Ileal Brush Border Membrane Vesicles Research Articles

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 μm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 μm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 μm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 μm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r12AS, fluorescence anisotropy of 12-AS).Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA.When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r12AS).In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.

Generation of a Novel Antibody Probe to the Apical Sodium-Dependent Bile Acid Transporter That Inhibits Ileal Bile Acid Absorption

Intestinal bile acid absorption is mediated by a sodium-dependent transporter located in the brush border apical membrane of ileocytes. The transmembrane topology and the role of individual amino acid residues in the bile acid transport process have been investigated by means of various experimental approaches, leading to multiple hypotheses. We raised a monoclonal antibody against a segment of the transporter comprising vicinal cysteine residues, in order to evaluate its functional role. A 14 amino acid peptide, corresponding to amino acids 104-117 of the transporter, was synthesized, and a monoclonal anti-peptide antibody was raised. In vitro uptake-inhibition studies in the presence of the monoclonal anti-peptide antibody were performed using ileal brush border membrane vesicles. Rabbit ileum was perfused in vivo with 5 mM taurocholic acid in the presence of the monoclonal antibody, and bile acid absorption inhibition was evaluated. The anti-peptide monoclonal antibody significantly reduced the in vitro uptake and in vivo absorption of taurocholic acid. The present data demonstrate the functional relevance of the 104-117 peptide segment and report the generation of a novel antibody against the apical sodium-dependent bile acid transporter (ASBT) that may be used as a therapeutic agent in hypercholesterolemia and in cholestatic pruritus.

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.

The Human Monocarboxylate Transporter MCT1: Gene Structure and Regulation

To the Editor: We read with concern the recent paper by Hadjiagapiou et al. ([4][1]) that examines the regulation of the monocarboxylate transporter isoform 1 (MCT1) gene promoter in Caco-2 cells and would like to make a number of comments with regard to 1 ) errors in their data and 2 ) misquotation

Inhibitory effects of choleretic hydroxyacetophenones on ileal bile acid transport in rats

The effects of the choleretic and cholesterol lowering compound, 2,4,6-trihydroxyacetophenone (THA) and its analog, 2,6-dihydroxyacetophenone (DHA), on ileal bile acid absorption were investigated in rats. THA inhibited taurocholate (TC) uptake into ileal brush-border membrane vesicles (BBMV), showing a maximum inhibition of 50%, whereas DHA completely inhibited TC uptake into ileal BBMV. THA exhibited competitive inhibition with a K i of 9.88 mM, while DHA showed non-competitive inhibition with a K i of 7.65 mM. Both total and ouabain-sensitive basolateral membrane (BLM) Na +–K +–ATPase activities, which are essential for maintenance of the Na +-gradient for bile acid transport, were inhibited by THA and DHA in a dose-dependent manner. The inhibition of BLM ATPase was uncompetitive with a K i of 10.1 and 5.0 mM for THA and DHA, respectively. Administration of THA or DHA (400 μmol/kg) twice a day, to hypercholesterolemic rats for 3 weeks caused similar and marked reductions in plasma cholesterol to 60% of the cholesterol-fed controls. The data suggest that the inhibitory actions of THA and DHA on two essential components of ileal bile acid recycling to liver could, in part, contribute to the cholesterol lowering effect of the hydroxyacetophenone compounds. These effects on decreasing bile acid recycling, in combination with their potent choleretic effect, accelerating biliary excretion of bile acids, are responsible for the effective cholesterol lowering capacities of these compounds.

Evaluation of absorption of heparin-DOCA conjugates on the intestinal wall using a surface plasmon resonance

We validated the application of the surface plasmon resonance (SPR) technique to reliably determine adhesion of drugs to the intestinal wall using heparin-DOCA conjugates, developed to enhance the oral absorption of poorly absorbed heparin. In this study, heparin conjugates, or deoxycholyl-heparin (H-DOCA) and bisdeoxycholyl-heparin (H-bis-DOCA), were synthesized by covalently coupling the synthesized succinimido deoxycholate (DOCA-NHS) or succinimido bis-deoxycholyl- l-lysine (DOCA-bis-NHS) to amine groups of heparin, and their physicochemical and biological properties were evaluated. To mimic the duodenal and ileal surfaces, duodenal and ileal brush border membrane (BBM) vesicles isolated from Sprauge–Dawley (SD) rats were immobilized onto a biosensor chip composed of dextran derivatives with modified lipophilic residues. The adhesion of heparin conjugates on the BBM surface was evaluated by measuring the SPR response signal. The adhesion of heparin conjugates was significantly dependent on the conjugated DOCA molecules: that is, they showed higher adhesion signal on the ileal BBM surface than that on the duodenal BBM surface. In particular, the solubilized heparin conjugates in DMSO solution presented significantly increased adhesion affinity on the ileal BBM surface. The adhesion of heparin conjugates on the intestinal surfaces was successfully assayed using the surface plasmon resonance technique with the sensor chip on which BBM vesicles were immobilized.

Transport properties of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells.

Electroneutral NaCl absorption in the intestine is mediated by parallel Na+/H+ and Cl-/HCO3- exchange. Mutations in the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea but the transport characteristics of human DRA have not been studied in a heterologous human expression system. A N-terminal enhanced green fluorescence protein (EGFP)-tagged human DRA construct was therefore expressed stably in HEK293 cells. Cl-/HCO3- exchange was assessed by measuring intracellular pH and intracellular Cl- using fluorescent dyes. Expression of DRA resulted in the appearance of EGFP fluorescence and DRA immunoreactivity consistent with a location in the plasma membrane and possibly structures below the plasma membrane. DRA mediated electroneutral Cl-/HCO3- exchange but OH- was not transported and SO4(2-)/HCO3- exchange was minimal. In the presence of 5% CO2/HCO3- the apparent affinity of DRA for Cl- in transfected HEK cells was 23-36 mM, which is lower than that reported for rabbit ileal brush border membrane vesicles and for oocytes injected with human DRA. DRA was inhibited by 4 mM DIDS (45+/-11%), by 50 microM tenidap (71+/-8%) and by 100 microM glibenclamide (59+/-22% inhibition of HCO3- transport and 79+/-3% inhibition of Cl- transport). The effects of DIDS and tenidap were not additive to those of glibenclamide.

Regulation of D-fructose transporter GLUT5 in the ileum of spontaneously hypertensive rats.

Abnormalities in carbohydrate metabolism and the insulin resistance status have been associated with hypertension. We have previously described alterations in the sodium-coupled sugar absorption in an experimental model of hypertension; in the present work, we studied the regulation of the sodium-independent, GLUT5-facilitated D-fructose intestinal transport in this pathology. Spontaneously hypertensive rats (SHR) and their normotensive, genetic control Wistar-Kyoto (WKY) rats, were used. Kinetic studies, carried out in ileal brush-border membrane vesicles (BBMVs), revealed a significant reduction (P < 0.05) in the maximal rate of transport (Vmax) for D-fructose in SHR, which, on the other hand, showed unaltered values for the Michaelis constant (Km) and the diffusion constant (Kd). Immunoblotting analysis revealed the existence of lower (P< 0.05) levels of GLUT5 in apical membranes from SHR, this reduction being similar to that of Vmax. Similarly, Northern blot studies on the abundance of GLUT5 mRNA from ileal enterocytes showed a decrease (P< 0.05) in hypertensive rats, following the same pattern mentioned above. Therefore, the impaired D-fructose intestinal absorption is another feature of SHR, and this decrease in D-fructose uptake correlates with a reduction in the abundance of the apical GLUT5 transporter, which is controlled at a transcriptional level.

Preparation and characterization of reconstructed small intestinal brush border membranes for surface plasmon resonance analysis.

To prepare the surface generated by small intestinal brush border membrane vesicles (BBMVs) for the surface plasmon resonance (SPR) analysis, which allows the real-time measurement of binding events occurring on the intestinal membrane. BBMVs were isolated from Sprague-Dawley rats, suspended in HEPES-buffered saline, and flowed over the surface of a SPR sensor chip composed of dextran derivatives modified with lipophilic residues. The surface coverage was determined from binding of bovine serum albumin to BBMV-immobilized sensor chip. The performance of BBMVs immobilized was evaluated by their interaction with otilonium bromide and bile salts. The stable BBMV surface was achieved when BBMV suspension was flowed over the sensor chip for 8 h at a rate of 2 microl/min. The flow of otilonium bromide resulted in an increased SPR signal because of its binding to calcium channel, which is known to be distributed over the gastrointestinal tact. When bile salts were flowed over ileal and duodenal BBMV surfaces, respectively, a slightly higher SPR signal was observed in the ileal BBMV surface, indicating the specific interaction of bile salts with bile acid transporters. BBMV surfaces may be useful for the estimation of binding events on the intestinal membrane by SPR analysis, especially for the drugs that are orally administrated.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B 0,+ and A in remnant small intestine after massive enterectomy

Background. Sodium-dependent brush-border nutrient transport is decreased 2 weeks after massive enterectomy. This down-regulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesize that glutamine (GLN) transport will be enhanced by earlier and longer growth factor infusion, with differential effects on the Na +-dependent GLN transport systems A, B 0,+, and B 0/ASCT2. Materials and methods. New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both EGF and GH, or neither for 2 weeks. Na +-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B 0,+, and B 0 was then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. Results. In nonresected animals, the relative contribution of the systems was similar in jejunum (A 9%, B 0,+ 20%, and B 0 71%) and ileum (A 13%, B 0,+ 27%, and B 0 60%). Na +-dependent GLN uptake was reduced by one half in resected untreated controls, primarily because of decreased B 0 activity. EGF or GH alone did not affect Na +-dependent GLN transport, but, as a combination, there was increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls ( P < 0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was a result of a 6.1–8.2-fold increase in system A as well as a 3.8–3.9-fold enhancement of system B 0,+ activity in remnant ileum and jejunum ( P < 0.01). Conclusions. Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B 0,+.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B 0,+ and A in remnant small intestine after massive enterectomy

Background Sodium-dependent brush border nutrient transport is decreased 2 weeks after massive enterectomy. This downregulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesized that glutamine (GLN) transport would be enhanced by earlier and longer growth factor infusion, with differential effects on the Na +-dependent GLN transport systems A, B 0,+, and B 0/ASCT2. Materials and methods New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both, or neither for 2 weeks. Na +-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B 0,+, and B 0 then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. Results In nonresected animals, the relative contribution of the systems was similar in jejunum (A, 9%, B 0,+, 20%; and B 0, 71%) and ileum (A, 13%; B 0,+, 27%; and B 0, 60%). Na +-dependent GLN uptake was reduced by half in resected, untreated controls, primarily because of decreased B 0 activity. EGF or GH alone did not affect Na +-dependent GLN transport, but as a combination, increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls ( P < 0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was due to a 6.1- to 8.2-fold increase in system A as well as a 3.8- to 3.9-fold enhancement of system B 0,+ activity in remnant ileum and jejunum ( P < 0.01). Conclusions Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B 0,+.

Transport of putrescine across duodenal, jejunal and ileal brush-border membrane of chicks ( Gallus domesticus)

Luminal polyamines and their absorption are essential for proliferation of the enterocytes and, therefore, nutrition, health and development of the animal. The transport systems that facilitate the uptake of putrescine were characterized in chick duodenal, jejunal and ileal brush-border membrane vesicles prepared by MgCl 2 precipitation from three-week-old chicks. An inwardly-directed Na + gradient did not stimulate putrescine uptake and, therefore, putrescine transport in chick intestine. In the duodenum, jejunum and ileum, kinetics of putrescine transport fitted a model with a single affinity component plus a non-saturable component. The affinity ( K t ) for [ 3H]putrescine transport across the brush-border membrane increased along the length of the small intestine. A model of intermediate affinity converged to the data obtained for [ 3H]putrescine transport with K t approximating 1.07 and 1.05 mM or duodenum and jejunum, respectively; and high affinity with a K t of 0.35 mM for the ileum. The polyamines cadaverine, putrescine, spermidine and spermine strongly inhibited the uptake of [ 3H]putrescine into chick brush-border membrane vesicles, more so for the jejunum and ileum than the duodenum. The kinetics of cadaverine, spermidine and spermine inhibition are suggestive of competitive inhibition of putrescine transport. These uptake data indicate that a single-affinity system facilitates the intestinal transport of putrescine in the chick; and the affinity of transporter for putrescine is higher in the ileum than in the proximal sections of the small intestine. In addition, this study shows that the ileum of chicks plays an important role in regulating cellular putrescine concentration.

Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.

Body and intestinal growth of broiler chicks on a commercial starter diet. 3. Development and characteristics of tryptophan transport

1. A study was conducted to characterise the development of amino acid transport in broiler chicks, using L-tryptophan as a model. The chicks were maintained on a broiler starter diet between hatch and 21 d of age. 2. There was a significant reduction in the rate of uptake of 0.04 mML-tryptophan with age in both the jejunum and ileum. Uptake was enhanced in the presence of 50 mM sodium chloride to different degrees depending on age and intestinal site. At both intestinal sites, uptake capacity increased with age while there was a reduction in uptake efficiency with age. 3. At a concentration of 25 mM, both sodium chloride and potasium chloride increased uptake by ileal brush-border membrane vesicles (BBMV) of 7-d-old chicks but uptake was reduced when potassium chloride was included at a concentration of 50 mM. In the presence of valinomycin, uptake by jejunal BBMV was stimulated by 25 mM sodium chloride. In the presence of both sodium chloride and potassium chloride and in the absence of valinomycin, uptake was increased by 42.6% but this was reduced to 23.4% when the ionophore was included in the buffer. 4. The Na+-independent uptake of L-tryptophan into jejunal vesicles of 21-d-old chicks was lower in the presence of D-tryptophan than in the presence of 2-aminobicyclo-{2,2,1}-heptane-2-carboxylic acid (BCH). The inclusion of BCH in the incubation medium at low concentrations significantly enhanced the uptake of 0.04 mM L-tryptophan by jejunal BBMV. 5. At similar concentration (0.04 mM) to L-tryptophan, lysine, methionine and alanine in the presence of Na + also stimulated L-tryptophan uptake. The uptake of L-tryptophan was reduced at a higher concentration, 25 mM, of these amino acids. 6. The study revealed a decline in rate of amino acid uptake and an increase in total uptake capacity with age. Tryptophan uptake was both Na + -independent and dependent, and occurred more in the ileum than in the jejunum. The uptake of L-tryptophan depended on the concentration of other amino acids and other factors in the diet and digesta.

Na(+)-dependent neutral amino acid transporter ATB(0) is a rabbit epithelial cell brush-border protein.

System B(0) activity accounts for the majority of intestinal and kidney luminal neutral amino acid absorption. An amino acid transport system, called ATB(0) (also known as ASCT2), with functional characteristics similar to those of system B(0), has been recently cloned. We generated polyclonal antibodies to human and rabbit ATB(0) COOH-terminal peptides and used Western blot analysis to detect ATB(0) protein in rabbit tissues, rabbit ileal brush-border membrane vesicles (BBMV), and HeLa cells transfected with plasmids containing ATB(0) cDNAs. Immunohistochemistry was used to localize ATB(0) in rabbit kidney and intestine. In Western blots of rabbit tissues, ATB(0) was a broad smear of 78- to 85-kDa proteins. In transfected HeLa cells, ATB(0) appeared as a smear consisting of 57- to 65-kDa proteins. The highest expression was found in the kidney. ATB(0) was enriched in rabbit ileal BBMV and in HeLa cells transfected with ATB(0) cDNAs. In the kidney and in the intestine, ATB(0) was confined to the brush-border membrane (BBM) of the proximal tubular cell and of the enterocyte, respectively. Tissue and intracellular distribution of ATB(0) protein parallels that of system B(0) activity. ATB(0) protein could be the transporter responsible for system B(0) in the BBM of epithelial cells.

Morphological and functional abnormalities in the ileum of rats with spontaneous hypertension: studies on SGLT1 protein.

Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Morphological and Functional Abnormalities in the Ileum of Rats with Spontaneous Hypertension: Studies on SGLT1 Protein

Background: Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. Methods: In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. Results: Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. Conclusion: Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters

The substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na+/bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na+/bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure–activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na+/bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na+/bile acid cotransporter. Surprisingly, the 3α-hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter.▪ The 3α-hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways.—Kramer, W., S. Stengelin, K-H. Baringhaus, A. Enhsen, H. Heuer, W. Becker, D. Corsiero, F. Girbig, R. Noll, and C. Weyland. Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. I. Transport studies with membrane vesicles and cell lines expressing the cloned transporters. J. Lipid Res. 1999. 40: 1604–1617.

Conductive pathways for chloride and oxalate in rabbit ileal brush-border membrane vesicles.

To evaluate the possibility that an apical membrane conductive pathway for oxalate is present in the rabbit distal ileum, we studied oxalate ([14C]oxalate) and chloride (36Cl) uptake into brush-border membrane vesicles enriched 15- to 18-fold in sucrase activity. Voltage-sensitive pathways for oxalate and chloride were identified by the stimulation of uptake provided by an inwardly directed potassium diffusion potential in the presence of valinomycin. Additionally, outwardly directed oxalate (or chloride) gradients stimulated [14C]oxalate (or 36Cl) uptake to a greater degree in the absence of valinomycin (when intracellular and extracellular potassium are equal) than in the presence of valinomycin. Voltage-dependent anion uptake was poorly saturable: apparent affinity constants were 141 +/- 17 and 126 +/- 8 mM for chloride and oxalate, respectively. Activation energies for the voltage-dependent uptake processes were low: 4.7 and 6.3 kcal/mol for chloride and oxalate, respectively. Sensitivity profiles of voltage-dependent chloride and oxalate uptake to anion transport inhibitors were similar. We conclude that an anion conductance is present in the apical membranes of ileal enterocytes and that this conductance is a candidate pathway for oxalate efflux from the enterocyte during transepithelial oxalate secretion.