Influence of oligopeptide transporter binding affinity upon uptake and transport of d-Asp(OBzl)-Ala and Asp(OBzl)-Sar in filter-grown Caco-2 monolayers

Abstract

The oligopeptide transporter, which is responsible for the absorption of various di/tripeptides and several peptidomimetic drugs across the intestinal epithelia, is expressed in mature Caco-2 monolayers. Using certain enzymatically stable dipeptides containing either l- or d-aspartic acid at the amino terminus, we investigated the relationship between a side-chain modified dipeptide's degree of binding affinity for the apically expressed Caco-2 oligopeptide transporter and its ability to undergo uptake and/or apical-to-basal transport. Two β-esterified dipeptides, d-Asp(OBzl)-Ala and Asp(OBzl)-Sar, possess markedly different affinities for the Caco-2 oligopeptide transporter (IC 50=2.62±0.35 and 0.014±0.007 mM, respectively) as determined using a [ 14C]Gly-Sar cellular uptake displacement assay. d-Asp(OBzl)-Ala undergoes rapid internalization into Caco-2 monolayers (14.33±1.00 nmol/mg protein) during a 15-min uptake study; additionally, d-Asp(OBzl)-Ala is efficiently transported in the apical-to-basal direction across Caco-2 monolayers (14.41±0.91 nmol/h/cm 2). Both uptake and transport of d-Asp(OBzl)-Ala are >90% inhibitable by the presence of a 20-fold molar excess of Gly-Pro in the apical chamber. Although Asp(OBzl)-Sar demonstrates a 187-fold lower IC 50 value than d-Asp(OBzl)-Ala, Asp(OBzl)-Sar does not achieve uptake or transport in parallel experiments. These data indicate that a side-chain modified, enzymatically stable dipeptide, d-Asp(OBzl)-Ala, is actively taken up into and transported across Caco-2 monolayers via the oligopeptide transporter. Additionally, the degree of affinity of a side-chain modified dipeptide for the Caco-2 oligopeptide transporter is not necessarily indicative of its ability to access the oligopeptide transporter-mediated uptake and transport pathway.