METABOLISM, UPTAKE, AND TRANSEPITHELIAL TRANSPORT OF THE DIASTEREOMERS OF VAL-VAL IN THE HUMAN INTESTINAL-CELL LINE, CACO-2

Purpose. The purpose of this study was to determine whether the bindin g of the diastereomers of Val-Val to the apical oligopeptide transport er(s) could be correlated with their cellular uptake and transepitheli al transport. Methods. The Caco-2 cell culture system was used for all experiments. The binding of the diastereomers of Val-Val was evaluate d by determining their ability to inhibit [H-3]cephalexin uptake. The stability of the diastereomers was determined in a homogenate of Caco- 2 cells and in the apical bathing solution over Caco-2 cell monolayers . The cellular uptake and transepithelial transport properties of the individual diastereomers were studied using Caco-2 cell monolayers. Re sults. 10 mM concentrations of L-Val-L-Val, L-Val-D-Val, D-Va-L-Val an d D-Val-D-Val inhibited cellular uptake of [H-3]cephalexin (0.1 mM) by 92%, 37%, 70%, and 18%, respectively. When the cellular uptake of Val -Val diastereomers (1 mM) were evaluated, the intracellular concentrat ions of L-Val-D-Val and D-Val-L-Val were 15 and 50 times higher, respe ctively, than that of D-Val-D-Val. The cellular uptake of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) (>95%), whereas Gly-P ro had no effect on the cellular uptake of D-Val-D-Val. L-Val-L-Val wa s not detected in the Caco-2 cells, probably due to its metabolic labi lity. When the transepithelial transport of the Val-Val diastereomers (1 mM) was determined, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val transp ort rates were similar. The transepithelial transport of L-Val-D-Val a nd D-Val-L-Val was inhibited by Gly-Pro (10 mM) 36% and 30%, respectiv ely, while Gly-Pro inhibited carnosine (1 mM) transepithelial transpor t by 65%. Gly-Pro had no effect on the transepithelial transport of D- Val-D-Val. Conclusions. These results suggest that the major transepit helial transport route of L-Val-D-Val, D-Val-L-Val and D-Val-D-Val is passive diffusion via the paracellular route. The binding of Val-Val d iastereomers to the oligopeptide transporter(s) is a good predictor of their cellular uptake, however, the binding is not a good predictor o f their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates efflux of the peptide a cross the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.