Rat Intestinal Brush-border Membrane Research Articles

Characterization of Monocarboxylate Transporter 6: Expression in Human Intestine and Transport of the Antidiabetic Drug Nateglinide

Monocarboxylate transporter (MCT) 6, encoded by SLC16A5, is a member of the monocarboxylate transporter family. Nateglinide, an oral hypoglycemic agent, quickly reaches the maximal serum concentration after its premeal administration. Although the functional existence of uptake systems for nateglinide in the intestine has been demonstrated, these transport systems have not yet been identified at the molecular level. The aim of this study was to demonstrate the localization of MCT6 in the human small intestine and characterize the transport properties of nateglinide via MCT6. Immunohistochemical analysis of the human small intestine revealed that anti-MCT6 antiserum stained the luminal side of the epithelial cells. When expressed in Xenopus laevis oocytes, MCT6-mediated uptake of [(14)C]nateglinide was sensitive to extracellular pH and membrane potential. Furthermore, the K(t) value of nateglinide (45.9 μM) for MCT6 was lower than those previously reported in Caco-2 cells and rat intestinal brush-border membrane vesicles. In addition, probenecid, fluorescein, valproic acid, and salicylic acid, which are inhibitors of nateglinide uptake in Caco-2 cells and rat intestine, did not inhibit the uptake of nateglinide via MCT6. These results suggest that MCT6 may play a role in the intestinal absorption of nateglinide, although other transporters are also likely involved.

A highly sensitive HPLC method for the assay of propantheline used to measure its uptake by rat intestinal brush border membrane vesicles.

A simple, sensitive, high-performance liquid chromatographic method for propantheline has been developed. Propantheline was quantitatively hydrolysed into xanthene-9-carboxylic acid in neutral or alkaline medium and the hydrolysate assayed by reversed phase high-performance liquid chromatography. This method measured to 2 pmol per injection and was used to investigate the uptake of the drug by rat intestinal brush border membrane vesicles. Propantheline was highly bound to the membrane and this binding was inhibited to varying extents by several quaternary ammonium compounds. Mepenzolate and methylbenactyzium inhibited it significantly, and neostigmine, butylscopolamine and N-methylnicotinamide inhibited it moderately. Choline, acetylcholine and thiamine had no effect.

Uptake of amino beta-lactam antibiotics into rat intestinal brush border membrane vesicles.

Uptake of amino beta-lactam antibiotics into rat intestinal brush border membrane vesicles has been examined for characterization of the transport of the antibiotics through the gut wall of the rat. The uptake of cephradine, cephalexin and ampicillin into membrane vesicles was similar, and there were no significant changes in the uptake in the presence of a NaCl or a KCl gradient. These results suggested that the carrier-mediated transport systems relating to amino acids and glucose were not concerned with the intestinal absorption mechanism of amino beta-lactam antibiotics.

Comparison of Transport Characteristics of Amino β-Lactam Antibiotics and Dipeptides Across Rat Intestinal Brush Border Membrane

The transport characteristics of amino beta-lactam antibiotics, ampicillin and cephradine, have been examined and compared with that of glycylglycine using brush border membrane vesicles isolated from rat small intestine. The initial rate of glycylglycine uptake was markedly stimulated in the presence of an inward H+ gradient compared with the uptake rates in the absence of an H+ gradient. With the same H+ gradient the stimulation of cephradine uptake was lower and ampicillin uptake was not altered. Cephradine uptake, however, was greater than that of glycylglycine in both vesicular conditions ((pH)i greater than (pH)o and (pH)i = (pH)o). Inhibitory effects of dipeptides, ampicillin and cephradine on the initial uptake of glycylglycine were also examined. Glycylglycine uptake was significantly decreased in the presence of L-phenylalanylglycine or carnosine. Ampicillin and cephradine did not alter the uptake of glycylglycine. These results suggest that the contribution of the inward H+ gradient to the permeation of ampicillin, cephradine and glycylglycine across the rat small intestinal brush border membranes is different for each of the substances examined.

Contribution of Passive Transport Mechanisms to the Intestinal Absorption of β-Lactam Antibiotics

The transport characteristics of aminopenicillins (ampicillin and amoxicillin), aminocephalosporins (cephalexin, cephradine and cefadroxil) and cefazolin have been compared with those of an actively transported substance (D-glucose) and a passively transported substance (L-glucose). Although the initial uptake of the aminocephalosporins was stimulated in the presence of an inward H+ gradient, there was no overshoot in the uptake of any of the drugs tested, even in the presence of an H+ gradient. Also, the time course and the degree of uptake of these drugs were similar to those of L-glucose, especially in the absence of an H+ gradient. These results suggest that the beta-lactam antibiotics tested, like L-glucose, pass through the rat intestinal brush border membrane mainly by passive diffusion. However, the differences in absorption between these drugs, like the differences in their disappearance from a proximal loop of rat intestine, cannot be explained by a simple permeation process alone.

The stimulative effect of diffusion potential on enoxacin uptake across rat intestinal brush-border membranes.

Evidence of a membrane potential dependence for enoxacin uptake by rat intestinal brush-border membrane vesicles has been found. The transient overshooting uptake of enoxacin disappeared in the voltage-clamped brush-border membrane vesicles in the presence of an outward H(+)-gradient. Momentary dissipation of the H(+)-gradient itself by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) did not affect the uptake of enoxacin. In contrast, enoxacin uptake was depressed by an interior positive K(+)-diffusion potential induced by valinomycin. Furthermore, not only the outward H(+)-gradient but also an inward Cl(-)-gradient caused a stimulating effect on enoxacin uptake, and the stimulation by the Cl(-)-gradient was dissipated by using voltage-clamped membrane vesicles. These results indicate that enoxacin transportation across the brush-border membrane is dependent on the ionic diffusion potential. On the other hand, neither Gly-Gly nor guanidine had any effect on enoxacin uptake by the membrane vesicles in the presence of an inward (for Gly-Gly) or outward (for guanidine) H(+)-gradient as a driving force for each transport system. Therefore, it seems that enoxacin transport through the intestinal epithelia does not participate in the carrier-mediated transport systems for Gly-Gly and guanidine.

H+ coupled transport of orally active cephalosporins lacking an α-amino group across brush-border membrane vesicles from rat small intestine

The effect of an inwardly directed H+ gradient' on the transport characteristics of ceftibuten, cefixime and analogues of ceftibuten in rat intestinal brush-border membrane vesicles have been investigated. In the presence of a transmembrane H+ gradient, ceftibuten and its analogues exhibited a peak to equilibrium overshoot and an accumulation in the vesicles against the concentration gradient. However, the uptake of cefixime and S-1006 [(6R, 7R)-(7-[(Z)-2-(2-aminothiazol-4-yl)-2-pentenoylamino]-8-oxo- 3- carbamoyloxy-methyl-5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxyl ic acid), which lacks a carboxyl group at position 4 of carboxyethylidene structure, exhibited no overshoot, although the equilibrium uptake was increased by a H+ gradient. The equilibrium uptake was dependent on the pH of the final incubation medium and the H+ gradient. These data suggested that the orally active cephalosporins were transported into rat intestinal brush-border membrane by the transmembrane H+ gradient and the pH of the medium.

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.

A structure-relationship study of the uptake of aliphatic polyamine compounds by rat intestinal brush-border membrane vesicles.

The effects of lipophilicity, ion-diffusion potential and membrane surface potential on the uptake of various aliphatic polyamine compounds by rat intestinal brush-border membrane vesicles (BBMV) have been investigated. A valinomycin-induced potassium-diffusion potential (inside-negative) stimulated the initial uptake of diamine compounds, and good correlation was observed between lipophilicity and the amount of diffusion-potential-dependent transport of the diamines. In contrast, because of their much lower lipophilicity, tri- and tetraamine compounds were not affected by the diffusion potential. Tetracaine, which can make the membrane surface potential more positive, inhibited the transport rate of 1,9-nonanediamine, spermidine and spermine by the BBMV. These data suggest that the transport mechanism of diamines is similar to that of monoamine compounds in respect to its dependence on ion-diffusion potential and on the membrane surface potential. The extent of the effect of ion-diffusion potential on the rate of transport of the diamines was closely related to the lipophilicity of the diamine. In contrast, only the surface potential contributed to the transport mechanism of lower lipophilic tri- and tetraamine compounds.

Ionic-diffusion potential-dependent transport of a new quinolone, sparfloxacin, across rat intestinal brush-border membrane.

The mechanism of uptake of sparfloxacin, a new quinolone, by intestinal brush-border membrane vesicles was investigated to clarify whether there is a common transport process for new quinolones mediated by the diffusion potential across the intestinal membrane bilayer. Sparfloxacin was taken up pH-dependently by rat intestinal brush-border membrane vesicles, behaviour analogous to that of organic cations including enoxacin and ciprofloxacin. Transient overshooting uptake of this quinolone was observed in the presence of an outward H+ gradient. Momentary dissipation of the H+ gradient by addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone did not affect the uptake of sparfloxacin, and a marked but incomplete reduction in the H+-sensitive overshooting uptake of sparfloxacin was apparent in the voltage-clamped brush-border membrane vesicles. Furthermore, a valinomycin-induced K+-diffusion potential (interior negative) and an inward C1--diffusion potential stimulated the initial uptake of sparfloxacin at pH 5.5. Sparfloxacin uptake was inhibited by tetracaine and imipramine. The inhibitory effect of these cations correlated well with changes in membrane surface charges induced by the presence of tetracaine or imipramine. These results indicate that sparfloxacin transport across the brush-border membrane depends upon the inside-negative ionic diffusion potential, that the H+- or K+-diffusion-potential-dependent uptake of sparfloxacin by intestinal brush-border membrane vesicles is affected by the membrane surface potential and that inhibition of sparfloxacin uptake originates from changes in the membrane surface potential caused by the organic cations.

Transport characteristics of ceftibuten, cefixime and cephalexin across human jejunal brush-border membrane

The transport characteristics of orally active cephalosporins, ceftibuten, cefixime and cephalexin have been examined using brush border membrane vesicles isolated from human jejunum. In the initial uptake of ceftibuten, the stimulation and overshoot phenomena were observed in the presence of an inward H+ gradient. Effects of H+ gradient on the uptake of cefixime and cephalexin were low and no overshoot was observed. These transport characteristics, especially uphill transport phenomena, were in agreement with previous results obtained from rat intestinal brush-border membrane vesicles and suggest that these beta-lactam antibiotics are absorbed by different transport systems, despite their similar molecular structures.

The inhibitory effects of cephalosporin and dipeptide on ceftibuten uptake by human and rat intestinal brush-border membrane vesicles.

The types of inhibitory effects caused by compound V (an analogue of ceftibuten) and alanylproline (dipeptide) on the uptake of ceftibuten by brush-border membrane vesicles (BBMV) prepared from human and rat small intestine were analysed. In the presence of an inward H(+)-gradient, the initial uptake rate of ceftibuten by both human and rat intestinal BBMV was concentration-dependent with apparent Km and Vmax values of 0.35 mM and 2.052 nmol (mg protein)-1 min-1 for human BBMV, and 0.50 mM and 3.056 nmol (mg protein)-1 min-1 for rat BBMV, respectively. For both human and rat BBMV, kinetic analysis by Dixon and Lineweaver-Burk plots demonstrated that the uptake of ceftibuten was competitively inhibited by compound V, whereas inhibition by alanylproline was noncompetitive or partially competitive. These results suggest that there is a stereospecific transport system which is common to ceftibuten and compound V, and that this system is not identical to the carrier system for the dipeptide, alanylproline.

The pH dependent uptake of enoxacin by rat intestinal brush-border membrane vesicles.

The mechanism of the intestinal transport of enoxacin, an orally active fluoroquinolone antibiotic, has been investigated using brush-border membrane vesicles isolated from rat small intestine. The initial rate and time-course of enoxacin uptake were considerably dependent upon the medium pH (pH 5.5 greater than pH 7.5) and upon the percent ionization of the carboxyl group (pKa 6.2, anionic charge), namely, the degree of uptake of cationic form was higher than that of the zwitterionic form. There was evidence of transport into the intravesicular space as shown by the effect of extravesicular medium osmolarity on enoxacin uptake at steady state (30 min). This transport across the brush-border membrane was stimulated by the valinomycin-induced K(+)-diffusion potential (interior negative) and an outward H(+)-diffusion potential. Furthermore, changing the pH of the medium from 5.5 to 7.5 significantly decreased the effect of valinomycin-induced K(+)-diffusion potential on the enoxacin uptake. These results suggest that the uptake behaviour of the cationic form of enoxacin plays an important role in the intestinal absorption process of enoxacin.

The effect of prostaglandin synthase inhibitor, aspirin on the rat intestinal membrane structure and function.

Aspirin at a dose of 50 mg/kg body weight was found to decrease the activity of the rat intestinal brush border membrane (BBM) - associated enzymes such as the sucrase, lactase, maltase and alkaline phosphatase. Aspirin treatment also led to a decrease in the microviscosity in the native as well as the benzyl alcohol treated membrane which might be due to the lipid peroxidative damage in the membrane. Physical correlation of the membrane oxidative damage was evident as the Fourier Transformation Infra Red (FTIR) study of the Aspirin treated membrane, which include an increased proportion of gauche to trans conformer, shift in the methylene C-H asymmetric and symmetric stretching frequencies, C = O double bond stretching, NH bending, antisymmetric (N)-CH3 bending, C-N stretching and antisymmetric CNC stretching while there was no change in the CH2 wagging and twisting as well as in NH-bending amide bond I and II. Aspirin treatment also caused an alteration in the glucose and histidine transport, as evident by a decreased Vmax value while the apparent Km remaining unchanged in the control and Aspirin-treated animals confirming that there was no change in the substrate affinity constant of the membrane transport proteins for the glucose and the basic amino acid, although the rate of transport decreased considerably. There was a decrease noted in the energy of activation of glucose and histidine transport when studied at different temperature but no change in the temperature of phase transition in the BBM with Aspirin treatment, thus implying that perhaps the thermotropic phase transition in the membrane may have relatively little effect on the transport processes. The result suggests an underlying molecular mechanism indicating the implied membrane damage by Aspirin, an important member of the non-steroidal antiinflammatory drug (NSAID) family which could possibly through an oxidative damage may lead to an altered molecular structure, physical state and biological functions of the intestinal membrane.

Oral administration of potassium dichromate inhibits brush border membrane enzymes and alters anti-oxidant status of rat intestine

Potassium dichromate (K2Cr2O7) is a soluble hexavalent chromium compound that is widely used in several industries. In the present work the effect of administration of K2Cr2O7 on rat intestinal brush border membrane(BBM) enzymes and anti-oxidant system was studied. Rats were given a single oral dose of K2Cr2O7 (100 mg/kg bodyweight) and sacrificed 6, 12, 24, 48 and 96 h after the treatment.Control animals were not given K2Cr2O7. The administration of K2Cr2O7 resulted in a reversible decline in the specific activities of several BBM enzymes. The decrease in the activities of these enzymes was due to changes in the maximum velocity while their affinities for the substrates remained unchanged. Lipid peroxidation increased while total SH groups decreased in K2Cr2O7-treated rats as compared to controls indicating increased oxidative stress in the intestinal mucosa. The activities of superoxide dismutase and glutathione-S-transferase increased while those of catalase, glutathione reductase, thioredoxin reductase and glucose-6-phosphate dehydrogenase decreased. The maximum changes in all the parameters studied above were 24 h after administration of K2Cr2O7 after which recovery took place,in most cases almost to control values after 96 h. These results show that oral administration of K2Cr2O7 to decrease in the activities of BBM enzymes, increase in oxidative stress and alters the activities of anti-oxidant enzymes in rat intestine.

Improved Intestinal Membrane Permeability of Hexose-Quinoline Derivatives via the Hexose Transporter, SGLT1

Intestinal membrane permeability is an important factor affecting the bioavailability of drugs. As a strategy to improve membrane permeability, membrane transporters are useful targets since essential nutrients are absorbed efficiently via specific transporters. For example, there are reports that intestinal hexose transporters could be used as a tool to improve permeability; however, there has been no direct evidence that the transporter protein, sodium/glucose cotransporter 1 (SGLT1), is involved in the transport of hexose analogs. Accordingly, we examined directly whether the intestinal membrane permeability of hexose analogs can be improved by utilizing SGLT1. Three hexose-quinoline derivatives were synthesized and their interactions with SGLT1 were evaluated. Among the three derivatives, the glucose-quinoline molecule exhibited an inhibitory effect on D-glucose uptake by both rat intestinal brush-border membrane vesicles (BBMVs) and Xenopus oocytes expressing SGLT1. In addition, significant uptake of the glucose-quinoline derivative by Xenopus oocytes expressing SGLT1 was observed by both an electrophysiological assay and direct measurement of the uptake of the compound, while the galactose-quinoline derivative did not show significant uptake via SGLT1. Thus, it was directly demonstrated that SGLT1 could be used as a tool for the improvement of intestinal membrane permeability of drugs by modification to the glucose analogs.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Alterations in the Lipid Profile and Membrane Dynamics of Rat Intestinal Brush Border Membrane Induced by Different Classes of Nonsteroidal Anti-Inflammatory Drugs

ABSTRACTNonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause gastrointestinal damage. New anti-inflammatory drugs have been developed in an attempt to improve their gastrointestinal side effect profile. Therefore, the objective of the present study was to compare the effect of three different NSAIDs, aspirin, nimesulide, and celecoxib, on the lipid profile and dynamics of rat intestinal brush border membranes (BBMs). Female Wistar rats were divided into four different groups viz: group I (control), group II (aspirin treated), group III (nimesulide treated), and group IV (celecoxib treated). Groups II, III, and IV received the corresponding drugs orally at a dose of 40 mg/kg body weight dissolved in water, while the control group received the vehicle only. After 28 days, all the treatment groups showed modification in the lipid profile of intestinal BBM as compared to control. Alterations in the intestinal membrane dynamics by fluidity studies showed a considerable increase, which correlated well with the changes in the lipid composition. It was suggested that NSAIDs such as aspirin, nimesulide, and celecoxib pose intestinal side effects due to initial changes in the composition and dynamics of the membranes. It was further concluded that newly discovered NSAIDs such as celecoxib have better safety profiles because of the less intense membrane effects, but studies are still required to comment decisively on the suitability of various NSAIDs depending upon their cyclooxygenase enzyme specificity.

Purification by p-aminobenzoic acid (PABA)-affinity chromatography and the functional reconstitution of the nateglinide/H + cotransport system in the rat intestinal brush-border membrane

(−)- N-( trans-4-Isopropylcyclohexanecarbonyl)- d-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a peptide-type bond and a carboxyl group in its structure. Recently, we have shown that nateglinide transport occurs via the ceftibuten/H + cotransport system, which is distinct from PepT1, and that the fluorescein/H + cotransport system is involved in the uptake of nateglinide. The aim of this study was to characterize the functional properties of the intestinal nateglinide transporter. In the first part of this study, we demonstrated that the ceftibuten/H + cotransport system is identical to the fluorescein/H + cotransport system. We succeeded in purification of the nateglinide transporter from brush-border membranes of the rat small intestine using p-aminobenzoic acid (PABA)-affinity chromatography. We then investigated the functional properties of the nateglinide transporter using proteoliposomes prepared from the PABA-affinity chromatography elute. We demonstrated that nateglinide, ceftibuten, and fluorescein are transported by the same transporter in the intestine.

H+-dependent transport mechanism of nateglinide in the brush-border membrane of the rat intestine.

(-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a carboxyl group and a peptide-type bond in its structure. Although nateglinide quickly reaches the maximal serum concentration after oral administration, nateglinide itself is not transported by PepT1 or MCT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. The uptake of nateglinide by rat intestinal brush-border membrane vesicles is associated with a proton-coupled transport system. Ceftibuten competitively inhibited H(+)-dependent nateglinide uptake. Glycylsarcosine (Gly-Sar), cephradine, and cephalexin did not significantly inhibit the uptake of nateglinide. The combination of Gly-Sar and nateglinide greatly reduced the uptake of ceftibuten. The effect of the combined treatment was significantly greater than that of Gly-Sar alone. Furthermore, nateglinide competitively inhibited H(+)-driven ceftibuten transporter-mediated ceftibuten uptake. Ceftibuten transport occurs via at least two H(+)-dependent transport systems: one is PepT1, and the other is the ceftibuten/H(+) cotransport system. On the other hand, we demonstrated that nateglinide transport occurs via a single system that is H(+) dependent but is distinct from PepT1 and may be identical to the ceftibuten/H(+) cotransport system.