Small Intestinal Brush Border Membrane Research Articles

Vitamin D and enterocyte brush border membrane calcium transport and fluidity in the rat

Prior studies of vitamin D repletion showed a threefold increase in the maximum rate (Vmax) for calcium uptake by brush border membrane vesicles, but did not differentiate saturable and nonsaturable uptake components. We studied the calcium uptake and fluidity response of intestinal brush border vesicles to vitamin D by treatment with 1α,25-dihydroxy-24,24-difluorocholecalciferol (24,24-F-1,25-(OH) 2 D 3). Treatment responses were measured by effects on (1) saturable and nonsaturable initial uptake rates of calcium by rat proximal small intestinal brush border membrane vesicles; (2) transmucosal calcium transport by everted duodenal sac; and (3) fluorescence anisotropy. Treatment of vitamin D-depleted weanlings increased the Vmax by 50% ( P < .05) in vesicles from the proximal 12 cm of small intestine from rats injected with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), but there was no response in rats not injected with EHDP or in vesicles from the proximal 30 cm of small intestine. Vitamin D-depleted weanlings were D-deficient based on serum 25-hydroxycalciferol (25-OH-D) concentration, but to produce 1α,25-dihydroxycalciferol [1,25-(OH) 2D] depletion, EHDP injection was required. Treatment of vitamin D-replete adult rats caused a 20% ( P < .05) increase in Vmax. Treatment did not affect the calcium concentration at half-Vmax ( K T ), the rate constant for nonsaturable uptake ( K D ), or vesicle fluidity measured as fluorescence anisotropy. Contrasting with these minimal effects of treatment on brush border Vmax, treatment increased transmucosal calcium transport by everted duodenal sac almost threefold in vitamin D-depleted weanlings administered EHDP. Thus, vitamin D actions on enterocyte calcium transport (1) at the brush border increase saturable but not nonsaturable uptake, and (2) produce the major transport response distal to the brush border. Despite previously described changes in membrane lipid, brush border fluidity is unaffected by vitamin D treatment.

The potential use of tomato lectin for oral drug delivery. 1. Lectin binding to rat small intestine in vitro

Tomato lectin, an apparently non-toxic dietary glycoprotein with a molecular mass of 71 kDa, was isolated from ripe tomato fruits. Its potential as an intestinal bioadhesive was examined using an in vitro organ culture system and Western blotting. The lectin was shown to bind avidly to rings of small intestine and this binding could be inhibited with specific competing sugars. After electrophoretic separation and transfer onto nitrocellulose, several of the proteins located on the small intestinal brush border membranes were found to bind tomato lectin, particularly those in the 160-116 kDa molecular mass range. These correspond to intestinal brush border digestive enzymes, which are known to be complex glycoproteins. The specific binding of tomato lectin to the enterocyte cell surface may give it potential for increasing the gastrointestinal residence time as a component of an oral drug formulation.

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.

Absorption of Monoacylglycerols by Small Intestinal Brush Border Membrane

The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.

Lipid peroxidation and electrogenic ion transport in the jejunum of the vitamin E deficient rat.

Increased concentrations of reactive oxygen species in children with depleted antioxidant defences have been implicated in a cycle of malnutrition, malabsorption, and infection leading to protracted diarrhoea. A rat model of chronic vitamin E deficiency has been used to study the effects of antioxidant depletion on jejunal structure and function in vitro. Basal intestinal short circuit current (Isc), a measure of net electrogenic ion movement across the intestinal epithelium, was greater in chronically vitamin E deficient jejuna than controls, as was the electrogenic secretory response to aminophylline and Escherichia coli STa but not to bethanechol. The galactose stimulated current was also greater in vitamin E deficient jejuna. Indices of lipid peroxidation (concentrations of thiobarbituric acid reactive substances and malondialdehyde) were increased in the vitamin E deficient small bowel. Small intestinal brush border membranes from vitamin E deficient animals displayed changes in both static and dynamic components of membrane fluidity measured by steady state fluorescence polarography. The results of these studies support the hypothesis that oxidative stress in subjects with compromised antioxidant defences results in small intestinal hypersecretion, which could predispose to or perpetuate protracted diarrhoea.

Specificity of riboflavin molecular groups for riboflavin binding to rat small intestinal brush border membrane.

The binding of riboflavin to rat small intestinal brush border membrane at equilibrium was formerly shown to have a saturable, specific component, prevailing at the intraluminal physiological concentrations of the vitamin. In this study, the specificity of riboflavin binding to rat small intestinal brush border vesicles was further investigated using structural analogues of riboflavin. The vesicles, prepared by Ca(2+)-precipitation, were incubated at 25 degrees C, for 20 min, in the presence of [3H]-riboflavin at physiological intraluminal concentrations for rat, and each analogue, at appropriate concentrations. Three groups of analogues were used, that were derived from the riboflavin molecule by modifying one of the following positions: the ribityl side chain, position 3, and position 8 of the isoalloxazine moiety. Group specificity was assessed by determining the inhibition potency of each analogue on the saturable component of riboflavin binding to the vesicles. Inhibition constants were calculated, according to Dixon, for lumiflavin, lumichrome, and for analogues substituted at position 8. Specific riboflavin binding was inhibited competitively by most of the analogues used. Substitutions at the ribityl side chain or at position 3 of the isoalloxazine moiety reduced the inhibition power. Substitutions at position 8 enhanced the inhibition power in direct proportion to the bulk of the substituents. We conclude that the ribityl side-chain and the NH group at position 3 are essential for recognition by the specific binding sites, whereas the methyl group at position 8 is important but not essential. The analogues that bind to specific membrane sites for riboflavin share specificity requirements with many riboflavin binding proteins, and are also good substrates for the intracellular phosphorylating enzyme flavokinase. Thus, the riboflavin-binding component in the membrane is likely to be a protein with high specificity. Cellular internalization of the membrane bound vitamin is probably achieved by phosphorylation of the vitamin bound to the inner side of the membrane.

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.

Inhibition of adhesion of enterotoxigenic Escherichia coli cells expressing F17 fimbriae to small intestinal mucus and brush-border membranes of young calves

Enterotoxigenic Escherichia coli strains expressing F17 fimbriae bind to the intestinal mucosa of young calves. F17 fimbriae recognize receptors present in the mucus layer and the brush-border membranes from duodenum, jejunum and ileum. The adhesion of E. coli F17 can be inhibited by several glycoproteins. Adhesion is also inhibited by pretreatment of mucus and brush-border membranes with sodium metaperiodate. The use of glycoconjugates as potential adhesion-blockers is further discussed.

Riboflavin uptake by rat small intestinal brush border membrane vesicles: A dual mechanism involving specific membrane binding

The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane vesicles. Vesicles were incubated at 25 degrees C in the presence of [3H]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [3H]-riboflavin uptake was unaffected by Na+ or K+ gradients. The 5 sec uptake rate plotted as a function of the initial concentration of [3H]-riboflavin in the medium (0.125 to 7.5 microM) revealed the presence of a dual mechanism, with a saturable component (apparent kinetic constants: 0.12 microM for Km and 0.36 pmol.mg-1 protein x 5 sec-1 for Jmax) prevailing at low concentrations (< 2 microM), and a nonsaturable component prevailing at higher concentrations. The presence of a carrier-mediated system for riboflavin was validated by countertransport experiments. At equilibrium, uptake was almost completely accounted for by membrane binding, whereas at earlier times the transport component accounted for about 30% of total uptake. The plot of [3H]-riboflavin binding at equilibrium as a function of its concentration in the medium was quite similar to that of the 5 sec uptake rate in both intact and osmotically shocked vesicles and demonstrated the occurrence of a saturable component: binding constants were 0.07 (Kd in microM), 0.54 (Bmax in pmol.mg-1 protein), and 0.11 (Kd), 1.13 (Bmax), respectively, indicating the existence of specific riboflavin binding sites. The specificity of riboflavin binding to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a specific riboflavin entry mechanism in enterocytes.

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.

Expression cloning of rat renal Na+/SO4(2-) cotransport.

Injection of rat kidney cortex mRNA into Xenopus laevis oocytes leads to a stimulation of Na(+)-dependent SO4(2-) uptake. Based on this information, we have isolated from a corresponding library a cDNA (NaSi-1) that is most likely related to a Na+/SO4(2-) cotransport system. NaSi-1 cRNA leads in a time- and dose-dependent manner to specific stimulation of Na(+)-dependent SO4(2-) uptake in oocytes. The apparent affinity constants of the NaSi-1 cRNA-expressed transport resemble those of Na+/SO4(2-) cotransport in brush-border membrane. The NaSi-1 cDNA contains 2239 bp [including a poly(A) tail] and encodes a protein of 595 amino acids (66.05 kDa); the hydropathy profile suggests at least eight membrane-spanning regions. In vitro translation of NaSi-1 cRNA results in a protein of the expected size and suggests glycosylation. Northern blot analysis shows signals of 2.3 and 2.9 kb in kidney (more abundant in cortex than in papilla/medulla) and in mucosa of small intestine of rats. The above data indicate that we have structurally identified a membrane protein involved in renal and small-intestinal brush-border membrane Na+/SO4(2-) cotransport.

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.

Dietary regulation of rat intestinal angiotensin-converting enzyme and dipeptidyl peptidase IV.

The small intestinal brush-border membrane contains several peptidases that are involved in the hydrolysis of dietary peptides containing proline. A high-proline (gelatin) diet was administered to one of several groups of rats to study its possible regulatory effect on levels of two prolyl peptidases, namely angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP IV). Groups of rats were maintained on isocaloric diets containing either low (4%), normal (17%), or high (50%) protein (casein) or high (50%) gelatin. After 7 days, brush-border membranes and total RNA were prepared from the small intestine. ACE activity was 3- to 10-fold higher in brush-border membranes from the gelatin group compared with the low-protein group. DPP IV exhibited a three- to sixfold increase. Immunoblot analysis of brush-border membrane-associated ACE protein indicated a six- to eightfold increase in the high-gelatin group. There was also a 1.5- to 3-fold increase in steady-state levels of ACE and DPP IV mRNA. These results suggest that a diet high in proline (gelatin) is particularly effective in increasing intestinal levels of these two enzymes.

Partition and distribution coefficients of solutes and drugs in brush border membrane vesicles

Partition and distribution coefficients (log P, log D) into rat small intestinal brush border membrane (BBM) were measured for a variety of ionizable and non-ionizable drugs and solutes using a novel technique. The log P values were compared with those determined with model solvents, octanol and propylene glycol dipelargonate (PGDP). Non-ionizable solutes with log P values up to 3.0 showed that octanol was a better model for partition into the BBM than PGDP. With one exception, BBM partition coefficients of greater than 3 were not observed, even for solutes with log P values in model solvents that were greater than 5. Liposomes prepared from BBM lipids, or synthetic lipid mixtures of similar composition to BBM, demonstrated similar trends in partition coefficients to the intact BBM. Two cationic drugs, Atenolol ® and Xamoterol ® were investigated for partition into BBM lipid liposomes. An apparent enhancement of log D with respect to octanol was attributed to a “surfactant-like” orientation in the membrane and an interaction of the ionized drug with anionic phospholipid head groups. The anionic drug Proxicromil ® shows the expected decrease in log D with increasing pH, at low NaCl concentrations. Changes in electrophoretic mobility of liposomes after incorporation of Proxicromil into them were consistent with the negative charge of the ionized drug being at the membrane surface. It was concluded that Proxicromil also associates with membranes in a “surfactant-like” orientation and that increased extraction with increasing NaCl concentrations is a result of ionic strength effects. Partition of solutes into BBM vesicles is more complex than into organic solvents and probably represents an important step in overall intestinal permeation of solutes.

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.

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter.

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.

ラット小腸刷子縁膜小胞におけるL-リジン取込みの完全飢餓の影響

ラット小腸刷子縁膜小胞におけるL-リジン取込みの完全飢餓の影響

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.

Intestinal disaccharidases in the house musk shrew, Suncus murinus: occurrence of sucrase deficiency

1. 1. Dissacharidase activities of the small-intestinal brush border membrane were studied in six laboratory lines of the house musk shrew, Suncus murinus 2. 2. Sucrase activity was detected in all shrews of one line, but not in any shrew of three lines. In the other two lines it was found in some shrews, but not in the others. 3. 3. Maltase, isomaltase, trehalase and lactase acitivities were found in all shrews of all the lines examined. 4. 4. Sucrase was normally associated with isomaltase to form an enzyme complex. 5. 5. Detergent-solubilized isomaltase, whether associated with sucrase or not, was inhibited by antibodies against rabbit sucrase-isomaltase to almost the same extent as the rabbit one, suggesting that isomaltase is not affected by a mutation(s) in sucrase.

The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa

Gp170 is a transmembrane glycoprotein that is overexpressed in multidrug-resistant tumor cells and is present in the apical plasma membrane domain of small intestinal mucosal cells. The function of Gp170 was studied in the small intestine of the rat. Jejunal and ileal brush border membrane vesicles, but not basolateral membrane vesicles, manifested adenosine triphosphate (ATP)-dependent transport of daunomycin, a substrate for Gp170, and contained a ~170-kilodalton protein that reacts with anti-Gp170 monoclonal antibody. Whereas ATP supported daunomycin transport, nonhydrolyzable ATP analogues were ineffective. ATP-dependent daunomycin transport by brush border vesicles was unidirectional (inside to outside) and temperature dependent and was blocked by Gp170 inhibitors but not by taurocholate or bromsulphalein glutathione. Studies using everted small intestine revealed transport of rhodamine 123, a Gp170 substrate, from the serosal surface through the mucosa and inhibition by Gp170 inhibitors. These results suggest that Gp170 in rat small intestinal brush border membrane vesicles is an ATP-dependent efflux pump responsible for the transport of Gp170 substrates into the small intestinal lumen. Gp170 may protect against exogenously derived potentially damaging hydrophobic cations and contribute to the rarity of small intestinal cancer in humans and many animals.