PH REGULATION IN NEURONS AND ASTROCYTES IN HYPOXIA

脑缺氧以及通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的变化调节哺乳动物CNS中的缺氧和缺血性损伤。因此，有必要了解一方面神经元和星形胶质细胞中的pHi调节与另一方面缺氧和/或高碳酸血症之间的复杂相互关系。该项目的重点是缺氧和/或高碳酸血症（急性或慢性）如何调节HCO 5运输和pHi;在中枢神经系统神经元和星形胶质细胞，以及成熟在这些反应中发挥的作用。克隆和 开发抗体的几个新的Na+耦合HCO 3转运蛋白，我们现在的立场，调查这些重要的问题与强大的分子工具？基因操作（敲除技术和敲除动物）、类型特异性抗体和单细胞PCR。后两个，我们将适用于确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，研究动态pHi生理。这种方法将使我们能够确定哪些分子是负责特定的变化pH生理？如由缺氧和/或高碳酸血症引起的。这四个具体目标是检查：（1）急性缺氧和/或高碳酸血症对HCO 3转运蛋白活性的影响。(2)慢性缺氧和/或高碳酸血症对HCO 3转运蛋白和可能与其相关的碳酸酐酶（CA）的表达和活性的影响。(3)HCO 3转运蛋白和CA的基线表达和活性的成熟。(4)HCO 3和CA对缺氧和/或高碳酸血症的反应成熟。我们将对培养的和新鲜分离的小鼠海马神经元和星形胶质细胞进行细胞实验。在目的（1）中使用非平衡溶液将使得可以一次改变[HCO 3]o、pHo和[CO2]o中的一个，并确定电池是否可以单独地感测这些参数中的每一个。此外，我们将从长期暴露于低氧和/或高碳酸环境的小鼠中分离组织，并使用北方和西方印迹、PCR和免疫细胞化学研究这些组织中的表达。拟议的工作不仅应该澄清如何缺氧和/或高碳酸血症？受成熟度的影响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.