uptake were compared (Figure 3B). The inhibition of active uptake by intravesicular KI compared with potassium gluconate controls (77%) was significantly greater than that caused by internal KCl (46%), confirming that I0 is more permeable in this system than
BBMV Cl0 Conductance Properties After 60-second incubations, active uptakes driven by NaSCN, NaI, NaBr, and NaCl were all sig-
Figure 1. Time course for sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs (n Å 10) in the absence (h, KCli/KClo) or presence (s, KCli/KGo) of an outwardly directed 100-mmol/L Cl0 gradient: open symbols indicate total uptake; filled symbols indicate passive uptake in the presence of phlorhizin.
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Figure 2. Effect of Cl0 gradients (100 mmol/L) on Na/-dependent glucose uptake ({SEM) into rat BBMVs at 60 seconds. Vesicles were loaded with internal/external KCl (100 mmol/L), potassium gluconate (KG, 105 mmol/L), or mannitol (M) as indicated. (A ) Effect of outwardly directed Cl0 gradient. *P õ 0.05; one-way ANOVA. (B ) Effect of inwardly directed Cl0 gradient (n Å 4). *P õ 0.05; paired Student’s t test.
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Figure 4. Time course for active sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs with (s, KCli/KGo) or without (h, KCli/ KClo) an outwardly directed 100-mmol/L Cl0 gradient in the absence (open symbols) or presence (filled symbols) of 100 mmol/L DIDS (n Å 4).
NPPB (500 mmol/L) and DPC (1 mmol/L) did reduce this inhibition (by 62% and 32%, respectively), but lower concentrations of DPC (300 mmol/L) had no effect (P ú 0.05). When the gradient for Cl0 was reversed, a stimulation of glucose uptake that was inhibited by 84% by 1 mmol/L DPC (Figure 5) was observed. Figure 3. Effect of anion gradients (100 mmol/L) on active glucose uptake ({SEM) into rat BBMVs. (A ) Uptake driven by external sodium anion salts or sodium gluconate (NaG) after 60 seconds. *P õ 0.05 vs. all other groups; one-way ANOVA, n Å 8. (B ) Sodium gluconate– driven uptake (60 seconds) with internal K/ anion salts or potassium gluconate (KG) and external mannitol (M). P õ 0.05 for groups noted on the graph; one-way ANOVA.
Glucose Uptake Into Human BBMVs After studies of the effect of Cl0 gradients on active glucose uptake in rat BBMVs, similar measurements were made in vesicles prepared from human small intestine. There was no significant difference (P ú 0.05) in active glucose uptake between jejunal and ileal vesicles prepared from control tissues under non-Cl0 gradient
Cl0. The selectivity sequence of anion permeation from the intravesicular compartment (relative to Cl0) was I0 (1.7) ¢ Br0 (1.5) ¢ Cl0 (1.0). Effects of Cl0 Channel Blockers To determine whether intrinsic BBMV Cl0 conductance represented the presence of a specific ion channel, the effect of the Cl0 channel blocker DIDS on the time course for rat BBMV glucose uptake was examined (Figure 4). No significant effect of 100 mmol/L DIDS on active uptake was observed at any time point in either the presence or absence of a Cl0 gradient (P ú 0.05 for all, paired Student’s t tests; n Å 4). To investigate further, the effects of other Cl0 channel blockers on peak (60 seconds) changes in uptake associated with outwardly or inwardly directed Cl0 gradients were studied (Figure 5). Substitution of HEPES buffer with TES did not alter the decrease in uptake associated with an outward Cl0 gradient (P ú 0.05), neither did the addition of 100 mmol/L D-FSK compared with its controls (P ú 0.05). / 5e1b$$0056
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Figure 5. Effect of 20 mmol/L TES, 100 mmol/L D-FSK, 500 mmol/ L NPPB, and 300 mmol/L or 1 mmol/L DPC2 on the change in sodium gluconate–driven glucose uptake ({SEM) into rat BBMVs caused by outward (replacement of KCli/KClo with KCli/KGo) or inward (replacement of Mi/Mo with Mi/KClo) 100-mmol/L Cl0 gradients at 60 seconds. *P õ 0.05 vs. controls ( ); one-way ANOVA or Student’s t test as appropriate.
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Figure 6. Sodium gluconate–driven glucose uptake into human BBMVs at 60 seconds in the absence (KCli/KClo: h, non-CF; j, CF) or presence (KCli/KGo : ) of an outwardly directed 100-mmol/L Cl0 gradient. (A ) Individual data for BBMVs prepared from jejunum (J) or distal ileum (DI). (B ) Mean uptakes ({SEM) for non-CF (jejunum and ileum combined) and CF BBMVs expressed as percentage of value obtained in the absence of a Cl0 gradient. *P õ 0.05, paired Student’s t test.
(KCli/KClo) conditions (Figure 6A). However, in each case, a decrease in active uptake was observed when a 100-mmol/L outwardly directed Cl0 gradient was applied (Figure 6A), with the mean value decreasing from 339 { 76 to 55 { 71 pmol glucose/mg protein (Figure 6B; P õ 0.05, n Å 5). In contrast, active glucose uptake into BBMVs prepared from the one CF tissue used in this study was unaffected by an outwardly directed Cl0 gradient (Figure 6A and B), although the basal value was within the range observed in non-CF tissues. The patient from whom this tissue was obtained was pancreatic insufficient and had the genotype DF508/R553X.
Discussion Because Na//glucose absorption is an electrogenic process, the presence of a Cl0 gradient in vesicles that also express a Cl0 channel would be expected to affect the rate of active glucose absorption through changes in membrane potential. Such an approach has been used to show the existence of an apical membrane Cl0 conduc/ 5e1b$$0056
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tance in proximal tubules from rat kidney cortex.14 The observations that glucose uptake into intestinal BBMVs was inhibited by the imposition of an outwardly directed Cl0 gradient and that a gradient in the opposite direction stimulated active glucose uptake are consistent with the simultaneous existence of a Cl0 conductance and the Na//glucose transporter in the same vesicles. Theoretically, this apparent colocalization could result from the fusion of apical membrane fragments from different cell types; however, there is no direct evidence that this occurs in such vesicle preparations. Endosomal vesicles can be induced to fuse together for the purposes of patch clamping by a process of dehydration and subsequent rehydration, but the same technique has been unsuccessful in fusing together apical membrane vesicles,15 suggesting that BBMVs do not readily fuse with other membranes. The most clearly identified Cl0 channel in the intestinal epithelium is CFTR. The importance of this protein in intestinal secretion is shown by the fact that secretory responses to a multitude of agonists are abolished in CF intestine (for review see Anderson et al.5). In contrast, airway epithelium, which is known to possess additional Cl0 channels, retains the facility for Ca2/-mediated secretion in CF.5 In the present study, I0 consistently had a larger permeability than Cl0 (a ratio of nearly 2:1) regardless of the direction of the imposed gradient, a sequence more typically quoted for the outwardly rectifying Cl0 channel (ORCC) than CFTR.16,17 However, the permeability sequence quoted for CFTR (Cl0 ú I0) has been shown to be dependent on both experimental conditions and the direction of imposed electrochemical gradients.18 Other electrophysiological properties of CFTR have also been shown to vary in response to experimental conditions19; more evidence is therefore required to establish channel identity. DIDS, a well-recognized ORCC blocker, had no significant effect on the inhibition of Na//glucose uptake caused by an outwardly directed Cl0 gradient in the present investigation. Similarly, substitution of HEPES buffer with TES in all experimental solutions had no effect on the size of this inhibition. HEPES has been reported to inhibit ORCC channel activity,20 and the fact that substitution with TES (a similar buffer that is not believed to inhibit ORCC activity) had no effect supports the conclusion that these channels are not present in rat BBMVs. D-FSK (100 mmol/L), which has been shown to inhibit Cl0 channel activity associated with cell volume regulation in response to hypotonic shock,21 similarly had no effect in this system. DPC and NPPB are also inhibitors of the ORCC at low concentrations22,23 but are known to block both WBS-Gastro
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ORCC and CFTR channel activity at higher concentrations.17,23,24 No effect of DPC on the Cl0-dependent inhibition of Na//glucose uptake occurred at 300 mmol/L, but a significant reduction was observed at 1 mmol/L, suggesting that CFTR may be present in rat BBMV membranes. That a high concentration of NPPB (500 mmol/L) similarly diminished the size of this response and that 1 mmol/L DPC reduced the stimulation of glucose uptake observed with the presence of a Cl0 gradient in the opposite direction support this conclusion. In BBMVs prepared from non-CF human small intestine, an outwardly directed Cl0 gradient inhibited active Na//glucose uptake in an analogous manner to vesicles prepared from rat small intestine. In contrast, glucose uptake in the one section of CF tissue obtained during this period of investigation was not affected by the imposition of a Cl0 gradient. The patient involved had a severe phenotype, was pancreatic insufficient, and had the genotype DF508/R553X. The latter mutation results in the production of an unstable messenger RNA that is not successfully translated,25 whereas DF508 CFTR is incorrectly processed within intracellular compartments.26 Because both mutations result in the absence of functional CFTR in the apical membrane, the most likely explanation for the inhibitory effect of a Cl0 gradient on electrogenic glucose uptake in non-CF BBMVs is that the channel responsible is CFTR, although this requires substantiation. It should be emphasized that control data were derived from paired experiments with Cl0 gradient–dependent decreases being observed in every sample. Moreover, a substantial (43%) inhibition was shown even in control vesicles with a basal rate of active glucose uptake lower than that of CF vesicles, indicating that a low level of transporter activity probably does not account for the failure to observe such an effect in the CF sample. We have previously shown that the basal rate of Na//glucose cotransport in vesicles prepared from CF intestinal biopsy specimens is not significantly different from those prepared from non-CF tissue, which suggests that the intrinsic activity of the transporter is not altered in this disease.27 Colocalization of a Cl0 channel with the Na//glucose cotransporter is a novel finding, especially because these proteins have traditionally been believed to be localized to different regions of the crypt/villus axis. The highest levels of the CFTR Cl0 channel within the small intestine have previously been shown to exist in crypt enterocytes, with only 3% expressed in non–crypt cells.6 However, it is unlikely that the results of the present study represent the colocalization of Cl0 channel activity and Na// glucose transport in crypt cells because the preparation of rat vesicles began with mucosal scrapes that did not / 5e1b$$0056
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seem to encompass crypt tissue. Although the finding that rigorous mucosal scraping does not remove crypt tissues is at first surprising, it is a consistent observation in our laboratory; similar results have been obtained using mouse small intestine. Other investigators have reported similar findings.28 Studies of the distribution of the brush border glucose transporter all report the highest levels of expression to be in the villi.7,8 Although CFTR expression has been reported to be ‘‘switched off’’ as enterocytes migrate past the crypt/villus boundary,29 in situ hybridization in human intestine has shown a gradation of CFTR messenger RNA expression from crypt to villus, with low levels still detected in the very tips.30 A more recent study using human jejunum has concluded that the majority of villus cells may in fact express CFTR.31 The same report also described changes in intracellular Cl0 levels in non-CF villus enterocytes consistent with a normal role for these cells in intestinal secretion. In addition to these observations, it has been reported that wild-type CFTR expression is necessary for the adenosine 3*,5*cyclic monophosphate–mediated inhibition of electroneutral NaCl absorption in mouse small intestine.32 This process is thought to involve the parallel activity of Na// H/ and Cl0/HCO30 exchangers33,34; because Na//H/ antiport has been reported to be localized only within villus enterocytes,35 this again indicates that at least some CFTR must reside in these cells. The concept that CFTR may be involved with the processes of both Cl0 secretion and NaCl absorption is also consistent with the effects of cholera toxin on intestinal transport. In normal small intestine, cholera toxin causes diarrhea not only by stimulating Cl0 secretion but also by inhibiting electroneutral NaCl absorption.36 In contrast, it has been reported that there is no change in net fluid transport after cholera toxin exposure in CF intestine,37 clearly illustrating the importance of wild-type CFTR in both of these processes. In summary, a Cl0 conductance seems to be colocalized with Na//glucose cotransport in both rat and human BBMVs prepared from small intestinal villus enterocytes. These data therefore lend weight to the hypothesis that part of the increase in Na//glucose cotransport in CF may be an indirect secondary effect of defects in Cl0 secretion. Moreover, the possibility that this channel represents CFTR raises interesting questions regarding the potential cellular relationship of these two proteins.
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Received June 19, 1996. Accepted December 12, 1996. Address requests for reprints to: Jacqueline Hardcastle, Ph.D., Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, England. Fax: (44) 114-2765413. e-mail: [email protected]. Supported by the Special Trustees of the Former United Sheffield hospitals and the University of Sheffield. The authors thank Dr. E. Debnam and Dr. S. P. Shirazi-Beechey for their assistance in developing the techniques for the preparation of brush border membrane vesicles.
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