PHYSIOLOGY AND MOLECULAR BIOLOGY OF AA METABOLISM AND CFTR MUTATION IN CF

CF 中 AA 代谢和 CFTR 突变的生理学和分子生物学

• 批准号: 3778551
• 负责人: A B MUKHERJEE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3778551
• 关键词: arachidonate; calcium; calcium flux; chloride channels; cyclic AMP; cystic fibrosis; eicosanoid metabolism; enzyme inhibitors; gene mutation; human genetic material tag; ionophores; neoplastic cell culture for noncancer research; phorbols; phospholipase A2; protein kinase C

This project investigates the mutation of the gene coding for a cAMP- regulated anion channel, the cystic fibrosis transmembrane regulator (CFTR) protein causes Cystic Fibrosis (CF). The deletion, Phe 508 (deltaF508), in CFTR is the most common mutation in CF. A major complication of CF is extensive and destructive inflammation of the airways. The secondary biochemical and functional alterations caused in CF epithelial cells by a defect in CFTR function are not yet completely understood. Recent evidence suggests that cells carrying the deltaF508 mutation in the homozygote state have altered acidification of intracellular organelles and defective c-AMP-induced membrane recycling. It has been suggested that the regulation of phospholipase A2 (PLA2)- catalyzed arachidonic acid release may be defective in CF lymphocytes and fibroblasts. Last year the group described the results of calcium ionophore A23187-induced arachidonic acid release in the following cell lines: T84 cells (a colon carcinoma line which expresses high level of normal CFTR; CFPAC-1 cells (a pancreatic carcinoma line from a delta F508 CF patient) and clones derived from CFPAC-1 cells after transfection with a retroviral vector containing the normal CFTR gene (CFPAC-PLJ-CFTR) or with vector only (CFPAC-PLJ). The project's data indicated that those lines containing a mutant CFTR and showing defective c-AMP-induced Cl- efflux have a 5-10 fold higher arachidonate release than that in the controls. This increased release is dependent upon extracellular Ca++ and is further enhanced by phorbol-12-myristate, 13-acetate(PMA, stimulator of PKC) but not by the inactive derivative 4` phorbol,12,13 didecanoae and is inhibited by staurosporine (a PKC inhibitor). An inhibitor of PLA2, quinacrine (200micromoles) abolishes arachidonate release in these cells. These data suggested that Ca++ and pKC-stimulated arachidonate release, possibly catalyzed by PLA2, is disregulated in cells carrying the delta F508 CFTR mutation in CF. These data indicated that an alteration of a Ca++, PKC-regulated arachidonate release raising the possibility that this abnormality may be due to activation of cPLA2. Although the basal activity of cPLA2 is equivalent in the control as well as in CF cells, there may be agonist induced enhancement of activity of this enzyme in CF cells. Should this be the case cPLA2 inhibition may regulate the abnormal arachidonate release and thereby controlling the severe inflammation commonly found in the respiratory tracts of CF patients.

该项目研究了 cAMP-编码基因的突变 调节阴离子通道，囊性纤维化跨膜调节器 (CFTR) 蛋白会导致囊性纤维化 (CF)。删除，Phe 508 CFTR 中的 (deltaF508) 是 CF 中最常见的突变。 一个专业 CF 的并发症是全身广泛的破坏性炎症 航空公司。 引起的继发性生化和功能改变 CF上皮细胞由CFTR功能缺陷尚未完全 明白了。最近的证据表明携带 deltaF508 的细胞 纯合子状态的突变改变了酸化 细胞内细胞器和有缺陷的 c-AMP 诱导的膜回收。 有人提出，磷脂酶 A2 (PLA2) 的调节- CF 淋巴细胞中催化花生四烯酸的释放可能有缺陷 成纤维细胞。 去年，该小组描述了钙的结果 离子载体 A23187 在以下细胞中诱导花生四烯酸释放 细胞系：T84 细胞（结肠癌细胞系，表达高水平 正常 CFTR； CFPAC-1 细胞（来自 delta F508 的胰腺癌细胞系） CF 患者）和 CFPAC-1 细胞转染后衍生的克隆 含有正常 CFTR 基因的逆转录病毒载体 (CFPAC-PLJ-CFTR) 或 仅使用矢量 (CFPAC-PLJ)。 该项目的数据表明，那些 含有突变 CFTR 并显示有缺陷的 c-AMP 诱导 Cl- 的品系 外排的花生四烯酸释放量比内排高 5-10 倍 控制。 这种增加的释放取决于细胞外 Ca++ 和 佛波醇 12-肉豆蔻酸酯、13-乙酸酯（PMA，刺激剂）进一步增强 PKC)，但不是由无活性衍生物 4` 佛波醇，12,13 didecanoae 产生，并且是 被星形孢菌素（PKC 抑制剂）抑制。 PLA2 抑制剂， 奎纳克林（200微摩尔）消除这些细胞中花生四烯酸的释放。 这些数据表明 Ca++ 和 pKC 刺激花生四烯酸释放， 可能由 PLA2 催化，在携带 delta 的细胞中不受调节 CF 中的 F508 CFTR 突变。 这些数据表明，a 的改变 Ca++、PKC 调节的花生四烯酸释放增加了这种可能性 异常可能是由于 cPLA2 的激活所致。 虽然基础 cPLA2 的活性在对照细胞和 CF 细胞中是相同的， CF 中该酶的活性可能是激动剂诱导的增强 细胞。 如果是这种情况，cPLA2 抑制可能会调节异常 花生四烯酸释放，从而控制严重炎症 常见于 CF 患者的呼吸道。