Mechanisms and Correction of Abnormal Bicarbonate Secretion by DRA in Diarrhea

DRA 治疗腹泻时碳酸氢盐异常分泌的机制及纠正

• 批准号: 9753444
• 负责人: MARK DONOWITZ
• 金额: $ 51.07万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-05 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753444
• 关键词: Acute; Affect; Anion Transport Proteins; Anions; Antidiarrheals; Area; Bicarbonates; Binding; Brush Border; Caco-2 Cells; Cell model; Cells; Cessation of life; Child; Cholera Toxin; Colon; Colon Carcinoma; Cyclic AMP; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dehydration; Development; Diarrhea; Dissociation; Drug Targeting; Early Endosome; Electrolytes; Functional disorder; Future; Goals; Grant; Human; Intestines; Ion Transport; Knowledge; Link; Location; Modeling; Molecular Chaperones; Outcome; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Physiology; Population; Process; Proteins; Public Health; Regulation; Reporting; Research; Role; SLC26A3 gene; Small Intestines; Stem cells; Testing; Time; Undifferentiated; Villus; absorption; antiporter; apical membrane; colon cancer cell line; diarrheal disease; drug development; human subject; ileum; innovation; link protein; monolayer; new therapeutic target; novel; trafficking; virtual

SUMMARY There remain major gaps in understanding regulation of human intestinal electrolyte transport under normal physiologic conditions and in diarrheal diseases. This proposal deals with the role of the brush border (BB) Cl/HCO3 antiporter, SLC26A3 (DRA) in intestinal Na, Cl and HCO3 transport and will resolve the gap in understanding of its acute regulation, intestinal cells involved and ways to reverse the abnormal transport in severe, cAMP related diarrheas. The long term goal of this project is to define the coordinated and dynamic changes in Na, Cl and HCO3 transport that occur in diarrhea so that they can be targeted by drugs to reverse the changes and treat most diarrheal diseases. The Aims of this proposal include to: I.Test the hypothesis that necessary steps in cAMP/cholera toxin inhibition of neutral Na and Cl absorption that involves DRA, which occurs at the same time as stimulation of DRA activity and the related HCO3 secretion, includes increased trafficking of DRA to the BB and reduction in the physical association of DRA with NHE3 that occurs under basal conditions and increased physical association with CFTR. II. Test the hypothesis that cAMP related changes in DRA and NHE3 activities and trafficking involve association with SNX27 in early endosomes and these changes can be reversed by retromer stabilizers, which identify a new drug target to treat diarrhea. To accomplish these studies we will use ileal and proximal colonic enteroids made from healthy human subjects grown as 2D monolayers and compare the results with those from the human colon cancer cell line, Caco-2 cells. Caco-2 cells are used for many studies of intestinal physiology and for drug development, including anti- diarrheal drugs. We predict that Caco-2 cells will give similar information regarding intestinal Na, Cl and HCO3 transport under basal conditions and in models of diarrhea; however it will be important to identify any differences that could be relevant to studies of intestinal function and drug development. These studies are innovative being among the first to study electrolyte transport in human enteroids as monolayers, to examine the interaction of DRA with NHE3 and CFTR in the same cell population with emphasis on their dynamic interactions with each other under basal conditions and with elevated cAMP, are the initial study of the role of the early endosomal protein SNX27 and the retromer in acute DRA regulation, and develop studies of SNX27/retromer as a potential drug target for future development to treat diarrhea. The expected outcomes of this proposal will be to a) understand the coordinated regulation of DRA, NHE3, and CFTR in a model of severe diarrhea that includes consideration of mechanism of HCO3 loss, a gap in understanding the pathophysiology of diarrhea, b) establish that drug therapy of diarrhea must consider the coordinated regulation of DRA with NHE3 and CFTR and not just the regulation of each transporter alone, and c) identify a new drug target in a diarrhea model in human intestine that can reverse the diarrhea related transport changes in DRA and NHE3, and that potentially could be developed into a drug to treat diarrhea.

摘要 在了解正常情况下人体肠道电解质转运的调节方面仍有很大差距。 在生理条件和腹泻疾病中。本提案涉及灌木丛边框的作用(BB) CL/HCO3逆向转运蛋白SLC26A3(DRA)在肠道钠、氯和HCO3转运中的作用 对其急性调节、肠道细胞参与及逆转异常转运的认识 严重，与营地有关的腹泻。该项目的长期目标是定义协调和动态的 腹泻时发生的钠、氯和HCO3转运的变化，使它们可以被药物靶向逆转 这些变化和治疗大多数腹泻疾病。这项提议的目的包括：一、检验假设 CAMP/霍乱毒素抑制中性钠和氯吸收的必要步骤涉及DRA，这是 在刺激DRA活性和相关的HCO3分泌的同时发生，包括增加 将DRA贩运到BB，并减少DRA与NHE3的物理联系 基础条件和与CFTR的物理联系增加。二、检验cAMP与 DRA和NHE3活性的变化和贩运涉及早期内体SNX27和 这些变化可以被逆转录稳定剂逆转，这种稳定剂确定了治疗腹泻的新药物靶点。至 为了完成这些研究，我们将使用来自健康人的回肠和近端结肠。 作为2D单层生长，并将结果与人结肠癌细胞系Caco-2的结果进行比较 细胞。Caco-2细胞被用于许多肠道生理研究和药物开发，包括抗肿瘤药物 止泻药。我们预测Caco-2细胞将提供关于肠道Na、Cl和HCO3的类似信息 在基本条件下和腹泻模型中的运输；然而，重要的是要识别 这些差异可能与肠道功能和药物开发的研究相关。这些研究是 创新的是最先将人类肠道中的电解质运输作为单分子层研究的人之一，以检查 DRA与NHE3和CFTR在同一细胞群体中的相互作用及其动态 基础条件下的相互作用以及与升高的cAMP的相互作用，是对 急性DRA调节中的早期内体蛋白SNX27及其逆聚体的研究进展 SNX27/Retrmer作为未来治疗腹泻的潜在药物靶点。的预期结果 本提案将有助于a)了解在以下模型中对DRA、NHE3和CFTR的协调监管 严重腹泻，包括对HCO3丢失机制的考虑，对 腹泻的病理生理学，b)确立了腹泻的药物治疗必须考虑协调 与NHE3和CFTR一起调节DRA，而不仅仅是调节每个转运体，以及c)确定 可以逆转腹泻相关转运变化的人肠道腹泻模型的新药靶点 在DRA和NHE3中，这种药物可能会被开发成治疗腹泻的药物。