TRANSPORT IN ISOLATED MEMBRANES OF BLOOD BRAIN BARRIER

描述（来自申请人摘要） 本提案的目的是确定 葡萄糖和氨基酸载体的组成膜中， 血脑屏障，使用我们开发的新技术。 两 内皮细胞的腔膜和腔外膜， 血脑屏障将被分离为囊泡， 葡萄糖和氨基酸的转运将在体外进行。 的 研究人员假设，分布，离子依赖性， 这些转运蛋白在管腔和近管腔中的动力学 膜有利于促进葡萄糖和必需氨基酸的递送 酸的大脑，但防止积累的非必需氨基酸 作为神经递质。 此外，研究人员建议 苯丙氨酸和色氨酸的过度刺激 在高氨血症期间， 通过与谷氨酰胺交换，谷氨酰胺在细胞外积累， 脑液 因此，提出了以下具体目标： 1)测定葡萄糖的分布和转运动力学 内皮细胞腔膜和腔外膜中的载体 包括血脑屏障。 这将通过探测 用细胞松弛素B分别检测细胞膜， 携带者，并使用特异性抗体来确定其身份。 转运蛋白的动力学特性将由以下决定： 测量腔囊泡和腔外囊泡中的葡萄糖摄取。 膜将来自牛脑微血管。 （二） 测定中性氨基酸的分布和转运动力学 在血脑屏障的单个膜中的载体。 的 中性氨基的极性排列、性质和分类 将在牛膜囊泡中测定酸转运蛋白 通过对运输的动力学测量。 3)确定是否跨刺激 谷氨酰胺对苯丙氨酸、色氨酸和亮氨酸转运的影响 解释了它们穿过血脑屏障的增强吸收， 高氨血症 苯丙氨酸、色氨酸和 亮氨酸将在已经预加载的膜囊泡中测量 与谷氨酰胺，以确定是否与谷氨酰胺交换增强其 摄取。 交换机制将被描述。

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.