ROLE OF NAK-ATPASE AND POLCARITY DEFECTS IN ADPKD CYST F

NAK-ATP酶和极性缺陷在 ADPKD 囊肿 F 中的作用

• 批准号: 3246321
• 负责人: PATRICIA D. WILSON
• 金额: $ 12.32万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-15 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246321
• 关键词: active sites; adenosinetriphosphatase; apical membrane; basolateral membrane; biotin; brush border membrane; enzyme biosynthesis; enzyme structure; enzyme substrate complex; fatty acylation; genetic library; genetic transcription; genetic translation; human tissue; immunocytochemistry; in situ hybridization; intracellular transport; isozymes; membrane proteins; membrane transport proteins; northern blottings; ouabain; pathologic process; polycystic kidney; potassium; protein signal sequence; protein transport; radiotracer; sodium; sodium potassium exchanging ATPase; tissue /cell culture; western blottings

A defined culture system for the analysis of human autosomal dominant polycystic kidney disease (ADPKD) epithelia has been devised and characterized by this investigator during the last grant period. Comparisons made of monolayer cultures derived from individually microdissected ADPKD cysts and age-matched normal proximal (PST) and distal (CCT) tubules showed several abnormalities of growth, basement membrane synthesis, hormone response, and enzyme activities in ADPKD cells. An alteration of major consequence for cystic enlargement was increased activity of NaK-ATPase and mislocation to the apical cell surfaces in ADPKD cells. This could be responsible for reversed vectorial transport of sodium into cystic tubule lumens and lead to osmotic fluid accumulation. This possibility was confirmed experimentally in ADPKD monolayers grown on permeable membrane supports separating apical and basal compartments since 80% of 22Na was transported from the basal to apical media. The present proposal will focus on delineating the precise and detailed nature of these NaK-ATPase and membrane polarity defects. The hypothesis that ADPKD the NaK-ATPase expressed by ADPKD epithelia is abnormal will be tested by examining the kinetics of catalytic activity; molecular subunit composition; rates of subunit synthesis, cellular processing and degradation; messenger RNA levels and translational activity in vitro. To examine the full extent of membrane polarity defects: the distribution and activity levels of apical and basolateral enzymes and NHS-biotin labelling patterns will be determined. The hypothesis that altered location of NaK- ATPase to apical membranes is caused by defective sorting mechanisms in ADPKD will be tested by examination of the putative peptide signal sequence; and by examination of patterns of fatty acid (3H-myristate, 3H- palmitate and 14C-ethanolamine) labelling. To determine to what extent apical membrane alterations are due to defects in fatty acid acylation, susceptibility to specific cleavage of thioester-palmitate, thioester-amide and glycosyl phosphatidylinositol bonds will be tested. Whole tissues, cultured normal and ADPKD epithelial cells, apical and basolateral membrane vesicles will be used and subjected to pulse chase and NHS-biotin labelling protein extraction, characterization by SDS-PAGE, Western blotting and immunoprecipitation, enzyme assays, electron immuno-and cytochemical localization; RNA extraction; Northern analysis; and in vitro translation.

一种用于人常染色体显性遗传基因分析的确定的培养系统 已经设计了多囊肾病（ADPKD）上皮细胞， 在最后一次授予期间，该研究者对其进行了表征。 比较了来自单个细胞的单层培养物 显微切割的ADPKD囊肿和年龄匹配的正常近端（PST）和远端 (CCT)肾小管显示出几种异常的生长，基底膜 ADPKD细胞中的激素合成、激素反应和酶活性。 一个 囊性增大的主要后果改变增加 多囊多囊肾病Na K-ATP酶活性与根尖细胞表面定位异常 细胞 这可能是负责反向矢量运输 钠进入囊小管腔并导致渗透液积聚。 这种可能性在ADPKD单层细胞中得到实验证实， 渗透膜支持分隔顶室和基底室， 80%的~（22）Na由基部向顶部转运。 本 建议将侧重于界定这些准确和详细的性质， NaK-ATPase和膜极性缺陷。 ADPKD的假设是， ADPKD上皮细胞表达的NaK-ATPase是否异常，将通过 检测催化活性的动力学;分子亚单位 组成;亚基合成率，细胞加工和 降解;信使RNA水平和体外翻译活性。 到 检查膜极性缺陷的全部范围：分布和 顶侧和基底侧酶的活性水平和NHS-生物素标记 模式将被确定。 假设改变了钠钾的位置- 顶端膜的ATP酶是由细胞中有缺陷的分选机制引起的。 将通过检查推定的肽信号来检测ADPKD 序列;并通过检查脂肪酸（3 H-肉豆蔻酸，3 H- 棕榈酸酯和14 C-乙醇胺）标记。 确定在何种程度上 顶端膜改变是由于脂肪酸酰化的缺陷， 对硫酯-棕榈酸酯、硫酯-酰胺 和糖基磷脂酰肌醇键。 整个组织， 培养的正常和ADPKD上皮细胞，顶膜和基底外侧膜 将使用囊泡并进行脉冲追踪和NHS-生物素标记 蛋白质提取，SDS-PAGE表征，Western印迹和 免疫沉淀、酶测定、电子免疫和细胞化学 定位; RNA提取;北方分析;和体外翻译。