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: 3246323
• 负责人: PATRICIA D. WILSON
• 金额: $ 13.01万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 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 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246323
• 关键词: active sites; adenosinetriphosphatase; apical membrane; basolateral membrane; biotin; brush border membrane; enzyme activity; 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; 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.

用于分析人类常染色体显性遗传的明确培养系统 多囊肾病 (ADPKD) 上皮细胞已被设计并 该研究人员在上一次资助期间所描述的特征。 对来自各个个体的单层培养物进行比较 显微解剖 ADPKD 囊肿和年龄匹配的正常近端 (PST) 和远端 (CCT) 肾小管显示多种生长异常，基底膜 ADPKD 细胞中的合成、激素反应和酶活性。 一个 囊肿扩大的主要后果的改变增加 ADPKD 中 NaK-ATP 酶的活性和顶端细胞表面的错位 细胞。 这可能是反向矢量传输的原因 钠进入囊性小管管腔并导致渗透液积聚。 这种可能性在 ADPKD 单层生长的实验中得到了证实 渗透膜支持分离顶室和基底室，因为 80% 的 22Na 从基底介质转运至顶端介质。 现在的 提案将侧重于描述这些内容的精确和详细性质 NaK-ATP酶和膜极性缺陷。 ADPKD 的假设 ADPKD 上皮表达的 NaK-ATPase 异常将通过以下方法进行检测 检查催化活性的动力学；分子亚基 作品;亚基合成、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 顶端和基底外侧酶的活性水平以及 NHS 生物素标记 模式将被确定。 改变 NaK- 位置的假设 ATP酶到达顶膜是由缺陷的分选机制引起的 ADPKD 将通过检查假定的肽信号来测试 顺序;并通过检查脂肪酸的模式（3H-肉豆蔻酸，3H- 棕榈酸酯和 14C-乙醇胺）标记。 以确定到什么程度 顶膜改变是由于脂肪酸酰化缺陷造成的， 对硫酯-棕榈酸酯、硫酯-酰胺的特异性裂解的敏感性 和糖基磷脂酰肌醇键将被测试。 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、蛋白质印迹和 免疫沉淀、酶测定、电子免疫和细胞化学 本土化; RNA提取；北方分析；和体外翻译。