Human Jejunal Brush-border Membrane Research Articles

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.

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles.

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of Na(+) but markedly stimulated by an outwardly directed H(+) gradient (pH 5.5(in)/pH 7.5(out)); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 +/- 0.08 microM and a maximal velocity of 1.00 +/- 0.47 pmol. mg protein(-1). 10 s(-1). Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a Na(+)-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV.

Mechanism of thiamin uptake in the human jejunal brush-border membrane vesicles

Mechanism of thiamin uptake in the human jejunal brush-border membrane vesicles

Functional expression of human intestinal Na +-dependent and Na +-independent nucleoside transporters in Xenopus laevis oocytes

We have shown previously that the human jejunal brush border membrane expresses both the N1 ( cif) and the N2 ( cit) Na +-dependent (concentrative) nucleoside transporters but not the Na +-independent (facilitative) nitrobenzylmercaptopurineriboside (NBMPR)-sensitive ( es) transporter (Patil SD and Unadkat JD, Am J Physiol, 272: 1314–1320, 1997). In the present study, we have demonstrated that when Xenopus laevis oocytes are microinjected with human jejunal mRNA, four nucleoside transporters are expressed simultaneously, namely the N1 and N2 Na +-dependent nucleoside transporters and the es and the NBMPR-insensitive ( ei) Na +-independent transporters. The expressed Na +-dependent nucleoside transporters showed substrate specificity identical to that previously described by us using jejunal brush border membrane vesicles (Patil SD and Unadkat JD, Am J Physiol, 272: 1314–1320, 1997). The expressed es and ei Na +-independent transporters demonstrated broad substrate selectivity with both purines and pyrimidines capable of inhibiting the uptake of guanosine and thymidine mediated by this transporter. The expressed Na +-dependent nucleoside transporters mediated the transport of their respective nucleoside substrates with a high affinity and a low capacity, whereas the es and the ei transporters mediated the transport of nucleosides with a low affinity and a high capacity. Collectively, these observations suggest that the Na +-independent nucleoside transporters are expressed in the basolateral membrane of the human jejunal epithelium. Based on these data, we hypothesize that the concentrative transporters in the brush border membrane and equilibrative transporters in the basolateral membrane are arranged in series in the human jejunal epithelium to allow efficient vectorial transport of nucleosides from the lumen to the blood. The simultaneous expression of four nucleoside transporters in X. laevis oocytes establishes a basis for molecular cloning of these four human nucleoside transporters.

Uptake of 6‐Mercaptopurine Riboside via the Nucleoside Transporter in the Human Intestinal Brush‐border Membrane

The transport systems of 6-mercaptopurine riboside (6-MPR), a nucleoside analogue, in the human jejunal brush-border membrane and in the human epithelial cell line, Caco-2, were investigated. The transport activities of 6-MPR were found to be dependent upon an inward Na+-gradient at pH 5·5. Trans-stimulation studies indicated that the uptakes of both [3H]adenosine and [3H]uridine were significantly increased by the presence of 6-MPR inside the human brush-border membrane vesicles. The uptake of 6-MPR from the apical side of Caco-2 monolayer was sensitive for an inward-directed Na+-gradient, and a greater uptake was observed at an acidic medium (pH 5·5>7·5). Moreover, both adenosine and uridine were significantly effective in inhibiting the 6-MPR uptake by Caco-2. These results indicate that an analogue of adenosine, 6-MPR, is able to be taken up via the Na+-gradient-dependent purine- and pyrimidine-nucleosides transport systems in the human intestinal brush-border membrane.

Transport of L-leucine hydroxy analogue and L-lactate in human small intestinal brush border membrane vesicles.

Nitrogen load can be reduced by substituting dietary protein intake with keto or hydroxy analogues of amino acids. To investigate their intestinal absorption, human jejunal brush border membrane vesicles were used to measure uptake of L-leucine hydroxy analogue (L-LHA) and L-lactate. Uptake assays were performed under voltage-clamped conditions in the presence of valinomycin and K+ inside and outside. Both inward directed H(+)- and Na(+)-gradients stimulated uptake of both substrates. The H(+)-gradient was the major driving force and led to an increased rate of L-LHA or L-lactate transport. The proton ionophore FCCP abolished the H(+)-gradient-driven but not the Na(+)-gradient-driven uptakes of both substrates. The H(+)-gradient-driven uptake of both substrates was trans-stimulated by L-LHA, D-LHA, L-valine hydroxy analogue or L-lactate, respectively. In the presence of a Na(+)-gradient the uptake of tracer L-lactate was trans-stimulated only after preloading the vesicles with L-lactate but not by L-LHA. It can be concluded that the H(+)-gradient-driven transport of L- or D-hydroxy analogues of branched chain amino acids and L-lactate across the human intestinal brush border membrane is mediated by the same carrier.

Fast sampling, rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10-40 microliters) with an incubation medium (0.2-1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4 sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15-20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na(+)-D-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl- l-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na + or K + gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K + diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na + or proton gradient and that transport occurs by carrier-mediated and diffusional processes.

Xylose transport in the human jejunum

D-Xylose transport in the human jejunum was studied in vivo using a standard intestinal perfusion technique, and also in vitro in human jejunal brush border membrane vesicles. Initial D-xylose concentrations were linearly related to D-xylose absorption rates, a finding consistent with passive diffusion. Perfusion of D-xylose with varying D-glucose concentrations were aimed at examining D-xylose-D-glucose jejunal cotransport. D-Xylose absorption rates from a 30 mM D-xylose perfusate did not change significantly when 10, 30, or 60 mM glucose were added (-3.0 +/- 0.62 vs -3.34 +/- 0.71, -3.82 +/- 0.81, and -4.56 +/- 0.72 mM/30 cm/hr, respectively; minus indicates net absorption) suggesting an absence of a cotransport system. In brush border membrane vesicles, xylose uptake was partially inhibited by D-glucose and phlorizin. These data suggest that jejunal D-xylose absorption, at concentrations used clinically, is by passive diffusion, which process completely overrides a minor D-glucose cotransport component. The D-xylose tolerance test, therefore, reflects jejunal mucosal surface area and mucosal permeability to D-xylose and not nutrient carbohydrate absorption.

Phosphate transport in human jejunal brush-border membrane vesicles

Phosphate transport was studied in isolated brush-border membrane vesicles prepared from human jejunum. Transport consisted of two components: passive diffusion across the brush border, and sodium-dependent, carrier-mediated uptake. Both carrier-mediated transport and passive diffusion of phosphate were significantly greater at pH 6.1 than at pH 7.4. Sodium-dependent uptake was saturable at both pH values; the apparent Km was not affected by pH, whereas Vmax was significantly greater at pH 6.1 (179.3 vs. 118.4 pM/mg per 15 s). Phosphate uptake was half-maximally stimulated by a sodium concentration of 34 mM at pH 7.4, and a sodium concentration of 76 mM at pH 6.1. At both pH values, Hill coefficients were > 1 and were unaffected by pH. Moreover, sodium-dependent phosphate uptake was electroneutral at pH 7.4 and electrogenic at pH 6.1. These data suggest that at acid and neutral pH, both monovalent and divalent phosphate are transported across the human jejunal brush border membrane. The transport of each phosphate molecule is associated with the cotransport of two sodium ions.

Na+ and H+ transport in human jejunal brush-border membrane vesicles.

We have examined pH gradient-driven Na+ uptake and Na+-driven H+ transport in brush-border membrane vesicles prepared from jejunal tissue obtained from organ donors by measuring the influx of 22Na and the fluorescence quenching of acridine orange (AO). Vesicle preparation by either Ca2+ or Mg2+ precipitation showed no difference in 22Na uptake or AO fluorescence quenching and dissipation. An outward H+ gradient [intravesicular pH (pHi) 5.5; extravesicular pH (pHo) 7.5] induced a Na+ uptake "overshoot" of threefold over equilibrium, whereas the absence of an H+ gradient (at either pH 5.5 or 7.5) did not produce an overshoot. Voltage clamping by Ki+ = Ko+ plus valinomycin reduced the overshoot by 50%. The initial rate of pH-driven Na+ uptake in voltage-clamped vesicles was related to [Nao+] (Km = 29 mM and Vmax = 9.5 nmol.mg protein-1.3 s-1). Amiloride inhibited this uptake in voltage-clamped vesicles (Ki = 99 microM). Dissipation of AO fluorescence quench in vesicles with a preformed internal acid gradient was hastened by Nao+ as well as voltage clamping in the absence of Na+. In vesicles without a pH gradient, internal Na+, as well as a diffusion potential (Ki+ 100; Ko+ 0 plus valinomycin) in the absence of Na+, induced AO quenching. External Na+ and Li+, but not choline, acted to dissipate AO quenching induced by a diffusion potential, and the rate of dissipation was unaffected by the presence of Cl-.Li+ and NH4+, but not Cs+, K+, Rb+, or choline+, inhibited pH gradient-driven 22Na uptake. We conclude that human jejunal brush-border membrane vesicles contain conductive pathways for both Na+ and H+ and an Na+-H+ exchanger.

Transport characteristics of l-glutamate in human jejunal brush-border membrane vesicles

Previous work using human jejunal brush-border membrane vesicles has demonstrated the existence of a distinct transport system in man for acidic amino acids. This system is energized by an inwardly directed Na + gradient and an outwardly directed K + gradient. These studies further characterize the transport of l-glutamate in the human jejunal brush-border membrane vesicles. Efflux studies were performed by loading the brush-border membrane vesicles with radiolabeled l-glutamate and sodium chloride. Extravesicular K + accelerated the efflux of l-glutamate when compared to extravesicular Na + or choline, indicating that potassium serves to recycle the carrier. Unlabeled extravesicular l-glutamate (but not d-glutamate) also enhanced the efflux of radiolabeled l-glutamate demonstrating that there is a bidirectional similarity to the transport system. The effect of pH on the transport system was also investigated by varying the intravesicular and extravesicular pH from 5.5 to 9. A pH environment of 6.5 produced the highest initial uptake rates as well as the greatest overshoots for transport of l-glutamate into brush-border membrane vesicles. The imposition of an inwardly directed pH gradient (5.5 outside, 7.5 inside) accelerated both the influx and efflux of l-glutamate. These results demonstrate that the l-glutamate carrier system in human jejunum appears to have similar energizing characteristics in either direction across the brush-border membrane. In addition, the system operates at an optimal pH of 6.5 and protonation of the system may enhance its mobility.

Transport of acidic amino acids by human jejunal brush-border membrane vesicles.

This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na+ gradient (extravesicular greater than intravesicular). Concentrative uptake (resulting in an "overshoot" phenomenon) of these dicarboxylic amino acids occurred when there was an outward K+ gradient (intravesicular greater than extravesicular). In addition, increasing K+ gradients (0-100 mM) resulted in enhanced uptake of L-glutamic acid. This K+ requirement is somewhat specific as Rb+ and Cs+ could enhance uptake to a limited extent, whereas Li+ and choline+ showed no enhancement. The presence of a K+ gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the Vmax. The presence of extravesicular anions having differing membrane permeabilities did not alter L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. Our studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na+ gradient and an outward K+ gradient.