Identification of a candidate membrane protein for the basolateral peptide transporter of rat small intestine.

A candidate protein for the basolateral peptide transporter of rat jejunum is described. Vascular perfusion of the photoaffinity label, [4-azido-D-phe]-L-ala (2.5mM), had no effect on the transepithelial transport of the non-hydrolysable dipeptide D-phe-L-gln (1mM) from the lumen, its mucosal accumulation or wash-out into the vascular perfusate. When the label was perfused luminally, the transepithelial transport of D-phe-L-gln was inhibited by 38% (P<0.001) and accumulation increased by 62% (P<0.05). These data are consistent with those of a basolateral transporter that is strongly asymmetric in its substrate binding and transport properties. Labelling of basolateral membrane vesicles with [4-azido-3,5-3H-D-phe]-L-ala revealed that the majority of label was incorporated into a single protein of M(r)112+/-2 kDa and pI 6.5. MALDI-TOF analysis of tryptic digests of the protein followed by database searches established that this protein was novel with no obvious similarity to PepT1, the apical membrane transporter.

Peptide mimics as substrates for the intestinal peptide transporter.

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

4-aminomethylbenzoic acid is a non-translocated competitive inhibitor of the epithelial peptide transporter PepT1.

1. 4-Aminomethylbenzoic acid, a molecule which mimics the special configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus Laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2. This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1-exp ressing oocytes and indirectly by its failure to trans-stimulate labelle d peptide efflux through PepT1 in oocytes and in renal membrane vessicle s. 3. However 4-aminiomethylbenzoic acid does reverse trans-stimulation through expressed PepT1 of labelled peptid efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4-aminomethyl benzoic acid competitively binding to the external surface of PepT1. 4. 4-Aminomethylbenzoic acid (the first molecule discovered to be a non-translocated competitive inhibitor of proton-coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro.

Four hydrolysis-resistant dipeptides (D-phenylalanyl-L-alanine, D-phenylalanyl-L-glutamine, D-phenylalanyl-L-glutamate and D-phenylalanyl-L-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of -1 and +1 were transported at substantially slower rates (18 +/- 1.3 and 8.4 +/- 1.3 nmol min(-1)(g dry wt.)(-1), respectively) than neutral D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine (87 +/- 0.2 and 197 +/- 14 nmol min(-1)(g dry wt.)(-1), respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO3 content of Krebs Ringer perfusate equilibrated with 95% 02/5% CO2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine at pH 6.8 was stimulated (P < 0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged D-phenylalanyl-L-glutamate by 306% (P < 0.01) and decreased that for positively charged D-phenylalanyl-L-lysine by 46% (P < 0.05). Increasing luminal pH to 8.0 inhibited D-phenylalanyl-L-alanine transport by 60%, whereas D-phenylalanyl-L-lysine transport was 60% faster.

A model for the kinetics of neutral and anionic dipeptide-proton cotransport by the apical membrane of rat kidney cortex.

1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala and anionic D-Phe-L-Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (Em) to be varied systemically and independently. From this data the kinetic constants K(m) and Vmax were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra- and extravesicular), but not at pH 7.4, the rate constants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the extent of this trans-stimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton-peptide coupling on the net charge carried by the substrate. Quantitatively a 50-fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex.

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of -1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14 +/- 0.16 and 2.15 +/- 0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton:peptide cotransport is different for each peptide (0:1, 1:1 and 2:1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

Dipeptide transport and hydrolysis in rat small intestine, in vitro.

A range of natural and mixed D-/L-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent Km values in the 0.1-1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of L-/L-dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed D-/L-dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; D-alanyl-L-phenylalanine accumulated within the rings to twice the medium concentration.

Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

1. Isolated jejunal loops of rat small intestine were perfused by a single pass of bicarbonate Krebs-Ringer solution containing either D- or L-phenylalanine or one of eight dipeptides formed from D- or L-alanine plus D- or L-phenylalanine. 2. At 0.5 mM L-phenylalanyl-L-alanine increased serosal phenylalanine appearance to forty times the control rate giving a value similar to that found with 0.5 mM free L-phenylalanine. No serosal dipeptide could be detected. 3. Perfusions with the two mixed dipeptides with N-terminal D-amino acids (D-alanyl-L-phenylalanine and D-phenylalanyl-L-alanine) gave rise to the appearance of intact dipeptides in the serosal secretions although there were substantial differences in their rates of absorption and subsequent hydrolysis. 4. L-Alanyl-D-phenylalanine was absorbed from the lumen three to five times as fast as L-phenylalanyl-D-alanine. At 1 mM L-alanyl-D-phenylalanine transferred D-phenylalanine across the epithelial layer at more than seven times the rate found with the same concentration of the free D-amino acid. 5. Perfusions with D-alanyl-D-phenylalanine or D-phenylalanyl-D-alanine showed that these two dipeptides are poor substrates for both transport and hydrolysis by the rat small intestine. 6. Analysis of mucosal tissue extracts after perfusion with the two mixed dipeptides with N-terminal D-amino acids revealed that both dipeptides were accumulated within the mucosa and suggested that exit across the basolateral membrane was rate limiting for transepithelial dipeptide transport.

The absorption of 6-mercaptopurine from 6-mercaptopurine riboside in rat small intestine: effect of phosphate.

The intestinal absorption of 6-mercaptopurine and its nucleoside 6-mercaptopurine riboside has been studied in the rat with the in situ dual luminal and vascular perfusion. 6-Mercaptopurine is an inactive prodrug that requires intestinal absorption, cellular uptake and intracellular anabolism for cytotoxic activity. Vascular and mucosal samples were analysed by high-performance liquid chromatography (HPLC) to assess the rate of vascular appearance and amounts of thiopurine and its nucleoside in the mucosa. With 5 mmol luminal 6-mercaptopurine/l, the drug is transported across the intestine unchanged at a rate of 0.053 +/- 0.006 mumol min-1 (g dry wt.)-1. At concentrations below 20 mmol/l, 6-mercaptopurine riboside is not transported across the intestine intact but is split by phosphorolysis in the intestinal mucosa. The rate of vascular appearance of 6-mercaptopurine [0.043 +/- 0.005 mumol min-1 (g dry wt.)-1] from 5 mmol luminal 6-mercaptopurine riboside/l did not differ significantly from that seen with 5 mmol luminal 6-mercaptopurine/l. When the lumen was perfused with 6-mercaptopurine riboside the riboside appeared in the tissue together with a higher mucosal concentration of 6-mercaptopurine than in perfusions with 6-mercaptopurine. Some metabolism of 6-mercaptopurine to 6-thioguanine was also observed; however, no 6-thioguanine appeared in the vascular effluent. Increasing the luminal phosphate concentration from 2 to 10 mmol/l increased mucosal phosphorolysis of 6-mercaptopurine riboside and more than tripled the rate of vascular appearance of 6-mercaptopurine; conversion of 6-mercaptopurine to 6-thioguanine was significantly inhibited. These results suggest that with a modest increase in luminal phosphate concentration, 6-mercaptopurine riboside can be a more effective substrate than the free drug for the oral delivery of 6-mercaptopurine.

Tripeptide transport in rat lung.

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

Purine nucleoside transport and metabolism in isolated rat jejunum.

1. The absorption and metabolism of purine nucleosides and their constituent bases has been investigated by perfusion through the lumen of isolated loops of rat jejunum. In control perfusions and those with luminal purines or purine nucleosides, high-performance liquid chromatography (HPLC) revealed uric acid as the only detectable purine in the mucosal epithelial layer and the serosal secretions unless the xanthine oxidase inhibitor allopurinol was present. 2. Adenosine (0.5 mM) was quantitatively deaminated to inosine in the lumen after perfusion for 30 min. 3. Luminal inosine and hypoxanthine (0.15-1.0 mM) increased the serosal uric acid concentration significantly (P < 0.001); at 0.5 and 1.0 mM the nucleoside gave a significantly greater (P < 0.01) rate of serosal uric acid appearance than the base. 4. Luminal guanosine (0.05-0.50 mM) and guanine (0.05-0.15 mM) increased the serosal uric acid concentration significantly (P < 0.001); with 0.15 mM nucleoside the serosal uric acid appeared significantly faster (P < 0.01) than it did from the base. 5. Luminal allopurinol (0.3 mM) inhibited xanthine oxidase by 80% and reduced serosal purine appearance significantly (P < 0.01) from luminal guanine, hypoxanthine and inosine. With allopurinol, guanosine (0.1 and 0.15 mM) and inosine (0.1-1.0 mM) gave significantly higher (P < 0.01) total serosal purine concentrations than their respective bases. 6. Inosine and guanosine were cleaved to their respective bases plus ribose phosphate by the action of a cytoplasmic nucleoside phosphorylase, which was found to have widely different Michaelis constants (Km; 318 +/- 45 and 41.4 +/- 3.6 microM for inosine and guanosine, respectively) and maximum velocities (Vmax; 79.3 +/- 4.0 and 20.5 +/- 0.05 mumol min-1 (mg protein)-1 for inosine and guanosine, respectively). 7. We conclude that hypoxanthine and guanine absorbed by rat small intestine are oxidized to uric acid which is released in the serosa. The corresponding nucleosides are split by phosphorolysis after absorption and the resulting purine bases are converted to uric acid which appears on the serosal side with similar quantities of ribose phosphate.

The transport and metabolism of the uridine mononucleotides by rat jejunum in vitro.

1. Both uridine 3'-monophosphate (3'-UMP) and uridine 5'-monophosphate (5'-UMP) when perfused through the lumen of isolated rat jejunum gave rise to uracil as the only transported pyrimidine appearing in the serosal medium; neither the nucleotide nor the nucleoside could be detected in the serosal fluid. 2. There was a low level of the nucleoside, uridine, in the luminal fluid after the nucleotide had passed through the jejunal segment. Luminal nucleoside appearance was more marked from the 3' form of the nucleotide. 3. The hydrolysis of the nucleotides to the nucleoside form occurred via a brush-border membrane enzyme, which had the same maximal velocity (Vmax) for the two nucleotides (699 +/- 35 and 747 +/- 10 nmol min-1 (mg protein)-1 for 3'-UMP and 5'-UMP, respectively) but a different Michaelis constant (Km) so that 3'-UMP (Km = 58 +/- 3 microM) hydrolysis is favoured over 5'-UMP hydrolysis (Km = 108 +/- microM) at lower concentrations. 4. At 0.05 mM, luminal 3'-UMP gave rise to a higher rate of serosal uracil appearance than luminal 5'-UMP, but at higher luminal concentrations (0.1-0.2 mM) the rate of serosal uracil appearance was the same from both nucleotides. 5. The transmural transport of uracil from the uridine mononucleotides is discussed with reference to the metabolism and compartmentalization of the small intestine responsible for the appearance of the free pyrimidine in the serosal fluid.

The specificity of pyrimidine nucleoside transport and metabolism by rat jejunum in vitro.

1. 5-Methyluridine perfused through the lumen of isolated loops of rat jejunum gave rise to serosal thymine as the major species transported across the epithelial layer; no serosal 5-methyluridine was detected. 2. At 0.1 mM-luminal 5-methyluridine there was enhanced transmural transport of thymine (P less than 0.001) when compared with either 0.1 mM-thymine or 0.1 mM-thymidine as the luminal substrate for thymine transport. 3. At low luminal concentrations (0.025 mM) thymine was a significantly better substrate (P less than 0.001) for the transport of the free pyrimidine than either of the two nucleosides (thymidine or 5-methyluridine). 4. Luminal deoxyuridine gave rise to uracil as the major species appearing in the serosal secretion. High luminal concentrations of deoxyuridine (0.5 and 1.0 mM) gave rise to low levels of the nucleoside in the serosal fluid. 5. As a consequence of the specificity of the mucosal phosphorolysis the ribonucleosides are favoured over the deoxyribonucleosides as substrates for transmural pyrimidine transport.

Transport and metabolism of 6-thioguanine and 6-mercaptopurine in mouse small intestine.

1. The transport of 6-thioguanine and 6-mercaptopurine has been studied with isolated jejunal loops of mouse small intestine. H.p.l.c. was used to identify and quantify the thiopurines and their metabolites in the serosal secretions. 2. When the lumen of the intestinal loops contained either 6-thioguanine or 6-mercaptopurine at a concentration of 1 mmol/l, the concentration of unmetabolized drug in the serosal secretions reached a maximum of 0.13 +/- 0.02 mmol/l (mean +/- SEM). 3. Analysis of the serosal secretions from the perfusions with either of the drugs revealed the appearance of an unknown compound which had the characteristics of a thiopurine and the same time course of appearance as the unmetabolized drug. Thus 6-thioguanine and 6-mercaptopurine are significantly metabolized during absorption in mouse intestine. 4. The unknown compound was identified as 6-thiouric acid, and with 1 mmol/l 6-thioguanine or 6-mercaptopurine in the lumen the concentration of this metabolite in the serosal secretions rose to a maximum of 0.13 +/- 0.01 and 0.18 +/- 0.03 mmol/l, respectively. At luminal drug concentrations of 0.1 mmol/l, the metabolite accounted for approximately 90% of the serosal thiopurine. 5. After an initial lag period of 20 min, linear rates of appearance in the serosal secretions were obtained for both the unmetabolized drugs and 6-thiouric acid. 6. Addition of the xanthine oxidase inhibitor oxypurinol at a luminal concentration of 0.3 mmol/l prevented the formation of 6-thiouric acid from 6-thioguanine. However, the inhibitor reduced the rate of 6-thioguanine appearance in the serosal secretions by 50%. 7. The conversion of 6-mercaptopurine to 6-thiouric acid was prevented when allopurinol or oxypurinol were added to the lumen. At a luminal drug concentration of 1 mmol/l, allopurinol increased the rate at which 6-mercaptopurine appeared in the serosal secretions by 90% compared with an increase of only 50% with oxypurinol. 8. The transport of water and glucose by the mouse intestinal loops was unaffected by 6-thioguanine or the xanthine oxidase inhibitors. However, 6-mercaptopurine caused significant reductions in the rate of water transport (30%) and glucose transport (39%). These effects were observed at a luminal drug concentration of 0.1 mmol/l and there was no further increase at a drug concentration of 1 mmol/l.

The transport and metabolism of naturally occurring pyrimidine nucleosides by isolated rat jejunum.

1. Uridine perfused through the lumen of isolated loops of rat jejunum over a concentration range of 0.1-1.0 mM gave rise to higher serosal concentrations of uracil than the equivalent luminal concentration of uracil (P less than 0.001). No serosal uridine could be detected. 2. Luminal thymidine over a concentration range of 0.1-0.5 mM gave rise to the same serosal concentration of thymine as the equivalent luminal concentration of thymine (P greater than 0.1). Low concentrations of serosal thymidine were detected. Both luminal thymidine and thymine gave rise to elevated levels of serosal uracil. 3. Luminal cytidine at concentrations of 0.1-0.5 mM was poorly transported and yielded low serosal concentrations of cytidine. No serosal cytosine was detected, although elevated levels of uracil were found in the serosal secretions. 4. Cytosine over a luminal concentration range of 0.1-0.5 mM gave rise to low concentrations of cytosine in the serosal secretions. These results were consistent with a passive diffusion model for cytosine transport. No increase in serosal uracil was detected. 5. The cleavage of uridine and thymidine to their respective pyrimidine bases occurred via a cytoplasmic nucleoside phosphorylase, which had a similar Michaelis constant (Km), (61.0 +/- 4.4 and 97.1 +/- 5.7 microM for uridine and thymidine, respectively) but a maximal velocity (Vmax) for uridine cleavage (320 +/- 32 nmol min-1 (mg protein)-1) 13 times that for thymidine cleavage (24.7 +/- 1.4 nmol min-1 (mg protein)-1). 6. The differences between the three pyrimidine nucleosides are discussed with reference to the interactions between their epithelial transport and metabolism.

Absorption of 5-fluorouracil and related pyrimidines in rat small intestine.

The transport of 5-fluorouracil, uracil and thymine has been studied with isolated jejunal loops of rat small intestine. High performance liquid chromatography was used to identify the pyrimidines and measure their concentrations. When the lumen of the intestine was perfused with 5-fluorouracil or uracil at 0.1 mmol/l or 0.2 mmol/l, the concentration in the serosal secretions was significantly higher than that in the lumen. For thymine the serosal concentration exceeded that in the lumen only at 0.1 mmol/l. Analysis of the mucosal tissue water at the end of the perfusion demonstrated that when the intestinal lumen was perfused with any one of the three pyrimidines at 0.1 mmol/l or 0.2 mmol/l the concentration within the tissue was significantly above that in the lumen. After an initial lag period linear rates of transport from the lumen to the serosal secretions were obtained for all three pyrimidines over a 10-fold concentration range from 0.1 mmol/l to 1 mmol/l. Uracil and thymine inhibited the transmural transport of 5-fluorouracil. The transport of 5-fluorouracil was also studied with a vascularly perfused preparation of rat small intestine. At 0.1 mmol/l the rate of transmural transport of the drug in this preparation was substantially higher than in the jejunal loops. This difference was eliminated by adding 5-fluorouracil to the vascular perfusate, suggesting that the higher transport rate in the vascularly perfused preparation was due to the lower serosal drug concentrations in the mesenteric circulation of the perfused intestine. At a concentration of 5 mmol/l 5-fluorouracil inhibited water transport in the isolated loops and transmural D-galactose transport in the vascular perfusions.

The transport of pyrimidines into tissue rings cut from rat small intestine.

1. At low concentrations (0.1 mM) the transport of uracil, 5-fluorouracil and thymine into jejunal tissue rings is an active process. 2. The transport of 5-fluorouracil into tissue rings cut from the duodenum and jejunum was greater than the transport into rings cut from the ileum. This difference was abolished by starving the rats for 48 h before the experiment. 3. The active transport can be abolished by replacing the Na+ in the incubation medium with either K+ or mannitol, or by increasing the concentration of the pyrimidine to 1.0 mM. 4. The accumulation of uracil or 5-fluorouracil into the jejunal rings was identical when determined by radioactive tracer or by high-performance liquid chromatography. 5. The apparent Michaelis constant (Km) for 5-fluorouracil transport into jejunal rings was 0.074 mM in the standard Na+ bicarbonate Krebs-Ringer solution and 0.394 mM in the K+-substituted bicarbonate Krebs-Ringer solution. 6. Both thymine and uracil inhibited the transport of 5-fluorouracil into jejunal tissue rings: however, cytosine and orotic acid did not.

The transport of uric acid across mouse small intestine in vitro.

The in vitro recirculation technique was used to study the uptake and transport of uric acid by the jejunum of mouse small intestine. Three components of the serosal secretions appeared to be endogenously derived nucleic acid derivatives; two of these were identified as uric acid and uracil. There was no detectable metabolism of uric acid by the intestine. Uric acid transported from the lumen appeared in the serosal fluid at a concentration higher than that in the lumen. The final serosal/luminal concentration ratio of about 1.18 for exogenous uric acid was found to be constant over the concentration range studied (0.01-0.1 mM). The presence of exogenous uric acid in the lumen did not affect the production of endogenous uric acid by the intestine and its release into the serosal secretions. Mucosal concentration of exogenous uric acid was below, but the total mucosal concentration (exogenous+endogenous) was above, that in the lumen. There was no evidence for the secretion of endogenous uric acid into the lumen. Oxypurinol significantly decreased the rate of serosal appearance of exogenous uric acid. Allopurinol did not affect the transport of exogenous uric acid from the lumen and there was negligible metabolism of allopurinol to oxypurinol by the tissue. Uracil did not affect the transport of exogenous uric acid from the lumen, or the serosal appearance of endogenous uric acid. Likewise uracil transport was unaffected by luminal uric acid.

D-galactose transport in rat intestinal brush border membrane vesicles studied with a molecular-sieve technique.

A technique using G-50 Sephadex molecular-sieve chromatography has been developed to study both the accumulation of transported solutes by rat intestinal brush border membrane vesicles and the wash-out of these solutes from pre-loaded vesicles. The extent to which D-galactose was taken up into the brush border membrane vesicles was dependent on the medium osmolarity and this dependence was significantly reduced (P less than 0.01) by pre-treating the vesicles with the non-ionic detergent Triton X-100. The transport of D-galactose into the brush border membrane vesicles was sodium dependent, electrogenic and phloridzin sensitive. It is, therefore, qualitatively similar to D-glucose transport into brush border membrane vesicles. The wash-out of D-galactose from pre-loaded brush border membrane vesicles was inhibited by phloridzin and accelerated by the presence of an outwardly directed sodium gradient. D-Galactose was released from the pre-loaded brush border membrane vesicles more slowly than D-glucose and this difference was consistent with the higher overshoot found for D-galactose during the transport of the sugars into the vesicles.