High-affinity Peptide Transporter Research Articles

Role of PEPT2 in peptide/mimetic trafficking at the blood-cerebrospinal fluid barrier: studies in rat choroid plexus epithelial cells in primary culture.

Recent studies have established the functional and molecular presence of a high-affinity peptide transporter, PEPT2, in whole tissue rat choroid plexus. However, the precise membrane location and directionality of PEPT2-mediated transport is uncertain at present. In this study, we examined the transport kinetics of a model dipeptide, glycylsarcosine (GlySar), along with the protein expression of PEPT2 using primary cell cultures of choroidal epithelium from neonatal rats. GlySar accumulation and transepithelial transport were 3 to 4 times higher when introduced from the apical as opposed to the basal side of the monolayers. GlySar apical uptake was also stimulated by an inwardly directed proton gradient. The uptake of GlySar was inhibited by di/tripeptides, carnosine, and alpha-amino cephalosporins but was unaffected by amino acids, cephalosporins lacking an alpha-amino group, and organic anions and cations. The Michaelis constant (K(m)) of GlySar was 59.6 microM for apical uptake and 1.4 mM for basal uptake; this is consistent with the high-affinity properties of PEPT2 at the apical membrane. Immunoblot analyses and immunofluorescent confocal microscopy demonstrated the presence of PEPT2, but not PEPT1, in rat choroid plexus epithelial cells. Moreover, PEPT2 was present in the apical and subapical regions of the cell but was absent in the basolateral membrane. These findings demonstrate, for the first time, that PEPT2 protein is present at the apical membrane of choroidal epithelial cells and that it is functionally active at this membrane surface. The results suggest that PEPT2 may have a role in the efflux of peptides and/or mimetics from cerebrospinal fluid to the blood.

Renal assimilation of short chain peptides: visualization of tubular peptide uptake.

Renal assimilation of short chain peptides plays an important role in systemic protein metabolism and amino acid homeostasis. The transepithelial peptide transport across the apical membrane of tubular cells is mediated almost exclusively by pH-dependent H(+)-peptide symport pathways. The current study was designed to identify by visualization functional peptide transport activity along the nephron structures. Visualization of peptide uptake was achieved by using the fluorescent dipeptide derivative D-Ala-Lys-AMCA and unlabelled cefadroxil and glycylglutamine as transport competitors to demonstrate specificity. To confirm these assays, rat specific cRNA probes were synthesized and non-isotopic high-resolution in-situ-hybridization and northern blot analysis were carried out to demonstrate the expression of the high-affinity peptide transporter PEPT2. The reporter molecule was accumulated by cells of the proximal tubulus but not in glomerular or endothelial cells. Inhibition studies revealed competitive inhibition of D-Ala-Lys-AMCA uptake by the betalactam cefadroxil and the dipeptide glycylglutamine. The control organs intestine and spleen did not show uptake of the systemically administered molecule. Non-isotopic mRNA in-situ-hybridization, using an antisense probe for rat PEPT2 confirmed up-take assays by identifying PEPT2 expression throughout segments of the straight proximal tubule at the inner cortex and outer stripe. We demonstrate for the first time renal in vivo transport activity of a dipeptide that allows cells that participate in peptide reabsorption to be visualized. This functional assay may be used to investigate renal peptide transport mechanisms and test new compounds that are transported via proton-driven peptide transporters.

Preferential recognition of zwitterionic dipeptides as transportable substrates by the high-affinity peptide transporter PEPT2

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. d-Phe-L-Gln (neutral), d-Phe-L-Glu (anionic), and d-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H +-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H +:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.

Distribution of peptide transporter PEPT2 mRNA in the rat nervous system.

The signaling action of neuropeptides in the brain is terminated by breakdown through extracellular peptidases and subsequent removal of the peptide fragments from the extracellular fluid via specific transporter proteins. Here we describe the anatomical distribution in the rat nervous system of the recently isolated high affinity peptide transporter PEPT2. Using nonisotopic in situ hybridization we demonstrate that PEPT2 mRNA is expressed in brain by astrocytes, subependymal cells, ependymal cells and epithelial cells of choroid plexus. Furthermore, PEPT2 is expressed in retina by Müller cells and in dorsal root ganglia by satellite cells. The mRNA levels of PEPT2 in astrocytes are moderate and relatively homogenous throughout the brain except for an area in ventral forebrain where PEPT2 levels are below average. PEPT2 mRNA expression is weakly upregulated in reactive astrocytes that were stimulated through an injection of the glutamatergic neurotoxin quinolinic acid. These data suggest that removal of neuropeptide fragments from brain extracellular fluid occurs via PEPT2 expressed in astrocytes, ependymal cells and choroid plexus epithelial cells.

Regulation of the high‐affinity H/peptide cotransporter in renal LLC‐PK1 cells

Di- and tripeptides and peptide mimetics such as β-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 μM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+]in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7 ± 2.5 μM to 10.2 ± 1.9 μM with no change in maximal velocity. J. Cell. Physiol. 178:341–348, 1999. © 1999 Wiley-Liss, Inc.

Regulation of the high-affinity H+/peptide cotransporter in renal LLC-PK1 cells.

Di- and tripeptides and peptide mimetics such as beta-lactam antibiotics are efficiently reabsorbed from the tubular lumen by a high-affinity peptide transporter. We have recently identified and characterized this H+-coupled high-affinity peptide transport system in the porcine proximal tubular cell line LLC-PK1. Here we describe for the first time the regulation of the renal high-affinity peptide cotransporter at the cellular level. Uptake of 5 microM 3H-D-Phe-L-Ala into LLC-PK1 cells was significantly increased by lowering [Ca2+]in and decreased by increasing [Ca2+] in. Moreover, it was shown that the [Ca2+]in effects on peptide transport activity were dependent on Ca2+ entry from the extracellular site (e.g., via a store-regulated capacitative Ca2+ influx). Protein kinase C (PKC) was found to transmit the effects of [Ca2+]in on peptide transport. Although we demonstrate by pHin measurements that the PKC inhibitor staurosporine did decrease the transmembrane H+ gradient and consequently should have reduced the driving force for peptide uptake, the only effect on transport kinetics of 3H-D-Phe-L-Ala observed was a significant decrease in Km from 22.7+/-2.5 microM to 10.2+/-1.9 microM with no change in maximal velocity.

Stoichiometry and Kinetics of the High-affinity H+-coupled Peptide Transporter PepT2

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (Ipss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited Ipss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant Ipss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized Vm, our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells.

The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptide D-[3H]Phe-L-Ala, 2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake of D-Phe-L-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport of D-Phe-L-Ala of 6.0, an apparent Km value for influx of 25.8 +/- 3. 6 microM, and affinities of differently charged dipeptides or the beta-lactam antibiotic cefadroxil to the binding site in the range of 20-80 microM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.

Peptide aminonitrogen transport by the lactating rat mammary gland

Recent studies have shown that the lactating mammary gland is able to utilize plasma-derived dipeptides for milk protein synthesis. However, it was not clear whether the peptides were hydrolysed followed by uptake of the constituent amino acids or were taken up intact. In view of this, we have designed experiments to investigate (a) whether the lactating rat mammary gland is capable of transporting hydrolysis-resistant dipeptides and (b) whether or not mammary cells are able to hydrolyse peptides, including glutathione, extracellularly. The uptake of the hydrolysis-resistant dipeptides d-[3H]Phe-l-Gln and d-[3H]Phe-l-Glu by the perfused rat mammary gland was low. Concomitant addition of l-Leu-l-Ala (50 mM) had no effect on the clearance of either labelled dipeptide suggesting that the small, albeit significant, uptake of the dipeptides is not via a high affinity peptide transporter (PepT1/PepT2). All anionic dipeptides tested (l-Glu-l-Ala, l-Asp-l-Ala, l-Ala-l-Asp, l-Asp-Gly, Gly-l-Asp and Gly-l-Glu) with the exception of d-Phe-l-Glu were able to trans-accelerate the efflux of labelled d-aspartate from preloaded rat mammary tissue (explants and perfused mammary gland). It appears that these peptides were being hydrolysed extracellularly followed by the uptake of free anionic amino acids via the mammary tissue high affinity, Na+-dependent anionic amino acid carrier operating in the exchange mode. Glutathione was able to trans-accelerate d-aspartate efflux from lactating rat mammary tissue in a fashion which was sensitive to the peptidase inhibitor acivicin. This suggests that γ-glutamyltranspeptidase hydrolyses glutathione to produce l-glutamate which is subsequently transported via the high-affinity anionic amino acid carrier. Hydrolysis of peptides followed by uptake of the constituent amino acids may provide an important source of amino acids for milk protein synthesis.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.