title: Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells.
abstract: The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H(+)/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [(14)C]glycylsarcosine with K(i) values of 0.19 +/- 0.01 mm and 0.07 +/- 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K(t)) but not the maximal velocity (V(max)) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [(14)C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [(14)C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [(14)C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [(14)C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells trade mark, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H(+)/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H(+)-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H(+)/peptide cotransporters.
authors.affiliation: Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenburg, Halle, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Neumann
authors.forename: Mandy
authors.initials: M
authors.lastname: Bruch
authors.forename: Sabine
authors.initials: S
authors.lastname: Gebauer
authors.forename: Matthias
authors.initials: M
authors.lastname: Brandsch
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004151:Dipeptides / Q000378:metabolism; D004594:Electrophysiology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D042541:Intracellular Space / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D000074011:Peptide Transporter 1; D051381:Rats; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; C489940:Slc15a1 protein, rat; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C006437:alafosfalin; C004194:glycylsarcosine; D000409:Alanine