Abstract

Several proline derivatives such as l-azetidine-2-carboxylic acid, cis-4-hydroxy-l-proline, and 3,4-dehydro-dl-proline prevent procollagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors. This study was performed to investigate whether the recently discovered human proton-coupled amino acid transporter 1 (hPAT1) is capable of transporting such pharmacologically relevant proline derivatives and also GABA analogs. Uptake of l-[³H]proline and [³H]glycine in Caco-2 cells was Na⁺-independent but strongly H⁺-dependent. The l-proline uptake was saturable and mediated by a single transport system (hPAT1) with an affinity constant of 2.0 ± 0.2 mM. The uptake of l-[³H]proline was inhibited by d-proline, trans-4-hydroxy-l-proline, cis-4-hydroxy-l-proline, cis-4-hydroxy-d-proline, 3,4-dehydro-dl-proline, l-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, d- and l-pipecolic acid, l-thiaproline, and many others. Apical uptake and transepithelial flux of l-[³H]proline across Caco-2 cell monolayers were strongly inhibited by proline derivatives in proportions corresponding to their respective affinity constants at hPAT1. The basolateral to apical flux of l-[³H]proline was only 8% of that in the opposite direction. Apical uptake of unlabeled l-proline, cis-4-hydroxy-l-proline, and l-azetidine-2-carboxylic acid was stimulated by an inside directed H⁺ gradient 2- to 3-fold. Total apical to basolateral flux of proline derivatives was moderately correlated with their inhibitory potency for l-[³H]proline uptake and flux inhibition. We conclude that 1) the substrate specificity of hPAT1 is very much broader than so far reported and 2) the system accepts therapeutically relevant proline and GABA derivatives. hPAT1 is a promising candidate for new ways of oral drug delivery.