Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells.

The mechanisms of carrier-mediated transport of gamma-aminobutyric acid (GABA) at the blood-brain barrier (BBB) were examined by investigating [3H]GABA uptake by isolated bovine brain capillaries, monolayers of primary cultured brain capillary endothelial cells (BCECs) attached to plates or suspended BCECs. The uptake of [3H]GABA was concentration-dependent and saturable. Nonlinear regression analysis of the original data indicated the existence of two distinct high and low-affinity GABA transporters on isolated brain capillaries or suspended BCECs, with Km1, Km2, Vm1 and Vm2 equal to 25.3 microM, 485.2 microM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 microM, 322.0 microM, 6.1 and 15.7 nmol/5 min/mg protein, respectively, for the suspended BCECs. In contrast, a single low-affinity transporter was found for monolayers of BCECs attached to plates with Km and Vm equal to 338.7 microM and 18.8 nmol/5 min/mg protein, respectively. Subcellular location of the two distinct transporters on BCECs is discussed, suggesting that the low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter is probably localized to the antiluminal membrane. Low temperature (4 degreesC) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [3H]GABA by isolated brain capillaries. The substitution of Na+ with choline+, K+ or Li+ with the counter anion Cl- almost completely abolished both uptakes. Substitution of Cl- with Br-, I-, F- or NO3- in the presence of Na+ significantly reduced both uptakes to different extents. Alanine, leucine, phenylalanine, arginine, glutamate and pyruvate had no obvious effect on either uptake. Probenecid, amino-oxyacetic acid, beta-alanine, taurine, betaine, and nipecotic acid significantly reduced both uptakes. These data suggested that both the GABA transporters at the BBB were temperature, metabolic energy, Na+ and Cl--dependent, and may be specific and different from the known monocarboxylic acid, GABA and other amino acid transporters, which may play a role in the disposition of GABA in the brain.