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: 3246322
• 负责人: PATRICIA D. WILSON
• 金额: $ 13.68万
• 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/3246322
• 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基因的确定培养系统