The brain is dependent upon a supply of various blood-borne nutrients for maintenance of essential metabolic pathways and for carrying out specialized functions. Amino acids, although not a major source of metabolic energy, are essential for brain metabolism and function. The long-term goals of this research are to characterize the molecular events whereby amino acids are transported transcellularly from the plasma to the interstitial fluid via the luminal and abluminal membranes of the brain endothelial cell. These goals include an understanding of the molecular identity of the transporters, the sites where transporters are located, and the alterations that occur under pathophysiological conditions. In this proposal three major hypotheses will be tested and are: 1) the high affinity, large neutral amino acid transporter (System L1) of the rat blood-brain barrier can be cloned from a brain microvessel cDNA library using complementation cloning, 2) the two major amino acid transporters of the blood-brain barrier, System y+ (basic amino acids) and System L1, are located in the luminal and abluminal membranes of the capillary endothelial cell, and the neutral and basic amino acid transport system component (System bo,+- associated protein) is also present, and 3) expression of the System L1 transporter is stimulated and the System y+ transporter is decreased in response to hyperammonemia in a rat model of hepatic encephalopathy. Rat brain microvessel mRNA will be isolated and used to prepare a microvessel cDNA library. The System L1 transporter of brain microvessels will be identified by complementation cloning and sequenced. The System L1 transporter will be expressed in a baculovirus expression system and its substrate specificity, kinetics, and biochemical properties will be determined. Peptides corresponding to 13-15 amino acids of the System L1 transporter and the previously cloned System y+ transporter and the System bo,+-associated protein will be synthesized and used to raise polyclonal antibodies. The cellular distribution and abundance of the amino acid transporters will be determined by immunocytochemistry and immunoelectron microscopy. Transporter specific cRNA antisense and sense riboprobes will be prepared and used to determine the tissue and cellular distribution of transporter mRNA by in situ hybridization. Alterations in expression of amino acid transporter message and protein in response to hyperammonemia will be evaluated using a rat model of hepatic encephalopathy. Research describing the molecular events of blood-brain amino acid transport may be valuable in understanding dysfunctions associated with liver disease, stroke, diabetes and diverse metabolic encephalopathies and may lead to treatments for minimizing these dysfunctions or for designing strategies for therapeutic drug delivery to the brain.
