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

The nature of protein breakdown products and peptidomimetic drugs such as b eta -lactams is crucial for their transmembrane transport across apical ent erocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-media ted uptake of oligopeptides, an ex vivo assay using the fluorophore-conjuga ted dipeptide derivative D-Ala-Lys-N-epsilon-7-amino-4-methyleoumarin-3-ace tic acid (D-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small int estine. D-Ala-Lys-AMCA was accumulated by enterocytes throughout all segmen ts of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibiti on studies revealed competitive inhibition by the beta -lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide gl ycylglutamine. Controls were performed using either the inhibitor diethylpy rocarbonate or an incubation temperature of 4 degreesC to exclude unspecifi c 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 intes tine and indicates that uptake assays using D-Ala-Lys-AMCA might be useful for characterizing PEPT1-specific substrates or inhibitors.