REGULATION OF SODIUM IN TIGHT EPITHELIA

紧密上皮细胞中钠的调节

• 批准号: 6896533
• 负责人: Douglas C. Eaton
• 金额: $ 35.29万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6896533
• 关键词: G protein; Xenopus oocyte; acid aminoacid ligase; aldosterone; apical membrane; enzyme activity; enzyme induction /repression; hormone regulation /control mechanism; ion transport; laboratory rabbit; laboratory rat; membrane activity; microarray technology; phosphatidylinositol 3 kinase; phosphatidylinositols; protein tyrosine kinase; renal tubular transport; respiratory epithelium; second messengers; sodium channel; tight junctions; tissue /cell culture; ubiquitin; voltage /patch clamp

The long term goal of this program is to examine the control and regulation of ion transport in epithelial tissue. In particular this project will use single channel and biochemical methods to examine the regulation of amiloride-blockade sodium channels in renal and lung epithelial cells. These channels are interesting because of their relative uniqueness among channels in transporting tissue and because of the interesting hormonal regulation of these channels. However, the mechanisms for regulation of these channels have not been completely described. Therefore, this project will further investigate the signaling cascades which regulate sodium channels in three sodium-transporting epithelial cell lines using patch clamp techniques supplemented by direct biochemical measurements. The specific aims for the proposed grant period will investigate four signaling cascades that regulate sodium transport. The aims are (1) further examine the regulation of sodium channels by heterotrimeric G protein signaling cascades; specifically, what is the nature of the interaction between Galpha-3 and ENAC; do the G protein alpha subunits activate Na channels directly or do they activate some other effector molecule closely associated with the inner surface of the apical membrane; and do G protein betagamma subunits alter ENaC activity? (2) Examine the regulation of sodium channels by small G protein signaling cascades. The activation of one small G protein, K-Ras2A, is required to sustain normal ENaC activity. Elements of the K-Ras signaling cascade appear to be closely associated with the cytosolic surface of the apical membrane since the cascade can be activated in excised, inside-out patches. Therefore, the mechanism of activation of K-Ras and the signaling molecules activated by K-Ras will be examined. 3) Examine the regulation of sodium channels by inositol lipids and inositol lipid kinases. Sodium channels in excised, inside-out patches require the presence of phosphatidylinositol-4,5-bis-phosphate (4,5-PIP/2) and A6 cells have the necessary enzymes to produce 4,5- pip/2. (4) Investigate the mechanisms by which aldosterone increases sodium channel activity. Demonstrate that the signaling cascade that begins with aldosterone activation of K-Ras and leads to the PI-3K- mediated production of 3,4,5-PIP3 involves activation of phosphatidylinositol-dependent kinase (PDK1/2), serum glucocorticoid- dependent kinase (SGK), and the ubiquitin ligase, Nedd4. Determine that these signaling molecules are activated by activation of PI-3-kinase and that 4-PIP-5-kinase is activated to produce 4,5-PIOP2 and subsequently 3,4,5-PIP/3. Finally, we will use commercially available gene chips to identify new aldosterone-induced genes.

该程序的长期目标是检查上皮组织离子转运的控制和调节。特别是，该项目将使用单个通道和生化方法来检查肾脏和肺上皮细胞中胺型阻滞钠通道的调节。这些通道很有趣，因为它们在运输组织中的通道之间的相对独特性以及这些通道的荷尔蒙调节有趣。 但是，调节这些通道的机制尚未完全描述。因此，该项目将进一步研究信号级联反应，该级联级联对钠转移的上皮细胞系中调节钠通道，并使用通过直接生物化学测量补充的斑块夹技术。拟议赠款期的具体目的将调查调节钠运输的四个信号级联。目的是（1）进一步检查通过异三聚体G蛋白信号级联对钠通道的调节；具体而言，Galpha-3与ENAC之间相互作用的性质是什么？ G蛋白α亚基直接激活Na通道，还是激活与顶膜内表面密切相关的其他效应分子； G蛋白Betagamma亚基是否会改变ENAC活性？ （2）通过小G蛋白信号级联反应检查钠通道的调节。需要一个小的G蛋白K-RAS2A的激活才能维持正常的ENAC活性。 K-RAS信号级联的元素似乎与根尖膜的胞质表面密切相关，因为可以在切除的内而外的斑块中激活级联反应。因此，将检查K-RAS激活的机理和由K-RAS激活的信号分子。 3）检查肌醇脂质和肌醇脂质激酶对钠通道的调节。切除的内而外斑块中的钠通道需要磷脂酰肌醇-4,5-双磷酸（4,5-PIP/2）的存在，并且A6细胞具有产生4,5- PIP/2的必要酶。 （4）研究醛固酮增加钠通道活性的机制。证明始于醛固酮激活K-RAS并导致PI-3K介导的3,4,5-PIP3的产生涉及磷脂酰糖苷依赖性激酶（PDK1/2），Serum依赖性葡萄糖糖苷 - 依赖性激酶激酶激活的信号传导级联（SGK）和泛素连接酶Nedd4。确定这些信号分子是通过激活PI-3-激酶激活的，并激活4-PIP-5-激酶以产生4,5-PIOP2，然后随后3,4,5-PIP/3。最后，我们将使用市售基因芯片来识别新的醛固酮诱导的基因。