GAT2/BGT-1 as a system responsible for the transport of gamma-aminobutyric acid at the mouse blood-brain barrier.
In this study, the gamma-aminobutyric acid (GABA) transporter at the blood-brain barrier (BBB) was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis, and the transport mechanism was characterized using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB) as an in vitro model of the BBB. gamma-Aminobutyric acid transport was studied by the cellular uptake of [ 3 H]GABA. [3H]GABA uptake by TM-BBB cells was Na (+)-, Cl(-)-, and concentration-dependent. The corresponding Michaelis-Menten constant was 679 +/- 80 micromol/L and the maximal uptake rate was 4,790 +/- 494 pmol/(mg protein x 5 minutes). [3H]GABA uptake by TM-BBB cells was significantly inhibited by betaine, beta-alanine, nipecotic acid, taurine, and quinidine, whereas probenecid, L-proline, creatine, and glycine had no effect. This type of inhibition is consistent with the predominant involvement of the GAT2/BGT-1 transporter in TM-BBB cells. RT-PCR analysis showed that GAT2/BGT-1 mRNA was expressed in TM-BBB cells, whereas Western blot analysis showed that TM-BBB cells and mouse brain capillaries express GAT2/BGT-1 protein. Moreover, confocal immunofluorescent microscopy of dual-labeled mouse brain sections demonstrated the colocalization of GAT2/BGT-1 and P-glycoprotein, a BBB-specific marker, on brain capillaries labeled with anti-GAT2/BGT-1 antibody and anti-P-glycoprotein antibody, respectively. These results are evidence that GAT2/BGT-1 is expressed at the BBB and is involved in GABA transport across the BBB.
Takanaga
H
H
Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Ohtsuki
S
S
Hosoya Ki
Terasaki
T
T
D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics; D002478:Cells, Cultured; D050485:GABA Plasma Membrane Transport Proteins; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D027361:Organic Anion Transporters; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D019459:Urothelium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C490439:Slc6a12 protein, mouse; D005680:gamma-Aminobutyric Acid; C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase