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Alfonso Lampen - One of the best experts on this subject based on the ideXlab platform.
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Cytotoxicity of peptide-coated silver nanoparticles on the human Intestinal Cell Line Caco-2
Archives of Toxicology, 2012Co-Authors: Linda Böhmert, Andreas F Thunemann, Birgit Niemann, Alfonso LampenAbstract:Silver nanoparticles are used in a wide range of consumer products such as clothing, cosmetics, household goods, articles of daily use and pesticides. Moreover, the use of a nanoscaled silver hydrosol has been requested in the European Union for even nutritional purposes. However, despite the wide applications of silver nanoparticles, there is a lack of information concerning their impact on human health. In order to investigate the effects of silver nanoparticles on human Intestinal Cells, we used the Caco-2 Cell Line and peptide-coated silver nanoparticles with defined colloidal, structural and interfacial properties. The particles display core diameter of 20 and 40 nm and were coated with the small peptide L-cysteine L-lysine L-lysine. Cell viability and proliferation were measured using Promegas CellTiter-Blue® Cell Viability assay, DAPI staining and impedance measurements. Apoptosis was determined by Annexin-V/7AAD staining and FACS analysis, membrane damage with Promegas LDH assay and reactive oxygen species by dichlorofluorescein assay. Exposure of proliferating Caco-2 Cells to silver nanoparticle induced decreasing adherence capacity and cytotoxicity, whereby the formation of reactive oxygen species could be the mode of action. The effects were dependent on particle size (20, 40 nm), doses (5-100 μg/mL) and time of incubation (4-48 h). Apoptosis or membrane damage was not detected.
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Analysis of proteomic changes induced upon Cellular differentiation of the human Intestinal Cell Line Caco‐2
Development growth & differentiation, 2011Co-Authors: Thorsten Buhrke, Imme Lengler, Alfonso LampenAbstract:The human Intestinal Cell Line Caco-2 is a well-established model system to study Cellular differentiation of human enterocytes of Intestinal origin, because these Cells have the capability to differentiate spontaneously into polarized Cells with morphological and biochemical features of small Intestinal enterocytes. Therefore, the Cells are widely used as an in vitro model for the human Intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of Intestinal Cellular differentiation. The proteome of proliferating Caco-2 Cells was compared with that of fully differentiated Cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and Cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon Cellular differentiation. In parallel, proteins associated with proliferation, Cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the Cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 Cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of Intestinal Cells.
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analysis of proteomic changes induced upon Cellular differentiation of the human Intestinal Cell Line caco 2
Development Growth & Differentiation, 2011Co-Authors: Thorsten Buhrke, Imme Lengler, Alfonso LampenAbstract:The human Intestinal Cell Line Caco-2 is a well-established model system to study Cellular differentiation of human enterocytes of Intestinal origin, because these Cells have the capability to differentiate spontaneously into polarized Cells with morphological and biochemical features of small Intestinal enterocytes. Therefore, the Cells are widely used as an in vitro model for the human Intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of Intestinal Cellular differentiation. The proteome of proliferating Caco-2 Cells was compared with that of fully differentiated Cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and Cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon Cellular differentiation. In parallel, proteins associated with proliferation, Cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the Cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 Cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis – diseases associated with an imbalanced differentiation process of Intestinal Cells.
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Cytotoxicity of AgPure silver nanoparticles in the human Intestinal Cell Line Caco-2
Toxicology Letters, 2011Co-Authors: Linda Böhmert, Andreas F Thunemann, Birgit Niemann, Ulf Hansen, Matthias Girod, Patrick Knappe, Alfonso LampenAbstract:Purpose: Engineered nanomaterials may exhibit properties differing significantly from those observed in the bulk materials, because of their small dimensions and large surface-to-volume ratio. Due to their unique qualities silver nanoparticles are used in a wide range of consumer products. Even the use of nanoscaled silver hydrosol for nutritional purposes has been requested. Despite the wide applications of silver nanoparticles, there is still lack of information concerning the impact on human health after oral application. Methods: To investigate the effects of silver nanoparticles on human Intestinal Cells, we monitored the reaction of Caco-2 Cells, as a model for the first Intestinal barrier of the human body, and AgPure silver nanoparticles. AgPure has already been used in consumer products. The radius of gyration, hydrodynamic radius and size distribution were determined by SAXS and DLS. We studied the effects of AgPure on Cell viability and proliferation by CellTiter-Blue assay, DAPI staining and xCelligence impedance measurement. Additionally, we tested for membrane damage with LDH assay, for apoptotic effects with Annexin-V/7AAD staining and for reactive oxygen species with dichlorofluorescein assay.Results: AgPure are spherical with metal core radii of 7.4 nm and hydrodynamic radii of 22 nm with stabiliser. The size is Gaussian-distributed with a polydispersity of 17 %. When proliferating Caco-2 Cells are exposed to AgPure, morphological abnormally adherence and particle dose- and time-dependant cytotoxicity was observed. However, apoptosis or membrane damage did not occur, but results of the dichlorofluorescein assay suggested the formation of reactive oxygen species as possible mechanism of cytotoxicity.
Vadivel Ganapathy - One of the best experts on this subject based on the ideXlab platform.
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Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human Intestinal Cell Line Caco-2.
The Journal of physiology, 2003Co-Authors: Zhong Chen, You Jun Fei, Catriona M H Anderson, Katherine A Wake, Seiji Miyauchi, Wei Huang, David T Thwaites, Vadivel GanapathyAbstract:The human orthologue of the H(+)-coupled amino acid transporter (hPAT1) was cloned from the human Intestinal Cell Line Caco-2 and its functional characteristics evaluated in a mammalian Cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the Intestinal tract. When expressed heterologously in mammalian Cells, hPAT1 mediated the transport of alpha-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na(+)-independent, but was energized by an inwardly directed H(+) gradient. hPAT1 interacted with glycine, L-alanine, L-proLine, alpha-aminoisobutyrate (AIB) and gamma-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the D-isomers of alanine and proLine. With serine and cysteine, though the L-isomers did not interact with hPAT1 to any significant extent, the corresponding D-isomers were recognized as substrates. With proLine and alanine, the affinity was similar for L- and D-isomers. However, with cysteine and serine, the D-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding L-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H(+)-coupled amino acid transport system in Caco-2 Cells. Furthermore, there was a high degree of correlation (r(2) = 0.93) between the relative potencies of various amino acids to inhibit the H(+)-coupled MeAIB transport measured with native Caco-2 Cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 Cells. These data suggest that hPAT1 is responsible for the H(+)-coupled amino acid transport expressed in the apical membrane of Caco-2 Cells.
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structure function and immunolocalization of a proton coupled amino acid transporter hpat1 in the human Intestinal Cell Line caco 2
The Journal of Physiology, 2003Co-Authors: Zhong Chen, You Jun Fei, Catriona M H Anderson, Katherine A Wake, Seiji Miyauchi, Wei Huang, David T Thwaites, Vadivel GanapathyAbstract:Throughout the animal and plant kingdoms, amino acids play vital roles in a variety of essential biological functions, including protein synthesis, neurotransmission, nitrogen metabolism and Cell growth. Many of these functions depend on the entry of amino acids into the Cells from the extraCellular medium, a process mediated by amino acid transporters in the plasma membrane. Amino acid transport systems have been identified, characterized and named based on distinct functional characteristics such as substrate specificity, ion coupling and exchange properties (Christensen, 1990; Palacin et al. 1998; Ganapathy et al. 2001). Over recent years, many amino acid transporters have been identified at the molecular level in plants, yeast and animals (Palacin et al. 1998; Ganapathy et al. 2001; Wipf et al. 2002). Historically, the Na+ gradient was recognized as the primary driving force for solute transport across the plasma membrane of mammalian Cells (Crane et al. 1961). However, subsequent work in different laboratories identified several solute transporters in mammalian Cell plasma membranes that are energized not by the Na+ gradient but by the H+ gradient. These include the peptide transporters (Ganapathy & Leibach, 1985, 1991, 1999) and the monocarboxylate transporters (Halestrap & Price, 1999). In the case of amino acids, even though most of the transport systems are either Na+-coupled or ion-independent, several studies have produced evidence for the presence of a H+-coupled amino acid transport system in the apical plasma membrane of mammalian epithelial Cells (Rajendran et al. 1987; Roigaard-Petersen et al. 1987; Jessen et al. 1988, 1989, 1991; Thwaites et al. 1993a, 1994, 1995a). In human Intestinal Cell (Caco-2) monolayers, the apical H+-coupled amino acid transport system transports a wide range of small, unbranched, zwitterionic amino acids including glycine, alanine, imino acids (proLine and hydroxyproLine), methylated analogues such as sarcosine, betaine, α-aminoisobutyric acid (AIB) and α-(methylamino)isobutyric acid (MeAIB), β-amino acids (β-alanine and β-taurine), γ-aminobutyric acid (GABA), and some d-amino acids such as d-serine and d-cycloserine (Thwaites et al. 1993a,b, 1994, 1995a,b,c, 2000; Thwaites & Stevens, 1999). H+-coupled l-proLine transport has been demonstrated in eel (Anguilla anguilla) Intestinal Cells, and is localized solely in the apical membrane (Ingrosso et al. 2000). Similarly, Na+-independent, pH-dependent transport in the mucosa-to-serosa direction for l-alanine has been demonstrated across lizard (Gallotia galloti) duodenal enterocytes (Diaz et al. 2000). In the rat small intestine, in the absence of extraCellular Na+, MeAIB transfer across the small intestine is stimulated 3-fold when luminal pH is reduced from pH 7.2 to 5.6, and this Na+-independent, H+-dependent MeAIB uptake is inhibited by β-alanine. In contrast, no H+-dependent MeAIB uptake could be measured in either guinea-pig or rabbit small intestine (L. K. Munck & B G. Munck, personal communication). Interestingly, the substrate specificity of the H+-coupled amino acid transporter in Caco-2 Cells and rabbit renal brush border membrane vesicles is similar to that described for the IMINO carrier in rat small intestine (Munck et al. 1994), whereas the IMINO carrier identified in either rabbit (Stevens & Wright, 1985; Munck & Munck, 1992) or guinea-pig (Munck & Munck, 1994) small intestine transports a different range of substrates. The presence of a H+-coupled amino acid transport system (system PAT) in the small Intestinal epithelium with such a broad range of transportable substrates provides a potential route for nutrient, osmolyte and drug transport across the initial barrier (i.e. the luminal brush border membrane) to solute absorption. In particular, this transport system transports a number of neuromodulatory agents such as d-serine and d-cycloserine (Thwaites et al. 1995a, c). d-Serine is the endogenous co-agonist for the activation of the N-methyl-d-aspartate receptor by glutamate (Mothet, 2001). The only other apically localized amino acid transporter that transports d-serine is ATB0,+, but this transporter is expressed predominantly in the distal regions of the Intestinal tract (Hatanaka et al. 2002). The Intestinal system PAT also transports GABA and its analogues, which function as GABA re-uptake inhibitors and GABA receptor agonists/ antagonists (Thwaites et al. 2000). The H+ gradient as the driving force in the small intestine for nutrient or drug absorption is physiologically relevant because such a gradient is present across the enterocyte apical membrane in the form of an ‘acid microclimate’ on the mucosal surface (Rawlings et al. 1987; Daniel et al. 1989). Many amino acid transport systems in yeast, plants and bacteria are also driven by the electrochemical H+ gradient and over recent years a large number of H+-coupled transporters have been cloned from these sources (Wipf et al. 2002). No mammalian Intestinal H+-coupled amino acid transporter has yet been identified at the molecular level. However, a recent study has reported on the isolation of a H+-coupled amino acid transporter from a rat hippocampal cDNA library (Sagne et al. 2001). This transporter was named rLYAAT1 (rat lysosomal amino acid transporter 1) due to its apparent lysosomal localization in rat brain. Subsequently, the mouse orthologue of LYAAT1 was cloned and its functional characteristics elucidated using the X. laevis expression system (Boll et al. 2002). These latter investigators named the transporter PAT1 (proton-coupled amino acid transporter 1) to describe the coupling of the transport system to the electrochemical H+ gradient. In the same report, these investigators also described the cloning of a second mammalian homologue (PAT2), which is energized by an electrochemical H+ gradient. The present study was undertaken to establish the molecular identity of the H+-coupled amino acid transporter expressed in the Intestinal Cell Line Caco-2.
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ς Receptor Ligand-Induced Up-Regulation of the H+/Peptide Transporter PEPT1 in the Human Intestinal Cell Line Caco-2
Biochemical and biophysical research communications, 1999Co-Authors: Takuya Fujita, Vadivel Ganapathy, Yoshikatsu Majikawa, Sayoko Umehisa, Naoki Okada, Akira Yamamoto, Frederick H. LeibachAbstract: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.
Kenichi Inui - One of the best experts on this subject based on the ideXlab platform.
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Efflux properties of basolateral peptide transporter in human Intestinal Cell Line Caco-2
Pflügers Archiv, 2004Co-Authors: Megumi Irie, Masahiro Okuda, Tomohiro Terada, Kenichi InuiAbstract:Small peptides and some pharmacologically active compounds are absorbed from the small intestine by the apical H^+-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter. Here we investigated the efflux properties of the basolateral peptide transporter in Caco-2 Cells using two strategies, efflux measurements and a kinetic analysis of transepithelial transport of glycylsarcosine (Gly-Sar). [^14C]Gly-Sar efflux through the basolateral membrane was not affected significantly by the external pH. Both approaches revealed that the basolateral peptide transporter was saturable in the efflux direction, and that the affinity was lower than that in the influx direction. For two peptide-like drugs, there was no difference in substrate recognition by the basolateral peptide transporter between the two sides of the membrane. Using the kinetic parameters of PEPT1 and the basolateral peptide transporter, a computational model of Gly-Sar transport in Caco-2 Cells was constructed. The simulation fitted the experimental data well. Our findings suggested that substrate affinity of the basolateral peptide transporter is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport. The behaviour of Gly-Sar in Caco-2 Cells could be predicted by a mathematical model describing the peptide transporters.
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Transport characteristics of grepafloxacin and levofloxacin in the human Intestinal Cell Line Caco-2.
European journal of pharmacology, 2001Co-Authors: Hiroaki Yamaguchi, Ikuko Yano, Hideyuki Saito, Kenichi InuiAbstract:Transport characteristics of grepafloxacin and levofloxacin across the apical membrane of Caco-2 Cells were examined. Both grepafloxacin and levofloxacin uptakes increased rapidly, and were temperature-dependent. Grepafloxacin and levofloxacin uptakes showed concentration-dependent saturation with Michaelis constants of 3.9 and 9.3 mM, respectively. Uptake of grepafloxacin and levofloxacin increased in Cl(-)-free and ATP depleted conditions, suggesting the involvement of an efflux transport system different from the uptake mechanism. However, cyclosporin A, a typical inhibitor of P-glycoprotein, did not affect the uptake of these drugs. Unlabeled grepafloxacin, unlabeled levofloxacin and quinidine inhibited the uptake of grepafloxacin and levofloxacin under Cl(-)-free conditions. Tetraethylammonium, cimetidine, p-aminohippurate, probenecid, amino acids, beta-lactam antibiotic or monocarboxylates did not inhibit the uptake of grepafloxacin and levofloxacin under the same conditions. In conclusion, our results suggested that grepafloxacin and levofloxacin uptakes were mediated by a specific transport system distinct from those for organic cations and anions, amino acids, dipeptides and monocarboxylates.
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Secretory mechanisms of grepafloxacin and levofloxacin in the human Intestinal Cell Line caco-2.
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Hiroaki Yamaguchi, Ikuko Yano, Yukiya Hashimoto, Kenichi InuiAbstract:Grepafloxacin and levofloxacin transport by Caco-2 Cell monolayers was examined to characterize the Intestinal behavior of these quinolones. The levels of transCellular transport of [14C]grepafloxacin and [14C]levofloxacin from the basolateral to the apical side were greater than those in the opposite direction. The unidirectional transport was inhibited by the presence of excess unlabeled quinolones, accompanied by increased accumulation. The inhibitory effects of cyclosporin A plus grepafloxacin on basolateral-to-apical transCellular transport and Cellular accumulation of [14C]grepafloxacin were comparable to those of cyclosporin A alone, indicating that the transport of grepafloxacin across the apical membrane was mainly mediated by P-glycoprotein. On the other hand, basolateral-to-apical transCellular transport of [14C]levofloxacin in the presence of cyclosporin A was decreased by unlabeled levofloxacin, grepafloxacin, and enoxacin, accompanied by significantly increased Cellular accumulation. The organic cation cimetidine, organic anion p -aminohippurate, and the multidrug resistance-related protein (MRP) modulator probenecid did not affect the transCellular transport of [14C]grepafloxacin or [14C]levofloxacin in the presence of cyclosporin A. The basolateral-to-apical transCellular transport of levofloxacin in the presence of cyclosporin A showed concentration-dependent saturation with an apparent Michaelis constant of 5.6 mM. In conclusion, these results suggested that basolateral-to-apical flux of quinolones was mediated by P-glycoprotein and a specific transport system distinct from organic cation and anion transporters and MRP.
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Functional characteristics of basolateral peptide transporter in the human Intestinal Cell Line Caco-2
The American journal of physiology, 1999Co-Authors: Tomohiro Terada, Yukiya Hashimoto, Hideyuki Saito, Kyoko Sawada, Kenichi InuiAbstract:The apical H+-coupled peptide transporter (PEPT1) and basolateral peptide transporter in human Intestinal Caco-2 Cells were functionally compared by the characterization of [14C]glycylsarcosine transport. The glycylsarcosine uptake via the basolateral peptide transporter was less sensitive to medium pH than uptake via PEPT1 and was not transported against the concentration gradient. Kinetic analysis indicated that glycylsarcosine uptake across the basolateral membranes was apparently mediated by a single peptide transporter. Small peptides and beta-lactam antibiotics inhibited glycylsarcosine uptake by the basolateral peptide transporter, and these inhibitions were revealed to be competitive. Comparison of the inhibition constant values of various beta-lactam antibiotics between PEPT1 and the basolateral peptide transporter suggested that the former had a higher affinity than the latter. A histidine residue modifier, diethyl pyrocarbonate, inhibited glycylsarcosine uptake by both transporters, although the inhibitory effect was greater on PEPT1. These findings suggest that a single facilitative peptide transporter is expressed at the basolateral membranes of Caco-2 Cells and that PEPT1 and the basolateral peptide transporter cooperate in the efficient transepithelial transport of small peptides and peptidelike drugs.
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functional characteristics of basolateral peptide transporter in the human Intestinal Cell Line caco 2
American Journal of Physiology-gastrointestinal and Liver Physiology, 1999Co-Authors: Tomohiro Terada, Yukiya Hashimoto, Hideyuki Saito, Kyoko Sawada, Kenichi InuiAbstract:The apical H+-coupled peptide transporter (PEPT1) and basolateral peptide transporter in human Intestinal Caco-2 Cells were functionally compared by the characterization of [14C]glycylsarcosine tra...
Zhong Chen - One of the best experts on this subject based on the ideXlab platform.
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Structure, function and immunolocalization of a proton-coupled amino acid transporter (hPAT1) in the human Intestinal Cell Line Caco-2.
The Journal of physiology, 2003Co-Authors: Zhong Chen, You Jun Fei, Catriona M H Anderson, Katherine A Wake, Seiji Miyauchi, Wei Huang, David T Thwaites, Vadivel GanapathyAbstract:The human orthologue of the H(+)-coupled amino acid transporter (hPAT1) was cloned from the human Intestinal Cell Line Caco-2 and its functional characteristics evaluated in a mammalian Cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the Intestinal tract. When expressed heterologously in mammalian Cells, hPAT1 mediated the transport of alpha-(methylamino)isobutyric acid (MeAIB). The cDNA-induced transport was Na(+)-independent, but was energized by an inwardly directed H(+) gradient. hPAT1 interacted with glycine, L-alanine, L-proLine, alpha-aminoisobutyrate (AIB) and gamma-aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the D-isomers of alanine and proLine. With serine and cysteine, though the L-isomers did not interact with hPAT1 to any significant extent, the corresponding D-isomers were recognized as substrates. With proLine and alanine, the affinity was similar for L- and D-isomers. However, with cysteine and serine, the D-isomers showed 6- to 8-fold higher affinity for hPAT1 than the corresponding L-isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H(+)-coupled amino acid transport system in Caco-2 Cells. Furthermore, there was a high degree of correlation (r(2) = 0.93) between the relative potencies of various amino acids to inhibit the H(+)-coupled MeAIB transport measured with native Caco-2 Cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco-2 Cells. These data suggest that hPAT1 is responsible for the H(+)-coupled amino acid transport expressed in the apical membrane of Caco-2 Cells.
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structure function and immunolocalization of a proton coupled amino acid transporter hpat1 in the human Intestinal Cell Line caco 2
The Journal of Physiology, 2003Co-Authors: Zhong Chen, You Jun Fei, Catriona M H Anderson, Katherine A Wake, Seiji Miyauchi, Wei Huang, David T Thwaites, Vadivel GanapathyAbstract:Throughout the animal and plant kingdoms, amino acids play vital roles in a variety of essential biological functions, including protein synthesis, neurotransmission, nitrogen metabolism and Cell growth. Many of these functions depend on the entry of amino acids into the Cells from the extraCellular medium, a process mediated by amino acid transporters in the plasma membrane. Amino acid transport systems have been identified, characterized and named based on distinct functional characteristics such as substrate specificity, ion coupling and exchange properties (Christensen, 1990; Palacin et al. 1998; Ganapathy et al. 2001). Over recent years, many amino acid transporters have been identified at the molecular level in plants, yeast and animals (Palacin et al. 1998; Ganapathy et al. 2001; Wipf et al. 2002). Historically, the Na+ gradient was recognized as the primary driving force for solute transport across the plasma membrane of mammalian Cells (Crane et al. 1961). However, subsequent work in different laboratories identified several solute transporters in mammalian Cell plasma membranes that are energized not by the Na+ gradient but by the H+ gradient. These include the peptide transporters (Ganapathy & Leibach, 1985, 1991, 1999) and the monocarboxylate transporters (Halestrap & Price, 1999). In the case of amino acids, even though most of the transport systems are either Na+-coupled or ion-independent, several studies have produced evidence for the presence of a H+-coupled amino acid transport system in the apical plasma membrane of mammalian epithelial Cells (Rajendran et al. 1987; Roigaard-Petersen et al. 1987; Jessen et al. 1988, 1989, 1991; Thwaites et al. 1993a, 1994, 1995a). In human Intestinal Cell (Caco-2) monolayers, the apical H+-coupled amino acid transport system transports a wide range of small, unbranched, zwitterionic amino acids including glycine, alanine, imino acids (proLine and hydroxyproLine), methylated analogues such as sarcosine, betaine, α-aminoisobutyric acid (AIB) and α-(methylamino)isobutyric acid (MeAIB), β-amino acids (β-alanine and β-taurine), γ-aminobutyric acid (GABA), and some d-amino acids such as d-serine and d-cycloserine (Thwaites et al. 1993a,b, 1994, 1995a,b,c, 2000; Thwaites & Stevens, 1999). H+-coupled l-proLine transport has been demonstrated in eel (Anguilla anguilla) Intestinal Cells, and is localized solely in the apical membrane (Ingrosso et al. 2000). Similarly, Na+-independent, pH-dependent transport in the mucosa-to-serosa direction for l-alanine has been demonstrated across lizard (Gallotia galloti) duodenal enterocytes (Diaz et al. 2000). In the rat small intestine, in the absence of extraCellular Na+, MeAIB transfer across the small intestine is stimulated 3-fold when luminal pH is reduced from pH 7.2 to 5.6, and this Na+-independent, H+-dependent MeAIB uptake is inhibited by β-alanine. In contrast, no H+-dependent MeAIB uptake could be measured in either guinea-pig or rabbit small intestine (L. K. Munck & B G. Munck, personal communication). Interestingly, the substrate specificity of the H+-coupled amino acid transporter in Caco-2 Cells and rabbit renal brush border membrane vesicles is similar to that described for the IMINO carrier in rat small intestine (Munck et al. 1994), whereas the IMINO carrier identified in either rabbit (Stevens & Wright, 1985; Munck & Munck, 1992) or guinea-pig (Munck & Munck, 1994) small intestine transports a different range of substrates. The presence of a H+-coupled amino acid transport system (system PAT) in the small Intestinal epithelium with such a broad range of transportable substrates provides a potential route for nutrient, osmolyte and drug transport across the initial barrier (i.e. the luminal brush border membrane) to solute absorption. In particular, this transport system transports a number of neuromodulatory agents such as d-serine and d-cycloserine (Thwaites et al. 1995a, c). d-Serine is the endogenous co-agonist for the activation of the N-methyl-d-aspartate receptor by glutamate (Mothet, 2001). The only other apically localized amino acid transporter that transports d-serine is ATB0,+, but this transporter is expressed predominantly in the distal regions of the Intestinal tract (Hatanaka et al. 2002). The Intestinal system PAT also transports GABA and its analogues, which function as GABA re-uptake inhibitors and GABA receptor agonists/ antagonists (Thwaites et al. 2000). The H+ gradient as the driving force in the small intestine for nutrient or drug absorption is physiologically relevant because such a gradient is present across the enterocyte apical membrane in the form of an ‘acid microclimate’ on the mucosal surface (Rawlings et al. 1987; Daniel et al. 1989). Many amino acid transport systems in yeast, plants and bacteria are also driven by the electrochemical H+ gradient and over recent years a large number of H+-coupled transporters have been cloned from these sources (Wipf et al. 2002). No mammalian Intestinal H+-coupled amino acid transporter has yet been identified at the molecular level. However, a recent study has reported on the isolation of a H+-coupled amino acid transporter from a rat hippocampal cDNA library (Sagne et al. 2001). This transporter was named rLYAAT1 (rat lysosomal amino acid transporter 1) due to its apparent lysosomal localization in rat brain. Subsequently, the mouse orthologue of LYAAT1 was cloned and its functional characteristics elucidated using the X. laevis expression system (Boll et al. 2002). These latter investigators named the transporter PAT1 (proton-coupled amino acid transporter 1) to describe the coupling of the transport system to the electrochemical H+ gradient. In the same report, these investigators also described the cloning of a second mammalian homologue (PAT2), which is energized by an electrochemical H+ gradient. The present study was undertaken to establish the molecular identity of the H+-coupled amino acid transporter expressed in the Intestinal Cell Line Caco-2.
Thomas Kissel - One of the best experts on this subject based on the ideXlab platform.
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evaluation of absorption enhancement for a potent cyclopeptidic ανβ3 antagonist in a human Intestinal Cell Line caco 2
European Journal of Pharmaceutical Sciences, 2000Co-Authors: Walter Kamm, Alfred Jonczyk, Tobias Jung, Gerd Luckenbach, Peter Raddatz, Thomas KisselAbstract:Abstract Different absorption enhancing principles for a potent cyclopeptidic ανβ3-antagonist (EMD 121974) were investigated in monolayers of a human Intestinal Cell Line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol–buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as β-cyclodextrin neither affected integrity of the Cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.
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Evaluation of absorption enhancement for a potent cyclopeptidic ανβ3-antagonist in a human Intestinal Cell Line (Caco-2)
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2000Co-Authors: Walter Kamm, Alfred Jonczyk, Tobias Jung, Gerd Luckenbach, Peter Raddatz, Thomas KisselAbstract:Different absorption enhancing principles for a potent cyclopeptidic alpha(nu)beta(3)-antagonist (EMD 121974) were investigated in monolayers of a human Intestinal Cell Line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol-buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as beta-cyclodextrin neither affected integrity of the Cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.
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Effects of Permeation Enhancers on the Transport of a Peptidomimetic Thrombin Inhibitor (CRC 220) in a Human Intestinal Cell Line (Caco-2)
Pharmaceutical research, 1996Co-Authors: Ute Werner, Thomas Kissel, Martin ReersAbstract:Purpose. The effects of five different permeation enhancer systems on the transport properties of a peptidomimetic thrombin inhibitor, CRC 220, were investigated in monolayers of a human Intestinal Cell Line (Caco-2).
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Heterogeneity in the human Intestinal Cell Line Caco-2 leads to differences in transepithelial transport
European Journal of Pharmaceutical Sciences, 1995Co-Authors: Elke Walter, Thomas KisselAbstract:Abstract When the Caco-2 Cell Line is used as a model for transepithelial transport pathways, striking differences in transepithelial electrical resistance and in permeability of hydrophilic marker substances were reported. In this study, Caco-2 Cell Lines from different laboratories have been tested for permeability characteristics using hydrophilic and hydrophobic substances. To elucidate the vast differences in permeability, the following properties were monitored: growth characteristics, morphological homogeneity, metabolic activity, viability, and actin. Using lectin binding experiments and alkaLine phosphatase staining, the degree of differentiation in Caco-2 clones was estimated. Our results are in agreement with observations made by others, demonstrating heterogeneity of the Caco-2 Cell Line. Caco-2 monolayers consist of several subpopulations. The variation in permeability characteristics of the Caco-2 Cell Line may be a result of a selection process promoted by the respective culture conditions yielding a varying composition of subpopulations.
