Abstract
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.
Highlights
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
4-aminophenylacetic acid (4-APAA) transport across the rat intact intestine was stimulated 18-fold by luminal acidification 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
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]
Summary
Rat 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]. Isolated cells were exposed to 20 mM HEPES-buffered Krebs solution containing either the dipeptide (Phe-Ala) or 4-APAA. Xenopus oocyte expression of PepT1 cRNA was as described [10, 11] with subsequent HPLC detection [3] of transported substrate and correction for transport in water-injected controls. For efflux assays 4.6 nl (110 fmol) of radiolabeled dipeptide (D-Phe-L-Gln, 0.1 Ci/l) was injected into each oocyte. Radiolabeled dipeptide influx was corrected for non-mediated transport by subtracting the transport of radiolabel seen in the presence of saturating unlabeled peptide (10 mM). The ENZFITTER program was used to calculate the Ki for mediated transport only
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