Inactivation of the intestinal uptake system for β-lactam antibiotics by diethylpyrocarbonate

Kramer, Werner; Girbig, Frank; Petzoldt, Evelyne; Leipe, Irina

doi:10.1016/0005-2736(88)90560-3

Cited by 30 publications

(6 citation statements)

References 25 publications

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“…3 and 4). The significance of His residues was also shown for the H ϩ gradient-dependent peptide transporters PepT1 and PepT2 in studies with renal and intestinal brush-border membrane vesicles, where transport activity was severely impaired following incubation with DEPC (21,29). Other examples include the Na ϩ /H ϩ exchanger (9,12), the organic cation/H ϩ antiporter (16) or the folate transporter (53).…”

Section: Discussionmentioning

confidence: 92%

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Leuthold¹,

Hagenbuch²,

Mohebbi³

et al. 2009

American Journal of Physiology-Cell Physiology

160

176

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Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.

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Section: Discussionmentioning

confidence: 92%

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Leuthold¹,

Hagenbuch²,

Mohebbi³

et al. 2009

American Journal of Physiology-Cell Physiology

160

176

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“…These compounds have been previously characterized as PEPT1 substrates and are of pharmacological interest because of their action as antibiotics (50) or ACE inhibitors (48). Moreover, the transport was blocked by diethylpyrocarbonate, which is known to act as an inhibitor for PEPT1 (23,44). Passive diffusion of D-Ala-Lys-AMCA into the cells was ruled out by the lack of staining of the enterocytes surrounding the cells and the lack of tissue fluorescence in the cells when tissues were incubated at 4°C as demonstrated previously (18).…”

Section: Discussionmentioning

confidence: 99%

“…1). Control experiments were performed by incubation in the presence of the transport inhibitor diethylpyrocarbonate (Sigma) (23,44), by incubation carried out at 4°C, and by omission of the fluorophore-labeled dipeptide.…”

Section: Methodsmentioning

confidence: 99%

Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1

Groneberg

Döring

Eynott

et al. 2001

American Journal of Physiology-Gastrointestinal and Liver Physi

112

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The nature of protein breakdown products and peptidomimetic drugs such as beta-lactams is crucial for their transmembrane transport across apical enterocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-mediated uptake of oligopeptides, an ex vivo assay using the fluorophore-conjugated dipeptide derivative D-Ala-Lys-N(epsilon)-7-amino-4-methylcoumarin-3-acetic acid (D-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small intestine. D-Ala-Lys-AMCA was accumulated by enterocytes throughout all segments of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibition studies revealed competitive inhibition by the beta-lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide glycyl-glutamine. Controls were performed using either the inhibitor diethylpyrocarbonate or an incubation temperature of 4 degrees C to exclude unspecific uptake. Immunohistochemistry for PEPT1 localized immunoreactivity to the enterocytes, with the highest intensity at the apical membrane. This is the first study that visualizes dipeptide transport across the mammalian intestine and indicates that uptake assays using D-Ala-Lys-AMCA might be useful for characterizing PEPT1-specific substrates or inhibitors.

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“…the transport of the peptide is coupled to the transfer of positive charge(s) from the external to the internal side of the membrane^addition of diand tripeptides to the extravesicular medium induces signi¢cant membrane potential depolarization (Verri et al 2008). Diethylpyrocarbonate (DEP), which e⁄ciently inhibits peptide transport in mammalian BBMV (Miyamoto, Ganapathy & Leibach 1986;Kramer, Girbig, Petzoldt & Leipe 1988;Kato, Maegawa, Okano, Inui & Hori 1989), also inhibits peptide transport in ¢sh BBMV (Verri et al 1992(Verri et al , 2000(Verri et al , 2008Thamotharan & Gomme et al 1996;Ma⁄a et al 1997Ma⁄a et al , 2003. Taken together, the BBMV data provide a strong biochemical basis for the understanding of the carrier-mediated mechanism that allows di-and tripeptide uptake across the apical barrier of teleost ¢sh enterocytes, and establish that a single carrier system with apparent K m values (K m,app ) (K m,app indicates the apparent concentration of di-or tripeptide that yields one-half of maximal in£ux) in the 0.1^10 mM range mediates the transport process.This system transports a large variety of di-and tripeptides, with remarkable di¡erences in substrate a⁄nity and maximal velocity depending on the particular amino acid composition of the peptide substrate used for assay.…”

Section: Introductionmentioning

confidence: 99%

Transport of di- and tripeptides in teleost fish intestine

Verri

Romano

Barca

et al. 2010

Aquaculture Research

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The initial observation of peptide absorption in fish intestine dates back to 1981, when, in rainbow trout (Oncorhynchus mykiss), the rate of intestinal absorption of the dipeptide glycylglycine (Gly‐Gly) was compared in vivo with the rate of absorption of its component amino acid glycine (Gly). The description of the identification of the underlying mechanisms that allow di‐ and tripeptide transport across the plasma membranes in fish was provided in 1991, when the first evidence of peptide transport activity was reported in brush‐border membrane vesicles of intestinal epithelial cells of Mozambique tilapia (Oreochromis mossambicus) by monitoring uptake of radiolabelled glycyl‐l‐phenylalanine (Gly‐l‐Phe). Since then, the existence of a carrier‐mediated, H+‐dependent transport of di‐ and tripeptides (H+/peptide cotransport) in the brush‐border membrane of fish enterocytes has been confirmed in many teleost species by a variety of biochemical approaches, providing basic kinetics and substrate specificities of the transport activity. In 2003, the first peptide transporter from a teleost fish, i.e. the zebrafish (Danio rerio) PEPtide transporter 1 (PEPT1), was cloned and functionally characterized in the Xenopus laevis oocyte expression system as a low‐affinity/high‐capacity system. PEPT1 is the protein in brush‐border membranes responsible for translocation of intact di‐ and tripeptides released from dietary protein by luminal and membrane‐bound proteases and peptidases. The transporter possesses affinities for the peptide substrates in the 0.1–10 mM range, depending on the structure and physicochemical nature of the substrates. After the molecular and functional characterization of the zebrafish transporter, the interest in PEPT1 in teleost fish has increased and approaches for cloning and functional characterization of PEPT1 orthologues from other fish species, some of them of the highest commercial value, are now underway. In this paper, we provide a brief overview of the transport of di‐ and tripeptides in teleost fish intestine by recalling the bulk of biochemical, biophysical and physiological observations collected in the pre‐cloning era and by recapitulating the more recent molecular and functional data.

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Inactivation of the intestinal uptake system for β-lactam antibiotics by diethylpyrocarbonate

Cited by 30 publications

References 25 publications

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1

Transport of di- and tripeptides in teleost fish intestine

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