Characteristics of transport of fluoresceinated methotrexate in rat small intestine

The small intestinal damage induced by the methotrexate (MTX) treatment results in malabsorption and diarrhea. The fluoresceinated methotrexate (F-MTX) may possibly be useful to study such effects of MTX on the small intestine. The purpose of this study is to characterize the transport of F-MTX in the small intestine in order to use it as a membrane transport and cellular marker of MTX. The transport of F-MTX in the rat small intestine (jejunum) was examined in the in vitro everted segments of the intestine. The uptake was pH-dependent and showed a maximal effect at pH 6.0, which was the same as the results of MTX previously reported. Further, it was temperature-dependent and was inhibited by metabolic inhibitors, dinitrophenol and sodium azide, and by MTX. The transport kinetics at pH 6.0 in the mucosal solution and at pH 7.4 in the serosal solution was saturable with Km of 0.48 +/- 0.23 microM and Vmax of 0.66 +/- 0.24 pmol/cm/min and in addition, the passive diffusion was observed there. These results suggested that the transport of F-MTX was energy-dependent and was mediated by the same transporter as that of MTX, although, in addition to it, other transport mechanism might contribute to the F-MTX transport. Therefore F-MTX will be of great use to investigate the MTX transport system in the normal and diseased states of small intestine, using various fluorescence techniques like visualization of membrane-associated transport proteins.     

Enhancement of glucose transport in small intestinal brush border membrane of retinol administered rat

Retinylpalmitate (200 IU/kg body weight) was administered intraperitoneally to rats once daily for 4 days. Brush border membrane vesicles (BBMVs) were prepared from small intestinal epithelium cells from along the crypt-villus axis. D-glucose uptake by BBMVs was examined under the inwardly directed Na+ gradient. The D-glucose uptake by BBMVs from the villus-tip and mid-villus cells of retinylpalmitate treated rats was significantly larger than that of control (corn oil treated) rats, respectively. Thus, retinol treatment of rats promoted the D-glucose transport in small intestinal brush border membrane. Interestingly, the enhancement of D-glucose transport was more prominent in villus-tip and mid-villus than in lower villus.     

Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics

The interaction of two renin inhibitors, S 86,2033 and S 86,3390, with the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86,3390 and 2.5 mM of S 86,2033 led to a half-maximal inhibition of the H(+)-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86,2033 and S 86,3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of S 86,3390 was stimulated by an inwardly directed H(+)-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86,2033 and 86,3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent Mr 127,000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and beta-lactam antibiotics.     

Sodium-cotransport systems in intestine and kidney of the winter flounder

Summary Brush-border membrane vesicles were isolated from the intestine and kidney of the winter flounder,Pseudopleuronectes americanus, and the transport ofd-glucose,l-alanine and sodium was examined by a rapid filtration technique.d-glucose,l-alanine, and sodium entered the same osmotically reactive space suggesting that uptake into vesicles represents transport across rather than binding to the membrane. d-glucose andl-alanine uptake by intestinal and renal brush-border membrane vesicles was stimulated by sodium as compared to potassium or choline. In the presence of a sodium chloride gradient, overshooting uptake was observed indicating a transient intravesicular accumulation ofd-glucose andl-alanine. The sodium-dependentd-glucose uptake was inhibited by phlorizin andd-galactose while the transport ofl-alanine was inhibited byl-phenylalanine. The sodium-dependent transport ofd-glucose andl-alanine was affected by the electrical potential difference across the vesicle membrane; the addition of valinomycin in the presence of an inwardly directed potassium chloride gradient inhibited sodium-dependent solute uptake, whereas replacing chloride or gluconate with more permeant anions, such as SCN^–, stimulated uptake. Similar results were obtained with intestinal and renal membranes; they document the presence of sodium/d-glucose and sodium/l-alanine cotransport systems in the brush-border membrane of intestine and kidney.Sodium uptake into brush border membrane vesicles from the flounder intestine and kidney was saturable (tracer replacement) and trans-stimulated (tracer coupling), indicating transport via facilitated diffusion systems. Additionally, sodium uptake was only slightly affected by superimposing diffusion potentials demonstrating that the majority of sodium transport was by electroneutral coupled processes. In both the intestinal and kidney brush-border membrane vesicles sodium uptake was inhibited by an inwardly directed proton gradient suggesting the presence of a sodium/proton exchange mechanism. In intestinal, but not in renal membrane preparations, sodium uptake was stimulated by chloride. Chloride stimulation was abolished after preincubation with furosemide indicating the presence of an additional coupled sodium-chloride transport in the intestinal brush-border membranes.The experiments were carried out at the Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USAAddress effective February 1, 1980: Albert Einstein College of Medicine, Department of Physiology, 1300 Morris Park Avenue, Bronx, New York 10461, USA     

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.     

Characteristics of tetraethylammonium transport in rabbit renal plasma-membrane vesicles.

Transport of [14C]tetraethylammonium (TEA), an organic cation, was studied in brush-border (BBMV) and basolateral (BLMV) membrane vesicles isolated from rabbit kidney cortex. In BBMV, the presence of an outwardly directed H+ gradient induced a marked stimulation of TEA uptake against its concentration gradient (overshoot phenomenon), whereas a valinomycin-induced inside-negative potential had no effect on TEA uptake. In BLMV, TEA uptake was significantly stimulated by the presence of an outwardly directed H+ gradient and by an inside-negative potential, but the effect of H+ gradient was absent when the vesicles were chemically 'voltage clamped'. In BBMV, internal H+ stimulated TEA uptake in a non-competitive manner by binding at a site with apparent pKa of 6.87. External H+ inhibited TEA uptake through a direct interaction with the putative H+/organic-cation exchanger at a site with apparent pKa of 6.78. Changing external pH while maintaining the pH gradient constant produced a result similar to that obtained by changing external pH alone. Increasing external H+ showed a mixed-type inhibition of TEA uptake. These results suggest that in the rabbit TEA transport across the basolateral membranes is driven by an inside-negative potential and that transport across the brush-border membrane is driven by a H+ gradient via an electroneutral H+/TEA antiport system.     

Transport of procainamide via H(+)/tertiary amine antiport system in rabbit intestinal brush-border membrane

Transport characteristics of procainamide in the brush-border membrane isolated from rabbit small intestine were studied by a rapid-filtration technique. Procainamide uptake by brush-border membrane vesicles was stimulated by an outward H(+) gradient (pH(in) = 6.0, pH(out) = 7.5) against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H(+) gradient was subjected to rapid dissipation by the presence of a protonophore, FCCP. An outward H(+) gradient-dependent procainamide uptake was not caused by H(+) diffusion potential. The initial uptake of procainamide was inhibited by other tertiary amines with N-dimethyl or N-diethyl moieties in their structures, such as triethylamine, dimethylaminoethyl chloride, and diphenhydramine, but not by tetraethylammonium and thiamine. Furthermore, procainamide uptake was stimulated by preloading the vesicles with these tertiary amines (trans-stimulation effect), indicating the existence of a specific transport system for tertiary amines. These findings indicate that procainamide transport in the intestinal brush-border membrane is mediated by the H(+)/tertiary amine antiport system that recognizes N-dimethyl or N-diethyl moieties in the structures of tertiary amines.     

Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles.

L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Presence of a potential-sensitive Na+ transport across renal brush-border membrane vesicles from rats of the Milan hypertensive strain

Sodium transport was measured in brush-border membrane vesicles prepared from kidney cortex of the Milan hypertensive strain (MHS) rats and the corresponding normotensive controls. In the presence of an outwardly directed proton gradient, 22Na was transiently accumulated in the vesicles. When a transmembrane electrical potential was imposed across membrane vesicles, both the accumulation ratio and the initial uptake were increased, indicating the presence of an electrogenic pathway for sodium in these membranes. The potential-dependent sodium uptake was significantly higher in MHS rats. Kinetic analysis give simple Michaelis Menten curves in the presence and in the absence of a membrane potential. In both conditions Jmax was significantly increased in MHS rats, whereas Km was the same for the two rat strains. Sodium uptake was inhibited by amiloride at concentrations that inhibit Na+-H+ exchange. The presence of the higher, potential-sensitive, sodium uptake in MHS is in agreement with studies on renal physiology which support the hypothesis that an increase in tubular sodium reabsorption may be the primary cause for the development of hypertension in this rat strain.     

Carrier-mediated transport system for choline and its related quaternary ammonium compounds on rat intestinal brush-border membrane.

The characteristics of the intestinal transport system for choline were investigated using isolated brush-border membrane vesicles from rat small intestine. In spite of the diminutive lipid solubility, the uptake of choline by membrane vesicles reflected smooth permeation into intravesicular space rather than the binding to the membrane surface. Physiological conditions, present in the intact intestine, such as an inward-directed Na+ or H+ gradient and inside negative membrane potentials, didn't directly involve in choline transport across the brush-border membrane. Moreover, an outward-directed H+ gradient had no significant effect on the time course of choline transport. However, in the absence of a driving-force, the initial uptake of choline exhibited a saturable manner. A kinetic analysis of the initial uptake rate gave an apparent Km of 159 microM. Furthermore, unlabeled choline caused both cis-inhibition and trans-stimulation for labeled choline transport, suggesting the existence of a carrier-mediated transport system for choline, classified as so-called 'facilitated diffusion'. Since tetramethylammonium, acetylcholine, and N1-methylnicotinamide caused both cis-inhibition and trans-stimulation, they appear to be accepted as the substrate of choline carrier. On the other hand, quaternary ammonium compounds (QACs) such as those which possessed hydrophobic parts in their molecules exhibited only cis-inhibition. They also inhibited Na(+)-dependent D-glucose transport, indicating that they influenced various carrier-mediated transport systems non-specifically due to interaction with the membrane. These findings strongly suggest that the choline transport system on the brush-border membrane of rat intestine recognizes only small molecular QACs as its substrate.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

p-Aminohippurate transport in basal-lateral membrane vesicles from rabbit renal cortex: stimulation by pH and sodium gradients

p-Aminohippuric acid (PAH) uptake was studied in basal-lateral membrane vesicles prepared from rabbit renal cortex. An outwardly directed hydroxyl gradient (pHo = 6.0, pHi = 7.6) stimulated PAH uptake slightly over that when the internal and external pH values were equal at 7.6. A 100 mM sodium gluconate gradient directed into the basal-lateral membrane vesicles increased PAH uptake about 2-fold over that when N-methyl-D-glucamine or potassium gluconate gradients were present. When hydroxyl and sodium gradients were simultaneously imposed (pHo = 6.0, pHi = 7.6 and 100 mM sodium gluconate extravesicularly) PAH uptake was stimulated greater than with the pH or Na+ gradient alone. In fact, an 'overshoot' was observed. Countertransport experiments showed that either intravesicular PAH or intravesicular PAH and Na+ could stimulate 3H-PAH uptake. Probenecid, an inhibitor of organic anion transport, inhibited both the hydroxyl-stimulated and Na+ gradient-stimulated PAH uptake but the greatest inhibition by probenecid was seen when the hydroxyl and sodium gradients were both present. Thus, it is proposed that the driving force for PAH accumulation across the basal-lateral membrane of the proximal tubule is a transport system which moves Na+ and PAH into the cell for an hydroxyl ion leaving the cell, i.e. a sodium-dependent anion-anion exchange system.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig,sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na⁺ gradient (Na _out ⁺ >Na _in ⁺ ). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na⁺ gradient. Unlike selenate uptake, selenite uptake was not Na⁺ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of⁷⁵Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na⁺-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na⁺-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na⁺ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of⁷⁵Se from⁷⁵Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.     

Transport of putrescine across duodenal,jejunal and ileal brush-border membrane of chicks (Gallus domesticus)

Luminal polyamines and their absorption are essential for proliferation of the enterocytes and, therefore, nutrition, health and development of the animal. The transport systems that facilitate the uptake of putrescine were characterized in chick duodenal, jejunal and ileal brush-border membrane vesicles prepared by MgCl2 precipitation from three-week-old chicks. An inwardly-directed Na+ gradient did not stimulate putrescine uptake and, therefore, putrescine transport in chick intestine. In the duodenum, jejunum and ileum, kinetics of putrescine transport fitted a model with a single affinity component plus a non-saturable component. The affinity (Kt) for [3H]putrescine transport across the brush-border membrane increased along the length of the small intestine. A model of intermediate affinity converged to the data obtained for [3H]putrescine transport with Kt approximating 1.07 and 1.05 mM or duodenum and jejunum, respectively; and high affinity with a Kt of 0.35 mM for the ileum. The polyamines cadaverine, putrescine, spermidine and spermine strongly inhibited the uptake of [3H]putrescine into chick brush-border membrane vesicles, more so for the jejunum and ileum than the duodenum. The kinetics of cadaverine, spermidine and spermine inhibition are suggestive of competitive inhibition of putrescine transport. These uptake data indicate that a single-affinity system facilitates the intestinal transport of putrescine in the chick; and the affinity of transporter for putrescine is higher in the ileum than in the proximal sections of the small intestine. In addition, this study shows that the ileum of chicks plays an important role in regulating cellular putrescine concentration.     

Evidence for tripeptide-proton symport in renal brush border membrane vesicles. Studies in a novel rat strain with a genetic absence of dipeptidyl peptidase IV

We have investigated the transport characteristics of L-phenylalanyl-L-prolyl-L-alanine in renal brush-border membrane vesicles isolated from Japan Fisher 344 rats. This particular rat strain genetically lacks dipeptidyl peptidase IV. Owing to the absence of this enzyme, the tripeptide was found to be completely resistant to hydrolysis by the renal brush-border membrane vesicles. Uptake of the tripeptide into these membrane vesicles in the presence of an inwardly directed Na+ gradient was slightly greater than in the presence of a K+ gradient, but there was no evidence for active transport. On the contrary, uptake was very rapid in the presence of an inside-alkaline transmembrane pH gradient, and accumulation of the tripeptide inside the vesicles against a concentration gradient could be demonstrated under these conditions. The uptake was drastically reduced by dissipation of the pH gradient. The uptake was stimulated by an inside-negative membrane potential and inhibited by an inside-positive membrane potential. Moreover, the uptake was greater in voltage-clamped membrane vesicles than in control vesicles. Many di- and tripeptides inhibited this pH gradient-stimulated uptake of Phe-Pro-Ala. The apparent dissociation constant for the tripeptide was 48 microM. High performance liquid chromatography analysis of the intravesicular content at the peak of the overshoot revealed that the tripeptide was transported across the membrane almost entirely in the intact form. These data provide the first direct evidence for the presence of an electrogenic tripeptide-proton symport in renal brush-border membranes.     

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.     

Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells

The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6. Enhanced accumulation of F-MTX at 4 degrees C suggests the existence of an active efflux system. MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the accumulation of F-MTX. Transcellular transport of F-MTX from the apical to the basolateral compartment was 2.5 times higher than the opposite direction. This vectorial transport was also reduced by MK-571, indicating the presence of Mrp-type transporter(s) on the basolateral membrane. Mrp3 mRNA was readily detectable, and the protein was localized on the basolateral membrane. Uptake of FMTX into membrane vesicles from IEC-6 cells and Spodoptera frugiperda-9 cells expressing rat Mrp3 were both ATP dependent and saturable as a function of the F-MTX concentration. Similar Km values (11.0 +/- 1.8 and 4.5 +/- 1.1 microM) and inhibition profiles by MK-571, estradiol-17beta-d-glucuronide, and taurocholate for the ATP-dependent transport of F-MTX into these vesicles were obtained. These findings suggest that the efflux of F-MTX is mediated by Mrp3 on the basolateral membrane of IEC-6 cells.     

Sodium ion/L-lactate co-transport in rabbit small-intestinal brush-border-membrane vesicles.

Uptake of L-lactate into rabbit jejunal brush-border-membrane vesicles prepared by a Ca2+-precipitation procedure was studied by a rapid filtration technique with L-[14C]-lactate as tracer. Transport of L-lactate into an intravesicular (osmotically reactive) space could be established. An inwardly directed NaCl gradient (outside 21 mM/inside 0mM) stimulated the uptake of L-lactate at 15 s 2-4-fold compared with that observed with an equal KCl gradient. A transient accumulation of L-lactate inside the vesicles (overshoot) was observed in the presence of an NaCl gradient. Gradients of LiCl, RbCl, CsCl or choline chloride were not able to replace NaCl in the stimulation of L-lactate uptake. L-Lactate uptake was saturable only in the presence of Na+. D-Lactate, DL-thiolactate (2-DL-mercaptopropionate), pyruvate and propionate inhibited the Na+-stimulated L-lactate uptake; D-lactate, thiolactate and pyruvate provoked trans-stimulation of L-lactate uptake. Artificially imposed diffusion potentials (inside negative) did not exert any effect on the Na+-dependent L-lactate uptake. The results are consistent with the existence of an electroneutral Na+/L-lactate co-transport system in the brush border of rabbit small intestine.