Biomedical subjects

Hannelore Daniel

Publications and source records attributed to Hannelore Daniel.

64 records · Page 4Linked to original sources

PubMed2002

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters.

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.

Alanine↗

PubMed2002

Mammalian peptide transporters as targets for drug delivery.

Peptide transporters are integral plasma membrane proteins that mediate the cellular uptake of dipeptides and tripeptides in addition to a variety of peptidomimetics. The carriers, which occur predominantly in the brush-border membranes of epithelial cells of the small intestine, lung, choroid plexus and kidney, contribute to absorption, distribution and elimination of their substrates. The cellular uptake of peptides and peptidomimetics involves the cotransport of protons down an inwardly directed, electrochemical proton gradient that provides the driving force and causes the electrogenicity of the translocation step. Peptide transporters represent excellent targets for the delivery of pharmacologically active compounds because their substrate-binding site can accommodate a wide range of molecules of differing size, hydrophobicity and charge.

Amino Acid Sequence↗

PubMed2002

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

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

Animals↗

PubMed2002

Defining minimal structural features in substrates of the H(+)/peptide cotransporter PEPT2 using novel amino acid and dipeptide derivatives.

The peptide transporter PEPT2, expressed in a variety of tissues, including kidney, lung, and the central nervous system, mediates the uphill transport of di- and tripeptides, as well as a variety of peptidomimetic drugs. To identify the essential molecular features of substrates that determine affinity and transport by PEPT2, we synthesized a series of amino acid derivatives as well as modified dipeptides. Kinetic constants for the interaction of test compounds with PEPT2 were obtained in a competition assay using Pichia pastoris yeast cells expressing mammalian PEPT2. The two-electrode voltage-clamp technique in Xenopus laevis oocytes was used to assess the substrate's electrogenic transport properties. Whereas omega bar-amino fatty acids showed no affinity for PEPT2, the introduction of a single carbonyl group into the backbone increased both affinity and transport currents more than 30-fold. omega bar-amino fatty acids, at their amino or carboxyl group coupled to an alanine residue, allowed us to determine the importance of the spatial position of functional groups within the molecule. Affinity and transport function declined by elongating the omega bar-amino acid chain when located in the N-terminal position, whereas the elongation in the carboxyl terminal with an N-terminal alanine caused less pronounced effects. The results clearly establish that a free N terminus, a correctly positioned backbone carbonyl group, and a carboxylic group that is in a suitable distance from the intramolecular carbonyl function and the amino terminal head group are the main features for substrate recognition and transport by PEPT2. This information provides the framework for a rational design of peptidomimetic drugs for delivery via PEPT2.

Amino Acids↗

PubMed2002

PEPT1-mediated cefixime uptake into human intestinal epithelial cells is increased by Ca2+ channel blockers.

Ca2+ channel blockers like nifedipine have been shown to increase the oral bioavailability of beta-lactam antibiotics, such as cefixime, in humans. The molecular mode of action of Ca2+ channel blockers on beta-lactam absorption, however, has not yet been defined. Using the Caco-2 human intestinal epithelial cell line, we assessed whether alterations in intracellular free Ca2+ ion (Ca2+in) concentrations by Ca2+ channel blockers or by Ca2+ ionophores affect [14C]cefixime absorption. Reduction of Ca2+in levels by Ca2+ channel blockers (nifedipine, verapamil, diltiazem, or bepridil) at concentrations of 100 microM led to 35 to 50% increases in the cellular uptake of 1 mM [14C]cefixime. Increases in Ca2+in levels by Ca2+ ionophores, on the other hand, led to 40% reductions in [14C]cefixime absorption. Nifedipine increased the V(max) of cefixime transport by 67%, whereas the K(m) of cefixime transport remained unaffected. By measuring the pH in Caco-2 cells loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein, we show that cefixime transport mediated by the intestinal H+-coupled peptide transporter PEPT1 leads to intracellular acidification. This acid load was reduced by nifedipine, although the Ca2+ channel blocker increased the level of H+ and cefixime cotransport. Increases in Ca2+in levels by ionomycin enhanced the decline in intracellular pH induced by cefixime alone, although ionomycin reduced the level of H+ and cefixime cotransport. In conclusion, our studies demonstrate that alterations of Ca2+in levels, e.g., by Ca2+ channel blockers, affect pH regulatory systems, such as apical Na+ and H+ exchange, and thereby alter the H+ gradient that serves as the driving force for uptake of beta-lactams into intestinal epithelial cells.

Caco-2 Cells↗

PubMed2002

Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein.

The lactating mammary gland utilizes free plasma amino acids as well as those derived by hydrolysis from circulating short-chain peptides for protein synthesis. Apart from the major route of amino acid nitrogen delivery to the gland by the various transporters for free amino acids, it has been suggested that dipeptides may also be taken up in intact form to serve as a source of amino acids. The identification of peptide transporters in the mammary gland may therefore provide new insights into protein metabolism and secretion by the gland. The expression and distribution of the high-affinity type proton-coupled peptide transporter PEPT2 were investigated in rat lactating mammary gland as well as in human epithelial cells derived from breast milk. By use of RT-PCR, PEPT2 mRNA was detected in rat mammary gland extracts and human milk epithelial cells. The expression pattern of PEPT2 mRNA revealed a localization in epithelial cells of ducts and glands by nonisotopic high resolution in situ hybridization. In addition, immunohistochemistry was carried out and showed transporter immunoreactivity in the same epithelial cells of the glands and ducts. In addition, two-electrode voltage clamp recordings using PEPT2-expressing Xenopus laevis oocytes demonstrated positive inward currents induced by selected dipeptides that may play a role in aminonitrogen handling in mammalian mammary gland. Taken together, these data suggest that PEPT2 is expressed in mammary gland epithelia, in which it may contribute to the reuptake of short-chain peptides derived from hydrolysis of milk proteins secreted into the lumen. Whereas PEPT2 also transports a variety of drugs, such as selected beta-lactams, angiotensin-converting enzyme inhibitors, and antiviral and anticancer metabolites, their efficient reabsorption via PEPT2 may reduce the burden of xenobiotics in milk.

Animals↗

PubMed2002

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

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

Animals↗

PubMed2001

Synthesis and characterization of high affinity inhibitors of the H+/peptide transporter PEPT2.

In this study, we describe the rational synthesis and functional analysis of novel high affinity inhibitors for the mammalian peptide transporter PEPT2. Moreover, we demonstrate which structural properties convert a transported compound into a non-translocated inhibitor. Starting from Lys[Z(NO(2))]-Pro (where Z is benzyloxycarbonyl), which we recently identified as the first competitive high affinity inhibitor of the intestinal peptide transporter PEPT1, a series of different lysine-containing dipeptide derivatives was synthesized and studied for interaction with PEPT2 based on transport competition assays in Pichia pastoris yeast cells expressing PEPT2 heterologously and in renal SKPT cells expressing PEPT2. In addition, the two-electrode voltage clamp technique in Xenopus laevis oocytes expressing PEPT2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. Synthesis and functional analysis of Lys-Lys derivatives containing benzyloxycarbonyl or 4-nitrobenzyloxycarbonyl side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by PEPT2 with K(i) values as low as 10 +/- 1 nm. This is the highest affinity of a ligand of PEPT2 ever reported. Moreover, based on the structure-function relationship, we conclude that the spatial location of the side chain amino protecting group in a dipeptide containing a diaminocarbonic acid and its intramolecular distance from the Calpha atom are key factors for the transformation of a substrate into an inhibitor of PEPT2.

Animals↗

PubMedDate not supplied

Genistein reverses changes of the proteome induced by oxidized-LDL in EA.hy 926 human endothelial cells.

Endothelial cells are primary targets for pro-atherosclerotic stressors such as oxidized LDL (ox-LDL). The isoflavone genistein, on the other hand, is suggested to prevent a variety of processes underlying atherosclerosis and cardiovascular diseases. By analyzing the proteome of EA.hy 926 endothelial cells, here we show, that genistein reverses the ox-LDL-induced changes of the steady-state levels of several proteins involved in atherosclerosis. These alterations caused by genistein are functionally linked to the inhibition of ox-LDL induced apoptosis.

Cell Line↗

PubMedDate not supplied

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1.

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.

Amino Acid Transport Systems↗