CFTR and acid-base transporters of intestinal epithelia

CFTR 和肠上皮酸碱转运蛋白

基本信息

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
项目类别：
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

项目摘要

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。阴离子分泌的病理改变和与之相伴的氯化钠吸收在囊性纤维化、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（通过耦合的 Na+/H+ - CI-/HCO3- 交换）的过程。研究表明，小肠上皮细胞顶膜中的主要阴离子传导蛋白 CFTR 和主要非营养性 Na+ 吸收蛋白 NHE3 对于这两个过程的正常功能和调节至关重要。然而，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，随着多功能阴离子交换剂家族 Slc26a 成员的发现，顶膜阴离子交换剂的身份已得到阐明，主要是 Slc26a3（在腺瘤中下调，DRA）和 Slc26a6（假定的阴离子转运蛋白-1，PAT-1））。这些 CI-/HCO3- 交换剂与 CFTR 和 NHE3 的相互作用最广泛地发生在绒毛上皮，但沿绒毛轴存在 CFTR（隐窝到绒毛）和 NHE3（绒毛到隐窝）表达的倒数梯度。因此，我们开发了测量完整绒毛上皮细胞转运蛋白活性的技术，使用 BCECF 比率微荧光法测量细胞内 pH (pHi)，单管微电极测量顶膜电位，以及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、DRA、CFTR 或 NHE3 单基因和双基因靶向缺失的小鼠进行研究，将获得最终结果。根据我们的初步研究，我们在具体目标 1 中假设 PAT-1 是十二指肠上绒毛上皮中主要的 CI-/HCO3- 交换剂，在酸激发期间提供 HCO3- 分泌并调节 pHi。据推测，PAT-1 独立发挥作用，并与 CFTR 和 NHE3 间接相关。在具体目标 2 中，我们假设 DRA 是十二指肠下部绒毛上皮中主要的 CI-/HCO3- 交换剂，对于刺激 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 DRA 是空肠上层绒毛上皮中主要的 CI-/HCO3- 交换器，它提供 CI-吸收并受到 NHE3 的密切调节。这些目标的完成将确定顶膜CI-/HCO3-交换器在HCO3-分泌和天然肠粘膜吸收NaCl的功能中的作用。了解这些生理过程对于肠道电解质和水运输疾病具有治疗意义。