Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo

Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide with anti-hepatitis activity that has been chemically synthesized. Previous experiments in rats showed that JBP485 was well absorbed by the intestine after oral administration. The human peptide transporter (PEPT1) is expressed in the intestine and recognizes compounds such as dipeptides and tripeptides. The purposes of this study were to determine if JBP485 acted as a substrate for intestinal PEPT1, and to investigate the characteristics of JBP485 uptake and transepithelial transport by PEPT1. The uptake of JBP485 was pH dependent in human intestinal epithelial cells Caco-2. And JBP485 uptake was also significantly inhibited by glycylsarcosine (Gly-Sar, a typical substrate for PEPT1 transporters), JBP923 (a derivative of JBP485), and cephalexin (CEX, a β-lactam antibiotic and a known substrate of PEPT1) in Caco-2 cells. The rate of apical-to-basolateral transepithelial transport of JBP485 was 1.84 times higher than that for basolateral-to-apical transport. JBP485 transport was obviously inhibited by Gly-Sar, JBP923 and CEX in Caco-2 cells. The uptake of JBP485 was increased by verapamil but not by cyclosporin A (CsA) and inhibited by the presence of Zn²⁺ or the toxic metabolite of ethanol, acetaldehyde (AcH) in Caco-2 cells. The in vivo uptake of JBP485 was increased by verapamil and decreased by ethanol in vivo, which was consisted with the in vitro study. PEPT1 mRNA levels were enhanced after exposure of the cells to JBP485 for 24 h, compared to control. In conclusion, JBP485 was actively transported by the intestinal oligopeptide transporter PEPT1. This mechanism is likely to contribute to the rapid absorption of JBP485 by the gastrointestinal tract after oral administration.     

Effects of JBP485 on the expression and function of PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells

To investigate the effect of JBP485 (an anti-inflammatory dipeptide) on PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells, the activity and expression of PEPT1 were examined. The effects of treatment with indomethacin and co-treatment with JBP485 were examined in terms of intestinal histological changes, MDA and MPO levels in rats; as well as LDH-release and oxidative stress in Caco-2 cells. Uptake of glycylsarcosine (Gly-Sar) by PEPT1 was determined by in vivo, in vitro and in situ studies. RT-PCR and Western blot were used to assess the expression of PEPT1 in rat intestine and Caco-2 cells. JBP485 caused a significant decrease in MDA and MPO levels, and improved the pathological condition of rat intestine, while attenuating Caco-2 cells damage induced by indomethacin. Uptake of Gly-Sar by PEPT1 was decreased by indomethacin treatment, whereas the Gly-Sar plasma concentration was markedly increased in JBP485 co-treated rats. Indomethacin down-regulated the expression of PEPT1 mRNA and protein in rat intestine and Caco-2 cells, and the effects were reversed after administration of JBP485. These results indicated that JBP485 not only improved intestinal injury and cell damage but also partially blocked the down-regulation of PEPT1 expression and function induced by indomethacin.     

Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells

Aims

The purpose of this study was to clarify the expression and function of peptide transporter 2 (PEPT2) in primary cultured alveolar type II epithelial cells and in transdifferentiated type I-like cells.

Main methods

Real-time PCR analysis, uptake study of [³H]Gly-Sar, and immunostaining were performed in alveolar epithelial cells.

Key findings

The expression of PEPT2 mRNA in type II cells isolated from rat lungs was highest at day 0, and decreased rapidly during culture of the cells. In accordance with this change, PEPT2 activity estimated as cefadroxil-sensitive [³H]Gly-Sar uptake also decreased along with transdifferentiation. The expression of PEPT2 protein in type II cells was confirmed by immunostaining and Western blot analysis. The uptake of [³H]Gly-Sar in type II cells was time- and pH-dependent. In contrast, minimal time-dependence and no pH-dependence of [³H]Gly-Sar uptake were observed in type I-like cells. The maximal [³H]Gly-Sar uptake was observed at pH 6.0, and the uptake decreased at higher pHs in type II cells. The uptake of [³H]Gly-Sar in type II cells was inhibited by cefadroxil in a concentration-dependent manner, the IC₅₀ value being 4.3 μM. On the other hand, no significant inhibition by cefadroxil was observed in type I-like cells. In addition, [³H]Gly-Sar uptake in type II cells was saturable, the Km value being 72.0 μM.

Significance

PEPT2 is functionally expressed in alveolar type II epithelial cells, but the expression decreases along with transdifferentiation, and PEPT2 would be almost completely lost in type I cells.     

Influence of proton and essential histidyl residues on the transport kinetics of the H+/peptide cotransport systems in intestine (PEPT 1) and kidney (PEPT 2)

The mechanism by which H⁺ alters the kinetics of the H⁺-coupled peptide transporters PEPT 1 and PEPT 2 was investigated in two different cell lines which differentially express these transporters, namely Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). The effects of H⁺ on the affinity and the maximal velocity of Gly-Sar uptake were analyzed in these cells under identical conditions. In both cells, H⁺ influenced only the maximal velocity of uptake and not the apparent affinity. The effects of H⁺ on the IC₅₀ values (i.e., concentration necessary to cause 50% inhibition) of the cationic dipeptide Ala-Lys and the anionic dipeptide Ala-Asp for inhibition of Gly-Sar uptake were also investigated. H⁺ did not change the IC₅₀ value for Ala-Lys but did decrease the IC₅₀ value for Ala-Asp considerably. The influence of diethylpyrocarbonate (DEP) on the kinetic parameters of PEPT 1 and PEPT 2 was then studied. Histidyl residues are the most likely amino acid residues involved in H⁺ binding and translocation in H⁺-coupled transport systems and DEP is known to chemically modify histidyl residues and block their function. DEP treatment altered the maximal velocity of Gly-Sar uptake but had no effect on its K_t (Michaelis-Menten constant) or the IC₅₀ values of Ala-Lys or Ala-Asp for the inhibition of Gly-Sar uptake. It is concluded that H⁺ stimulates PEPT 1 and PEPT 2 primarily by increasing the maximal velocity of the transporters with no detectable influence on the substrate affinity.     

Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells.

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H(+)/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [(14)C]glycylsarcosine with K(i) values of 0.19 +/- 0.01 mm and 0.07 +/- 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K(t)) but not the maximal velocity (V(max)) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [(14)C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [(14)C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [(14)C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [(14)C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells trade mark, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H(+)/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H(+)-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H(+)/peptide cotransporters.     

sigma Receptor ligand-induced up-regulation of the H(+)/peptide transporter PEPT1 in the human intestinal cell line Caco-2.

We determined the effects of (+)pentazocine, a selective sigma(1) ligand, on the uptake of glycylsarcosine (Gly-Sar) in the human intestinal cell line Caco-2 which expresses the low affinity/high capacity peptide transporter PEPT1. Confluent Caco-2 cells were treated with various concentrations of (+)pentazocine for desired time (mostly 24 hr). The activity of PEPT1 was assessed by measuring the uptake of [(14)C]Gly-Sar in the presence of a H(+) gradient. (+)Pentazocine increased the uptake of [(14)C]Gly-Sar mediated by PEPT1 in a concentration- and time-dependent manner. Kinetic analyses have indicated that (+)pentazocine increased the maximal velocity (V(max)) for Gly-Sar uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). In addition, semi-quantitative RT-PCR revealed that treatment of (+)pentazocine increased PEPT1 mRNA in Caco-2 cells in a concentration-dependent manner. These data suggest that sigma(1) receptor ligand (+)pentazocine up-regulates PEPT1 in Caco-2 cells at the level of increased mRNA, causing an increase in the density of the transporter protein in the cell membrane.     

Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters

In this study we described the design, rational synthesis and functional characterization of a novel radiolabeled hydrolysis-resistant high-affinity substrate for H(+)/peptide cotransporters. L-4,4'-Biphenylalanyl-L-Proline (Bip-Pro) was synthesized according to standard procedures in peptide chemistry. The interaction of Bip-Pro with H(+)/peptide cotransporters was determined in intestinal Caco-2 cells constitutively expressing human H(+)/peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively expressing rat H(+)/peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the [(14)C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (K(i) = 24 microm and 3.4 microm, respectively) in a competitive manner. By employing the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by both peptide transporters although to a much lower extent than the reference substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h when incubated with intact cell monolayers. Bip-[(3)H]Pro uptake into SKPT cells was linear for up to 30 min and pH dependent with a maximum at extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled Bip-Pro but also by known peptide transporter substrates such as dipeptides, cefadroxil, Ala-4-nitroanilide and delta-aminolevulinic acid, but not by glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis-Menten constant (K(t)) of 7.6 microm and a maximal velocity (V(max)) of 1.1 nmol x 30 min(-1) x mg of protein(-1). Hence, the uptake of Bip-Pro by PEPT2 is a high-affinity, low-capacity process in comparison to the uptake of Gly-Sar. We conclude that Bip-[(3)H]Pro is a valuable substrate for both mechanistic and structural studies of H(+)/peptide transporter proteins.     

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.     

Inhibitory effect of supramolecular polyrotaxane-dipeptide conjugates on digested peptide uptake via intestinal human peptide transporter

The effect of polyrotaxane-dipeptide (Val-Lys) conjugates on the uptake of a model dipeptide (Gly-Sar) was examined via human peptide transporter (hPEPT1) on HeLa cells. Here, Val-Lys groups are introduced to alpha-CDs, which are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups (polyrotaxane). The Gly-Sar uptake via hPEPT1 was significantly inhibited in the polyrotaxane conjugates, and this inhibitory effect was not explained by the sum of interaction between hPEPT1 and alpha-CD-Val-Lys conjugates. Further, the inhibition was significantly greater than those observed in dextran-Val-Lys conjugates. Therefore, our data clearly suggests that supramolecular structure in the polyrotaxane conjugates contributes considerably to the inhibitory effect via multivalent binding of Val-Lys groups with hPEPT1.     

Organic anion transporters involved in the excretion of bestatin in the kidney

Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.     

Possible role of PEPT1 in gastrointestinal hormone secretion

Oligopeptides originating from ingested meal stimulate the secretion of various gastrointestinal hormones, but the mechanism is unknown. In this study, we show that transfection of oligopeptide transporter 1 (PEPT1) in STC-1 cells, a murine enteroendocrine cell line, evokes di-peptide-stimulated hormone secretion in a pH-dependent manner. Measurement of membrane potentials shows that PEPT1- transfected STC-1 cells are depolarized by di-peptide glycyl-glycine but not by glycine monomer. Glycyl-glycine stimulation induces a rise in the intracellular calcium concentration in PEPT1-transfected STC-1 cells. The secretion induced by glycyl-glycine in PEPT1-transfected STC-1 cells was blocked by nifedipine, a Ca(2+) channel blocker, suggesting that the secretion is triggered by Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. These data suggest that PEPT1 mediates oligopeptide-induced hormone secretion in enteroendocrine cells.     

USP18 Sensitivity of Peptide Transporters PEPT1 and PEPT2

USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (I_gly-gly). Coexpression of USP18 significantly increased I_gly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal I_gly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased I_gly-gly. Coexpression of USP30 similarly increased I_gly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

The objective was to determine whether protective effects of JBP485 on biliary obstruction induced by alpha-naphthylisothiocyanate (ANIT) are mediated by the organic anion transporters Oat1, Oat3 and the multidrug resistance-associated protein Mrp2. The ANIT-induced increases in bilirubin (BIL), alanine aminotransferase (ALT) and aspartate transaminase (AST) in rat serum were inhibited significantly by oral administration of JBP485. The plasma concentration of JBP485 which is the substrate of Oat1 and Oat3 determined by LC–MS/MS was markedly increased after intravenous administration in ANIT-treated rats, whereas cumulative urinary excretion of JBP485 in vivo and the uptake of JBP485 in kidney slices were decreased remarkably. RT-PCR and Western blot showed the decreased expression of Oat1 and Oat3, increased expression of Mrp2 in ANIT-induced rats, meanwhile, the expression levels of Mrp2 and Oat1 were up-regulated after administration of JBP485. The up-regulation of Mrp2 and Oat1 was associated with a concomitant increase in urinary BIL after treatment with JBP485 in ANIT-treated rats. The mechanism for JBP485 to restore liver function might be related to improvement of the expression and function for Oat1 and Mrp2 as well as facilitation of urinary excretion for hepatoxic substance.     

Effect of Janus Kinase 3 on the Peptide Transporters PEPT1 and PEPT2

The tyrosine kinase Janus kinase 3 (JAK3) contributes to signaling regulating the proliferation and apoptosis of lymphocytes and tumor cells. Replacement of lysine by alanine in the catalytic subunit yields the inactive ^K851AJAK3 mutant that underlies severe combined immune deficiency. The gain-of-function mutation ^A572VJAK3 is found in acute megakaryoplastic leukemia and T cell lymphoma. The excessive nutrient demand of tumor cells requires upregulation of transporters in the cell membrane including peptide transporters PEPT1 and PEPT2. The carriers further accomplish intestinal peptide transport. Little is known about signaling regulating peptide transport. The present study explored whether PEPT1 and PEPT2 are upregulated by JAK3. PEPT1 or PEPT2 was expressed in Xenopus oocytes with or without additional expression of JAK3, and electrogenic peptide (glycine–glycine) transport was determined by dual-electrode voltage clamp. PEPT2-HA membrane protein abundance was analyzed by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide-induced current in Ussing chamber experiments. In PEPT1- and PEPT2-expressing oocytes, but not in water-injected oocytes, the dipeptide gly–gly generated an inward current, which was significantly increased following coexpression of JAK3. The effect of JAK3 on PEPT1 was mimicked by ^A568VJAK3 but not by ^K851AJAK3. JAK3 increased maximal peptide-induced current in PEPT1-expressing oocytes but rather decreased apparent affinity of the carrier. Coexpression of JAK3 enhanced the PEPT2-HA protein abundance in the cell membrane. In JAK3- and PEPT1-expressing oocytes, peptide-induced current was blunted by the JAK3 inhibitor WHI-P154, 4-[(3′-bromo-4′-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (22 μM). In intestinal segments gly–gly generated a current which was significantly smaller in JAK3-deficient mice (jak3 ^?/?) than in wild-type mice (jak3 ^+/+). In conclusion, JAK3 is a powerful regulator of peptide transporters PEPT1 and PEPT2.     

H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant (Kt) value of 1.1 mM. Several dipeptides, cefadroxil, and delta-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 +/- 29 omega x cm(2). The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.     

Mechanisms of 5-aminolevulinic acid uptake at the choroid plexus

5-Aminolevulinic acid (5-ALA) is a precursor of porphyrins and heme that has been implicated in the neuropsychiatric symptoms associated with porphyrias. It is also being used clinically to delineate malignant gliomas. The blood-CSF barrier may be an important interface for 5-ALA transport between blood and brain as in vivo studies have indicated 5-ALA is taken up by the choroid plexuses whereas the normal blood-brain barrier appears to be relatively impermeable. This study examines the mechanisms of 5-[(3)H]ALA uptake into isolated rat lateral ventricle choroid plexuses. Results suggest that there are two uptake mechanisms. The first was a Na(+)-independent uptake system that was pH dependent (being stimulated at low pH). Uptake was inhibited by the dipeptide Gly-Gly and by cefadroxil, an alpha-amino-containing cephalosporin. These properties are the same as the proton-dependent peptide transporters PEPT1 and PEPT2, which have recently been shown to transport 5-ALA in frog oocyte expression experiments. Choroid plexus uptake was not inhibited by captopril, a PEPT1 inhibitor, suggesting PEPT2-mediated uptake. The presence of PEPT2 and absence of PEPT1 in the choroid plexus were confirmed by western blotting. The second potential mechanism was both Na(+) and HCO(3)(-) dependent and appears to be an organic anion transporter, although it is possible that removal of Na(+) and HCO(3)(-) may indirectly affect PEPT2 by affecting intracellular pH. The presence of PEPT2 and a putative Na(+)/HCO(3)(-)-dependent organic anion transporter is important not only for an understanding of 5-ALA movement between blood and brain but also because these transporters may affect the distribution of a number of drugs between blood and CSF.     

Measurement of electric current evoked by substrate transport via bi-directional H+/oligopeptide transporter over-expressed in HeLa cells: electrogenic efflux and existence of a newly observed channel-like state

In the present study, we measured an electric current induced by substrate transport in a HeLa cell over-expressing a human intestinal di/tri-peptide transporter using the whole-cell patch-clamp technique. Gly-Sar, a typical substrate, induced an inward current associated with its uptake, which showed concentration-dependency following Michaelis-Menten-type kinetics with an apparent K(0.5) of 1.3mM as well as voltage-dependency. An outward current accompanying the efflux of Gly-Sar was also observed after washing out the cell. This outward current was voltage-dependent and was reduced by the inward proton gradient. In the case of hydrophobic dipeptides such as Gly-Phe and Gly-Leu, a distinctive current was observed: after washing out the cells, no outward current was observed, but rather, an 'inward leak' current was sustained in spite of the absence of transportable substrate. This leaky current was abolished by the perfusion of Gly-Sar and subsequent washing. It is considered that the hydrophobic substrate sticks within the substrate-binding site and causes the newly observed state, or the 'inward leak' current.     

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.