RATE OF NA+ ENTRY DETERMINES CHRONIC ADAPTATION OF ENAC

NA 进入率决定 ENAC 的长期适应

• 批准号: 6294727
• 负责人: JONATHAN J LEBOWITZ
• 金额: $ 4.56万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6294727
• 关键词: cell line; cellular polarity; fluorescence microscopy; postdoctoral investigator; protein structure function; renal tubular transport; sodium channel; sodium potassium exchanging ATPase

We intend to study the regulation of ENaC and the basolateral Na/K- ATPase under conditions of chronic up regulation or down regulation of apical Na+ entry. Although many studies have looked at short term regulation, few studies address chronic regulation of these transporters. Furthermore, the advent of an in vitro model represents a novel approach in examining these phenomena. Our model uses A6 cells--a cell line derived from Xenopus collecting duct (CD)--grown on semi-permeable supports. These cells, the standard model of the CD, will be subjected to two experimental conditions: 1. "short-circuiting" as a model of increased apical Na+ entry; 2. Na+ depletion, where A6 cells are exposed to sodium depleted media, ablating apical Na+ entry. Preliminary data has already revealed changes in the expression of ENaC and Na/K-ATPase subunits when subjected to these different conditions. We hope to characterize the effects of apical sodium entry on the transcription, translation, and post- translational regulation of these channels using biochemical, electrophysiologic, and fluorescent microscopic techniques. In this way, we hope to derive a better understanding of the molecular basis for such entities as diuretic resistance and obstructive uropathy--clinical correlates for our model.

我们打算研究在顶端Na+进入长期上调或下调的条件下ENaC和基底外侧Na/K-ATP酶的调节。尽管许多研究着眼于短期调节，但很少有研究涉及这些转运蛋白的长期调节。此外，体外模型的出现代表了研究这些现象的一种新方法。我们的模型使用 A6 细胞（一种源自非洲爪蟾集合管 (CD) 的细胞系），生长在半渗透性支持物上。这些细胞，CD的标准模型，将受到两种实验条件的影响： 1.“短路”作为增加顶端Na+进入的模型； 2. Na+耗尽，其中A6细胞暴露于钠耗尽的培养基，消除顶端Na+进入。初步数据已经揭示了在这些不同条件下 ENaC 和 Na/K-ATPase 亚基的表达发生变化。我们希望利用生化、电生理学和荧光显微镜技术来表征顶端钠进入对这些通道的转录、翻译和翻译后调节的影响。通过这种方式，我们希望更好地理解利尿抵抗和梗阻性尿病等实体的分子基础——与我们的模型的临床相关性。