Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of -alanine as well as -fluoro--alanine (FAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na⁺-dependent transport with high affinity for -alanine and GABA with Michaelis constant (K_m) of 35.3 and 22.5 µM, respectively. A second Na⁺-dependent kinetic component with K_m >1 mM was also identified. The sigmoidal profile of -alanine uptake with respect to Na⁺ shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 ± 0.4 indicates that at least two sodium ions are cotransported with -alanine. The flux of -alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na⁺, reduced the intracellular concentrative accumulation of -alanine to passive diffusion level, indicating that both Na⁺ and Cl are essential for the activity of this transporter. The transport of -alanine was inhibited by GABA, hypotaurine, -aminoisobutyric acid, and FAL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and -aminoisobutyric acid did not affect -alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of -alanine and FAL and their effect on the cellular reuptake of GABA.

uridine; liver; 5-fluorouracil