Rat Small Intestinal Brush-border Membrane Research Articles

In vitro and in vivo digestibility of prebiotic galactooligosacharides synthesized by β-galactosidase from Lactobacillus delbruecki subsp. bulgaricus CRL450.

The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by β-galactosidase (β-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-β-gal) from lactose, lactulose and lactitol. In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-β-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general β (1→6) linked products showed higher resistance compared to β (1→3) oligosaccharides. In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Uptake mechanism of trientine by rat intestinal brush-border membrane vesicles.

The uptake characteristics of trientine by rat intestinal brush-border membrane vesicles were studied. The uptake characteristics of trientine were similar to those of the physiological polyamines with respect to the excessive accumulation in vesicles, the pH dependency, the temperature dependency and the ineffectiveness of K+ diffusion potential (inside negative). The initial uptake of trientine was saturable with a K(m) value of 1.13 mM, which was larger than that of spermine and spermidine. Furthermore, the uptake rate of trientine was dose-dependently inhibited by spermine and spermidine. Spermine competitively inhibited the uptake of trientine with a Ki value of 18.6 microM, and it was close to the K(m) value for spermine (30.4 microM). These data suggested that the uptake of trientine was similar to that of spermine and spermidine in rat small intestinal brush-border membrane vesicles, and these polyamines seem to inhibit the absorption of trientine from the gastrointestinal tract.

Stabilization of enzyme-susceptible glucoside bonds of phloridzin through conjugation with poly(γ-glutamic acid)

We designed and prepared poly(γ-glutamic acid)s (γ-PGA) bearing phloridzin, which is an inhibitor of Na +/glucose cotransporter 1 (SGLT1), via a non-biodegradable ω-amino triethylene glycol linker. Properties of γ-PGA-phloridzin conjugates (PGA-PRZ) were examined because our previous research revealed that PGA-PRZ with a 15% phloridzin content suppressed an increase in the blood glucose level after oral administration of d-glucose in rats, even though intact phloridzin scarcely affected the glucose-induced hyperglycemic effect. In uptake experiments using rat small intestinal brush-border membrane vesicles (BBMVs), the conjugation resulted in a 10-fold increase in the inhibitor concentration giving half-maximum inhibition of SGLT1-mediated d-glucose uptake, indicating that the inhibitory effect on the uptake was considerably reduced. On the other hand, β-glucosidase-susceptible glucoside bonds of phloridzin were stabilized through conjugation with γ-PGA. d-glucose, which is essential for the inhibition of SGLT1, was not released from PGA-PRZ with a phloridzin content of greater than 15% incubated with BBMVs, despite the immediate release of d-glucose from intact phloridzin. It was strongly indicated that the improved stability resulted in the difference in pharmacological activities between the conjugate and phloridzin. We also concluded that the toxic phloretin was not released from the conjugates. These results suggest that γ-PGA-phloridzin conjugates have potential as oral anti-diabetic drugs with high safety.

Characterization of binding between the rat small intestinal brush-border membrane and dietary proteins in the sensory mechanism of luminal dietary proteins.

Dietary proteins are recognized by the gastrointestinal tract to display physiological functions, however, the sensory mechanism of the intestinal mucosa is not known. We examined binding properties between the rat small intestinal brush-border membrane (BBM) and proteins by using a surface plasmon resonance biosensor. BBM and solubilized BBM prepared from the rat jejunum bound to casein immobilized on the sensor surface, but not to bovine serum albumin. The ileal BBM showed less binding to casein than the jejunal BBM. Solubilized BBM binding to immobilized alpha-casein was slightly inhibited by aminopeptidase inhibitors, but still more inhibited by addition of casein with the inhibitors. Guanidinated casein inhibited the solubilized BBM binding to alpha-casein more strongly than casein (casein sodium and alpha-casein) inhibited. Trypsinization of solubilized BBM abolished its binding activity to alpha-casein. These results indicate that some membrane protein, but not aminopeptidases, contained in BBM interacts with dietary proteins, and that guanidinated casein has a higher affinity for BBM than intact casein. These binding intensities for proteins were closely correlated to physiological responsiveness, and are possibly involved in a sensory system for dietary protein in the intestine.

Transport mechanisms of a glycoside, p-nitrophenyl- β- d-glucopyranoside, across rat small intestinal brush-border membranes

We examined the mechanism of p-nitrophenyl- β- d-glucopyranoside ( p-NP- β- d-Glc) transport in brush-border membrane vesicles from rat small intestine. The initial uptake rate showed an overshoot phenomenon in the presence of an inwardly directed sodium-ion concentration gradient. The overshoot disappeared when the sodium-ion concentration gradient was replaced with a potassium ion concentration gradient. d-Glucose and p-NP- β- d-Glc analogues inhibited the uptake, whereas uridine, leucine and disaccharide did not. Data on the concentration dependence of p-NP- β- d-Glc uptake indicated that two carrier-mediated systems are involved. The uptake via the high-affinity site required an inwardly directed sodium-ion concentration gradient, while the uptake via the low-affinity site proceeded such a gradient. d-Glucose competitively inhibited the initial uptake of p-NP- β- d-Glc via the high-affinity site with a K i value of 301 μM. The p-NP- β- d-Glc is transported in the small intestine via both the same carrier-mediated transport system that takes up d-glucose and a distinct low-affinity carrier-mediated transport system.

Absence of vacuolar H+-ATPases from rat small intestine brush-border membranes.

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.

Contribution of hydrophobicity of nonionic detergents to membrane lipid fluidity and disopyramide uptake by rat intestinal brush-border membrane vesicles.

The contribution of hydrophobicity of different types of detergents to disopyramide uptake by rat small intestinal brush-border membrane vesicles was studied in relation to their membrane lipid fluidity and the physicochemical parameters of the detergents, i.e., hydrophile-lipophile balance (HLB). Span-, Tween-type detergents or glycerol esters at non-solubilizing concentrations (0.01-0.05% (w/v)) decreased the extent of maximum uptake of the drug in the presence of outward H(+)-gradient, but not in the absence of the gradient. The fluorescence anisotropy of the vesicles using diphenylhexatriene (DPH), as reflected by its incorporation into the membrane inner lipid layer, decreased with the addition of all detergents used. In contrast, that of the vesicles using trimethylammoniumphenyl phenylhexatriene (TMA-DPH), which reflected its incorporation into the membrane outer lipid layer, increased depending on the concentration of Tween-type detergents except for Tween 81 and Tween 85, glycerol esters (MO-500, MO-750, ML-500 and ML-750); it decreased with the addition of Span-type detergents, Tween 81 and glycerol ester (MO-310). Therefore, the membrane lipid fluidity change of the outer leaflet, rather than the inner lipophilic domain, of the membrane vesicles caused by the detergents was found to be dependent on the hydrophobicity, but not on the type of detergent. This seems to correlate with the inhibitory effects on the facilitated uptake of the drug by the membrane vesicles.

Transport characteristics of S-1090, a new oral cephem, in rat intestinal brush-border membrane vesicles.

Elucidating the transport characteristics of S-1090, a new orally active cephalosporin in rat small intestinal brush-border membranes. A rapid filtration technique. The uptake of S-1090 was stimulated by an inwardly directed H(+)-gradient, but did not show overshooting uptake. To investigate the transport system, the inhibitory and countertransport effects of various compounds on S-1090 uptake were examined. Although the dipeptides and tripeptides composed of amino acids with aliphatic side chains did not inhibit the uptake of S-1090, those having histidine, proline or tryptophan as the N-terminal amino acid showed an inhibitory effect. Among the oral cephems tested, ceftibuten showed marked inhibition, while cefaclor and cephalexin had no inhibitory effect. Countertransport effects on S-1090 uptake were observed only when the vesicles were preloaded with histidyl peptides such as His-Gly or His-Ala, while other compounds which exhibited inhibition had no countertransport effect. Based on the above results, there seems to be heterogeneity (multiplicity) in the oligopeptide transport system which may depend on the structure of the N-terminal amino acid. S-1090 may be dominantly transported via a system that recognizes peptides having histidine as the N-terminal amino acid.

Renin inhibitor: transport mechanism in rat small intestinal brush-border membrane vesicles.

The transport characteristics of the renin inhibitor ((3S,4S)-4-[N-morpholinoacetyl-(1-naphthyl)-L-alanyl-N-methyl-(4-t hiazolyl)-L- alanyl]amino-3-hydroxy-5-cyclohexyl-1-(4-pyridyl)-1-pentanone; CH3-18) in rat small intestinal brush-border membrane vesicles (BBMV) were examined by a rapid filtration technique. The uptake of CH3-18 was markedly stimulated by an inwardly directed H+ gradient (pH 7.5 inside, pH 5.5 outside) and showed an uphill transport. It was competitively inhibited by tripeptides and tetrapeptides, but not by amino acids or dipeptides. A countertransport effect on the uptake of CH3-18 was observed in the vesicle preloaded with a tripeptide. Effects of the fragments of several renin inhibitors were evaluated by their inhibitory and countertransport effects on the uptake of CH3-18. The morpholino group at the N-terminal was found to be important for the uptake of CH3-18.

The diversity of Na +-independent uptake systems for polyamines in rat intestinal brush-border membrane vesicles

Na +-independent uptake rate and binding to the membrane surface of polyamines (spermine, spermidine and putrescine) have been characterized using rat small intestinal brush-border membrane vesicles. The uptake of spermine and spermidine was saturable ( K m  30.4 μM and 148.1 μM, respectively), however, putrescine uptake was not saturable up to 8 mM. In contrast, the values of binding to the membrane surface of all polyamines were not saturable in the present studies. In Dixon plot analysis, spermine competitively inhibited the uptake rate of spermidine with a K i value of 33.8 μM, while the putrescine inhibitory effect on the spermidine uptake rate was non-competitive ( K i  3.28 mM). These uptake systems were not affected by the valinomycin-induced K +-diffusion potential (inside negative). These results suggested that there were two different Na +-independent uptake systems for spermine and spermidine, as well as for putrescine, on this membrane. However, they were not the same as the electric potential-dependent uptake system for monocationic compounds. Furthermore, this uptake system for spermine and spermidine might not be a carrier protein, because the intravesicular spermine exhibited no trans-stimulation effect on the uptake of spermidine.

Inactivation of rat small intestinal brush-border membrane alkaline phosphatase by oxygen free radicals

Background: To date, the potential effects of free radicals on the activities of intestinal brush-border membrane enzymes have received little attention. Therefore, we conducted a series of experiments to examine the effects of free radicals on various enzymatic activities of rat small intestinal brush-border membranes. Methods: An in vitro Fe2+/ascorbate oxygen-radical generating system and rat small intestinal brush-border membranes were used for this purpose. The rate of lipid peroxidation was used as an index of free radical damage. In addition, fluorescence polarization, fatty acid analyses, membrane delipidation techniques, and studies with antioxidants and metal cofactors were used. Results: Increased free radical formation was associated with the inhibition of alkaline phosphatase activity, with no change in the activities of sucrase, maltase, leucine aminopeptidase, and γ-glutamyl transpeptidase. Generation of free radicals also markedly decreased membrane lipid fluidity and altered fatty acid composition. Catalase, reduced glutathione, or α-tocopherol treatment reduced lipid peroxidation as well as inactivation of enzyme activity. The use of artificial fluidizers, Arrhenius plots, and membrane delipidation studies failed to show a relationship between the inactivation of this enzyme and changes in membrane lipid composition or fluidity. Conclusions: The inactivation of alkaline phosphatase by free radicals appears to involve its direct oxidation.

Effect of streptozotocin-induced diabetes on the handling of phosphate by the rat small intestine.

The effect of chemically-induced diabetes on the handling of phosphate (Pi) by rat jejunal enterocytes has been investigated in the presence of a Na- or a choline-gradient. Pi uptake was significantly increased in both gradients. The Pi efflux rate constants for enterocytes from diabetic rats were similar to those of control rats. The effect of diabetes on both the protein and alkaline phosphatase isoenzymes of the rat small intestinal brush-border membranes was examined using SDS-PAGE. The patterns given by membranes from rats 14 days after the induction of diabetes were no different from those of controls.

N-linked neutral sugar chains of aminopeptidase N purified from rat small intestinal brush-border membrane

Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high mannose type sugar chains (7% of the total neutral sugar chains) are also included. The unique feature of the complex-type sugar chains is the most of them contain terminal N-acetylglucosamine residues and blood group H antigenic determinants in their outer chain moieties, and bisecting N-acetylglucosamine residues in their trimannosyl cores. Both the type 1H and type 2H determinants are found, but the former is mainly expressed at the distal portions of the outer chain moieties of the oligosaccharides.

Correction of enhanced Na(+)-H+ exchange of rat small intestinal brush-border membranes in streptozotocin-induced diabetes by insulin or 1,25-dihydroxycholecalciferol.

Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.

Iron uptake from transferrin and lactoferrin by rat intestinal brush-border membrane vesicles

Interaction of 59Fe-labeled rat transferrin, human lactoferrin, and bovine lactoferrin with rat small intestinal brush-border membrane vesicles was investigated with the use of a rapid filtration technique. Specific binding of 59Fe-labeled rat transferrin and bovine lactoferrin to brush-border membrane vesicles from suckling and adult rats was identified. In contrast, no binding of human lactoferrin occurred. The presence of transferrin receptors on the brush-border membrane of suckling rats was confirmed by immunoblotting, and the molecular mass of the receptor was 96 kDa under nonreducing conditions. Scatchard plot analysis indicated 2.4 x 10(14) binding sites/mg of membrane protein with an affinity constant (Ka) of 4.9 x 10(6) M-1 for rat milk transferrin and 2.2 x 10(14) binding sites/mg of membrane protein with a Ka of 3.2 x 10(6) M-1 for bovine lactoferrin. Bovine lactoferrin competitively inhibited the binding of rat transferrin to the brush-border membrane vesicles. Deglycosylation of rat transferrin and bovine lactoferrin had no influence on the binding of these proteins. The results suggested that bovine lactoferrin bound to the receptor for rat transferrin on the brush-border membrane and that the polypeptide chain rather than the glycan moiety is responsible for the interaction of these proteins with the rat brush-border membrane.

Dietary triacylglycerol modulates sodium-dependent d-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent d-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as ( n − 3) unsaturated fatty acids and lower percentages of monounsaturated and ( n − 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0–20:5, 16:0–22:6 and 18:0–20:5 and lower levels of 18:0–18:2 and 16:0–18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na +-dependent d-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity ( V max) than butter fat membrane vesicles but a similar K m for glucose.

Transport characteristics of ceftibuten, a new oral cephem, in rat intestinal brush-border membrane vesicles: relationship to oligopeptide and amino beta-lactam transport.

Ceftibuten undergoes H+-coupled uphill transport across rat small intestinal brush-border membrane vesicles. The effects of amino acids, peptides, folate, and beta-lactams on the uptake of ceftibuten were examined. Uptake of ceftibuten was competitively inhibited by dipeptides or tripeptides. A counter-transport effect on ceftibuten uptake was observed in the vesicle preloaded with these peptides, and the transport was temporarily against a concentration gradient (overshooting). On the other hand, ceftibuten uptake was not changed by amino acids and a tetrapeptide. Therefore, ceftibuten is predominantly transported via the oligopeptide transport system in the brush-border membranes. The relationship of ceftibuten transport to folate and other oral antibiotics was also investigated. Cyclacillin, cephradine, and cefadroxil exhibited both inhibitory and countertransport effects, but folate, cefaclor, and cephalexin showed only a slight inhibitory effect. As the transport of cefaclor showed no uphill uptake in the presence of a H+ gradient and its H+ stimulated uptake was small, a H+ gradient-independent carrier-mediated system seems to participate in its transport. These findings suggest that two different carrier-mediated transport systems, H+ gradient dependent and independent, may exist for oral cephems.

Membrane fluidity and lipid composition of rat small intestinal brush-border membranes during postnatal maturation

Fluidity and lipid composition of rat small intestinal brush-border membranes (BBM) were studied during maturation in five age groups: newborns, sucklings (1–3 weeks), weaned (4–6 weeks), juveniles (8–10 weeks), and adults (12 weeks). Brush-border membrane fluidity was measured by steady-state fluourescence polarization. Fluorescent probes used were: 1,6-diphenyl-1,3,5-hexatriene, 1-(4-(trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene, and a set of n-(9- antrhoyloxy) fatty acids. Fluorescence anisotropy measured with all fluorophores was increased in adult versus newborn rats ( P < 0.004). The weight ratio of saturated to cis- unsaturated fatty acids increased from birth to the suckling age ( P < 0.0004). The cholesterol to phospholipid molar ratio increased from birth to the weaned age ( P < 0.0001). Cholesterol to protein ratio and phospholipid to protein ratio decreased after the weaned age ( P < 0.004). The results not only describe maturational changes of brush-border membranes but also give a better understanding of the correlations between biophysical and biochemical data in biological membranes.

Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes

An earlier study (Biochim. Biophys. Acta 46 (1961) 205–216) failed to detect the enzymatic synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via a transmethylation pathway in rat small-intestinal microsomal membranes. This pathway was therefore assumed to be absent from this organ. Recently, however, in our laboratory it has been demonstrated that this pathway for the synthesis of phosphatidylcholine is present in rat colonic brush-border and basolateral membranes. It was therefore of interest to examine whether phospholipid methylation activity was present in rat small-intestinal brush-border membranes. The results of the present experiments demonstrate for the first time that this pathway for the synthesis of phosphatidylcholine exists in these plasma membranes. Evidence to support the enzymatic nature of this reaction include: (1) loss of activity by heat denaturation and at 0°C, (2) significant inhibition by S- adenosyl- l- homocysteine and (3) saturation kinetics. The predominant product of this brush-border membrane phospholipid methyltransferase is phosphatidyl- N-monomethylethanolamine. This enzymatic activity has an apparent K m for S- adenosyl- l- methionine of 40 μM, a V max of 8.4 pmol/mg protein per 5 min, and a pH optimum of 8.0.