Basolateral glycylsarcosine (Gly-Sar) transport in Caco-2 cell monolayers is pH dependent

OBJECTIVES: Transepithelial di/tripeptide transport in enterocytes occurs via the apical proton-coupled peptide transporter, hPEPT1 (SLC15A1) and a basolateral peptide transporter, which has only been characterized functionally. In this study we examined the pH dependency, substrate uptake kinetics and substrate specificity of the transporter. METHODS: We studied the uptake of [(14) C]Gly-Sar from basolateral solution into Caco-2 cell monolayers grown for 17-22 days on permeable supports, at a range of basolateral pH values. KEY FINDINGS: Basolateral Gly-Sar uptake was pH dependent, with a maximal uptake rate at a basolateral pH of 5.5. Uptake of Gly-Sar decreased in the presence of the protonophore nigericin, indicating that the uptake was proton-coupled. The uptake was saturable, with a maximal flux (Vmax ) of 408 ± 71, 307 ± 25 and 188 ± 19 pmol/cm(2) /min (mean ± S.E., n = 3) at basolateral pH 5.0, 6.0 and 7.4, respectively. The compounds Gly-Asp, Glu-Phe-Tyr, Gly-Glu-Gly, Gly-Phe-Gly, lidocaine and, to a smaller degree, para-aminohippuric acid were all shown to inhibit the basolateral uptake of Gly-Sar. CONCLUSIONS: The study showed that basolateral Gly-Sar transport in the intestinal cell line Caco-2 is proton-coupled. The inhibitor profile indicated that the transporter has broad substrate specificity.