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.

在人体气道中，局部核苷酸释放启动旁分泌和自分泌途径，通过刺激粘液分泌、离子转运和纤毛跳动来调节粘液清除。尽管 CFTR 活性对于有效的粘液纤毛清除显然很重要，但我们对将物理压力转化为 CFTR 激活的机制知之甚少。我们假设 ATP 释放到气道表面后，ATP 代谢为腺苷，腺苷通过 A2B 受体发挥作用，调节 CFTR 功能。我们假设 ATP 释放到气道表面后，ATP 代谢为腺苷，腺苷通过 A2B 受体发挥作用，调节 CFTR 功能。我们将在新鲜切除的组织和 CalU3 细胞中测试这一假设，CalU3 细胞是一种浆液细胞模型，表达易于检测的 CFTR 功能和蛋白质。 A2B 受体刺激腺苷酸环化酶以增加细胞内 cAMP，我们之前表明 cAMP 依赖性蛋白激酶 A 必须锚定在顶膜上，才能通过腺苷有效调节 CFTR。腺苷酸环化酶和磷酸二酯酶之间的平衡控制亚细胞区室中 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定亚型也在靠近 CFTR 的顶膜处划分。我们将在从 CalU3 细胞中切除的顶膜斑块中功能性地测试这一假设，并将确定所涉及的每种蛋白质的特定亚型。我们发现蛋白激酶 A 需要蛋白激酶 C 活性来调节 CFTR。因此，我们将测试 PKC 必须存在于顶膜才能通过腺苷有效调节 CFTR 的假设，我们将鉴定所涉及的特定 PKC 同工酶。我们还将鉴定将 PKC 靶向气道顶端细胞表面的蛋白质。该实验将提供对调节气道细胞心尖区室中 cAMP 局部浓度的信号转导途径的深入了解。由于 cAMP 是 CFTR 的主要调节因子，因此这些实验将深入了解气道上皮中 C1 转运的调节，并表明 cAMP 效率如何调节粘膜纤毛清除的其他成分。