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 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.
Liu
M
M
Departments of Internal Medicine (Oncology) and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Russell
R L
RL
Beigelman
L
L
Handschumacher
R E
RE
Pizzorno
G
G
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000502:physiology*; D002460:Cell Line; D002712:Chlorides / Q000502:physiology; D004285:Dogs; D050485:GABA Plasma Membrane Transport Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000502:physiology; D014529:Uridine / Q000378:metabolism; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000378:metabolism*
D002352:Carrier Proteins; D002712:Chlorides; D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; C092004:Slc6a13 protein, rat; D015091:beta-Alanine; C032348:alpha-fluoro-beta-alanine; D012964:Sodium; D014529:Uridine