Hannelore Daniel

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Minimal Molecular Determinants of Substrates for Recognition by the Intestinal Peptide Transporter

Proton-dependent electrogenic transporters for di- and tripeptides have been identified in bacteria, fungi, plants, and mammalian cells. They all show sequence-independent transport of all possible di- and tripeptides as well as of a variety of peptidomimetics. We used the mammalian intestinal peptide transporter PEPT1 as a model to define the molecular basis for its multisubstrate specificity. By employing computational analysis of possible substrate conformations in combination with transport assays using transgenic yeast cells and Xenopus laevis oocytes expressing PEPT1, the minimal structural requirements for substrate binding and transport were determined. Based on a series of medium chain fatty acids bearing an amino group as a head group (ω-amino fatty acids, ω-AFA), we show that electrogenic transport by PEPT1 requires as a minimum the two ionized head groups separated by at least four methylene groups. Consequently, a > 500 pm < 630 pm distance between the two charged centers (carboxylic carbon and amino nitrogen) is sufficient for substrate recognition and transport. Removal of either the amino group or the carboxyl group in ω-AFA maintained the affinity of the compound for interaction with the transporter but abolished the capability for electrogenic transport. Additional groups in the ω-AFA backbone that provide more hydrogen bonding sites appear to increase substrate affinity but are not essential. The information provided here does (a) explain the capability of the peptide carrier for sequence-independent transport of thousands of different substrates and (b) set the molecular basis for a rational drug design to increase the absorption of peptide-based drugs mediated by PEPT1.

Stereoselective uptake of β-lactam antibiotics by the intestinal peptide transporter

1 The stereoselective transport of β‐lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco‐2, by use of D‐ and L‐enantiomers of cephalexin and loracarbef as substrates. 2 The L‐isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H+‐symporter in Caco‐2 cells than the D‐isomers. This was demonstrated by inhibition of the influx of the β‐lactam, [3H]‐cefadroxil. 3 By measurement of the substrate‐induced intracellular acidification in Caco‐2 cells loaded with the pH‐sensitive fluorescent dye BCECF (2′,7′‐bis(2‐carboxyethyl)‐5‐(6)‐carboxy‐fluorescein), it was demonstrated for the first time that L‐isomers of β‐lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4 Efficient proton‐coupled transport of L‐β‐lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepTl from rabbit small intestine. 5 Both cell systems therefore express a stereoselective transport pathway for β‐lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide‐derived drugs.

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

Abstract. The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung

Background: Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Methods: Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the β-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. Results: PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative d-Ala-l-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. Conclusions: These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics.

Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di‐ and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate‐H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity‐type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1‐401) is derived from PepT2 whereas the carboxyterminal region (residues 402‐707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two‐electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino‐terminal region.

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.

1 To gain information on the mode of operation of the renal proton‐coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2 Using differently charged glycyl‐dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH‐dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2–50 μm) between pH 5.5 and 7.4 and membrane potentials of ±0 to −50 mV. 3 Maximal substrate‐evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pH0). Potential‐dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4 The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from –50 to –150 mV substrate affinities decrease 10‐ to 50‐fold whereas apparent Imax increases almost 10‐fold. 5 Even at saturating H+/H3O+ and dipeptide concentrations the I–V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6 These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

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.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.

SUMMARY H+/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H+/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (Km,app) of 0.806±0.161 mmol l-1. Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H+/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)+ RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H+/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0°C for the former and 25°C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

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 dipeptided-[3H]Phe-l-Ala, 2) by cellular accumulation of the fluorescent dipeptided-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake ofd-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 ofd-Phe-l-Ala of 6.0, an apparent K m value for influx of 25.8 ± 3.6 μM, and affinities of differently charged dipeptides or the β-lactam antibiotic cefadroxil to the binding site in the range of 20-80 μM. 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.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.