ACTIVATION OF CFTR MUTANTS IN VIVO FOR CF THERAPY

体内 CFTR 突变体的激活用于 CF 治疗

• 批准号: 2770609
• 负责人: Mitchell L Drumm
• 金额: $ 24.68万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770609
• 关键词: amiloride; beta antiadrenergic agent; chloride channels; chlorine; clinical research; cystic fibrosis; disease /disorder model; gene expression; gene mutation; human subject; ion transport; laboratory mouse; membrane potentials; metabolism disorder chemotherapy; nonhuman therapy evaluation; phosphodiesterase inhibitors; phosphorylation; protein biosynthesis; tissue /cell culture

DESCRIPTION (Taken directly from the application) lt is generally accepted that the symptoms of cystic fibrosis are in part, if not wholly, due to defective electrolyte transport mediated by CFTR, although the contribution of CFTR as a chloride channel or as a regulator of other ion channels in pathogenesis is unclear. The hypothesis to be examined in this proposal is that increasing the activity of CFTR mutants is likely to have therapeutic potential for CF patients. As a measure of predicted efficacy, we will measure electrogenic chloride transport across CF tissues in response to various drugs which activate CFTR. First, as a model for human CF airway tissues, the inferior nasal turbinate of CF patients will be exposed to beta-adrenergic agonists and class-specific phosphodiesterase inhibitors, the combination of which has been found to induce CFTR-mediated chloride efflux from primary CF nasal epithelial cells. The ability of these compounds, in the presence of amiloride, to increase nasal potential difference will be measured as an indicator of chloride secretion. Patients of various genotypes will be studied. As a more versatile model system for CF, transepithelial chloride transport will be measured across tissues from mice carrying CF mutations. Nasal potential difference measurements will be made to examine transport in the airway and short circuit current across the jejunum measured in response to beta-adrenergic agonists and class-specific phosphodiesterase inhibitors. The second focus of this application is to examine the effects of chronic exposure to these potential therapeutic compounds on electrolyte transport. Cells expressing CFTR will be cultured in the presence of compounds found to induce transepithelial chloride transport and the effects on CFTR mRNA and protein will be measured, as well as short circuit current. The mechanism by which these compounds increase chloride transport will also be investigated. To discriminate between the mechanisms of increasing channel activity and increasing channel number, the quantity of CFTR in the plasma membrane will be measured by cell-surface biotinylation before and after stimulation. The level of CFTR phosphorylation will also be measured by metabolic labeling of CFTR with 32P in the presence and absence of stimulation. Comparison of the amount in the membrane with the amount phosphorylated should indicate the contribution of CFTR activity and quantity. If beta-adrenergic agonists and class-specific phospho-diesterase inhibitors can significantly increase chloride transport, it is important to carry out studies on mechanism to determine if the process can be further improved.

描述（直接取自应用程序） 一般认为囊性纤维化的症状部分是， 如果不是全部，由于CFTR介导的电解质转运缺陷， 尽管CFTR作为氯离子通道或作为细胞增殖调节剂的作用， 其他离子通道的发病机制尚不清楚。 假设是 在这项建议中检查的是，增加CFTR突变体的活性是 可能对CF患者具有治疗潜力。 作为衡量 预测的功效，我们将测量产电氯离子转运， CF组织响应于激活CFTR的各种药物。 一是作为 人CF气道组织模型，CF的下鼻甲 患者将暴露于β-肾上腺素能受体激动剂， 磷酸二酯酶抑制剂，已经发现它们的组合 诱导CFTR介导氯离子从原代CF鼻上皮细胞流出。 这些化合物在阿米洛利存在下， 将测量鼻电位差作为氯化物的指标 分泌物。 将研究不同基因型的患者。 作为一个更 CF的多功能模型系统，跨上皮氯离子转运将是 在携带CF突变的小鼠的组织中测量。 鼻电位 将进行差异测量以检查气道中的运输， 响应于以下测量的空肠短路电流： β-肾上腺素能激动剂和类别特异性磷酸二酯酶抑制剂。 本申请的第二个重点是检查慢性 暴露于这些潜在的治疗化合物对电解质转运的影响。 表达CFTR的细胞将在存在被发现与CFTR结合的化合物的情况下培养。 诱导跨上皮氯离子转运以及对CFTR mRNA和 将测量蛋白质以及短路电流。 这些化合物增加氯离子转运的机制也将 追究 为了区分增加的机制， 通道活动和增加通道数量、CFTR的数量 质膜将通过细胞表面生物素化进行测量， 刺激后。 还将测量CFTR磷酸化水平 通过在存在和不存在下用32 P代谢标记CFTR， 刺激. 膜中的量与 磷酸化应表明CFTR活性的贡献， 数量。 如果β-肾上腺素能激动剂和类特异性磷酸二酯酶 抑制剂可以显著增加氯离子转运，重要的是 进行机制研究，以确定该过程是否可以进一步 提高