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.

总结 在理解正常情况下人类肠道电解质转运的调节方面仍然存在重大差距 生理条件和牙周病。这一建议涉及刷状边框（BB）的作用 Cl/HCO 3逆向转运蛋白，SLC 26 A3（α）在肠道Na，Cl和HCO 3转运中起重要作用，并将解决肠道Na，Cl和HCO 3转运中的差距。 了解其急性调节，肠细胞参与和如何扭转异常运输， 重度cAMP相关性充血。该项目的长期目标是确定协调和动态的 钠，氯和HCO 3运输的变化发生在腹泻，使他们可以有针对性的药物逆转 这些变化并治疗大多数腹泻疾病。本提案的目的包括：一、检验假设 这是cAMP/霍乱毒素抑制中性Na和Cl吸收的必要步骤， 在刺激呼吸活动和相关HCO 3分泌的同时发生，包括增加 运输到BB和减少在NHE 3下发生的NHE 3与NHE 3的物理关联 基础条件和与CFTR的物理关联增加。二.检验cAMP相关的假设 NHE 3和NHE 3活性和运输的变化涉及与早期内体中SNX 27的结合， 逆转录酶稳定剂可以逆转这些变化，从而确定了治疗腹泻的新药物靶点。到 为了完成这些研究，我们将使用从健康人受试者制备的回肠和近端结肠肠样组织 作为2D单层生长，并将结果与来自人结肠癌细胞系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- 治愈性药物。我们预测，Caco-2细胞将提供类似的信息，肠钠，氯和HCO 3 在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 这些差异可能与肠道功能和药物开发的研究有关。这些研究 作为第一批研究人类肠道细胞单层电解质转运的创新者， 在同一细胞群中，NHE 3和CFTR的相互作用，重点是它们的动态 在基础条件下和升高的cAMP下相互作用，是对 早期内体蛋白SNX 27和逆转录酶在急性胰腺炎的调节，并开展研究， SNX 27/retromer是未来开发治疗腹泻的潜在药物靶点。的预期成果 本建议将是a）理解在一个模型中协调调节NHE 3和CFTR， 严重腹泻，包括考虑HCO 3损失的机制，在理解的差距， B）确立腹泻的药物治疗必须考虑协调 用NHE 3和CFTR调节E2，而不仅仅是单独调节每种转运蛋白，以及c）鉴定一种 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 在大肠杆菌和NHE 3中，它有可能被开发成治疗腹泻的药物。