CFTR and acid-base transporters of intestinal epithelia

CFTR 和肠上皮酸碱转运蛋白

• 批准号: 7147394
• 负责人: LANE L CLARKE
• 金额: $ 28.91万
• 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 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7147394
• 关键词: acid base balance; apical membrane; bicarbonates; chloride channels; cystic fibrosis; duodenum; fluorescent dye /probe; fluorimetry; gastrointestinal absorption /transport; gastrointestinal epithelium; genetically modified animals; immunocytochemistry; ion transport; jejunum; laboratory mouse; laser capture microdissection; membrane transport proteins; microelectrodes; protein structure function; sodium hydrogen exchanger; tissue /cell culture

DESCRIPTION (provided by applicant): Regulation of electrolyte and water transport is a major function of the intestinal epithelium. Pathological alterations in anion secretion and coupled NaCI absorption play a significant role in the intestinal manifestations of cystic fibrosis, diarrheaI disease and duodenal ulcer disease. In this proposal, the processes of transepithelial HCO3- secretion across the duodenum and electroneutral NaCI absorption (via coupled Na+/H+ - CI-/HCO3- exchange) across the jejunum will be specifically addressed. Studies have shown that CFTR, the major anion conductance, and NHE3, the major non-nutrient Na+ absorptive protein, in the apical membrane of small intestinal epithelia are essential to normal function and regulation of both processes. However, our current understanding of these transport processes requires extension to include interactions with the apical membrane CI-/HCO3- exchangers. The identity of the apical membrane anion exchangers has been elucidated in recent years with the discovery of members in the multifunctional anion exchanger family Slc26a, primarily Slc26a3 (down-regulated in adenoma, DRA) and Slc26a6 (putative anion transporter-1, PAT-1). The interaction of these CI-/HCO3- exchangers with CFTR and NHE3 occur most extensively at the villous epithelium, but there are reciprocal gradients for CFTR (crypt to villus) and NHE3 (villus to crypt) expression along the villous axis. Therefore, we have developed techniques to measure the activity of transporters in epithelial cells in intact villi using BCECF ratio microfluorimetry to measure intracellular pH (pHi), single-barreled microelectrodes to measure apical membrane potential, and laser capture dissection coupled with quantitative real-time PCR and immunohistochemistry to measure mRNA and protein expression. Definitive results will be achieved through the opportunities afforded by studies using mice with single and double gene-targeted deletions of PAT-1, DRA, CFTR or NHE3. Based on our Preliminary Studies, we hypothesize in Specific Aim 1 that PAT-1 is the major CI-/HCO3- exchanger in the upper villous epithelium of the duodenum where it provides HCO3- secretion and regulates pHi during acid challenge. It is postulated that PAT-1 acts independently and has indirect associations with CFTR and NHE3. In Specific Aim 2, we hypothesize that DRA is the major CI-/HCO3- exchanger in the lower villous epithelium of the duodenum where it is critical to stimulated HCO3- secretion and is closely regulated by CFTR. In Specific Aim 3, we hypothesize that DRA is the major CI-/HCO3- exchanger in the upper villous epithelium of the jejunum where it provides CI- absorption and is closely regulated by NHE3. Completion of these aims will establish the roles of the apical membrane CI-/HCO3- exchangers in the functions of HCO3- secretion and NaCI absorption by native intestinal mucosa. Understanding these physiological processes has therapeutic implications for diseases of intestinal electrolyte and water transport.

描述（由申请方提供）：电解质和水转运的调节是肠上皮的主要功能。阴离子分泌和偶联NaCl吸收的病理改变在囊性纤维化、十二指肠溃疡病和十二指肠溃疡病的肠道表现中起重要作用。在该提案中，将具体讨论跨十二指肠的跨上皮HCO3-分泌和跨空肠的电中性NaCl吸收（通过耦合Na +/H +-Cl-/HCO3-交换）的过程。研究表明，CFTR，主要的阴离子电导，和NHE 3，主要的非营养Na+吸收蛋白，在小肠上皮细胞的顶端膜的正常功能和调节这两个过程是必不可少的。然而，我们目前对这些运输过程的理解需要扩展到包括与顶端膜Cl-/HCO3-交换器的相互作用。近年来，随着多功能阴离子交换剂家族Slc26a成员的发现，主要是Slc26a3（在腺瘤中下调，腺瘤）和Slc26a6（推定的阴离子转运蛋白-1，PAT-1），顶端膜阴离子交换剂的身份已经得到阐明。这些Cl-/HCO 3-交换剂与CFTR和NHE 3的相互作用最广泛地发生在绒毛上皮中，但CFTR（隐窝到绒毛）和NHE 3（绒毛到隐窝）表达沿绒毛轴沿着存在相互梯度。因此，我们已经开发了技术来测量转运蛋白在完整绒毛上皮细胞中的活性，使用BCECF比率显微荧光测定法来测量细胞内pH值（pHi），单管微电极来测量顶端膜电位，激光捕获解剖结合定量实时PCR和免疫组织化学来测量mRNA和蛋白质表达。通过使用PAT-1、NHE3、CFTR或NHE3的单基因和双基因靶向缺失的小鼠进行的研究所提供的机会，将获得成功的结果。基于我们的初步研究，我们在特定目标1中假设PAT-1是十二指肠上绒毛上皮中的主要Cl-/HCO3-交换器，在酸挑战期间，PAT-1在其中提供HCO3-分泌并调节pHi。据推测，PAT-1独立发挥作用，并与CFTR和NHE 3间接相关。在具体目标2中，我们假设CFTR是十二指肠下绒毛上皮中的主要Cl-/HCO3-交换器，在那里它对刺激HCO3-分泌至关重要，并受到CFTR的密切调节。在具体目标3中，我们假设NHE3是空肠上绒毛上皮中的主要Cl-/HCO3-交换器，在那里它提供Cl-吸收并受到NHE3的密切调节。这些目标的完成将确立顶端膜Cl-/HCO3-交换器在HCO3-分泌和NaCl被天然肠粘膜吸收的功能中的作用。了解这些生理过程对肠道电解质和水转运疾病具有治疗意义。