AMINO ACID TRANSPORT IN HYPOXIC-ISCHEMIC ENCEPHALOPATHY

细胞外神经递质谷氨酸浓度的增加，如在缺氧缺血中所见，导致神经元损伤。在缺氧缺血性事件中，谷氨酸最有可能从突触前神经元和周围的神经胶质细胞释放出来。谷氨酸释放到神经突触连接的确切机制仍有待阐明，尽管已发表的证据暗示氨基酸转运体的反向功能。我们的总体假设是，ASCT氨基酸转运体参与了在缺氧缺血性状态下观察到的谷氨酸介导的兴奋性毒性，通过催化谷氨酸外排进入神经突触连接处，只有在与病理状况（如缺氧缺血性脑病（HIE））相关的较低pH值下。ASCT1在生理pH下是一种中性的氨基酸转运蛋白，但在较低的pH下，它的底物特异性会改变，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。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是否负责在缺氧缺血条件下谷氨酸的外排来验证这一假设。体外模型系统将包括新生儿神经元原代培养中ph依赖性d -天冬氨酸（一种谷氨酸类似物）外排的研究。此外，阴离子氨基酸转运蛋白和ASCT转运蛋白在HeLa细胞中的过表达，以证明哪一种转运蛋白能够介导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ASCT1转运蛋白的表达，无论是在体外培养的神经元还是在体内完整的脑组织中，以了解其在神经元损伤后可能通过生物合成或降解进行调节。

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.