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Hannelore Daniel - One of the best experts on this subject based on the ideXlab platform.
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Delta-aminolevulinic Acid Transport by Intestinal and Renal Peptide Transporters and Its Physiological and Clinical Implications
2016Co-Authors: Clin J. Invest, Wolfgang Clauss, Hannelore DanielAbstract:Delta-aminolevulinic acid (ALA) is the precursor of por-phyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photo-dynamic 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 in-testinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane trans-port into epithelial cells of intestine and kidney are un-known. Here we demonstrate that ALA uses the intestinal and renal apical Peptide Transporters for entering into epi-thelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevi
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taste and move glucose and Peptide Transporters in the gastrointestinal tract
Experimental Physiology, 2015Co-Authors: Hannelore Daniel, Tamara ZietekAbstract:New Findings What is the topic of this review? Nutrient absorption in the gastrointestinal tract requires membrane proteins embedded in the apical membrane of epithelial cells that allow bulk quantities of nutrients, such as monosaccharides and amino acids, to be moved into epithelial cells. Very recently, a new function of the Transporters as nutrient sensors mediating Peptide hormone release from enteroendocrine cells has been discovered. What advances does it highlight? The review covers recent advances in membrane transporter functions for the absorption and sensing of dietary Peptides and sugars and their putative interplay. Nutrient Transporters are integral membrane proteins responsible for uptake into enterocytes and release of nutrients into the circulation. Absorption of food breakdown products, such as fatty acids, monosaccharides or amino acids, requires high-capacity Transporters. In the case of glucose, amino acids and Peptides, the Transporters are electrogenic in nature, coupling substrate flux to ion movement. While glucose absorption is mediated by the Na+-dependent SGLT1 protein, uptake of short-chain Peptides is mediated by the H+-coupled PEPT1 protein. Interestingly, both Transporters were recently shown to fulfil an additional role as intestinal ‘sensors’ in enteroendocrine cells, mediating the release of gastrointestinal Peptide hormones into the circulation. Sensing of d-glucose and of di- and triPeptides is particularly relevant for the secretion of the incretins glucose-dependent insulinotrophic polyPeptide and glucagon-like Peptide 1 that promote insulin output from β-cells and mediate β-cell protection. In addition to these sensing pathways, a variety of G-protein-coupled receptors are involved in sensing of intestinal contents. d-Glucose is sensed not only by SGLT1 but also by the sweet taste receptor T1R2/3 expressed in enteroendocrine cells. Activation of T1R2/3 increases SGLT1 levels and intestinal glucose absorption. Although T1R2/3 ligands, such as artificial sweeteners, were shown to elicit incretin secretion from enteroendocrine cell lines or in vitro in tissue preparations, convincing data that this is also relevant in vivo are missing to date. However, there is growing interest in targeting intestinal sensory pathways, involving receptors but also the ‘transceptors’ PEPT1 and SGLT1, by use of drugs or food constituents to elicit the beneficial effects of incretins on the pancreas and metabolic control.
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high affinity interaction of sartans with h Peptide Transporters
Drug Metabolism and Disposition, 2009Co-Authors: Ilka Knütter, Hannelore Daniel, Gabor Kottra, Wiebke Fischer, Matthias BrandschAbstract:Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble diPeptide derivatives, we investigated whether they are transported by Peptide Transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/Peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited Peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of diPeptides and cefadroxil.
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transport of angiotensin converting enzyme inhibitors by h Peptide Transporters revisited
Journal of Pharmacology and Experimental Therapeutics, 2008Co-Authors: Ilka Knütter, Hannelore Daniel, Claudia Wollesky, Gabor Kottra, Martin G Hahn, Wiebke Fischer, Katja Zebisch, Reinhard H H Neubert, Matthias BrandschAbstract:Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/Peptide Transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the diPeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to Peptide Transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that Peptide Transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.
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Peptide Transporters and their roles in physiological processes and drug disposition
Xenobiotica, 2008Co-Authors: Isabel Rubioaliaga, Hannelore DanielAbstract:1. The Peptide Transporters belong to the Peptide transporter (PTR) family and serve as integral membrane proteins for the cellular uptake of di- and triPeptides in the organism. By their ability also to transport peptidomimetics and other substrates with therapeutic activities or precursors of pharmacologically active agents, they are of considerable importance in pharmacology. 2. PEPT1 is the low-affinity, high-capacity transporter and is mainly expressed in the small intestine, whereas PEPT2 is the high-affinity, low-capacity transporter and has a broader distribution in the organism. 3. Targeted mouse models have revealed PEPT2 to be the dominant transporter for the reabsorption of di- and triPeptides and its pharmacological substrates in the organism, and for the removal of these substrates from the cerebrospinal fluid. Moreover, the Peptide Transporters undergo physiological and pharmacological regulation and, of great interest, are present in disease states where PEPT1 exhibits ectopic expression in colonic inflammation. 4. The paper reviews the structural characteristics of the Peptide Transporters, the structural requirements for substrates, the distribution of the Peptide Transporters in the organism, and finally their regulation in the organism in healthy and pathological situations.
Ken-ichi Inui - One of the best experts on this subject based on the ideXlab platform.
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recent advances in structural biology of Peptide Transporters
Current Topics in Membranes, 2012Co-Authors: Tomohiro Terada, Ken-ichi InuiAbstract:Abstract Peptide Transporters localized at brush-border membranes of intestinal and renal epithelial cells mediate the membrane transport of di- and triPeptides, and play important roles in protein absorption and the conservation of Peptide-bound amino nitrogen. Peptide-like drugs that show structural similarities to di- and triPeptides are also recognized by Peptide Transporters. The energy for transport of small Peptides and Peptide-like drugs is provided by the proton gradient across the cell membrane. Since the cloning of H + /Peptide cotransporter (PEPT1, SLC15A1), there have been advances in the molecular biology, biochemistry, biophysics and structural determination of PEPT1. By integrating these advances, much effort has been made to understand the relationship between structure and function. In silico experimental strategies are classified as (1) construction of kinetic models, (2) computer modeling of PEPT1 structure and (3) homology modeling of PEPT1 with crystal structures of bacterial Transporters. The hypotheses regarding the structure–function relationship produced by these strategies have been confirmed by in vitro mutagenesis including cysteine-scanning mutagenesis. Recently, the crystal structure of PepT So , a functionally similar prokaryotic homolog of the mammalian Peptide Transporters from Shewanella oneidensis , was classified, and the previous hypotheses regarding the structure–function relationship of PEPT1 have been re-evaluated. This review highlights the recent advances in our knowledge of the structural biology of PEPT1.
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inhibitory effect of zinc on the absorption of β lactam antibiotic ceftibuten via the Peptide Transporters in rats
Drug Metabolism and Pharmacokinetics, 2008Co-Authors: Miyako Okamura, Tomohiro Terada, Toshiya Katsura, Ken-ichi InuiAbstract:Zinc is an essential metal ion for the body, and is widely used for nutritional and clinical purposes. Previously, we showed that zinc inhibits the transport of glycylsarcosine via the intestinal Peptide transporter PEPT1 in the human intestinal cell line Caco-2. In this study, we examined the effect of zinc on the activity of Peptide Transporters in rats using the oral beta-lactam antibiotic ceftibuten as a model drug. The plasma ceftibuten concentration after intraintestinal administration was decreased in the presence of zinc. The maximum plasma concentration (C(max)) was significantly decreased and the time required to reach C(max) (T(max)) was prolonged by zinc coadministration. The plasma ceftibuten concentration after iron coadministration or two hours after zinc administration was not affected. The in situ loop technique revealed 50% inhibition of ceftibuten absorption by zinc. In conclusion, zinc inhibits the transport activity of PEPT1 in vivo as well in vitro.
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section a molecular structural and cellular biology of drug Transporters Peptide Transporters structure function regulation and application for drug delivery
Current Drug Metabolism, 2004Co-Authors: Tomohiro Terada, Ken-ichi InuiAbstract:Proton-coupled Peptide Transporters, localized at brush-border membranes of intestinal and renal epithelial cells, play important roles in protein absorption and the conservation of Peptide-bound amino nitrogen. These Transporters also have significant pharmacological and pharmacokinetic relevance to the transport of various Peptide-like drugs such as beta-lactam antibiotics. The identification and molecular characterization of H(+)/Peptide coTransporters (PEPT1 and PEPT2) have facilitated the clarification of many aspects of these Transporters such as the structure/function relationship and regulation. Recent findings that intestinal PEPT1 can transport l-valine ester prodrugs such as valacyclovir provided a major step forward toward the development of novel drug delivery systems. It has been demonstrated that Peptide Transporters, which have a similar substrate specificity to PEPT1 and PEPT2, but possess other distinct functional properties, are localized at basolateral membranes of intestinal and renal epithelial cells. This review highlights the recent advances in our knowledge of the cellular and molecular nature of PEPT1, PEPT2 and the basolateral Peptide Transporters.
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decreased expression of glucose and Peptide Transporters in rat remnant kidney
Drug Metabolism and Pharmacokinetics, 2004Co-Authors: Nobuhiko Nakamura, Hideyuki Saito, Satohiro Masuda, Kazushige Takahashi, Masahiro Okuda, Ken-ichi InuiAbstract:The loss of renal mass induces tubular hypertrophy as well as glomerular sclerosis and results in the end stage of renal disease. However, there is little information about adaptation of tubular glucose and Peptide reabsorption under conditions of chronic renal failure. In the present study, we performed functional and molecular analyses focused on the tubular reabsorption of filtered glucose and small Peptides using 5/6 nephrectomized rats at 16 weeks, as a model of chronic renal failure. Sixteen weeks after 5/6 nephrectomy or sham treatment, the brush-border membranes and total RNA were obtained from the renal cortex to evaluate the uptake of Na(+) gradient-dependent D-glucose and H(+) gradient-dependent glycylsarcosine. The amounts of SGLT and PEPT mRNA levels were quantified by competitive PCR. The urinary glucose/creatinine ratio was markedly higher in nephrectomized rats than in sham-operated controls. Na(+)-dependent glucose uptake by the isolated renal brush-border membrane vesicles was markedly decreased in nephrectomized rats compared with that in sham-operated controls. However, H(+)-dependent Peptide transport, another secondary active transport system in the brush-border membranes, was maintained. In addition, kinetic analysis revealed that both SGLT1 (high-affinity type)- and SGLT2 (low-affinity type)-mediated Na(+)/glucose uptake had markedly decreased Vmax values, but not Km values. Furthermore, competitive PCR demonstrated that the mRNA expression levels of SGLT2, PEPT1 and PEPT2, but not SGLT1, were markedly depressed. These findings suggested that loss of SGLT2 during chronic renal failure implies a high risk of renal glucosuria.
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distinct transport characteristics of basolateral Peptide Transporters between mdck and caco 2 cells
Pflügers Archiv: European Journal of Physiology, 2001Co-Authors: Kyoko Sawada, Tomohiro Terada, Hideyuki Saito, Ken-ichi InuiAbstract:The cellular polarity of [14C]glycylsarcosine (Gly-Sar) transport in Madin-Darby canine kidney (MDCK) cells was compared with that in the human intestinal cell line Caco-2. In MDCK cells, [14C]Gly-Sar accumulation was greater at the basolateral side than at the apical side, and there was little net transcellular transport. In Caco-2 cells, [14C]Gly-Sar accumulation was greater at the apical side and unidirectional transcellular transport occurred from the apical to basolateral side. Efflux of [14C]Gly-Sar from MDCK cells to either side was negligible, whereas that from Caco-2 cells was significantly faster to the basolateral side. The basolateral Peptide transporter in MDCK cells possessed a similar substrate specificity, but a much higher substrate affinity, than that in Caco-2 cells. The basolateral Peptide transporter in MDCK cells did not accumulate Gly-Sar during hypertonic stress. These findings indicate that the basolateral Peptide transporter in MDCK cells is involved in the cellular uptake of small Peptides, but not in the extrusion of small Peptides to the extracellular space.
Frederick H Leibach - One of the best experts on this subject based on the ideXlab platform.
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transport of valganciclovir a ganciclovir prodrug via Peptide Transporters pept1 and pept2
Journal of Pharmaceutical Sciences, 2000Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Vadivel Ganapathy, Wei Huang, Youjun Fei, Malliga E GanapathyAbstract:In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal Peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal Peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal Peptide transporter PEPT1. This prodrug is also recognized by the renal Peptide transporter PEPT2 with high affinity.
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valacyclovir a substrate for the intestinal and renal Peptide Transporters pept1 and pept2
Biochemical and Biophysical Research Communications, 1998Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Wei Huang, Hong Wang, Frederick H LeibachAbstract:Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a Peptide bond in its structure. We studied the interaction of valacyclovir with the Peptide Transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express Peptide Transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned Peptide Transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the Peptide Transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the Peptide Transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned Peptide Transporters. We conclude that valacyclovir is a substrate for the Peptide Transporters PEPT1 and PEPT2 and that a Peptide bond is not a prerequisite for recognition as a substrate by the Peptide Transporters.
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identification of a potential substrate binding domain in the mammalian Peptide Transporters pept1 and pept2 using pept1 pept2 and pept2 pept1 chimeras
Biochemical and Biophysical Research Communications, 1998Co-Authors: Youjun Fei, Vadivel Ganapathy, Jincai Liu, Takuya Fujita, Rong Liang, Frederick H LeibachAbstract:Abstract The mammalian Peptide Transporters PEPT1 and PEPT2 are energized by a transmembrane electrochemical H+gradient and exhibit similar broad substrate specificity. These Transporters however differ in their affinity for substrates, PEPT1 being a low-affinity transporter and PEPT2 being a high-affinity transporter. To identify the substrate binding domain in PEPT1 and PEPT2 which is responsible for the differing affinities, we constructed a series of PEPT1-PEPT2 and PEPT2-PEPT1 chimeras using anin vivorestriction site-independent procedure and determined their substrate affinities. A comparison of these kinetic data for different chimeras with those of the wild-type PEPT1 and PEPT2 in conjunction with the specific structural PEPT1/PEPT2 crossover regions in these chimeras has led to the identification of a putative substrate binding site, which is comprised of the transmembrane domains 7, 8 and 9 of the Transporters.
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interaction of anionic cephalosporins with the intestinal and renal Peptide Transporters pept 1 and pept 2
Biochimica et Biophysica Acta, 1997Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Puttur D Prasad, Bryan Mackenzie, Frederick H LeibachAbstract:The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the renal Peptide transporter (PEPT 2) and the intestinal Peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the diPeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the Peptide Transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).
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Peptide Transporters in the intestine and the kidney
Annual Review of Nutrition, 1996Co-Authors: Frederick H Leibach, Vadivel GanapathyAbstract:Even though the existence of a transport process for intact Peptides in the brush border membrane of intestinal and renal absorptive epithelial cells has been known for almost three decades, it is only recently that the molecular nature of the proteins responsible for the transport process has been elucidated. Two Peptide Transporters, PEPT 1 and PEPT 2, have been cloned. The cloned Transporters catalyze active transport of intact di- and triPeptides and utilize a transmembrane electrochemical H+ gradient as the driving force. The characteristic of H+ coupling makes PEPT 1 and PEPT 2 unique among the Transporters thus far identified in mammalian cells. In addition, the Peptide Transporters have immediate pharmacologic relevance because a number of Peptide-like drugs are recognized as substrates by these Transporters. Recently, cultured cell lines of intestinal and renal origin that express PEPT 1 and PEPT 2 have been identified. These cell lines are likely to facilitate studies on the regulatory aspects of the Peptide Transporters.
Matthias Brandsch - One of the best experts on this subject based on the ideXlab platform.
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pharmaceutical and pharmacological importance of Peptide Transporters
Journal of Pharmacy and Pharmacology, 2010Co-Authors: Matthias Brandsch, Ilka Knütter, Eva BossedoeneckeAbstract:Peptide transport is currently a prominent topic in membrane research. The transport proteins involved are under intense investigation because of their physiological importance in protein absorption and also because Peptide Transporters are possible vehicles for drug delivery. Moreover, in many tissues Peptide carriers transduce peptidic signals across membranes that are relevant in information processing. The focus of this review is on the pharmaceutical relevance of the human Peptide Transporters PEPT1 and PEPT2. In addition to their physiological substrates, both carriers transport many β-lactam antibiotics, valaciclovir and other drugs and prodrugs because of their sterical resemblance to di- and triPeptides. The primary structure, tissue distribution and substrate specificity of PEPT1 and PEPT2 have been well characterized. However, there is a dearth of knowledge on the substrate binding sites and the three-dimensional structure of these proteins. Until this pivotal information becomes available by X-ray crystallography, the development of new drug substrates relies on classical transport studies combined with molecular modelling. In more than thirty years of research, data on the interaction of well over 700 di- and triPeptides, amino acid and Peptide derivatives, drugs and prodrugs with Peptide Transporters have been gathered. The aim of this review is to put the reports on Peptide transporter-mediated drug uptake into perspective. We also review the current knowledge on pharmacogenomics and clinical relevance of human Peptide Transporters. Finally, the reader's attention is drawn to other known or proposed human Peptide-transporting proteins.
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the bioactive diPeptide anserine is transported by human proton coupled Peptide Transporters
FEBS Journal, 2010Co-Authors: Stefanie Geissler, Madlen Zwarg, Ilka Knütter, Fritz Markwardt, Matthias BrandschAbstract:The bioactive diPeptide derivative anserine (β-alanyl-1-N-methyl-l-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H+/Peptide coTransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [14C]glycylsarcosine ([14C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hypertensive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anserine inhibited [14C]Gly-Sar uptake with Ki values of 1.55 mm (Caco-2) and 0.033 mm (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, Ki values of 0.65 mm (hPEPT1) and 0.18 mm (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [14C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal Peptide transporter and are transported by hPEPT1 in an active, electrogenic H+ symport. As PEPT1 is the predominant transport system for di- and triPeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of Peptide Transporters, such as prodrugs, that can be administered orally.
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transport of drugs by proton coupled Peptide Transporters pearls and pitfalls
Expert Opinion on Drug Metabolism & Toxicology, 2009Co-Authors: Matthias BrandschAbstract:The pharmaceutical relevance of proton-coupled Peptide Transporters is currently under intense investigation in many laboratories. Studies have shown that these membrane proteins, expressed in inte...
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high affinity interaction of sartans with h Peptide Transporters
Drug Metabolism and Disposition, 2009Co-Authors: Ilka Knütter, Hannelore Daniel, Gabor Kottra, Wiebke Fischer, Matthias BrandschAbstract:Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble diPeptide derivatives, we investigated whether they are transported by Peptide Transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/Peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited Peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of diPeptides and cefadroxil.
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transport of angiotensin converting enzyme inhibitors by h Peptide Transporters revisited
Journal of Pharmacology and Experimental Therapeutics, 2008Co-Authors: Ilka Knütter, Hannelore Daniel, Claudia Wollesky, Gabor Kottra, Martin G Hahn, Wiebke Fischer, Katja Zebisch, Reinhard H H Neubert, Matthias BrandschAbstract:Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/Peptide Transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the diPeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to Peptide Transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that Peptide Transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.
Vadivel Ganapathy - One of the best experts on this subject based on the ideXlab platform.
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transport of valganciclovir a ganciclovir prodrug via Peptide Transporters pept1 and pept2
Journal of Pharmaceutical Sciences, 2000Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Vadivel Ganapathy, Wei Huang, Youjun Fei, Malliga E GanapathyAbstract:In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal Peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal Peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal Peptide transporter PEPT1. This prodrug is also recognized by the renal Peptide transporter PEPT2 with high affinity.
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valacyclovir a substrate for the intestinal and renal Peptide Transporters pept1 and pept2
Biochemical and Biophysical Research Communications, 1998Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Wei Huang, Hong Wang, Frederick H LeibachAbstract:Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a Peptide bond in its structure. We studied the interaction of valacyclovir with the Peptide Transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express Peptide Transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned Peptide Transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the Peptide Transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the Peptide Transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned Peptide Transporters. We conclude that valacyclovir is a substrate for the Peptide Transporters PEPT1 and PEPT2 and that a Peptide bond is not a prerequisite for recognition as a substrate by the Peptide Transporters.
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identification of a potential substrate binding domain in the mammalian Peptide Transporters pept1 and pept2 using pept1 pept2 and pept2 pept1 chimeras
Biochemical and Biophysical Research Communications, 1998Co-Authors: Youjun Fei, Vadivel Ganapathy, Jincai Liu, Takuya Fujita, Rong Liang, Frederick H LeibachAbstract:Abstract The mammalian Peptide Transporters PEPT1 and PEPT2 are energized by a transmembrane electrochemical H+gradient and exhibit similar broad substrate specificity. These Transporters however differ in their affinity for substrates, PEPT1 being a low-affinity transporter and PEPT2 being a high-affinity transporter. To identify the substrate binding domain in PEPT1 and PEPT2 which is responsible for the differing affinities, we constructed a series of PEPT1-PEPT2 and PEPT2-PEPT1 chimeras using anin vivorestriction site-independent procedure and determined their substrate affinities. A comparison of these kinetic data for different chimeras with those of the wild-type PEPT1 and PEPT2 in conjunction with the specific structural PEPT1/PEPT2 crossover regions in these chimeras has led to the identification of a putative substrate binding site, which is comprised of the transmembrane domains 7, 8 and 9 of the Transporters.
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interaction of anionic cephalosporins with the intestinal and renal Peptide Transporters pept 1 and pept 2
Biochimica et Biophysica Acta, 1997Co-Authors: Malliga E Ganapathy, Vadivel Ganapathy, Puttur D Prasad, Bryan Mackenzie, Frederick H LeibachAbstract:The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the renal Peptide transporter (PEPT 2) and the intestinal Peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the diPeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the Peptide Transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).
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Peptide Transporters in the intestine and the kidney
Annual Review of Nutrition, 1996Co-Authors: Frederick H Leibach, Vadivel GanapathyAbstract:Even though the existence of a transport process for intact Peptides in the brush border membrane of intestinal and renal absorptive epithelial cells has been known for almost three decades, it is only recently that the molecular nature of the proteins responsible for the transport process has been elucidated. Two Peptide Transporters, PEPT 1 and PEPT 2, have been cloned. The cloned Transporters catalyze active transport of intact di- and triPeptides and utilize a transmembrane electrochemical H+ gradient as the driving force. The characteristic of H+ coupling makes PEPT 1 and PEPT 2 unique among the Transporters thus far identified in mammalian cells. In addition, the Peptide Transporters have immediate pharmacologic relevance because a number of Peptide-like drugs are recognized as substrates by these Transporters. Recently, cultured cell lines of intestinal and renal origin that express PEPT 1 and PEPT 2 have been identified. These cell lines are likely to facilitate studies on the regulatory aspects of the Peptide Transporters.