MOLECULAR BIOLOGY OF BLOOD BRAIN AMINO ACID TRANSPORT

血脑氨基酸转运的分子生物学

• 批准号: 2431229
• 负责人: LESTER Richard DREWES
• 金额: $ 22.55万
• 依托单位: UNIVERSITY OF MINNESOTA DULUTH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2431229
• 关键词: RNA; aminoacid transport; ammonia; blood brain barrier; brain metabolism; disease /disorder model; gene expression; hepatic coma /encephalopathy; immunocytochemistry; in situ hybridization; laboratory rat; membrane transport proteins; molecular cloning; nucleic acid probes; transfection; vascular endothelium

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.

描述： 大脑依赖于各种血源性物质的供应 维持基本代谢途径的营养素 执行专门职能。 氨基酸，虽然不是主要成分 代谢能量的来源，对大脑新陈代谢至关重要 功能。 这项研究的长期目标是表征 氨基酸跨细胞转运的分子事件 从血浆通过管腔和近管腔到间质液 脑内皮细胞膜。 这些目标包括 了解转运蛋白的分子特性、位点 运输商所在的位置以及发生的变化 病理生理条件。 在这个提案中，三个主要假设 将被测试并且是：1)高亲和力、大中性氨基酸 可以克隆大鼠血脑屏障的转运蛋白（系统 L1） 使用互补克隆从脑微血管 cDNA 文库中提取，2) 血脑屏障的两个主要氨基酸转运蛋白系统 y+（碱性氨基酸）和系统 L1，位于管腔和 毛细血管内皮细胞的近腔膜和中性细胞膜 和碱性氨基酸转运系统组件（系统bo，+-相关 蛋白）也存在，并且 3）系统 L1 转运蛋白的表达 受到刺激，系统 y+ 转运蛋白响应减少 肝性脑病大鼠模型中的高氨血症。 大鼠大脑 将分离微血管 mRNA 并用于制备微血管 cDNA 图书馆。 脑微血管的 L1 系统转运蛋白将是 通过互补克隆进行鉴定并测序。 系统L1 转运蛋白将在杆状病毒表达系统中表达，其 底物特异性、动力学和生化特性将 决定。 对应于系统13-15个氨基酸的肽 L1 转运蛋白和之前克隆的 System 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 免疫电子显微镜。 转运蛋白特异性 cRNA 反义和 将制备有义核糖探针并用于确定组织和 通过原位杂交观察转运蛋白 mRNA 的细胞分布。 氨基酸转运蛋白信息和蛋白质表达的改变 将使用大鼠模型评估对高氨血症的反应 肝性脑病。 描述分子事件的研究 血脑氨基酸转运对于理解可能很有价值 与肝病、中风、糖尿病和多种疾病相关的功能障碍 代谢性脑病，并可能导致最小化治疗 这些功能障碍或用于设计治疗药物策略 输送到大脑。