Intestinal Oligopeptide Transporter Research Articles

Dipeptide Derivatives of AZT: Synthesis, Chemical Stability, Activation in Human Plasma, hPEPT1 Affinity, and Antiviral Activity

5'-O-Dipeptide ester prodrugs of antiviral zidovudine (AZT) were designed to target the human intestinal oligopeptide transporter, hPEPT1, and were evaluated for their stability at pH 7.4 in buffer and in human plasma, affinity toward hPEPT1, cytotoxicity, and antiretroviral activity. The dipeptide esters of AZT undergo cyclization in buffer at pH 7.4 to release the parent drug at a rate that depends on the size of the side chains of the peptide carrier; the prodrug is considerably more stable if bulky beta-branched amino acids such as Ile and Val are present, particularly as C-terminal residues. Incubation in human plasma showed that most of the dipeptide esters of AZT release the parent drug through two aminopeptidase-mediated pathways: 1) stepwise cleavage of each of the amino acids and 2) direct cleavage of the dipeptide-drug ester bond. However, the plasma hydrolysis of Gly-Gly-AZT and Phe-Gly-AZT showed only direct cleavage of the dipeptide-drug ester bond. Substrate half-lives in plasma were again remarkably high when hydrophobic beta-branched amino acids (Val, Ile) were present. The esters were also good substrates for the intestinal oligopeptide transporter hPEPT1 in vitro, with Val-Gly-AZT and Val-Ala-AZT presenting the highest affinity toward the transporter (IC(50): 0.20 and 0.15 mM, respectively). The AZT dipeptide esters were assayed against the IIIB and ROD strains of HIV, and their cytotoxicity was evaluated in MT-4 cells. The selectivity index of the prodrugs was two- to threefold higher than that of AZT for all compounds analyzed. These results point to the potential of dipeptide-based carriers for the development of effective antiviral drug-delivery systems. Val-Ala-AZT appears to combine chemical stability with good affinity for the hPEPT1 transporter and an improved cytotoxicity/antiretroviral index relative to AZT.

Abnormal Expression of the Peptide Transporter PepT1 in the Colon of Massive Bowel Resection Rat: A Potential Route for Colonic Mucosa Damage by Transport of fMLP

The aim of this study was to investigate the effect of abnormal intestinal oligopeptide transporter (PepT1) on rat colon inflammation by transportion of N-formyl-methionyl-leucyl-phenylalanine (fMLP). We induced upregulation of PepT1 in the colon of rats by 80% small bowel resection and examined colonic PepT1 di-tripeptide transport activity. By Western blot analysis, PepT1 was clearly detected in the colon of bowel resection rats, while it was absent from the colon of bowel transection and reanastomosis rats. The experiment with cephalexin colon perfusion showed that the arterial cephalexin concentration in resection rats was five to nine times that in transection rats. Inhibition of PepT1 by Gly-Gly completely abolished cephalexin absorption from the colon of resection rats. We found that 10 microM fMLP perfusion in the colon of resection rats for 4 hr significantly increased myeloperoxidase (MPO) activity and caused colon wall damage. In contrast, 10 muM fMLP perfusion in the colon of transection rats did not induce any inflammation. A 5 mM Gly-Gly perfusion completely inhibited the MPO activity and colonic wall damage induced by 10 muM fMLP. We conclude that colonic PepT1 induced by bowel resection may provide a mechanism for oligopeptide transport and may serve as a potential cause of colonic mucosa damage by transport of the bacterial product fMLP in rat colon.

PepT1 Mediates Colon Damage by Transporting fMLP in Rats with Bowel Resection

The intestinal oligopeptide transporter (PepT1) is responsible for the absorption of nutrient di-tripeptide and mediates the transportation of bacterial product N-formylmethionylleucyl-phenylalanine (fMLP) that induces intestinal inflammation. PepT1 is absent from normal colon epithelia but is abnormally expressed in the colon of patients with inflammatory bowel diseases. This study was designed to investigate the role of PepT1 in colonic inflammation and its relation with fMLP. PepT1 mRNA expression was analyzed by RT-PCR and oligopeptide transportation was quantified by the luminal perfusion with cephalexin in rats following 80% small bowel resection. The inflammatory effects of PepT1-mediated fMLP transport were also investigated by the measurement of myeloperoxidase (MPO) activity, histological analysis, and the use of Gly-Gly (Glycine-Glycine, a competitive PepT1 blocker). The overexpression of PepT1 was observed, peaking at 2 weeks post-operation, with its levels declining to 12% at week 4. The ability of oligopeptide transport was correlated with the PepT1 levels. The fMLP perfusion into the colon of rats with bowel resection resulted in an increased MPO activity and mucosal inflammation at week 2, but not at week 4 post-operation. The administration of Gly-Gly (50 mm) competitively inhibited PepT1, reducing the fMLP-associated MPO activation and colonic mucosal damage. The colonic PepT1 overexpression induced by bowel resection increases the transport of bacterial product fMLP and hence causes colonic inflammation, which may serve as a previously unrecognized pathway resulting in colon mucosa damage.

Human PEPT1 Pharmacophore Distinguishes between Dipeptide Transport and Binding

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.

Amino Acid Ester Prodrugs of the Antiviral Agent 2-Bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole as Potential Substrates of hPEPT1 Transporter

Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) were synthesized and evaluated for their affinity for hPEPT1, an intestinal oligopeptide transporter. Assays of competitive inhibition of [(3)H]glycylsarcosine (Gly-Sar) uptake in HeLa/hPEPT1 cells by the amino acid ester prodrugs of BDCRB suggested their 2- to 4-fold higher affinity for hPEPT1 compared to BDCRB. Further, promoieties with hydrophobic side chains and l-configuration were preferred by the hPEPT1 transporter.

Pharmacokinetics of novel dipeptide ester prodrugs of acyclovir after oral administration: intestinal absorption and liver metabolism.

The amino acid prodrug of acyclovir (ACV), valacyclovir (VACV), is an effective antiherpetic drug. Systemic availability of ACV in humans is 3 to 5 times higher after oral administration of VACV. Enhanced bioavailability of VACV has been attributed to its carrier-mediated intestinal absorption via hPEPT1 peptide transporter followed by rapid and complete conversion to ACV. An earlier report suggested that the dipeptide ester prodrugs of ACV possess high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of oral herpes virus infections. In the present study, we have examined the bioavailability of a series of dipeptide prodrugs of ACV after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The area under plasma-concentration time curves expressed as minutes microgram milliliter(-1) for total concentration of VACV (208.4 +/- 41.2), and the dipeptide prodrugs Gly-Val-ACV (GVACV) (416.1 +/- 140.9), Val-Val-ACV (VVACV) (147.7 +/- 89.3), and Val-Tyr-ACV (VYACV) (180.7 +/- 81.2) were significantly higher than that of ACV (21.2 +/- 5.2) upon intestinal absorption. Interestingly, the bioavailability of ACV after administration of GVACV was approximately 2-fold higher than VACV. There was significant metabolism by hepatic first pass effect of the dipeptide prodrugs as evident by the higher levels of ACV obtained after systemic absorption compared with intestinal absorption of GVACV and VVACV. The dipeptide prodrugs of ACV exhibited higher systemic availability of regenerated ACV upon oral administration and thus seem to be promising drug candidates in treatment of genital herpes infections.

Regulation of expression of the intestinal oligopeptide transporter (Pept-1) in health and disease.

The abundance of the oligopeptide transporter (Pept-1) in the brush-border membrane of the intestinal epithelium is the central mechanism for regulation of transport of products of protein digestion (dipeptides and tripeptides) and peptidomimetic drugs (for example, beta-lactam antibiotics). Within the past few years, there has been substantial progress in identifying the factors controlling this regulation and the mechanisms of their actions. The purpose of this report is to review this progress. The studies of individual substrates and hormones in a human intestinal cell line (Caco-2) have shown that dipeptides, certain amino acids, insulin, and leptin increase and epidermal growth factor and triiodothyronine decrease the membrane population of Pept-1. In the case of dipeptides, epidermal growth factor, and thyroid hormone, there are parallel changes in the gene expression brought about by alteration of transcription and/or stability of Pept-1 mRNA. In contrast, the treatment with insulin and leptin does not induce any alteration in the Pept-1 gene expression, and the mechanism of increased protein expression appears to be increased trafficking from a preformed cytoplasmic pool to the apical membrane. In vivo studies in rats have shown modulation of protein and gene expressions of the intestinal oligopeptide transporter during the day and during development and in nutritional and metabolic alterations, such as high-protein diet, fasting, and diabetes. Patients with intestinal diseases, such as ulcerative colitis, Crohn's disease, and short-bowel syndrome, may have induction of the Pept-1 expression in their colon. Finally, pharmacological studies have shown that the expression of Pept-1 can be upregulated by agents such as 5 fluorouracil and downregulated by agents such as cyclosporine. In conclusion, the above studies have produced a wealth of new information on regulation of a key transporter in the intestine. This information may have useful applications in nutritional and pharmacological treatments, for example, in diabetic patients needing enteral nutrition or in ulcerative colitis patients needing the suppression of the intestinal inflammation.

Interactions of the Dipeptide Ester Prodrugs of Acyclovir with the Intestinal Oligopeptide Transporter: Competitive Inhibition of Glycylsarcosine Transport in Human Intestinal Cell Line-Caco-2

The oligopeptide transporter may be exploited to enhance the absorption of drugs by synthesizing their dipeptide ester prodrugs, which may be recognized as its substrates. Various dipeptide esters of acyclovir (ACV), an antiviral nucleoside analog, were synthesized. Enzymatic hydrolysis and affinity of the prodrugs toward the human intestinal peptide transporter hPEPT1 were studied using the human intestinal Caco-2 cell line. Affinity studies were performed by inhibiting the uptake of [(3)H]glycylsarcosine by the prodrugs. The uptake of glycylsarcosine was found to be saturable at higher concentrations and was competitively inhibited by the prodrugs of ACV. All prodrugs except Tyr-Gly-ACV demonstrated a higher affinity (1.41-4.96 mM) toward hPEPT1 than cephalexin (8.19 +/- 2.12 mM), which was used as a positive control. Two prodrugs, Gly-Val-ACV and Val-Val-ACV, showed comparable affinity to Val-ACV, an amino acid prodrug of ACV recognized by PEPT1/PEPT2. The permeability of Gly-Val-ACV (2.99 +/- 0.59 x 10(-6) cm/s) across Caco-2 was comparable with that of Val-ACV (3.01 +/- 0.21 x 10(-6) cm/s) and was significantly inhibited (63%) in presence of glycylsarcosine. The transport of GVACV across Caco-2 was saturable at higher concentrations, and the parameters were calculated as K(m) 3.16 +/- 0.31 mM and V(max) 0.014 +/- 0.00058 nmol cm(-2) min(-1). Overall, the results suggest that the dipeptide prodrugs of ACV have a high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of ocular and oral herpesvirus infections, because cornea and intestinal epithelia seem to express the oligopeptide transporters.

Regulation of oligopeptide transporter (Pept-1) in experimental diabetes.

The knowledge of expression and biology of the intestinal oligopeptide transporter (Pept-1) in a metabolic disorder such as diabetes may have nutritional and pharmacological implications. To study this problem, rats were made diabetic by streptozotocin injection, and Western and Northern blot analyses and nuclear run-on assay were used to determine the protein and gene expressions of Pept-1 and its rate of transcription, respectively. Uncontrolled diabetes for 96 h increased the activity of Pept-1 in the brush-border membrane of intestinal mucosa. Studies of Michaelis-Menten constant, maximal velocity, and protein expression of Pept-1 indicated that an increase in the abundance of this transporter was responsible for the increased activity. Studies of the gene expression showed that uncontrolled diabetes increased the abundance of mRNA encoding Pept-1 without altering its rate of transcription. Lastly, studies of the specificity of the above effect showed that uncontrolled diabetes similarly affected the protein and gene expressions of Pept-1 located in the kidney. In conclusion, the data show that 1) uncontrolled diabetes has a tropic effect on Pept-1 and 2) the effect is systemic, and its molecular mechanism appears to be an increase in the stabilization of mRNA encoding Pept-1.

Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line

The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the stabilized dipeptides d-Glu–Ala and d-Asp–Ala as pro-moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding d-Glu–Ala and d-Asp–Ala model prodrugs’ affinity for hPepT1 in Caco-2 cells. A second aim was to investigate the transepithelial transport and hydrolysis parameters for d-Asp(BnO)–Ala and d-Glu(BnO)–Ala across Caco-2 cell monolayers. In the present study, all investigated d-Asp–Ala and d-Glu–Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used to establish a QSAR of our benzyl alcohol modified model prodrugs, aided at elucidating the observed differences in model prodrug affinity for hPepT1; additionally, these data suggest that the hydrophobicity of the side-chain model drug is the major determinant in the compounds affinity for hPepT1. Transepithelial transport studies performed using Caco-2 cells of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala showed that the K m for transepithelial transport was not significantly different for the two compounds. The maximal transport rate of the carrier-mediated flux component does not differ between the two model prodrugs either. The transepithelial transport of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala follows simple kinetics, and the release of benzyl alcohol is pH-dependent, but unaffected by 1 mM of the esterase inhibitor Paraoxon in 80% human plasma and Caco-2 cell homogenate.

Model prodrugs for the intestinal oligopeptide transporter: model drug release in aqueous solution and in various biological media

The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a target for increasing intestinal transport of low permeability compounds by creating prodrugs designed for the transporter. Model ester prodrugs using the stabilized dipeptides d-Glu-Ala and d-Asp-Ala as pro-moieties for benzyl alcohol have been shown to have affinity for hPepT1. Furthermore, in aqueous solution at pH 5.5 to 10, the release of the model drug seems to be controlled by a specific base-catalyzed hydrolysis, indicating that the compounds may remain relatively stable in the upper small intestinal lumen with a pH of ∼6.0, but still release the model drug at the intercellular and blood pH of ∼7.4. Even though benzyl alcohol is not a low molecular weight drug molecule, these results indicate that the dipeptide prodrug principle is a promising drug delivery concept. However, the physico-chemical properties such as electronegativity, solubility, and log P of the drug molecule may also have an influence on the potential of these kinds of prodrugs. The purpose of the present study is to investigate whether the model drug electronegativity, estimated as Taft substitution parameter ( σ*) may influence the acid, water or base catalyzed model drug release rates, when released from series of d-Glu-Ala and d-Asp-Ala pro-moieties. Release rates were investigated in both aqueous solutions with varying pH, ionic strength, and buffer concentrations as well as in in vitro biological media. The release rates of all the investigated model drug molecules followed first-order kinetics and were dependent on buffer concentration, pH, ionic strength, and model drug electronegativity. The electronegativity of the model drug influenced acid, water and base catalyzed release from d-Asp-Ala and d-Glu-Ala pro-moieties. The model drug was generally released faster from d-Asp-Ala- than from the d-Glu-Ala pro-moieties. In biological media the release rate was also dependent on the electronegativity of the model drug. These results demonstrate that the model drug electronegativity, estimated as Taft ( σ*) values, has a significant influence on the release rate of the model drug.

Functional and molecular expression of intestinal oligopeptide transporter (Pept-1) after a brief fast

The intestinal oligopeptide transporter, cloned as Pept-1, has major roles in the assimilation of dietary proteins and absorption of peptidomimetic medications. The initial aim of the present experiment was to investigate whether the functional expression of this transporter is affected by dietary intake. Functional expression was determined as the rate of uptake of glycylglutamine (GlyGln) by brush-border membrane vesicles (BBMVs) prepared from the jejunum of fed and fasted rats. Surprisingly, the rate of dipeptide uptake was greatly increased after 1 day of fasting. The subsequent aim of the experiment became the investigation of the mechanism of this alteration in transport, which showed that 1 day of fasting increased (1) the maximal GlyGln uptake (V max) by twofold without changing the K m of GlyGln uptake by BBMVs, (2) the amount of intestinal oligopeptide transporter (Pept-1) protein by threefold in the brush-border membrane, and (3) the abundance of Pept-1 mRNA by threefold in the intestinal mucosa. We conclude that 1 day of fasting increases dipeptide transport in rat intestine by increasing the population of Pept-1 in the brush-border membrane. The mechanism appears to be an increase in Pept-1 gene expression.

Hormonal regulation of oligopeptide transporter pept-1 in a human intestinal cell line.

The intestinal oligopeptide transporter (cloned as Pept-1) has major roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. In this experiment, we investigated whether insulin has such a role. We used a human intestinal cell monolayer (Caco-2) as the in vitro model of human small intestine and glycylglutamine (Gly-Gln) as the model substrate for Pept-1. Results showed that addition of insulin at a physiological concentration (5 nM) to incubation medium greatly stimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blocked when genistein, an inhibitor of tyrosine kinase, was added to incubation medium. Studies of the mechanism of insulin stimulation showed the following. 1) Stimulation occurred promptly (30-60 min) after exposure to insulin. 2) There was no significant change in the Michaelis-Menten constant of Gly-Gln transport, but there was a nearly twofold increase in its maximal velocity. 3) Insulin effect persisted even when Golgi apparatus, which is involved in trafficking of newly synthesized Pept-1, was dismantled. 4) However, there was complete elimination of insulin effect by disruption of microtubules involved in trafficking of preformed Pept-1. 5) Finally, with insulin treatment, there was no change in Pept-1 gene expression, but the amount of Pept-1 protein in the apical membrane was increased. In conclusion, the results show that insulin, when it binds to its receptor, stimulates Gly-Gln uptake by Caco-2 cells by increasing the membrane population of Pept-1. The mechanism appears to be increased translocation of this transporter from a preformed cytoplasmic pool.

Overexpression of human intestinal oligopeptide transporter in mammalian cells via adenoviral transduction.

Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligopeptide transporter (hPepT1). This study characterizes the expression of hPepT1 in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction. A recombinant replication-deficient adenovirus carrying the hPepT1 gene was made and used as a vector for the expression of hPepT1. The increase in the uptake permeability of cephalexin and Gly-Sar was determined. The effects of time, dose, apical pH, and substrate specificity were evaluated. A significant increase in the uptake permeability of Gly-Sar and cephalexin was found in all three cell lines after viral transduction. The increase of Gly-Sar permeability in Hela. IEC-18, and Caco-2 cells was 85-, 46-, and 15-fold respectively. Immunoblotting using an antibody against hPepT1 detected high levels of a 85-98-kDa protein in all three infected cell lines. Substrate permeability was dependent on time of infection, inward pH gradients, and multiplicity of infection (MOI). Decreased infectivity and lower hPepT1 expression were observed in differentiated Caco-2 cells. The uptake was inhibited by dipeptides and beta-lactam antibiotics but not amino acids. Adenoviral infected Hela cells displayed a pronounced level of hPepT1 expression with a low background and high specificity to dipeptides. These features make this system a useful tool for screening of potential substrates. The success of overexpression of hPepT1 in Caco-2 and IEC-18 cells may lead to a novel approach in improving oral absorption of peptides and peptidornimetic drugs.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide.

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na(+)-dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na(+)-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na+- dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na+-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes. (Hepatology 1996 Aug;24(2):380-4)

Intestinal Oligopeptide Transporter: From Hypothesis to Cloning

Several decades ago it was hypothesized that there is an active intestinal oligopeptide transporter that allows large-scale absorption of dipeptides and tripeptides. After a period of uncertainty, this hypothesis was recently validated by the cloning of an intestinal oligopeptide transporter that appears to belong to a superfamily of proton-dependent transporters.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day-old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

Molecular modeling study of structural requirements for the oligopeptide transporter.

The intestinal oligopeptide transporter (OPT) mediates the absorption of di-/tripeptides, beta-lactam antibiotics, angiotensin converting enzyme (ACE) inhibitors and renin inhibitors. This suggests that the targeting of molecules to this transporter could result in orally-absorbed drugs. Results from a recent study with renal brush border membrane vesicles (BBMV) suggested that an alpha-NH2 group is required for interaction with the renal OPT. In general, structural requirements for interaction with the renal and intestinal OPT are similar. However, these recent findings do not agree with earlier studies, which showed that an alpha-NH2 group is not essential for interaction with the intestinal OPT. Thus, it appears that the renal and intestinal OPT may differ in their recognition of compounds containing an alpha-NH2 group. In this study, molecular modeling was used to determine the tridimensional structures of various cephalosporins for which Ki values had been determined using renal BBMV. All cephalosporins which interact with the OPT have two, energetically equivalent, conformations. Most compounds which do not interact with the OPT cannot adopt the two conformations. A key factor which influences the conformation seems to be the substituent group at the alpha position; an electron drawing group at that position alters the common conformations. For the OPT substrates, the distances between the -NH2 and -COOH groups are comparable to those of the tripeptide, GlyGlyGly; and the distances between -NH2 and carbonyl group in the beta-lactam ring are close to the distance between N-terminal and C-terminal in the dipeptide, GlyGly. The corresponding distances in cephamycin C (in which a -NH2 group is located in a different position) and the tetrapeptide, GlyGlyGlyGly, are longer than those in alpha-NH2 cephalosporins and GlyGlyGly. Cephamycin C and the tetrapeptide have low affinity for the renal OPT, suggesting that the distances between functional groups are critical for affinity. The alpha-NH2 group had no effect on the conformations of the molecules. We concluded that the alpha-NH2 group may interact directly with the renal oligopeptide transporter. Whether this is unique to the renal transporter or could be applied to the intestinal transporter will require further investigation.

TISSUE AND INTRA-CELLULAR DISTRIBUTION OF INTESTINAL OLIGOPEPTIDE TRANSPORTER AND ITS FUNCTIONAL CHARACTERIZATION

Function and tissue and intra-cellular distributions of rat intestinal oligopeptide transporter, rat PepT1 cloned as a homologue of rabbit PepT1, were studied. Functional characteristics of a rat PepT1 were evaluated by measuring transport activities expressed in Xenopus laevis oocytes injected with complementary RNA for PepT1 synthesized by in vitro transcription. Rat PepT1 exhibited a pH-dependent transport of [14C]glycylsarcosine and the transport was specifically inhibited by di- and tripeptides but not by their constitutive amino acids or tetra- and pentapeptides. Rat PepT1 also transported β-lactam antibiotics with pH-dependent and stereoselective manners. Furthermore, transport activities expressed in oocytes injected with intestinal total mRNA were abolished by the hybridization of the mRNA with an antisense oligonucleotide against PepT1. Polyclonal antibody against PepT1 was prepared and used for immunohistochemical analysis of PepT1. PepTi was strongly detected in intestinal epithelium, especially in the brush-border side. Accordingly, PepT1 was shown to function in the uptakes of oligopeptides and peptide-like drugs from intestinal lumen to epithelial cells.