beta-alanine and alpha-fluoro-beta-alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2

Studies on the compartmentalization of uridine catabolic metabolism in live r have indicated accumulation of beta-alanine as well as alpha-fluoro-beta- alanine (F beta AL) for 5-fluorouracil in the hepatocytes. Using preparatio ns of rat hepatocytes we were able to identify a Na+-dependent transport wi th high affinity for beta-alanine and GABA with Michaelis constant (K-m) of 35.3 and 22.5 mu M, respectively. A second Na+-dependent kinetic component with K-m >1 mM was also identified. The sigmoidal profile of beta-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 beta-alanine. The flux of beta-alanine was also shown to be chlorine dependent. The substitution of t his anion with gluconate, even in the presence of Na+, reduced the intracel lular concentrative accumulation of beta-alanine to passive diffusion level , indicating that both Na+ and Cl- are essential for the activity of this t ransporter. The transport of beta-alanine was inhibited by GABA, hypotaurin e, beta-aminoisobutyric acid, and F beta AL in a competitive manner. Howeve r, concentrations up to 1 mM of L- and D-alanine, taurine, and a-aminoisobu tyric acid did not affect beta-alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepat ocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa b and corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 t ransfected Madin-Darby canine kidney cell extract, confirming the extraneur al localization of this transporter. In view of these results, the neurotox ic effects related to the administration of uridine and 5-fluorouracil coul d be explained with the formation of beta-alanine and F beta AL and their e ffect on the cellular reuptake of GABA.