TRANSPORT IN ISOLATED MEMBRANES OF BLOOD BRAIN BARRIER

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DESCRIPTION (From applicant's abstract) The purpose of this proposal is to determine the characteristics of glucose and amino acid carriers in the constituent membranes of the blood-brain barrier, using a new technique that we have developed. Both the luminal and abluminal membranes of endothelial cells that constitute the blood-brain barrier will be isolated as vesicles, and measurements of glucose and amino acid transport will be made in vitro. The investigators hypothesize that the distribution, ion dependency, and kinetics of these transport proteins in the luminal and abluminal membranes favor facilitated delivery of glucose and essential amino acids to the brain, but prevent accumulation of nonessential amino acids that serve as neurotransmitters. Furthermore, the investigators propose that the disproportionate stimulation of phenylalanine and tryptophan transport across the blood-brain barrier during hyperammonemia is caused by exchange with glutamine, which has accumulated in the extracellular fluid of the brain. Thus, the following specific aims are proposed: 1) Determine the distribution and the transport kinetics of glucose carriers in the luminal and abluminal membranes of endothelial cells comprising the blood-brain barrier. This will be done by probing the respective membranes with cytochalasin B to quantify the number of carriers, and to use specific antibodies to determine their identity. The kinetic characteristics of the transporter will be determined by measuring glucose uptake in both luminal and abluminal vesicles. Membranes will be derived from bovine cerebral microvessels. 2) Determine the distribution and transport kinetics of neutral amino acid carriers in the individual membranes of the blood-brain barrier. The polar arrangement, characteristics, and classification of neutral amino acid transport proteins will be determined in bovine membrane vesicles by kinetic measurements of transport. 3) Determine if trans-stimulation of phenylalanine, tryptophan, and leucine transport by glutamine can account for their enhanced uptake across the blood-brain barrier during hyperammonemia. Transport kinetics of phenylalanine, tryptophan, and leucine will be measured in membrane vesicles that have been pre-loaded with glutamine, to determine if exchange with glutamine enhances their uptake. The exchange mechanism will be characterized.