Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

Lister, Norma; Sykes, A. P.; Bailey, Patrick D.; Boyd, C. A. R.; Bronk, J. R.

doi:10.1113/jphysiol.1995.sp020656

Cited by 48 publications

(28 citation statements)

References 15 publications

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“…17,24,26,27) We also examined the absorption and assimilation of Hypdipeptides by using a vascular perfusion technique in the intestine. When Hyp-Gly and Pro-Hyp were injected into the jejunum, perfusate free Hyp did not increase, but the peptide form of Hyp was absorbed and gradually Crude material from the pooled perfusates was subjected to solid-phase extraction (SPE) with a Sep-Pak Light C18 cartridge.…”

Section: Discussionmentioning

confidence: 99%

“…In transport studies on small oligopeptides, labelled compounds are often used and/or the measurement of substrates or products is carried out by HPLC analyses. 16,17,26,27) The resolution and identification of Hyp-dipeptides by reversed-phase HPLC are not specific and sensitive. Therefore, we used LC-MS/MS analyses of the perfusates to check the level of intact dipeptide transport across the intestinal wall.…”

Section: Discussionmentioning

confidence: 99%

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Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestinein Situ

Liu

Sugita

Nihei

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestinein Situ

Liu

Sugita

Nihei

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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“…Rat intestinal loops in vitro and vascularly perfused small intestine in situ were used to measure transepithelial fluxes in the intact small intestine as described previously (3,7). Luminal pH was changed using a previously published protocol (8).…”

Section: Methodsmentioning

confidence: 99%

“…The substrate, 4-aminophenylacetic acid (4-APAA), 1 was selected on the basis of its chemical structure, it being a potential mimic of a dipeptide (D-Phe-LAla) (Fig. 1) which previously we have shown to be an excellent substrate for epithelial peptide transport (3,4).…”

Section: -Aminophenylacetic Acid (4-apaa)mentioning

confidence: 99%

Peptide Mimics as Substrates for the Intestinal Peptide Transporter

Temple¹,

Stewart²,

Meredith³

et al. 1998

Journal of Biological Chemistry

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4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated.4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter.These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.The rapid uptake of intact small peptides across the brushborder membrane of the small intestinal epithelium is the major route for absorption of dietary protein ␣-amino nitrogen (1). Hitherto, it has been thought that a number of chemical features, for example free amino and carboxyl termini, are essential in contributing to substrate interaction with, and translocation through, the intestinal peptide transporter.These features include the presence of a peptide bond within the substrate molecules. Indeed a major review (1) states that "it is the presence of peptide bonds which make di-and tripeptide acceptable to the peptide transport systems." Although previous work (e.g. Ref.2) has shown that molecules lacking this feature can inhibit transport of peptides (presumably by substrate binding), we describe here for the first time rapid transport of a small totally non-peptidic substrate through the intestinal peptide transporter. The substrate, 4-aminophenylacetic acid (4-APAA), 1 was selected on the basis of its chemical structure, it being a potential mimic of a dipeptide (D-Phe-LAla) (Fig. 1) which previously we have shown to be an excellent substrate for epithelial peptide transport (3, 4). EXPERIMENTAL PROCEDURESRat renal brush-border membrane vesicles were prepared as described previously (5), and initial rates of labeled peptide transport (influx, efflux) were determined by rapid filtration (4, 6). Rat intestinal loops in vitro and vascularly perfused small intestine in situ were used to measure transepithelial fluxes in the intact small intestine as described previously (3, 7). Luminal pH was changed using a previously published protocol (8). Isolated murine enterocytes were prepared by enzymatic digestion using haluronidase, and i...

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“…The absence of appropriate model substrates has impeded the analysis of peptide transport (a process of very substantial physiological importance in many epithelia, Matthews, 1991;Meredith & Boyd, 1995a). However, the systematic studies of Lister, Sykes, Bailey, Boyd & Bronk (1995) have shown that the presence of a D-amino acid at the N-terminal of an oligopeptide confers hydrolysis resistance but does not substantially reduce transepithelial transport across rat small intestine in vitro. As a direct consequence of these studies we have used two such labelled molecules (the neutral D-Phe-L-Ala and the anionic D-Phe-L-Glu) to probe the mechanisms of peptide transport.…”

mentioning

confidence: 99%

A model for the kinetics of neutral and anionic dipeptide‐proton cotransport by the apical membrane of rat kidney cortex.

Temple

Bailey

Bronk

et al. 1996

The Journal of Physiology

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1. Kinetics of influx (mediated through peptide-proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D-Phe-L-Ala and anionic D-Phe-L-Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (Em) to be varied systemically and independently. From this data the kinetic constants Km and Vmax were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5-5 (intraand extravesicular), but not at pH 7 4, the rate constants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D-Phe-L-Glu (but not D-Phe-L-Ala) trans-stimulated efflux of both peptides to a similar extent; the extent of this transstimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton-peptide coupling on the net charge carried by the substrate. Quantitatively a 50-fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.The analysis of transport systems has been critically dependent on the development of appropriate (i.e. specific, stable and hydrolysis resistant) model substrates; this has been true for example in both the amino acid and sugar transport field. The absence of appropriate model substrates has impeded the analysis of peptide transport (a process of very substantial physiological importance in many epithelia, Matthews, 1991; Meredith & Boyd, 1995a). However, the systematic studies of Lister, Sykes, Bailey, Boyd & Bronk (1995) have shown that the presence of a D-amino acid at the N-terminal of an oligopeptide confers hydrolysis resistance but does not substantially reduce transepithelial transport across rat small intestine in vitro. As a direct consequence of these studies we have used two such labelled molecules (the neutral D-Phe-L-Ala and the anionic D-Phe-L-Glu) to probe the mechanisms of peptide transport. Our studies have been carried out on isolated apical membrane vesicles from mammalian renal cortex, a preparation which is very well-characterized m...

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Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity.

Cited by 48 publications

References 15 publications

Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestinein Situ

Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestinein Situ

Peptide Mimics as Substrates for the Intestinal Peptide Transporter

A model for the kinetics of neutral and anionic dipeptide‐proton cotransport by the apical membrane of rat kidney cortex.

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