G PROTEIN REGULATION OF CL SECRETION IN AIRWAY EPITHELIA

囊性纤维化（CF）引起的肺部疾病的发病机制是由于 部分原因是囊性纤维化跨膜传导突变 调节子（CFTR），cAMP刺激的Cl（CFTR）通道。 无法 cAMP刺激Cl（CFTR）分泌，增加Na+的速率 CF患者气道中的重吸收有助于 呼吸道脱水和肺部疾病的发病机制。 最近，研究表明，使用钠通道的气雾剂治疗 抑制剂阿米洛利改善粘膜纤毛清除率， 肺活量下降的症状 气雾剂治疗旨在纠正 CF患者的Cl分泌缺陷也可能对肺有益， 疾病 然而，增加cAMP刺激的Cl （CFTR）分泌的CF患者的气道还没有被证实。 鉴定 本研究提出的长期目标是 因此，应用是开发一种药理学方法， 刺激CF患者中cAMP激活的Cl通道（CFTR）， 气溶胶疗法 我们发展这种方法的战略是 基于我们的初步实验，证明异源三聚体 G蛋白抑制人气管环腺苷酸激活的氯通道（CFTR） 上皮细胞和用A1灭活抑制性G蛋白 腺苷受体拮抗剂恢复Cl的cAMP激活（CFTR） 渠道 我们建议检验腺苷产生的假设， 并由气道上皮细胞释放，激活A1腺苷 刺激抑制性G蛋白的受体。 G蛋白，在 开启cAMP激活的Cl（CFTR）通道。 磷酸化 蛋白激酶A介导的Cl（CFTR）通道可解除G蛋白抑制 而在CF细胞中则没有。 我们的具体目标是：1） 使用全细胞表征Cl（CFTR）通道的G蛋白调节 和单通道膜片钳技术。 我们将鉴定G蛋白 通过北方印迹和西方印迹分析及其细胞定位 通过共聚焦免疫细胞化学显微镜：2）阐明信号 涉及Cl的G蛋白调节的转导途径（CFTR） 使用全细胞和单通道膜片钳技术的通道。 我们 将确定G蛋白是否通过磷脂酶抑制Cl（CFTR）通道 A2和花生四烯酸; 3）开发一种治疗方法， 用A1受体拮抗剂灭活抑制性G蛋白， 应用膜片钳技术增加CF细胞Cl（CFTR）分泌 和测量氯离子通过人气道单层的转运 细胞 由于A1腺苷拮抗剂无毒且有效， 在nuM浓度下，A1拮抗剂是有希望的候选物， CF患者的气雾剂药物治疗。 我们预计我们的研究 将增加我们对调节 气道上皮细胞中cAMP激活的Cl（CFTR）通道， 阐明A1腺苷受体与G蛋白相互作用的机制 在调节Cl（CFTR）通道方面。 最后，我们预计， 研究将导致开发基于气溶胶的治疗方法， 刺激CF气道的Cl（CFTR）分泌。

The pathogenesis of lung disease induced by Cystic Fibrosis (CF) is due in part to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-stimulated Cl (CFTR) channel. The inability of cAMP to stimulate Cl (CFTR) secretion and the increased rate of Na reabsorption in the airway of patients with CF contributes to the dehydration of the airway and to the pathogenesis of lung disease. Recently, it was shown that aerosol therapy with the sodium channel inhibitor amiloride improved mucociliary clearance and slowed the rate of decline in lung vital capacity. Aerosol therapy designed to correct defective Cl secretion in CF patients may also be beneficial for lung disease. However, an effective treatment to increase CAMP-stimulated Cl (CFTR) secretion by the airway of patients with CF has not been identified. The long-term objective of the research proposed in this application, therefore, is to develop a pharmacological approach to stimulate CAMP-activated Cl (CFTR) channels in CF patients using aerosol-based therapy. Our strategy to develop such an approach is based on our preliminary experiments demonstrating that heterotrimeric G proteins inhibit cAMP-activated Cl (CFTR) channels in human tracheal epithelial cells and that inactivating inhibitory G proteins with A1 adenosine receptor antagonists restores cAMP-activation of Cl (CFTR) channels. We propose to test the hypothesis that adenosine, produced and released by airway epithelial cells, activates an A1 adenosine receptor that stimulates inhibitory G proteins. The G proteins, in turn, inactivate cAMP-activated Cl (CFTR) channels. Phosphorylation of Cl (CFTR) channels by protein kinase A may relieve G protein inhibition in normal but not in CF cells. Our specific aims are to: 1) Characterize G protein regulation of Cl (CFTR) channels using whole-cell and single channel patch clamp techniques. We will identify G proteins by Northern blot and Western blot analysis and their cellular location by confocal immunocytochemical microscopy: 2) Elucidate the signal transduction pathways involving G protein regulation of Cl (CFTR) channels using whole-cell and single channel patch clamp techniques. We will determine if G proteins inhibit Cl(CFTR) channels via phospholipase A2 and arachidonic acid and 3) Develop a therapeutic approach, based on inactivating inhibitory G proteins with A1 receptor antagonists, to increase Cl (CFTR) secretion by CF cells using patch clamp techniques and measurements of Cl transport across monolayers of human airway cells. Because A1 adenosine antagonists are nontoxic and are effective in nuM concentrations, A1 antagonists are promising candidates for aerosol pharmacotherapy for CF patients. We anticipate that our studies will increase our understanding of the signalling pathways regulating cAMP-activated Cl (CFTR) channels in airway epithelial cells and elucidate the mechanism of A1 adenosine receptor-G protein interaction in regulating Cl (CFTR) channels. Finally, we anticipate that our studies will lead to the development of an aerosol-based treatment for stimulating Cl (CFTR) secretion by CF airways.