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

Abstract

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.

hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes

“…5 Interestingly, hPepT1 expression is normally restricted to the small intestine, where bacteria are far fewer in number, and was not detected in the normal esophagus, stomach or colon. [6][7][8] However, we found that hPepT1 is expressed in colon tissues affected with inflammatory bowel disease 9 and it has been shown that hPepT1 is upregulated in the colon of patients with short-bowel syndrome, 10 suggesting that hPepT1 may be involved in intestinal pathological states. 9 As fMLP transport across basolateral membranes is rate limiting, 4 it seems logical that hPepT1 expression in the presence of a significant luminal concentration of n-formyl peptides would lead to the accumulation of these bacterial peptides in the cytoplasm.…”

Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1

“…Cefaclor (1 mM) and glycyl-sarcosine (Gly-Sar, 10 mM) are known PepT1 substrates (37). ␤-Ala-Lys-Ne-AMCA labeled peptide, a known PepT1-transportable substrate used to measure peptide uptake into the cell cytosol (13), was synthesized by J. R. Reeve [University of California Los Angeles (UCLA), School of Medicine].…”

Deletion of the Intestinal Peptide Transporter Affects Insulin and TOR Signaling in Caenorhabditis elegans

“…Even if PEP-2 were expressed from this mutant allele, it would lack the N-terminal six transmembrane domains required for substrate binding and transport (29) and therefore pep-2(lg601) most likely represents a strong loss-of-function or null allele. To demonstrate that the pep-2(lg601) mutant strain has lost its capability for transport of di-and tripeptides, animals were exposed to the fluorescent dipeptide conjugate ␤-Ala-Lys-AMCA that was previously shown to be a representative substrate of PEPT1 (30). Efficient uptake of the reporter molecule into intestinal epithelial cells of wild type animals was indicated by a strong fluorescence of all intestinal cells, whereas the gut lumen lacked staining suggesting complete and rapid intestinal peptide absorption (Fig.…”