MOLECULAR BIOLOGY OF BLOOD BRAIN AMINO ACID TRANSPORT

描述：大脑依赖于各种血液来源的供应。 维持基本代谢途径的营养素， 履行专门职能。 氨基酸，虽然不是主要的 代谢能量来源，是大脑代谢所必需的， 功能 这项研究的长期目标是描述 氨基酸跨细胞转运的分子事件 从血浆经由管腔和腔外 脑内皮细胞的膜。 这些目标包括 了解转运蛋白的分子特性， 运输者所在的地方，以及发生在 病理生理条件。 在这份提案中，有三个主要假设 将被测试的是：1）高亲和力，大的中性氨基酸 大鼠血脑屏障转运蛋白（系统L1）可以被克隆 使用互补克隆从脑微血管cDNA文库，2） 血脑屏障的两种主要氨基酸转运体，系统 y+（碱性氨基酸）和系统L1位于管腔中， 毛细血管内皮细胞的近腔膜和中性 和碱性氨基酸转运系统组分（系统bo，+-相关 蛋白质）也存在，以及3）系统L1转运蛋白的表达 受到刺激，系统y+转运蛋白减少， 肝性脑病大鼠模型高氨血症 大鼠脑 分离微血管mRNA并用于制备微血管cDNA 图书馆 脑微血管的系统L1转运蛋白将被 通过互补克隆鉴定并测序。 系统L1 转运蛋白将在杆状病毒表达系统中表达，并且其 底物特异性、动力学和生物化学性质将被 测定 对应于系统的13-15个氨基酸的肽 L1转运蛋白和先前克隆的系统y+转运蛋白以及 系统bo，+-相关蛋白将被合成并用于提高 多克隆抗体。 细胞的分布和丰度， 氨基酸转运蛋白将通过免疫细胞化学测定， 免疫电镜 转运蛋白特异性cRNA反义和 将制备有义核糖核酸探针并用于确定组织和 通过原位杂交检测转运蛋白mRNA的细胞分布。 氨基酸转运体信息和蛋白表达的改变 将使用以下大鼠模型评价对高氨血症的反应： 肝性脑病 研究描述的分子事件 血脑氨基酸转运可能对理解 与肝脏疾病、中风、糖尿病和各种疾病相关的功能障碍 代谢性脑病并可能导致最小化的治疗 这些功能障碍或设计治疗药物的策略 输送到大脑。

DESCRIPTION: 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.