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: 9981963
• 负责人: MARK DONOWITZ
• 金额: $ 16.03万
• 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: 2019-08-08 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981963
• 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.

概括 对正常情况下人体肠道电解质转运的调节的理解仍然存在重大差距 生理状况和腹泻疾病。该提案涉及刷状边框（BB）的作用 Cl/HCO3 逆向转运蛋白，SLC26A3 (DRA) 在肠道 Na、Cl 和 HCO3 转运中将解决 了解其急性调节、涉及的肠道细胞以及逆转异常转运的方法 严重的 cAMP 相关腹泻。该项目的长期目标是定义协调和动态的 腹泻时发生的 Na、Cl 和 HCO3 转运发生变化，因此药物可以针对这些变化进行逆转 改变并治疗大多数腹泻疾病。该提案的目的包括： I. 检验假设 cAMP/霍乱毒素抑制中性 Na 和 Cl 吸收的必要步骤涉及 DRA，其中 与刺激 DRA 活性和相关的 HCO3 分泌同时发生，包括增加 DRA 向 BB 的运输以及 DRA 与 NHE3 物理联系的减少（发生在以下情况） 基础条件和与 CFTR 增加的身体关联。二.检验 cAMP 相关的假设 DRA 和 NHE3 活动和运输的变化涉及早期内涵体中与 SNX27 的关联， 这些变化可以通过逆转录体稳定剂来逆转，从而确定治疗腹泻的新药物靶点。到 为了完成这些研究，我们将使用由健康人类受试者制成的回肠和近端结肠肠类 生长为 2D 单层，并将结果与​​人类结肠癌细胞系 Caco-2 的结果进行比较 细胞。 Caco-2 细胞用于许多肠道生理学研究和药物开发，包括抗 止泻药。我们预测 Caco-2 细胞将提供有关肠道 Na、Cl 和 HCO3 的类似信息 基础条件下和腹泻模型中的转运；然而，重要的是要确定任何 可能与肠道功能和药物开发研究相关的差异。这些研究是 创新是第一个研究人类肠类单层电解质运输的人之一，以检查 DRA 与 NHE3 和 CFTR 在同一细胞群中的相互作用，重点是它们的动态 在基础条件下以及与升高的 cAMP 之间相互作用，是对 早期内体蛋白 SNX27 和逆转录酶在急性 DRA 调节中的作用，并开展以下研究 SNX27/retromer 作为未来开发治疗腹泻的潜在药物靶点。预期成果 该提案将 a) 了解 DRA、NHE3 和 CFTR 在以下模型中的协调监管： 严重腹泻，包括考虑 HCO3 损失的机制，理解上的差距 腹泻的病理生理学，b) 确定腹泻的药物治疗必须考虑协调 DRA 与 NHE3 和 CFTR 的调节，而不仅仅是每个转运蛋白的单独调节，并且 c) 确定 人肠道腹泻模型中的新药物靶点可以逆转腹泻相关的转运变化 在 DRA 和 NHE3 中，它有可能被开发成治疗腹泻的药物。