Neutral amino acid transport by the blood-brain barrier. Membrane vesicle studies.
Endothelial cell membranes, the site of the blood-brain barrier, were obtained from the capillaries of cow brain. The luminal and abluminal membranes were separated by centrifugation on a discontinuous Ficoll gradient. Electron microscopy revealed that the membrane preparations consisted almost entirely of sealed vesicles. The release of latent enzyme activity showed that both membrane preparations were primarily right side out. Radiolabeled L-phenylalanine uptake by luminal vesicles was proportional to membrane protein concentration, with less than 10% binding. Transport was by a high affinity carrier (Km 11.8 +/- 0.1 microM, asymptotic standard error) that showed little or no stereospecificity, and was independent of Na+ or H+ gradients. Transport was inhibited by L-tryptophan, L-leucine, 2-aminobicyclo[2,2,1]heptane-2-carboxylate and D-phenylalanine, but not by N-(methylamino)-isobutyrate. Abluminal membranes showed an additional component in which a Na+ gradient accelerated the transport of both phenylalanine and N-(methylamino)-isobutyrate. These studies demonstrate the utility of membrane vesicles as a model to characterize the transport properties of the distinct membranes of the polar endothelial cells that form the blood-brain barrier.
Sánchez del Pino
M M
MM
Department of Physiology and Biophysics, University of Health Sciences/Chicago Medical School, Illinois 60064.
Hawkins
R A
RA
Peterson
D R
DR
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001812:Blood-Brain Barrier; D002196:Capillaries; D002250:Carbon Radioisotopes; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002501:Centrifugation, Zonal; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D010042:Ouabain / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D014316:Tritium; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000378:metabolism
D000596:Amino Acids; D002250:Carbon Radioisotopes; D014316:Tritium; D015091:beta-Alanine; C012924:2,2-dimethyl-beta-alanine; D010649:Phenylalanine; D010042:Ouabain; D012964:Sodium