Under certain experimental conditions transepithelial dipeptide transport across human intestinal epithelial (Caco-2) cell monolayers is Na⁺ dependent. In contrast, the cloned intestinal di/tripeptide carrier PepT1 functions as a H⁺-coupled, Na⁺-independent transport system (Fei et al. 1994). A model to account for this apparent Na⁺ dependence has been proposed in which PepT1 and the apically located Na⁺-H⁺ exchanger (NHE3) function in a co-operative manner to optimise dipeptide absorption (NHE3 being responsible for the maintenance of the H⁺-electrochemical gradient required for PepT1 activity) (Ganapathy & Leibach, 1985). This Na⁺-dependent component of dipeptide transport can be inhibited by both pharmacological (S1611) and physiological (VIP) regulators of NHE3 (Thwaites et al. 2000; Kennedy & Thwaites 2001). The aim of this study was to investigate the relationship between hPepT1 and NHE3 at apical pH values (pH 6.5) typical of those measured at the surface of the mammalian small intestine (McEwan et al. 1988).

Caco-2 cell monolayers (passage 99-124) were grown on permeable supports and used 14-17 days post-seeding. Apical [¹⁴C] Gly-Sar (0.1 mM, 0.5 µCi ml^-1) uptake was determined (1-180 min, 37 °C) in modified Krebs-Ringer solution (containing either 137 mM NaCl or choline chloride, pH 5.0-8.0) in the presence or absence of the NHE3 selective inhibitor S1611 (3 µM). Results are expressed as a percentage of the uptake at apical pH 5.5-5.6 (mean ± S.E.M. (n), ANOVA). The pH-dependent profile of Gly-Sar uptake over short incubation periods (1 min) resembled that determined using Xenopus laevis oocytes expressing rabbit PepT1 (Fei et al. 1994). Maximal peptide uptake was observed at pH 5.3-5.6 and was reduced to 46 ± 5 (10) % when apical pH was increased to pH 6.5 (P < 0.001 versus pH 5.6). As incubation time increased the pH-dependent profile of Gly-Sar uptake changed (the peak moving towards more alkali pH) and after 180 min there was no significant difference in the uptake at apical pH 5.5 or 6.5 (100 ± 4 (15) %, P > 0.05 versus pH 5.5). Inhibition of NHE3 activity led to a decrease in Gly-Sar uptake (at 180 min) at apical pH 6.5 (30 ± 4 (15) % (P < 0.001 versus pH 5.5) and 52 ± 2 (11) % (P < 0.01 versus pH 5.5) in the absence of extracellular Na⁺ or presence of S1611, respectively). Modulation of pH-dependent apical-to-basolateral Gly-Sar transport (180 min) followed a similar pattern. In conclusion, at apical pH 6.5 a functional interplay between hPepT1 and NHE3 is required to allow optimal dipeptide absorption to proceed over time periods (3-4 h) required to accomplish absorption of a meal across the intestinal wall.

This work was supported by the BBSRC (grant number 13/D09145). D.J.K. is supported by a studentship from the University of Newcastle upon Tyne Faculty of Medicine. S1611 was a kind gift from H.J. Lang, Aventis Pharma Deutschland GmbH, Frankfurt/Main, Germany.