The cloned intestinal di/tripeptide transporter hPepT1 is a H⁺-coupled, Na⁺-independent carrier. However, for optimal dipeptide uptake to occur in intact intestinal epithelia extracellular Na⁺ is required to allow functional coupling between the Na⁺/H⁺ exchanger NHE3 and hPepT1 (Kennedy et al. 2002; Thwaites et al. 2002). The Na⁺-dependent component of dipeptide uptake in Caco-2 cell monolayers is reduced in the presence of the NHE3 inhibitor S1611 whereas S1611 has no effect on dipeptide uptake in hPepT1-expressing oocytes (Kennedy et al. 2002). The aim of the present study was to investigate whether or not other pharmacological NHE inhibitors are able to modulate dipeptide uptake via the same mechanism as S1611.

Caco-2 cells (passage 102-118) were cultured on permeable supports and used 14-17 days post seeding (Thwaites et al. 1999). Apical uptake (37 °C) of Gly-Sar, leucine or arginine (all 10-100 µM, 0.5 µCi ml^-1) was determined using Na⁺ and Na⁺-free (choline chloride) modified Krebs-Ringer solution (pH 5.0-7.4). Xenopus laevis were killed humanely and oocytes removed. Gly-Sar (88 µM, 5 µCi ml^-1) uptake (40 min, pH 6.5 in the presence of Na⁺) was determined in X. laevis oocytes 3 days after injection with 50 ng hPepT1 cRNA.

One hundred micromolar EIPA inhibits NHE3 (Orlowski, 1993; Thwaites et al. 1999) and has the same effect as S1611 (3 µM) on Gly-Sar uptake across the apical membrane of Caco-2 cell monolayers (P > 0.05, ANOVA). However, at higher (e.g. 500 µM) EIPA concentrations there was an additional S1611-insensitive effect (P < 0.05 versus S1611). In the presence of Na⁺, Gly-Sar uptake (pH 6.5) was reduced (P < 0.001) by EIPA (500 µM) from 32.6 ± 2.6 (10) to 12.4 ± 1.0 pmol cm^-2 (15 min)^-1 (11) (means ± S.E.M. (n)). Even in the absence of extracellular Na⁺ (where NHE3 is inactive) Gly-Sar uptake was reduced (P < 0.05) by EIPA (500 µM) (from 16.8 ± 0.8 (11) to 11.3 ± 0.7 pmol cm^-2 (15 min)^-1 (11)). EIPA (500 µM) had no effect (P > 0.05) on either arginine or leucine uptake. In the absence of Na⁺, EIPA (500 µM) inhibited Gly-Sar uptake in a pH-dependent manner being maximal at pH 5.0-6.0. In addition, EIPA (500 µM) inhibited (P < 0.001) Gly-Sar uptake into hPepT1-expressing oocytes whereas S1611 was without effect.

In conclusion, high concentrations of EIPA (e.g. 500 µM) are able to inhibit dipeptide uptake via a Na⁺-independent, non-NHE3 mediated pathway. This pharmacological inhibition may be either direct on hPepT1 or indirect by a reduction in the H⁺-driving force for hPepT1 activity.

This work was supported by the BBSRC (grant 13/D17277).