Dipeptide Cotransporter Research Articles

PepT1 expressed in immune cells has an important role in promoting the immune response during experimentally induced colitis

We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-γ were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site–leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.

Dual role of the Na+/H+ exchanger isoform 3 for PEPT1‐mediated H+/dipeptide cotransport in native murine intestine

This study investigates the molecular function of NHE3 in H+‐dipeptide transport regulation in the native intestine. Luminal application of the dipeptide Gly‐Sar resulted in a strong increase in murine small intestinal fluid absorption in vivo, Isc response in chambered jejunal mucosa, and a decrease in pHi in BCECF‐loaded enterocytes in isolated jejunal villi. Genetic ablation of PEPT1 abolished Gly‐Sar‐induced fluid absorption, Isc response, and enterocyte acidification. Genetic ablation, pharmacological or second‐messenger‐mediated inhibition of NHE3 also reduced Gly‐Sar‐induced fluid absorption in vivo and Isc response in vitro, but enhanced enterocyte acidification. Genetic ablation of Slc26a6 also enhanced enterocyte acidification, but did not alter either Gly‐Sar induced fluid absorption or Isc response. The transmembrane proton gradient was irrelevant for sustained H+/dipeptide transport rates, but the luminal Na+ concentration, enterocyte Na+/K+ ATPase activity, and luminal membrane potential were essential determinants. PEPT1 is the only H+/dipeptide uptake pathway, and NHE3 function is essential for sustained H+/dipeptide uptake rates in the native intestine. We speculate that NHE3 is the major Na+‐uptake pathway into enterocytes during dipeptide‐induced volume increase, and therefore essential to the maintenance of membrane potential negativity.

C. elegans NHX‐2 influences nutrient uptake and insulin signaling

Disruption of the nhx-2 gene, which codes for an intestinal Na/H exchanger, leads either to larval arrest or, for those animals that make it through larval development, to an increased lifespan; both effects are mediated through a mechanism resembling starvation. We hypothesized that the proton gradient established by NHX-2 is functionally coupled to intestinal H+/dipeptide cotransport by OPT-2. We find that NHX-2 RNAi causes intracellular acidification and inhibits the uptake of fluorescently labeled dipeptide substrate. We have initiated measurements of luminal pH. We also hypothesized that pHi coordiantes cell growth with nutrient availability. To explore this hypothesis, we performed a two gene loss-of-function analysis using nhx-2 RNAi combined with insulin signaling mutants. We assessed larval development, post-adult lifespan, and intestinal fat stores. We find that larval arrest is insulin signaling/daf-16 dependent, however, lifespan extension requires a daf-16 independent pathway that utilizes protein kinases associated with insulin signaling. Furthermore, since dauer formation is regulated by nutrient availability, we examined the pHi of the intestine during entry into and recovery from dauer phase. We find that dauer recovery is NHX-2 dependent and results in a transient acidification of the intestine. Our data suggest a complex regulatory network where pHi influences insulin signaling to coordinate nutrient availability and cell growth. Finally, the initial results from a global RNAi screen for intestinal acidification identified several gene products that act in the mitochondria, suggesting that intestinal pHi may provide a link between caloric restriction, mitochondria function, and insulin signaling.

Inhibition of the putative anion transporter (PAT‐1, Slc26a6) impairs the ability of villous epithelial cells to regulate intracellular pH (pH i ) during peptide transport

The upper villus is exposed to acid challenge from H+ influx via the H+ dipeptide cotransporter PEPT1. Intracellular carbonic anhydrase activity facilitates diffusive H+ movement to help maintain a transmembrane ion gradient allowing for maximal absorption through PEPT1. Other studies have revealed that carbonic anhydrase II (CAII) can interact with the apical membrane Cl−/HCO3− exchanger PAT-1. Therefore, we hypothesized that CAII functions with PAT-1, the predominant Cl−/HCO3− exchanger of the upper duodenal villus, to form a HCO3− transport metabolon at the apical membrane to regulate pHi during PEPT1 transport activity. Measurements of pHi by BCECF microfluoroscopy were conducted in wild-type (WT) and PAT-1 knockout (KO) upper duodenal villous epithelium exposed to the dipeptide glycyl-sarcosine (gly-sar) in Kreb's HCO3− Ringers. During gly-sar exposure, inhibition of luminal Cl−/HCO3− exchange in WT epithelium with niflumic acid resulted in greater acid influx (NFA = −0.83 ± 0.94; control = −7.05± 0.89 mM/min, n = 3; p<0.05). Furthermore, a greater acid influx was observed in the PAT-1 KO as compared to WT during gly-sar exposure (−0.07 ± 0.79 vs. −9.54 ± 3.40 mM/min, n = 3, p<0.05). These observations suggest that PAT-1 provides protection against intracellular acidification in the upper villous epithelium, perhaps by operating in reverse mode as a base-loading mechanism for the cell. Supported by NIH.

Mechanism of inhibition of proton: Dipeptide co-transport during chronic enteritis in the mammalian small intestine

Amino acids, a critical energy source for the intestinal epithelial cells, are more efficiently assimilated in the normal intestine via peptide co-transporters such as proton:dipeptide co-transport (such as PepT1). Active uptake of a non-hydrolyzable dipeptide (glycosarcosine) was used as a substrate and PepT1 was found to be present in normal villus, but not crypt cells. The mRNA for this transporter was also found in villus, but not crypt cells from the normal rabbit intestine. PepT1 was significantly reduced in villus cells also diminished in villus cell brush border membrane vesicles both from the chronically inflamed intestine. Kinetic studies demonstrated that the mechanism of inhibition of PepT1 during chronic enteritis was secondary to a decrease in the affinity of the co-transporter for the dipeptide without an alteration in the maximal rate of uptake ( V max). Northern blot studies also demonstrated unaltered steady state mRNA levels of this transporter in the chronically inflamed intestine. Proton dipeptide transport is found in normal intestinal villus cells and is inhibited during chronic intestinal inflammation. The mechanism of inhibition is secondary to altered affinity of the co-transporter for the dipeptide.

Further evidence that epidermal growth factor enhances the intestinal adaptation following small bowel transplantation

Recent reports indicate that epidermal growth factor (EGF) plays a crucial role for graft adaptation in rat model of small bowel transplantation (SBT). The administration of EGF enhances intestinal cell proliferating rate and the recovery of mucosal structure. However, the effect of EGF on biological functions including glucose absorption in intestinal graft remains to be elucidated. SBT was performed in the two-step procedure. On the first step, intestinal graft (30-cm jejunum) from Brown Norway rats was exteriorized through abdominal wall as a Thiry-Vella loop in recipient Lewis rats for one week. On the second surgery (POD 7), recipient jejunum was replaced orthotopically by the graft, and transplanted rats were treated intraperitoneally with EGF or its vehicle for 3 days. Analyses of histology and biological functions in the graft were done at POD 14. EGF increased both levels of villus height and crypt depth in the graft of transplanted groups. EGF enhanced the glucose absorption as well as the induction of sodium glucose cotransporter 2- to 3-fold in transplanted groups. Further, EGF stimulated the activities of disaccharidase (maltase and sucrase) and the induction of dipeptide cotransporter. These results demonstrate that EGF enhances the structural and functional adaptation of intestinal grafts after SBT. EGF may be useful therapy for patients following intestinal transplantation.

In vitro studies on the effects of Saccharomyces boulardii and Bacillus cereus var. toyoi on nutrient transport in pig jejunum

The probiotics Saccharomyces boulardii and Bacillus cereus var. toyoi are nonpathogenic microbes which have been shown to affect certain functions of the mucosal barrier in pig jejunum such as electrogenic ion transport capacity and paracellular permeability. The present studies were performed to investigate potential effects of the probiotics on jejunal nutrient transport such as sodium‐dependent glucose transport or proton‐dependent dipeptide transport. For this purpose the in vitro Ussing‐chamber technique was applied in order to examine net electrogenic ion flux rates (short circuit currents, Isc) across isolated intact jejunal epithelia in the absence and presence of either 10 mmol/l glucose (mucosal side) or two‐fold application of 5 mmol/l glycyl‐ l‐sarcosine or glycyl‐ l‐glutamine to the mucosal bathing solution. Brush border membrane vesicles (BBMV) were prepared in order to characterize kinetic parameters (Vmax, Km) of Na‐dependent glucose transport. Intestinal tissues were obtained from growing pigs in a weight range between 23 and 33 kg. All animals were fed twice daily and received 0.8–0.9 kg/day of a standard diet. After a 9‐ to 10‐day adaptation period the diets for treated animals were either supplemented for 8 days with 1.7×107 colony‐forming units (CFU)/g feed of S. boulardii or for 3 weeks with 106 CFU/g feed B. cereus var. toyoi. Under basal conditions Isc values were not affected by different treatment protocols (controls: 0.74 ± 0.04 µeq/cm2 per h, n=9; S. boulardii: 0.74 ± 0.12 µeq/cm2 per h, n=7; B. cereus 0.68 ± 0.09 µeq/cm2 per h, n=5). Irrespective of dietary treatment, the addition of glucose resulted in significant increases of Isc indicating substantial onset of electrogenic net Na/glucose cotransport. Maximal Isc values occurred within 30 min and reached 2.79 ± 0.41 µeq/cm2 per h in control epithelia. This was significantly lower than found in S. boulardii (4.47 ± 0.43 µeq/cm2 per h, p &lt; 0.05) and B. cereus var. toyoi tissues (4.45 ± 0.31 µeq/cm2 per h, p &lt; 0.05). Gt values were 22.4 ± 1.3 mS/cm2 in control animals and were significantly lower as shown in S. boulardii (p &lt; 0.01) and B. cereus var. toyoi (p &lt; 0.01)‐treated animals (28.4 ± 1.3 and 29.9 ± 0.8 mS/cm2, respectively). Vmax values of Na‐dependent glucose uptake into BBMV differed significantly between controls (0.64 ± 0.08 nmol/mg protein per 10 s; n=5), S. boulardii (0.89 ± 0.06 nmol/mg protein per 10 s; n=5, p &lt; 0.05) and B. cereus var. toyoi preparations (1.08 ± 0.05 nmol/mg protein per 10 s; n=3, p &lt; 0.01). Km values were not significantly affected (control: 0.31 ± 0.04 mmol/l, S. boulardii: 0.29 ± 0.05 mmol/l, B. cereus var. toyoi: 0.21 ± 0.01 mmol/l). Irrespective of dietary treatment, application of the dipeptide model substances glycyl‐ l‐sarcosine or glycyl‐ l‐glutamine resulted in significant increases of Isc indicating marked stimulation of electrogenic net H+/dipeptide cotransport. Highest Isc responses occurred in B. cereus var. toyoi preparations and lowest were found in control tissues. However, these differences were not significant. Gt values were not affected by different dietary treatments. The results clearly demonstrate that oral administration of either S. boulardii or B. cereus var. toyoi stimulates Na‐dependent glucose absorption in pig jejunum.

Regulation of proton: Dipeptide co-transport by glucocorticoids during chronic enteritis

Regulation of proton: Dipeptide co-transport by glucocorticoids during chronic enteritis

Apical uptake of radiolabelled ochratoxin A into Madin–Darby canine kidney cells

Uptake of ochratoxin A (OTA) across the apical cell membrane of collecting duct cells is the first step in reabsorption and partly mediated by proton–dipeptide cotransport. As the remaining part of apical OTA uptake remained unclear, we studied the characteristics of apical uptake of tritium-labelled OTA ( 3H-OTA) in MDCK-C11 cells in detail. Uptake of 3H-OTA was pH- and temperature-dependent and led to intracellular accumulation of OTA. Lowering pH led to an increase and lowering temperature (4°C) to a decrease of OTA uptake. Besides dipeptides, the β-lactam antibiotics cephalexin and ceftibuten inhibited the 3H-OTA uptake also confirming the role of the proton–dipeptide cotransporter. In addition, substrates of organic anion transporter, taurocholate and methotrexate, inhibited 3H-OTA uptake in part. Aspartylphenylalanine methyl ester (aspartame) had no inhibitory effect on 3H-OTA uptake. Uptake of OTA was not dependent on sodium. Sixty minutes of preincubation with phorbol 12-myristate 13-acetate (PMA) led to increased apical uptake of OTA. The PMA effects were inhibited by ethylisopropylamilorid (EIPA). We conclude that apical uptake of OTA occurs by Na +-independent transport. One part of the uptake is mediated by proton–dipeptide cotransport (30%, dipeptide-inhibitable), by organic anion transporter (20%, taurocholate-inhibitable) and by diffusion (20%, responsible for uptake at 4°C). The remaining part occurs by as yet unidentified but pH-dependent transport mechanisms. An acidic urine in distal parts of the nephron provides thus the main risk for OTA uptake leading to its reabsorption and consequently alkalinisation of the urine should help to prevent this reabsorption.

5'-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter.

General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5' -amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleoside antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycly ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5' -amino acid ester prodrugs was characterized in three different experimental systems; in situ rat perfusion model, CHO/hPEPT1 cells and Caco-2 cells. Testing 5' -amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPT1, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H+/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5' - amino acid ester prodrugs enhanced the transcellular transport of the parent drug. This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.

Apical-to-basolateral transepithelial transport of Ochratoxin A by two subtypes of Madin-Darby canine kidney cells

In this study we investigated the transepithelial transport of Ochratoxin A (OTA), a potent nephrotoxin, across monolayers of two collecting duct-derived cells clones (Madin-Darby canine kidney cells (MDCK)-C7 and MDCK-C11 cells, resembling principal and intercalated cells, respectively) either from the apical to the basolateral side or vice versa. We cultured cells on permeable supports and compared the transport rates of OTA, p-aminohippuric acid (PAH) and fluorescein-labelled inulin. Monolayers of both cell clones translocated OTA from the apical to the basolateral side but not in the opposite direction. Transport rate across MDCK-C11 cell monolayers was 2.9-fold the transport rate across MDCK-C7 cell monolayers. OTA transport was temperature-dependent being reduced from 77.5 pmol/cm 2 per h to 10.1 pmol/cm 2 per h in MDCK-C11 and from 27.0 pmol/cm 2 per h to 7.6 pmol/cm 2 per h in MDCK-C7 cells when temperature was decreased from 37°C to 4°C. In both cell clones, the dipeptides carnosine and glycylsarcosine but not the amino acids glycine or phenylalanine had an inhibitory effect on OTA transport. In both cell clones, transepithelial transport of OTA was dependent on the apical pH (p K a of OTA=7.1). In an environment mimicking the transepithelial in vivo pH gradient to some extent with more acidic pH on the apical side than on the basolateral side, transport was 4-fold higher in both cell clones as compared to conditions when pH was 7.4 in both bath solutions. In the absence of a pH gradient, transport rates were similar to that at 4°C. Apical uptake of [ 3H]OTA was inhibited by carnosine and by glycylsarcosine and the uptake of [ 3H]carnosine was inhibited by OTA. Our results indicate that OTA is transported across the apical membrane of MDCK cells by both non-ionic diffusion and by a H +-dipeptide cotransporter. Thus, reabsorption of OTA in the collecting duct contributes to the observed long half life of OTA in the mammalian body.

Transport characteristics of ceftibuten, a new cephaloporin antibiotic, via the apical H+/dipeptide cotransport system in human intestinal cell line Caco-2: regulation by cell growth.

The intestinal epithelial cell line Caco-2 possesses the H+/dipeptide cotransport system responsible for uptake of oral cephalosporins. In this study, the transport characteristics of ceftibuten were examined from the viewpoint of cell growth in the Caco-2 cells. The uptake of cephalosporins by Caco-2 cell monolayers grown on plastic dishes was measured and analyzed kinetically. The uptake of ceftibuten was increased by lowering pH of the incubation medium and was inhibited by excess dipeptide. The transport activity of ceftibuten was dependent on the duration of analysis revealed that the development of ceftibuten uptake was due to not only a decrease in Km but also to an increase in Vmax value. The uptake of ceftibuten is mediated by the apical H+/dipeptide cotransport system which is regulated by cell growth and/or differentiation in the Caco-2 cells.

Bestatin transport in rabbit intestinal brush-border membrane vesicles

The effect of papain treatment on bestatin uptake by rabbit intestinal brush-border membrane vesicles (BBMVs) was studied. Papain treatment of BBMVs effectively diminished aminopeptidase activity but not bestatin uptake by a H +/dipeptide cotransporter. Bestatin uptake by BBMVs was composed of two saturable components, and after papain treatment the high-affinity component disappeared while the low-affinity component was retained. These findings suggest that high- and low-affinity components represent bestatin binding to aminopeptidase and the true uptake by the H +/dipeptide cotransporter, respectively.

Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2.

The localization and transport characteristics of dipeptide transporters of the intestinal epithelial cell line Caco-2 were examined by measuring the intracellular accumulation and transcellular flux of Bestatin, a dipeptide-like anticancer agent. When added to the apical surface of Caco-2 monolayers grown on microporous membrane filters, Bestatin was accumulated in the cells and was transported unidirectionally to the basolateral side. The cellular uptake of Bestatin from the basolateral as well as from the apical surface was inhibited by excess dipeptides. Bestatin accumulation from the apical surface was dependent on the pH of the incubation medium with an optimal pH of 6.0, whereas uptake from the basolateral surface was insensitive to the medium pH. Kinetic parameters for Bestatin uptake also indicated that the basolateral and apical dipeptide transporters could be distinguished from each other. A sulfhydryl reagent, p-chloromercuribenzene sulfonate, inhibited Bestatin accumulation from both surfaces, although the inhibitory effect on the basolateral transport was greater than that on the apical transport. These findings suggest not only that dipeptide transporters exist on both the apical and basolateral membranes of Caco-2 cells but also that the basolateral dipeptide transporter is distinct from the apical H(+)-dipeptide cotransporter.

Transport of bestatin in rat renal brush-border membrane vesicles

Bestatin [(2 S,3 R)-3-amino-2-hydroxy-4-phenylbutanoyl- l-leucine] is a dipeptide, comprising l-leucine and an unusual β-ammo acid. We studied its transport mechanism in rat renal brush-border membrane vesicles. Uptake of cephradine, an aminocephalosporin, by isolated brush-border membrane vesicles was trans-stimulated and cis-inhibited by bestatin, indicating that these drugs are transported via the same transport system(s). The uptake of bestatin was trans-stimulated by preloading the vesicles with glycylsarcosine, and was cis-inhibited by substrates for the H +/dipeptide cotransport system. Bestatin inhibited tetraethylammonium (an organic cation) uptake, and bestatin uptake was cis-inhibited by substrates for the H +/organic cation antiport system. In addition, bestatin uptake was stimulated by an outward H + gradient (the driving force for the H +/organic cation antiport system). These findings suggest that bestatin, in spite of being a dipeptide, is transported via not only the H +/dipeptide cotransport system but also the H +/organic cation antiport system in rat renal brush-border membrane.

Expression of human intestinal dipeptide transporter in Xenopus laevis oocytes

The human colon adenocarcinoma cell line Caco-2 retains the H +/dipeptide cotransporter. To identify the structure of the human dipeptide transporter, we have examined the expression of the transporter in Xenopus laevis oocytes injected with Caco-2 poly(A) +RNA, by monitoring the uptake of bestatin, a dipeptide-like anticancer agent. The bestatin uptake in the poly(A) +RNA-injected oocytes was inhibited by excess glycyl- l-leucine, and showed pH dependence (optimal pH of 5.5–6.0). These observations suggest that the human intestinal dipeptide transporter can be expressed functionally in Xenopus oocytes.

Factors affecting the microclimate pH in rat jejunum.

1. Single-barrelled pH-sensitive microelectrodes filled with liquid ion exchanger were used to study the layer of microclimate pH in the vicinity of the surface of rat jejunum in vitro. 2. During perfusion with a Na+-containing solution of pH 7.30, a layer having a pH gradient ranging from 7.30 (pH of the luminal bulk phase) to 6.05 +/- 0.03 (pH of the deepest region) was detected in eighteen different animals. The thickness of the layer was estimated to be 600-700 microns. No regional difference was seen along the height of the villus. 3. The addition of D-glucose to the perfusion solution significantly augmented the acidity of the deepest region without changing the thickness of the layer. On the other hand, the elimination of Na+ from the perfusion solution caused a significant reduction of the pH gradient. The lowest pH changed from a control value of 6.18 +/- 0.15 (n = 13 measurements from three animals) to 6.46 +/- 0.06 (n = 13). The gradient was sensitive to amiloride in the presence of Na+, K+, Ca2+ and Cl- had no significant effect on the microclimate pH. 4. Depletion of the surface mucus by treatment with dithiothreitol significantly raised the pH of the deepest region. 5. Glycylglycine and L-carnosine were found to reduce the microclimate pH gradient significantly, while glycine did not. 6. These results indicate that H+ secretion by the Na+-H+ antiport and the formation of mucus layer are important factors for maintaining the microclimate pH layer, and that H+-coupled co-transport, such as H+-dipeptide co-transport, causes a significant diminution of the microclimate pH gradient.