AMINO ACID TRANSPORT IN HYPOXIC-ISCHEMIC ENCEPHALOPATHY

缺氧缺血性脑病中的氨基酸转运

• 批准号: 6650272
• 负责人: MICHAEL D WEISS
• 金额: $ 12.34万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6650272
• 关键词: acidity /alkalinity; aminoacid transport; antisense nucleic acid; aspartate; central nervous system disorders; cerebral ischemia /hypoxia; complementary DNA; disease /disorder model; gene expression; glia; glutamate transporter; hypoxia neonatorum; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; neurons; neurotoxins; newborn animals; oligonucleotides; protein structure function; tissue /cell culture; western blottings

An increase in the extracelluar concentration of the neurotransmitter glutamate, as seen in hypoxia-ischemia, results in neuronal damage. Glutamate is most likely released from the presynaptic neuron and, possibly surrounding glial cells, during a hypoxic-ischemic event. The exact mechanism by which glutamate is released into the neurosynaptic junction remains to be elucidated, although there is published evidence to implicate reverse function of an amino acid transporter. Our global hypothesis is that an ASCT amino acid transporter contributes to glutamate-mediated excitotoxicity observed in hypoxic-ischemic states by catalyzing glutamate efflux into the neurosynaptic junction only at the lower pH values associated with pathological conditions, such as hypoxic ischemic encephalopathy (HIE). ASCT1 is a neutral amino acid transporter at physiologic pH, but at lower pH shifts its substrate specificity to accept anionic amino acids such as glutamate. These lower pH values would exist during HIE. The immediate goal of the experiments outlined is to test this hypothesis by determining if ASCT1, by itself or with other glutamate transporters, is responsible for the efflux of glutamate under hypoxic- ischemic conditions. In vitro model systems will include investigation of pH-dependent D-aspartate (a glutamate analog with regard to transport) efflux from primary cultures of neonatal neurons. In addition, overexpression of both anionic amino acid transporters and ASCT transporters in HeLa cells to document which of these transporters is capable of mediating pH-dependent D-aspartate efflux. Secondly, we will use both in situ hybridization and immunohistochemistry to document the expression of the ASCT1 transporter protein after hypoxic-ischemic events, both in neuronal cultures in vitro and in intact brain tissue in vivo, to gain a understanding of its possible modulation by biosynthesis or degradation following neuronal injury.

如缺氧缺血时所见，神经递质谷氨酸的细胞外浓度增加会导致神经元损伤。在缺氧缺血事件期间，谷氨酸很可能从突触前神经元释放，并且可能在神经胶质细胞周围释放。尽管有已发表的证据表明氨基酸转运蛋白的反向功能，但谷氨酸释放到神经突触连接的确切机制仍有待阐明。我们的总体假设是，ASCT 氨基酸转运蛋白仅在与病理条件（例如缺氧缺血性脑病 (HIE)）相关的较低 pH 值下催化谷氨酸流出到神经突触连接，从而导致缺氧缺血状态下观察到的谷氨酸介导的兴奋性毒性。 ASCT1 在生理 pH 下是一种中性氨基酸转运蛋白，但在较低 pH 下，其底物特异性会发生变化，以接受阴离子氨基酸（例如谷氨酸）。 HIE 期间会存在这些较低的 pH 值。这些实验的直接目标是通过确定 ASCT1 本身或与其他谷氨酸转运蛋白一起是否负责缺氧缺血条件下谷氨酸的流出来检验这一假设。体外模型系统将包括对新生儿神经元原代培养物中 pH 依赖性 D-天冬氨酸（一种与运输相关的谷氨酸类似物）流出的研究。此外，在 HeLa 细胞中过量表达阴离子氨基酸转运蛋白和 ASCT 转运蛋白，以记录这些转运蛋白中的哪一种能够介导 pH 依赖性 D-天冬氨酸外流。其次，我们将使用原位杂交和免疫组织化学来记录缺氧缺血事件后 ASCT1 转运蛋白在体外神经元培养物和体内完整脑组织中的表达，以了解其可能通过生物合成进行的调节或神经元损伤后的降解。