AMINO ACID TRANSPORT IN HYPOXIC-ISCHEMIC ENCEPHALOPATHY

细胞外神经递质谷氨酸浓度的增加，如缺氧缺血所见，会导致神经元损伤。谷氨酸很可能是在缺氧缺血期间从突触前神经元释放出来的，也可能是在神经胶质细胞周围释放出来的。谷氨酸被释放到神经突触连接的确切机制仍未阐明，尽管已公布的证据表明氨基酸转运体具有反向功能。我们的总体假设是，ASCT氨基酸转运体仅在与病理条件相关的较低pH值时，如缺氧缺血性脑病(HIE)，通过催化谷氨酸流出到神经突触连接来参与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体，但在较低pH时，其底物专一性改变为接受谷氨酸等阴离子氨基酸。这些较低的pH值在HIE期间会存在。概述的实验的直接目标是通过确定ASCT1本身或与其他谷氨酸转运体一起在缺氧缺血条件下负责谷氨酸的外流来检验这一假说。体外模型系统将包括研究依赖于pH的D-天冬氨酸(一种谷氨酸类似物)从新生神经元原代培养中外流的情况。此外，在HeLa细胞中过表达阴离子氨基酸转运体和ASCT转运体，以证明这些转运体中的哪一种能够调节pH依赖的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的方法来证明ASCT1转运蛋白在缺氧缺血事件后的表达，包括体外培养的神经元和体内完整的脑组织，以了解神经元损伤后生物合成或降解对其可能的调节作用。

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.