CFTR AND DUODENAL ANION TRANSPORT

CFTR 和十二指肠阴离子转运

• 批准号: 6680916
• 负责人: LANE L CLARKE
• 金额: $ 21.75万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680916
• 关键词: bicarbonates; chloride channels; chloride ion; cystic fibrosis; duodenum; fluorescent dye /probe; gastrointestinal absorption /transport; gastrointestinal epithelium; gene targeting; genetically modified animals; immunologic assay /test; ion transport; laboratory mouse; microelectrodes; protein structure function; secretion; tissue /cell culture; voltage /patch clamp

DESCRIPTION (Adapted from the Applicant's Abstract): The central role that the cystic fibrosis transmembrane conductance regulator (CFTR) plays in gastrointestinal anion transport physiology is evident from the intestinal and pancreatic manifestations of the genetic disease cystic fibrosis (CF). CFFR is essential to normal Cl- and HCO3- secretion across epithelial surfaces. Loss of this function in CF has been closely associated with disease processes such as distal obstructive syndrome, meconium ileus, pancreatic insufficiency, gallbladder disease and duodenal ulceration. Furthermore, the inability of the senescent epithelial cells to unload base may interfere with apoptosis, leading to necrotic cell death and accompanying inflammatory reactions that underlie CF pathogenesis. The present proposal focuses on the role that CFTR plays in mechanisms of Cl- and HCO3- secretion in the duodenum. Although anion secretion across this intestinal segment maintains a critical barrier to gastric effluent, little is known of the electrochemical gradients for anion secretion, the identity of anion exchange proteins that coordinate activity with CFTR, or the distribution of these processes along the crypt-villus axis. Studies will be performed on intact duodenum from genetically-altered CF mouse models because they accurately reproduce human CF intestinal disease. Knockout mouse models of other anion transport proteins will be used to dissect the mechanism of Cl- and HCO3- secretion in a physiological setting. We will test several hypotheses predicted from our proposed model of duodenal anion secretion. Microelectrodes and fluorescent dye markers will be used to investigate the hypothesis that CFTR functions as both a Cl- and HCO3- conductance. RT-PCR and immunoblots of normal and CF duodenal epithelia will be used to identify the effect of CFTR activity on anion transport protein expression along the crypt-villus axis. In addition, membrane vesicle and transepithelial flux studies will be used to test the hypothesis that the anion exchanger AE2 provides an alternative Cl- uptake mechanism for transepithelial Cl- secretion whereas the anion exchanger DRA, possibly driven by carbonic anhydrase, operates in parallel with CFTR for transepithelial HCO3- secretion. The successful completion of our proposed studies will firmly establish the mechanisms involved in Cl- and HCO3- secretion across the duodenum. The results will further define the role of CFTR in these processes and therefore have important implications for our understanding of intestinal pathophysiology in CF disease.

描述（改编自申请人的摘要）： 囊性纤维化跨膜电导调节因子（CFTR）在 胃肠道阴离子转运生理学从肠道和 遗传性疾病囊性纤维化（CF）的胰腺表现。 CFFR 为 对于上皮表面正常的 Cl- 和 HCO3- 分泌至关重要。损失 CF 中的这一功能与疾病过程密切相关，例如 远端阻塞综合征、胎粪性肠梗阻、胰腺功能不全、 胆囊疾病和十二指肠溃疡。此外，由于无法 衰老上皮细胞卸载碱基可能会干扰细胞凋亡，导致 导致 CF 的坏死细胞死亡和伴随的炎症反应 发病。本提案重点关注 CFTR 在以下方面发挥的作用： 十二指肠中 Cl- 和 HCO3- 分泌的机制。虽然负离子分泌 穿过这个肠段维持着胃的关键屏障 流出物，对阴离子分泌的电化学梯度知之甚少， 与 CFTR 协调活性的阴离子交换蛋白的身份，或 这些过程沿隐窝-绒毛轴的分布。研究将 在转基因 CF 小鼠模型的完整十二指肠上进行 因为它们准确地再现了人类 CF 肠道疾病。淘汰赛小鼠 其他阴离子转运蛋白的模型将用于剖析该机制 生理环境中 Cl- 和 HCO3- 的分泌。我们将测试几个 根据我们提出的十二指肠阴离子分泌模型预测的假设。 微电极和荧光染料标记将用于研究 假设 CFTR 同时充当 Cl- 和 HCO3- 电导。逆转录聚合酶链反应和 正常十二指肠上皮和 CF 十二指肠上皮的免疫印迹将用于识别 CFTR 活性对阴离子转运蛋白表达的影响 隐窝-绒毛轴。此外，膜囊泡和跨上皮通量 研究将用于检验阴离子交换剂 AE2 的假设 为跨上皮 Cl- 分泌提供了另一种 Cl- 吸收机制 而阴离子交换剂 DRA，可能由碳酸酐酶驱动， 与 CFTR 并行进行跨上皮 HCO3- 分泌。这 成功完成我们拟议的研究将牢固地确立 涉及十二指肠 Cl- 和 HCO3- 分泌的机制。结果 将进一步定义 CFTR 在这些流程中的作用，因此 对我们理解肠道病理生理学具有重要意义 囊性纤维化疾病。