Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1.

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac-Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-D-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr-NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (Ki = 1.81+/- 0.37 mM). Ac-Phe-NH2 had a very weak interaction with PepT1 (Ki = 16.8+/-5.64 mM); neither Phe nor Phe-NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (Ki = 0.10+/-0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94+/-0.38 mM). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH2 interacted weakly with PepT1 (Ki = 8.41+/-0.11 and 9.97+/-4.01 mM, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.

Proton-coupled oligopeptide transport by rat renal cortical brush border membrane vesicles: a functional analysis using ACE inhibitors to determine the isoform of the transporter.

We demonstrate that the angiotensin-converting enzyme inhibitors enalapril and captopril inhibit the transport of D-Phe-L-Gln into PepT1-expressing Xenopus oocytes and into rat renal cortical brush border membrane vesicles (BBMV). The kinetics of inhibition are competitive. Enalapril and captopril are not substrates for PepT2 (Boll et al., Proc. Natl. Acad. Sci. 93 (1996) 284-289). Therefore we conclude that in rat renal cortical BBMV this neutral dipeptide is transported via PepT1.

Peptide mimics as substrates for the intestinal peptide transporter.

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.

4-aminomethylbenzoic acid is a non-translocated competitive inhibitor of the epithelial peptide transporter PepT1.

1. 4-Aminomethylbenzoic acid, a molecule which mimics the special configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus Laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles. 2. This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1-exp ressing oocytes and indirectly by its failure to trans-stimulate labelle d peptide efflux through PepT1 in oocytes and in renal membrane vessicle s. 3. However 4-aminiomethylbenzoic acid does reverse trans-stimulation through expressed PepT1 of labelled peptid efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4-aminomethyl benzoic acid competitively binding to the external surface of PepT1. 4. 4-Aminomethylbenzoic acid (the first molecule discovered to be a non-translocated competitive inhibitor of proton-coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.

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

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 K(m) 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 (intra- and 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 trans-stimulation 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.

Substrate-charge dependence of stoichiometry shows membrane potential is the driving force for proton-peptide cotransport in rat renal cortex.

The proton dependence of the transport of three labelled, hydrolysis-resistant synthetic dipeptides carrying a net charge of -1, 0 or +1 has been investigated in a brush border membrane vesicle preparation obtained from rat renal cortex. Cross-inhibition studies are consistent with the transport of all peptides studied being through a single system. The extent and time course of uptake in response to an inwardly directed electrochemical gradient of protons differed for each peptide. For the cationic peptide D-Phe-L-Lys this gradient did not stimulate the initial rate of uptake, while for the neutral dipeptide D-Phe-L-Ala and the anionic peptide D-Phe-L-Glu stimulation was observed. However, the effect on D-Phe-L-Glu was more marked than that on D-Phe-L-Ala and the proton activation differed for these two peptides. The calculated Hill coefficients for the two proton-dependent peptides were 1.14 +/- 0.16 and 2.15 +/- 0.10 for D-Phe-L-Ala and D-Phe-L-Glu, respectively, providing evidence that the stoichiometry of proton:peptide cotransport is different for each peptide (0:1, 1:1 and 2:1 for D-Phe-L-Lys, D-Phe-L-Ala and D-Phe-L-Glu respectively); studies on energetics are compatible with this conclusion. The physiological and molecular implications of this model are discussed, as are the applicability of the conclusions to secondary active transport systems more generally.

Calcitonin and insulin in isobutylcyanoacrylate nanocapsules: protection against proteases and effect on intestinal absorption in rats.

One of the major limiting steps for the absorption of peptide drugs from the intestine is proteolytic degradation. To slow this degradation, human calcitonin was trapped in polyacrylamide nanoparticles, and human calcitonin and insulin were encapsulated with polyisobutylcyanoacrylate. Human calcitonin trapped in polyacrylamide nanoparticles showed no delayed release characteristics and thus would not provide protection from proteases. Proteolytic degradation of human calcitonin and insulin in polyisobutylcyanoacrylate nanocapsules was slower than the free peptides in solution. The plasma pharmacokinetic profiles were consistent with increased survival time of the peptides in the intestine, with higher plasma concentrations of the peptides in the later time samples compared with the controls. However, the nanocapsules gave no significant overall enhancement of peptide absorption. This led to the conclusion that the nanocapsules released the peptides into the intestinal lumen, with small amounts then being absorbed but the rest largely degraded.