TRANSPORT IN ISOLATED MEMBRANES OF BLOOD BRAIN BARRIER

描述（来自申请人的摘要） 该提案的目的是确定 组成膜中的葡萄糖和氨基酸载体 血脑屏障，使用我们开发的新技术。 两者都 内皮细胞的腔膜和近腔膜构成 血脑屏障将被分离为囊泡，并进行测量 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、离子依赖性和 这些转运蛋白在腔内和腔外的动力学 膜有利于葡萄糖和必需氨基酸的输送 酸对大脑，但防止非必需氨基酸的积累 充当神经递质。 此外，研究人员建议 苯丙氨酸和色氨酸的不成比例的刺激 高氨血症期间穿过血脑屏障的转运是由于 通过与细胞外积累的谷氨酰胺交换 大脑的液体。 因此，提出以下具体目标： 1) 确定葡萄糖的分布和运输动力学 内皮细胞腔膜和近腔膜中的载体 包括血脑屏障。 这将通过探测来完成 用细胞松弛素 B 分别对膜进行定量 携带者，并使用特异性抗体来确定其身份。 转运体的动力学特性将由下式确定 测量腔内和腔外囊泡中的葡萄糖摄取。 膜将取自牛脑微血管。 2） 确定中性氨基酸的分布和运输动力学 血脑屏障各个膜中的载体。 的 中性氨基的极性排列、特征和分类 将测定牛膜囊泡中的酸转运蛋白 通过运输动力学测量。 3) 确定是否存在反刺激 通过谷氨酰胺转运苯丙氨酸、色氨酸和亮氨酸可以 解释了它们在过程中穿过血脑屏障的增强摄取 高氨血症。 苯丙氨酸、色氨酸和苯丙氨酸的转运动力学 将在已预加载的膜囊泡中测量亮氨酸 与谷氨酰胺，以确定与谷氨酰胺的交换是否增强它们 吸收。 交换机制将被表征。

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.