AMINO ACID TRANSPORT IN HYPOXIC-ISCHEMIC ENCEPHALOPATHY

如在缺氧缺血中所见，神经递质谷氨酸的细胞外浓度增加导致神经元损伤。在缺氧缺血事件期间，谷氨酸最有可能从突触前神经元释放，并且可能从周围的神经胶质细胞释放。谷氨酸被释放到神经突触接头的确切机制仍有待阐明，尽管有已发表的证据表明氨基酸转运蛋白的反向功能。我们的总体假设是，ASCT氨基酸转运蛋白通过仅在与病理条件（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处，导致在缺氧缺血状态下观察到的谷氨酸介导的兴奋性毒性。ASCT 1在生理pH下是中性氨基酸转运蛋白，但在较低pH下改变其底物特异性以接受阴离子氨基酸如谷氨酸。这些较低的pH值将在HIE期间存在。概述的实验的直接目标是通过确定ASCT 1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测试该假设。体外模型系统将包括研究新生神经元原代培养物的pH依赖性D-天冬氨酸（一种转运方面的谷氨酸类似物）外排。此外，阴离子氨基酸转运蛋白和ASCT转运蛋白在HeLa细胞中的过度表达，以记录这些转运蛋白中的哪一种能够介导pH依赖性D-天冬氨酸外排。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ASCT 1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或降解进行的调节神经元损伤后。

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.