PH REGULATION IN NEURONS AND ASTROCYTES IN HYPOXIA

缺氧时神经元和星形胶质细胞的 PH 调节

• 批准号: 6910143
• 负责人: Walter F Boron
• 金额: $ 25.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-17 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6910143
• 关键词: acid base balance; antibody; astrocytes; bicarbonates; carbonate dehydratase; central nervous system; digital imaging; gene targeting; genetically modified animals; growth /development; hippocampus; hypercapnia; hypoxia; immunocytochemistry; ion transport; laboratory mouse; laboratory rabbit; neurons; neurophysiology; northern blottings; polymerase chain reaction; tissue /cell culture; western blottings

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.

大脑缺氧以及经常伴随的细胞外酸中毒和高碳酸血症会导致细胞内 pH (pHi) 调节的变化。相反，pH 值的变化会调节哺乳动物中枢神经系统的缺氧和缺血性损伤。因此，有必要一方面了解神经元和星形胶质细胞的 pHi 调节，另一方面了解缺氧和/或高碳酸血症之间复杂的相互关系。该项目重点关注缺氧和/或高碳酸血症（急性或慢性）如何调节 HCO5 转运和 pHi；中枢神经系统神经元和星形胶质细胞的作用，以及成熟在这些反应中所起的作用。克隆并 开发了几种新的 Na+ 偶联 HCO3 转运蛋白的抗体，我们现在能够利用强大的分子工具——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 来研究这些重要问题。在使用数字成像技术和 pH 敏感荧光染料动态研究 pHi 生理学后，我们将立即将后两者应用于已识别的神经元和星形胶质细胞。这种方法将使我们能够确定哪些分子负责由缺氧和/或高碳酸血症引起的 pHj 生理学的特定变化。四个具体目标是检查以下因素的影响： (1) 急性缺氧和/或高碳酸血症对 HCO3 转运蛋白活性的影响。 (2) 慢性缺氧和/或高碳酸血症对其可能相关的 HCO3 转运蛋白和碳酸酐酶 (CA) 的表达和活性产生影响。 (3) HCO3 转运蛋白和 CA 的基线表达和活性的成熟。 (4) HCO3 和 CAs 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。在目标 (1) 中使用非平衡溶液将可以一次改变一个 [HCO3]o、pHo 和 [CO2]o，并确定细胞是否可以单独感知这些参数中的每一个。此外，我们将从长期暴露于缺氧和/或高碳酸血症环境的小鼠中分离组织，并使用 Northern 和 Western 印迹、PCR 和免疫细胞化学研究这些组织中的表达。这项工作不仅应该阐明受成熟影响的缺氧和/或高碳酸血症如何影响神经元和星形胶质细胞的 pHi 调节，还应该在分子水平上阐明这些变化是如何发生的。