Autocrine regulation of CFTR

CFTR 的自分泌调节

• 批准号: 6576225
• 负责人: Monroe JACKSON Stutts
• 金额: $ 7.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576225
• 关键词: adenosine; adenylate cyclase; apical membrane; autocrine; biological signal transduction; chloride channels; cyclic AMP; cystic fibrosis; human tissue; isozymes; phosphodiesterases; phosphorylation; protein kinase A; protein kinase C; protein protein interaction; purinergic receptor; respiratory airway clearance; respiratory epithelium; secretion; tissue /cell culture

In human airways local nucleotide release initiates paracrine and autocrine pathways that regulate mucus clearance by stimulating mucus secretion, ion transport, and ciliary beating. Although CFTR activity is clearly important for efficient mucociliary clearance, little is known about the mechanisms that transduce physical stresses into activation of CFTR. We hypothesize that following release of ATP onto the airway surface, ATP is metabolized to adenosine, which acts via A2B receptors to regulate CFTR function. We hypothesize that following release of ATP onto the airway surface, ATP is metabolized to adenosine, which acts via A2B receptors to regulate CFTR function. We will test this hypothesis in freshly excised tissues and in CalU3 cells, a serous cell model that expresses readily detectable CFTR function and protein. A2B receptors stimulate adenylate cyclase to increase intracellular cAMP, and we previously showed that cAMP dependent protein kinase A must be anchored at the apical membrane for efficient regulation of CFTR by adenosine. The balance between adenylate cyclase and phosphodiesterase controls the local concentration of cAMP, and protein kinase A activity, in subcellular compartments. Therefore, we hypothesize that specific isoforms of adenylate cyclase and phosphodiesterase are also compartmentalized at the apical membrane in close proximity with CFTR. We will test this hypothesis functionally in excised apical membrane patches from CalU3 cells and will determine the specific isoforms of each protein that are involved. We find that protein kinase C activity is required for protein kinase A to regulate CFTR. Therefore, we will test the hypothesis that PKC must be present at the apical membrane for efficient modulation of CFTR by adenosine, we will identify the specific PKC isozyme(s) involved. We will also identify proteins that target PKC to the apical cell surface in airway. The experiments will provide an in-depth understanding of a signal transduction pathway of a signal transduction pathway that regulates the local concentration of cAMP in the apical compartment of airway cells. Since cAMP is the dominant regulator of CFTR, the experiments will provide insights into the regulation of C1-transport in airway epithelial and will suggest how cAMP efficiency regulates other components of mucociliary clearance.

在人类气道中，局部核苷酸释放引发了旁分泌和自分泌途径，这些途径通过刺激粘液分泌，离子转运和纤毛跳动来调节粘液清除率。尽管CFTR活性对于有效的粘膜缩减清除显然很重要，但对将物理应力转化为CFTR激活的机制知之甚少。我们假设将ATP释放到气道表面后，将ATP代谢为腺苷，该腺苷通过A2B受体起作用以调节CFTR功能。我们假设将ATP释放到气道表面后，将ATP代谢为腺苷，该腺苷通过A2B受体起作用以调节CFTR功能。我们将在新鲜切除的组织和Calu3细胞中检验这一假设，这是一种表达可检测到的CFTR功能和蛋白质的浆液细胞模型。 A2b受体刺激腺苷酸环化酶增加细胞内cAMP，我们先前表明，cAMP依赖性蛋白激酶A必须锚定在顶端膜上，以通过腺苷对CFTR有效调节。腺苷酸环化酶和磷酸二酯酶之间的平衡控制着亚细胞区室中cAMP的局部浓度和蛋白激酶A活性。因此，我们假设腺苷酸环化酶和磷酸二酯酶的特异性同工型在与CFTR近距离接近的顶端膜中也被分隔一。我们将在CALU3细胞中切除的顶膜斑块中在功能上检验该假设，并确定涉及的每种蛋白质的特定同工型。我们发现蛋白激酶A需要蛋白激酶C活性来调节CFTR。因此，我们将检验以下假设：PKC必须存在于顶部膜上，以通过腺苷对CFTR有效调节，我们将确定所涉及的特定PKC同工酶。我们还将确定靶向PKC到气道顶端细胞表面的蛋白质。该实验将对信号转导途径的信号转导途径有深入的理解，该途径调节气道细胞顶部区室中cAMP的局部浓度。由于CAMP是CFTR的主要调节剂，因此实验将提供有关气道上皮中C1-Transport的调节的见解，并将表明CAMP效率如何调节其他组件的其他组件。