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

Abstract

Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of beta-alanine as well as alpha-fluoro-beta-alanine (FbetaAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for beta-alanine and GABA with Michaelis constant (Km) of 35.3 and 22.5 microM, respectively. A second Na+-dependent kinetic component with Km >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 this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of beta-alanine to passive diffusion level, indicating that both Na+ and Cl- are essential for the activity of this transporter. The transport of beta-alanine was inhibited by GABA, hypotaurine, beta-aminoisobutyric acid, and FbetaAL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and alpha-aminoisobutyric acid did not affect beta-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 beta-alanine and FbetaAL and their effect on the cellular reuptake of GABA.