MOLECULAR BIOLOGY OF BLOOD BRAIN AMINO ACID TRANSPORT

血脑氨基酸转运的分子生物学

• 批准号: 2271153
• 负责人: LESTER Richard DREWES
• 金额: $ 23.09万
• 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 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2271153
• 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：大脑依赖于各种血液的供应 维持基本代谢途径的营养物质和 履行专门职能。氨基酸，虽然不是主要的 代谢能量的来源，对大脑新陈代谢和 功能。这项研究的长期目标是描述 氨基酸通过细胞运输的分子事件 从血浆通过管腔和腔隙进入间质液体 脑部内皮细胞的膜。这些目标包括 了解转运蛋白的分子同一性、位点 传输器所在的位置，以及在 病理生理条件。在这项提议中，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)的克隆 用互补克隆技术从脑微血管文库中提取2) 血脑屏障系统的两个主要氨基酸转运体 Y+(碱性氨基酸)和L1系统位于腔内和 毛细血管内皮细胞的腔膜和中性 和碱性氨基酸转运系统组件(系统bo，+-相关 蛋白质)，以及3)系统L1转运体的表达 被刺激并且系统y+转运体被减少以响应 肝性脑病大鼠模型中的高氨血症。大鼠脑 将分离出微血管mRNA，并用于制备微血管cDNA 图书馆。脑微血管的系统L1转运体将是 通过互补克隆和测序鉴定。系统L1 转运蛋白在杆状病毒表达系统中的表达及其 底物专一性、动力学和生化特性将是 下定决心。与该系统的13-15个氨基酸对应的多肽 L1传输器和先前克隆的系统y+传输器和 系统bo，+相关蛋白将被合成并用于提高 多克隆抗体。细菌的细胞分布和丰度 氨基酸转运体将通过免疫细胞化学和 免疫电子显微镜。转运蛋白特异性cRNA反义和 将准备并使用正义核探针来确定组织和 原位杂交法检测转运蛋白基因在细胞内的分布。 氨基酸转运蛋白信息和蛋白表达的变化 对高氨血症的反应将使用大鼠模型进行评估 肝性脑病。描述分子事件的研究 血-脑氨基酸转运可能对了解 与肝病、中风、糖尿病相关的功能障碍 代谢性脑病，并可能导致治疗最小化 这些功能障碍或用于设计治疗药物的策略 输送到大脑。