CFTR and acid-base transporters of intestinal epithelia

CFTR 和肠上皮酸碱转运蛋白

• 批准号: 7261910
• 负责人: LANE L CLARKE
• 金额: $ 29.03万
• 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/7261910
• 关键词: Acids; Address; Affect; Anions; Area; Bicarbonates; Cell membrane; Co-Immunoprecipitations; Condition; Coupled; Coupling; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytophotometry; Disease; Dissection; Down-Regulation; Duodenal Ulcer; Duodenum; Electrolytes; Epithelial Cells; Epithelium; Family; Gene Targeting; Immunohistochemistry; Intestinal Diseases; Intestinal Mucosa; Intestines; Laboratories; Lasers; Localized; Measures; Membrane Potentials; Messenger RNA; Microelectrodes; Mucous Membrane; Mus; Nutrient; Physiological; Physiological Processes; Play; Polymerase Chain Reaction; Process; Proteins; Reagent; Recycling; Regulation; Research Personnel; Role; Second Messenger Systems; Small Intestines; Surface; Techniques; Testing; Therapeutic; Time; Transport Process; Villous; Villus; Water; Water Movements; absorption; adenoma; apical membrane; base; intestinal epithelium; jejunum; mRNA Expression; member; programs; protein expression; putative anion transporter 1; second messenger

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（由申请人提供）：电解质和水运输的调节是肠上皮的主要功能。阴离子分泌和NACI吸收的病理改变在囊性纤维化，腹泻病和十二指肠溃疡病的肠道表现中起重要作用。在此提案中，将专门解决了跨甲库纳姆（Na+/h+ - ci-/hco3-交换）跨十二指肠和电鼻Naci吸收的跨跨性别HCO3-分泌的过程。研究表明，在小肠上皮的顶膜中，CFTR（主要阴离子电导率）和NHE3（主要非营养素Na+吸收性蛋白）对于正常功能和两个过程的调节都是必不可少的。但是，我们目前对这些运输过程的理解需要扩展，以包括与顶膜CI-/HCO3-交换器的相互作用。近年来，随着在多功能阴离子交换器家族SLC26A中发现成员，主要是SLC26A3（主要是在腺瘤，DRA）和SLC26A6（推定的阴离子Transporter-1，Pat-1，Pat-1，Pat-1）。这些CI-/HCO3-交换器与CFTR和NHE3的相互作用最广泛地发生在绒毛上皮，但是CFTR（crypt to Villus）和NHE3（绒毛）（绒毛至隐窝）沿绒毛轴的表达有相互梯度。因此，我们开发了使用BCECF比率的微氟映体在完整绒毛中测量转运蛋白在上皮细胞中的活性的技术，以测量细胞内pH（PHI），单管的微电极，以测量顶端膜势，并捕获激光捕获量的实时pcremno pcremno和Immenmunne pcremsistrna coctecter cotection caption和Imbotna cothem pcremno cockem insirna cocrna for srimno pcremsistry for Mernistry for Mermistry for Mermistry for Mermistry for Mermistry for Mermyrs in Cryrna。通过使用PAT-1，DRA，CFTR或NHE3的单一和双基因靶向缺失的小鼠提供的研究提供的机会，将实现确定的结果。基于我们的初步研究，我们在特定的目标1中假设PAT-1是十二指肠上部绒毛上皮中的主要CI-/HCO3-交换器，在该上皮提供了HCO3-在其中提供HCO3-分泌，并在酸挑战期间调节PHI。据推测，PAT-1独立起作用，并与CFTR和NHE3有间接关联。在特定的目标2中，我们假设DRA是十二指肠下部绒毛上皮中的主要CI-/HCO3-交换器，在刺激HCO3-分泌至关重要，并由CFTR密切调节。在特定的目标3中，我们假设DRA是空肠上绒上皮上皮的主要Ci-/HCO3-交换器，它提供了CI-吸收，并且受NHE3的密切调节。这些目的的完成将确定顶端膜CI-/HCO3-交换器在HCO3-分泌和NACI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运输的疾病具有治疗意义。