PH REGULATION IN NEURONS AND ASTROCYTES IN HYPOXIA

脑缺氧，以及经常伴随的细胞外酸中毒和高碳酸血症，可导致细胞内pH(Phi)调节的变化。相反，pH的变化调节了哺乳动物中枢神经系统的缺氧性和缺血性损伤。因此，了解神经元和星形胶质细胞的phi调节与低氧和/或高碳酸血症之间的复杂相互关系是至关重要的。本项目主要研究低氧和/或高碳酸血症(急性或慢性)如何调节中枢神经系统神经元和星形胶质细胞的HCO5转运和phi；以及成熟在这些反应中所起的作用。在克隆和 在为几个新的钠偶联HCO3转运体开发出抗体之后，我们现在能够利用强大的分子工具--遗传操作(基因敲除技术和基因敲除动物)、类型特异性抗体和单细胞聚合酶链式反应(Single-cell PCR)来研究这些重要问题。后两者，我们将应用于鉴定的神经元和星形胶质细胞后，立即使用数字成像技术和pH敏感的荧光染料来动态研究Phi生理学。这种方法将允许我们确定哪些分子负责由低氧和/或高碳酸血症引起的PHJ生理的特定变化。这四个特定目的是为了研究：(1)急性缺氧和/或高碳酸血症对HCO3转运体活性的影响。(2)慢性低氧和/或高碳酸血症对HCO3转运体和碳酸氢酶(CA)表达和活性的影响可能与其相关。(3)HCO3转运蛋白和CA的基础表达和活性成熟。(4)HCO3和CaS对低氧和/或高碳酸血症反应的成熟。我们将对培养的和新分离的小鼠海马神经元和星形胶质细胞进行细胞实验。在AIM(1)中使用非平衡溶液，可以一次改变[HCO3]o、PHO和[CO2]o，并确定细胞是否可以单独检测这些参数中的每一个。此外，我们将分离长期暴露在低氧和/或高二氧化碳环境中的小鼠的组织，并使用Northern和Western印迹、聚合酶链式反应和免疫细胞化学研究这些组织的表达。这项拟议的工作不仅应该阐明受成熟度影响的低氧和/或高碳酸血症如何影响神经元和星形胶质细胞的phi调节，这项工作还应该在分子水平上阐明这些变化是如何发生的。

Brain hypoxia, and the extracellular acidosis and hypercapnia that often accompany it, can lead to changes in the regulation of intracellular pH (pHi). Conversely, changes in pH modulate hypoxic and ischemic injury in the mammalian CNS. It is therefore essential to understand the complex interrelationships among pHi regulation in neurons and astrocytes on the one hand, and hypoxia and/or hypercapnia on the other. This project focuses on how hypoxia and/or hypercapnia (acute or chronic) modulate HCO5 transport and pHi; in CNS neurons and astrocytes, and the role that maturation plays in these responses. Having cloned and developed antibodies for several new Na+-coupled HCO3 transporters, we are now in the position to investigate these important problems with powerful molecular tools?genetic manipulations (knockdown techniques and knockout animals), type-specific antibodies, and single-cell PCR. The latter two we will apply to identified neurons and astrocytes immediately after using digital imaging techniques and pH-sensitive fluorescent dyes to study dynamically pHi physiology. This approach will permit us to determine which molecules are responsible for specific changes in pHj physiology?as induced by hypoxia and/or hypercapnia. The four specific aims are to examine the effect of: (1) Acute hypoxia and/or hypercapnia on the activity of HCO3 transporters. (2) Chronic hypoxia and/or hypercapnia on the expression and activity of HCO3 transporters and carbonic anhydrases (CAs) with which they may be associated. (3) Maturation on the baseline expression and activity of HCO3 transporters and CAs. (4) Maturation on the response of HCO3 and CAs to hypoxia and/or hypercapnia. We will perform the cellular experiments on both cultured and freshly-dissociated hippocampal neurons and astrocytes from mice. The use of out-of-equilibrium solutions in aim (1) will make it possible to change [HCO3]o, pHo and [CO2]o one at a time and determine whether the cells can individually sense each of these parameters. In addition, we will isolate tissues from mice chronically exposed to an environment of hypoxia and/or hypercapnia, and study expression in these tissues using northern and western blotting, PCR and immunocytochemistry. The proposed work should not only clarify how hypoxia and/or hypercapnia?as influenced by maturation?affect pHi regulation in neurons and astrocytes, the work should also clarify at a molecular level how these changes take place.