TRANSGENIC MODELS OF RESPIRATORY CONTROL

呼吸控制的转基因模型

• 批准号: 2901361
• 负责人: DAVID E MILLHORN
• 金额: $ 20.83万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-10 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2901361
• 关键词: PC12 cells; animal genetic material tag; biological models; calcium transporting ATPase; calmodulin dependent protein kinase; carotid body; cell membrane; endoplasmic reticulum; enzyme activity; genetically modified animals; hypercapnia; laboratory mouse; model design /development; neuroregulation; phosphoprotein phosphatase; pulmonary respiration; respiratory hypoxia; sarcoplasmic reticulum; tissue /cell culture

DESCRIPTION: The primary compensation for arterial hypoxemia is hyperventilation which is mediated by the O2-sensitive cells in the carotid body. The carotid body O2 chemoreceptors play a critical role in the maintenance of O2-homeostasis. The mechanisms by which the O2-sensitive (type I) cells in the carotid body detect a reduction in O2 tension and transduce this signal into the appropriate cellular responses leading to hyperventilation remains, for the most part, unknown. It is known, however, that type I cells express an O2-sensitive K channel that is inhibited by reduced O2 tension which, in turn, causes membrane depolarization and an increase in intracellular free Ca2+. We have shown that hypoxia-induced expression of the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, requires an increase in cytosolic Ca2+ and activation of calmodulin (CaM) in type I cells and in PC12 cells, an O2-sensitive cell line. We also found that neutralization of CaM in catecholamine cells in transgenic mice prevents hyperventilation and increased carotid body activity during hypoxia. In the proposed studies we shall investigate the molecular basis by which the Ca2+/CaM signal transduction system regulates carotid body function in transgenic mice during hypoxia. We hypothesize that an increase in intracellular free Ca2+ and activation of Ca2+/CaM target enzymes (CaM-KI, CaM-KII and CaIN) are involved in this critical process. We further hypothesize that calcium pumps (SERCA2, SERCA3 and PMCA2) play a major role in regulating the level of cytosolic Ca2+ during hypoxia. This is an important function which couples the level of intracellular free Ca2+ with the prevailing hypoxic stimulus. The Specific Aims of the proposed research are: 1) Determine the role of CaM-activated kinases and phosphatases in mediating the cellular responses to hypoxia in wild-type and genetically modified PC12 cells, 2) Determine the role of CaM activated-kinases and phosphatases in mediating the carotid body and ventilatory response to hypoxia in transgenic mice in which these pathways have been neutralized by a novel genetic approach, and 3) Determine the role of the CaM-sensitive plasma membrane and the sarco(endo)plasmic Ca2+-ATPases in mediating the cellular response to hypoxia. Studies are performed in the O2-sensitive PC12 cells, type I cells, transgenic mice and gene knockout mice. Findings from the proposed research should provide much needed information concerning the cellular and molecular basis of O2 chemosensitivity.

描述：动脉低氧血症的主要代偿是 颈动脉氧敏感细胞介导的过度换气 尸体。颈动脉小体O2化学感受器在 维持O2-动态平衡。氧敏感的机制 (I型)颈动脉小体中的细胞检测到氧分压降低和 将这一信号转化为适当的细胞反应，从而 在很大程度上，过度通风仍然是未知的。然而，众所周知的是， I型细胞表达O2敏感的K通道，该通道被 氧分压降低，进而导致膜去极化和 细胞内游离钙离子浓度升高。我们已经证明，低氧诱导 编码酪氨酸羟化酶(TH)的基因的表达 多巴胺生物合成中的限速酶，需要增加 I型细胞胞浆内钙离子和钙调素(CaM)的激活 PC12细胞，一种对O2敏感的细胞系。我们还发现，中和 转基因小鼠儿茶酚胺细胞中的CaM可防止过度换气和 低氧时颈动脉小体活动增加。在建议的研究中，我们 研究钙/钙调素信号的分子基础 信号转导系统对转基因小鼠颈动脉体部功能的调节 在缺氧期间。我们假设细胞内游离钙离子的增加 Ca~(2+)/CaM靶标酶(CaM-KI、CaM-KII和CaIN)的激活是 参与了这一关键过程。我们进一步假设钙 泵(SERCA2、SERCA3和PMCA2)在调节水平方面起主要作用 低氧时细胞内钙离子浓度的变化。这是一个重要的功能，它 细胞内游离钙离子水平与普遍的低氧 刺激。拟议研究的具体目标是：1)确定 钙调素激活的激酶和磷酸酶在细胞内的调节作用 野生型和转基因PC12细胞对低氧的反应 确定CaM激活的激酶和磷酸酶在介导中的作用 转基因小鼠颈动脉小体及对低氧的呼吸反应 这些途径已经被一种新的遗传方法中和了，并且 3)确定钙调素敏感的质膜和 Sarco(Endo)胞浆Ca~(2+)-ATPase在介导细胞免疫应答中的作用 缺氧。研究是在氧敏感的PC12细胞中进行的，类型为I型 细胞、转基因小鼠和基因敲除小鼠。建议的调查结果 研究应该提供关于细胞和 O2化疗敏感性的分子基础。