Small Intestinal Brush-border Membrane Vesicles 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.

Hydrolysis and transglycosylation activities of glycosidases from small intestine brush-border membrane vesicles

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC–MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties.

Characteristics of cefdinir uptake by rabbit small intestinal brush-border membrane vesicles.

Aminocephalosporins with peptide-like structures have been shown to be absorbed by the intestinal peptide carrier. We investigated the transport mechanism of cefdinir, an oral monocarboxylic acid cephalosporin, using rabbit small intestinal brush-border membrane vesicles. Transport of cefdinir showed a slow and almost linear uptake rate for concentrations up to 30 mM with and without and inward H+ gradient. No overshoot phenomenon was observed in the presence of an inward H+ gradient. The uptake rate increased only slightly with decreasing extravesicular pH, and a protonophore had little effect on the uptake. Aminocephalosporins such as cephalexin only slightly inhibited cefdinir uptake even in the presence of an inward H+ gradient, and vice-versa. Monocarboxylic acids such as acetic acid and salicylic acid had little effect on cefdinir uptake. These findings suggest that in contrast with other oral cephalosporins cefdinir uptake through the brush-border membrane is slow and involves a mechanism similar to passive diffusion.

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.

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na +-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3- O-methyl- d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na +-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na +/K +-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na +-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na +/K +-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na +-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.

Evidence for a carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles.

Recent studies from our laboratory have demonstrated the presence of a pH-dependent, amiloride-sensitive, electroneutral carrier-mediated exchange for thiamine absorption in the human small intestinal brush-border membrane vesicles. However, the mechanism of thiamine transport across the human small intestinal basolateral membrane is not understood. The present study was aimed to characterize the mechanism of thiamine transport across the basolateral membranes of the human jejunum. Basolateral membrane vesicles (BLMV) were purified from mucosal scrapings of organ donors, utilizing a Percoll continuous density gradient centrifugation technique. The results showed [3H] thiamine uptake into BLMV to be: (1) markedly stimulated in the presence of an outwardly directed H+ gradient (pH 5.5in/7.5out); (2) significantly inhibited by amiloride in a dose-dependent manner; (3) sensitive to temperature and medium osmolarity and insensitive to changes in membrane potential; (4) not influenced by the addition of 1 mM Mg(2+)-ATP, inside and outside the vesicles in the presence of Na+ and K+; (5) inhibited by structural analogs-amprolium, oxythiamin, and unlabeled thiamine (100 microM); (6) not affected by organic cations, eg, TEA, N-methyl-nicotinamide (NMN), and choline; and (7) saturable as a function of concentration (apparent Km of 0.76 +/- 0.21 microM and a V(max) of 1.38 +/- 0.35 pmol/mg protein/10 sec). These results indicate the presence of a proton gradient-dependent specialized carrier-mediated exchange mechanism for thiamine transport across the human jejunum basolateral membranes.

PH-dependent inhibitory effects of angiotensin-converting enzyme inhibitors on cefroxadine uptake by rabbit small intestinal brush-border membrane vesicles and their relationship with hydrophobicity and the ratio of zwitterionic species.

The inhibitory effects of five angiotensin-converting enzyme (ACE) inhibitors on the uptake of an aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush-border membrane vesicles were examined in the presence of an inward H+ gradient. Dixon plot analysis showed that all these ACE inhibitors inhibited the uptake of cefroxadine, which is transported by a H+/oligopeptide transporter in the membrane, in the order of enalapril<quinapril, benazepril<temocapril, trandolapril at extravesicular pH 6.0. These drugs, except for enalapril, which are relatively hydrophobic, exhibited a mix of competitive and noncompetitive inhibition. We also examined the inhibitory effects of quinapril and temocapril at extravesicular pH 5.5, at which the ratio of the zwitterionic species of the drugs increased. The inhibition occurred in a nearly competitive manner at this pH and the inhibitory effects were stronger than at pH 6.0. Regression analysis of the inhibitory effects suggested that the affinity of these ACE inhibitors for the transporter was regulated by the hydrophobicity of these ACE inhibitors and the ratio of the zwitterionic species of the drugs.

Uptake of Enalapril by Rabbit Small Intestinal Brush-Border Membrane Vesicles.

Uptake of the angiotensin-converting enzyme (ACE) inhibitor enalapril by rabbit small intestinal brush border membrane vesicles was examined. In the presence of an inward H+ gradient the uptake of this peptide mimetic drug was accelerated and an overshoot phenomenon was observed. The uptake was more stimulated by higher H+ gradient. Initial uptake rate was saturable in the presence of an inward H+ gradient, with apparent Km value of 4.2 mM. These findings suggest the involvement of the H+-coupled carrier-mediated transport in the uptake of enalapril. The uptake was inhibited by ACE inhibitor trandolapril but hardly or only slightly inhibited by aminocephalosporins.

Transport of phenethicillin into rat intestinal brush border membrane vesicles: role of the monocarboxylic acid transport system

Transport characteristics of phenethicillin (PEPC) was examined using brush border membrane vesicles (BBMV) prepared from rat small intestine. Uptake of PEPC in the presence of an inwardly directed H + gradient (pH in 7.5 and pH out 5.5) exhibited an overshoot phenomenon, indicating that PEPC is transported uphill into BBMV. Uptake of PEPC was inhibited by various monocarboxylic acids, not by oligopeptides or the β-lactams which are the substrates of the oligopeptide transporter. Moreover, when the uptake of l-lactic acid, a model substrate of the monocarboxylic acid transporter(s), was measured in the presence of PEPC, the uptake of l-lactic acid was inhibited by PEPC in a competitive manner. It was concluded that PEPC is transported into rat small intestinal BBMV mainly via the monocarboxylic acid transporter(s), not via the oligopeptide transporter.

Uptake of doxifluridine by rabbit small intestinal brush-border membrane vesicles.

Characteristics of the transport of doxifluridine (5'-DFUR) in rabbit small intestinal brush-border membrane vesicles were examined. 5'-DFUR was rapidly incorporated and a linear relation was found between the concentration of 5'-DFUR and its uptake rate in the concentration range tested (0.1-10 mM). The uptake rate increased at an acidic pH, at which 5'-DFUR is present mostly in an undissociated form. However, the uptake rate was not changed either by the extravesicular Na+ concentration or by the inside negative membrane potential. Nucleosides, uridine and adenosine only slightly inhibited the uptake, even at a fifty-fold concentration, and nucleobases, uracil and thymine had no effect. These results suggest the possibility of the major involvement of passive diffusion of the undissocated form of 5'-DFUR in the uptake of the drug through the brush-border membrane at a relatively high concentration.

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.

The immunohistochemical localization of the non-specific lipid transfer protein (sterol carrier protein-2)in rat small intestine enterocytes

A 13 kDa protein was isolated from rabbit small intestine brush-border membrane vesicles that was postulated to be involved in intestinal phosphatidylcholine (PC[jand cholesterol uptake. This protein has cholesterol and PC-transfer activity in vitro (Turnhofer, H. et al. (1991) Biochim. Biophys. Acta 1064, 275–286) and has a molecular mass and isoelectric point similar to that of the non-specific lipid transfer protein (nsL-TP, identical to sterol carrier protein-2). In addition, the first 28 N-terminal amino acid residues of the 13 kDa protein are nearly identical to nsL-TP from different species (Lipka, G. et al. (1995) J. Biol. Chem. 270, 5917–5925). In view of its possible role in intestinal lipid absorption, the localization of nsL-TP in rat small intestine was investigated using immunohistochemistry and immunoblotting. It is shown that nsLTP is predominantly localized in a subapical zone of the enterocyte but not in the brush-border membrane, thereby excluding a role in lipid uptake of this protein at the level of the plasma membrane. nsL-TP co-localized with the peroxisomal marker PMP70, underscoring earlier observations that nsL-TP is a peroxisomal protein. nsL-TP was found to be present along the entire length of the small intestine. The 58 kDa cross-reactive protein that was recently identified as a peroxisomal thiolase was shown to be present only in a small segment approximately halfway down the jejunum. The close apposition of the peroxisomes with the apical membrane and the discrete distribution of the 58 kDa protein may indicate that these organelles play a role in the intracellular processing of absorbed lipids.

Characteristics of penicillin transport in small intestinal brush-border membrane vesicles: [formula omitted], S. Hammond∗ & P. D. Fairclough, Dept. Gastroenterology, St. Bartholomew's Hospital, Charterhouse Square, London, UK, ∗Glaxo SpA, Verona, Italy

Characteristics of penicillin transport in small intestinal brush-border membrane vesicles: [formula omitted], S. Hammond∗ & P. D. Fairclough, Dept. Gastroenterology, St. Bartholomew's Hospital, Charterhouse Square, London, UK, ∗Glaxo SpA, Verona, Italy

L-Alanine transport in small intestine brush-border membrane vesicles of obese rats

Membrane vesicles from the small intestine brush border were obtained and used to determine the possible effects of genetic or nutritional obesity on l-alanine uptake. Membrane vesicles from Zucker fa fa obese rats and cafeteria diet-fed Zucker Fa/? rats showed the same characteristics as those of standard diet-fed lean animals. All preparations showed sodium-dependent transport as the main pathway for l-alanine uptake within the substrate concentration range tested. The apparent substrate affinity constant ( K rmm ) values and the pattern of inhibition of Na +-dependent l-alanine uptake by other amino acids ( l-leucine and l-glutamine), suggests that system B involved in the transport of dipolar amino acids (formerly named Neutral Brush Border System) participates in the Na +-dependent transport of l-alanine. The affinity constant ( K m) for l-alanine was essentially the same for all the groups studied (in the range of 10 mM). However, there was a higher ( P < 0.05) maximal capacity ( V max) in preparations from diet-induced obese animals (cafeteria diet) than that of genetically obese rats. These results indicate that either nutritional or genetic obesity may modify the capacity but not the affinity of transport systems for l-alanine uptake in the brush border of rat small intestine.

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.

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na(+)-dependent system A and the Na(+)-independent system L without producing any changes in either the Na(+)-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in Vmax of system A without any change in Kt. Starvation produces an increase in Vmax of system L of 1.7 times without any change in Kt. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na(+)-dependent L-alanine uptake and Na(+)-independent cycloleucine uptake by small-intestinal brush-border membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.