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

Abstract

The mechanisms of carrier-mediated transport of γ-aminobutyric acid (GABA) at the blood–brain barrier (BBB) were examined by investigating [ 3 H ]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 [ 3 H ]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 K m1, K m2, V m1 and V m2 equal to 25.3 μM, 485.2 μM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 μM, 322.0 μM, 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 K m and V m equal to 338.7 μM 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°C) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [ 3 H ]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 NO 3 − 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, β-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.