Diarrheal Disease: A Physiologic Approach to Treatment

腹泻病：生理治疗方法

基本信息

批准号：
7856333
负责人：
MARK DONOWITZ
金额：
$ 0.82万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-06-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7856333
关键词：
Abbreviations Accounting Actinin Acute Address Adrenergic Agonists Affect Agonist Algorithms Apical Area Back Binding Biochemical Biological Assay Brush Border C-terminal CSNK1A1 gene Ca(2+)-Calmodulin Dependent Protein Kinase Caco-2 Cells Carrier Proteins Cartoons Catalytic Domain Cell membrane Chimera organism Chronic Complex Cyclic AMP Cyclic GMP Databases Dependence Diarrhea Digestion Digestive Physiology Disease Dominant-Negative Mutation EGF gene Endocytosis Exocytosis Family Fasting Figs - dietary Human Immunoprecipitation Individual Intestines Knowledge Left Ligands Link Lipase Molecular Multiprotein Complexes Mutagenesis Mutation Oryctolagus cuniculus Pharmaceutical Preparations Pharmacotherapy Phosphatidylinositols Phospholipase C Phosphorylation Phosphotransferases Physiological Proteins Recycling Regulation Role SRC gene Signal Transduction Site Small Interfering RNA Sodium Tertiary Protein Structure Testing Time absorption base calmodulin-dependent protein kinase II dimer genetic regulatory protein in vivo inhibitor/antagonist insight knock-down public health relevance scaffold sodium-hydrogen exchanger regulatory factor trafficking

项目摘要

DESCRIPTION (provided by applicant): The underpinning of this proposal is that there is an urgent need for new pharmacotherapy of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate the brush border Na/H exchanger NHE3 under both the basal state and in the inhibited state as occurs in most diarrhea. A starting point in achieving this stimulation is to understand how NHE3, which accounts for the majority of intestinal Na absorption at least in the fasting state, is regulated both normally as part of digestive physiology and as inhibited in diarrhea. In this proposal we will expand understanding of acute NHE3 regulation by examining the mechanisms of action of several proteins that we have found both bind to the NHE3 C-terminus and regulate NHE3 activity. These proteins are 1) CaM kinase II, 2) CK2, 3) PLC3. The major hypothesis to be tested in this proposal is that NHE3 activity is determined under both basal and elevated Ca2+ conditions by coordinated and dynamic interactions with these C-terminal binding partners, which not only regulate NHE3 but also affect each others' association with and regulation of NHE3 in a coordinated manner. Aim I studies each of these proteins expressed in polarized intestinal Na absorptive cells (Caco-2) and uses transport assays correlated with biochemical approaches including siRNA and pharmacologic and molecular (dominant-negative) inhibitors with molecularly modified NHE3 or the associating proteins to determine the consequences of NHE3 physical association with these scaffolded proteins on NHE3 basal, stimulated and inhibited activity and on NHE3 complexes involved in its regulation. Aim II tests the hypothesis that a common domain in the NHE3 C-terminus with which all of the proteins in Aim I associate, represents part of "a switch" which moves NHE3 back and forth between a fully stimulated state through basal activity to inhibition, which defines NHE3 function as part of digestion. This will be examined by determining whether association with NHE3 of the proteins studied in Aim I changes in a reproducible and time dependent manner with changes in regulation of NHE3 activity; whether knockdown and pharmacologic/molecular inhibitors of each of these NHE3 associating proteins alter NHE3 association with the other interacting proteins; and the consequences of eliminating interactions with single or multiple of these associating proteins including by mutagenesis approaches. Public Health Relevance: The basis of this proposal is that there is an urgent need for new drug treatment of acute and chronic diarrheal diseases, and this might be achieved by determining ways to stimulate a specific intestinal sodium transport protein, the brush border Na/H exchanger NHE3, under both the basal state and in the inhibited state as occurs in most diarrheal diseases. This study examines the mechanism by which several proteins which bind to the cytoplasmic part of NHE3 affect its transport activity under basal conditions and when NHE3 is stimulated or inhibited to mimic changes which occur during digestion and in diarrheal diseases. In addition, the coordinated association with NHE3 of these proteins which all bind to a small area of NHE3, will be examined to determine if together they explain how NHE3 changes its activity during digestion and in diarrhea.

描述（由申请人提供）：该提案的基础是急需针对急性和慢性腹泻疾病的新药物疗法，这可以通过确定在两种条件下刺激刷状缘Na/H交换器NHE3的方法来实现。基础状态和大多数腹泻中发生的抑制状态。实现这种刺激的第一步是了解 NHE3（至少在禁食状态下占肠道 Na 吸收的大部分）如何在正常情况下作为消化生理学的一部分进行调节，并在腹泻时受到抑制。在本提案中，我们将通过检查几种蛋白质的作用机制来扩展对急性 NHE3 调节的理解，我们发现这些蛋白质既与 NHE3 C 末端结合又调节 NHE3 活性。这些蛋白质是 1) CaM 激酶 II、2) CK2、3) PLC3。本提案要测试的主要假设是，NHE3 活性在基础和高 Ca2+ 条件下通过与这些 C 端结合伙伴的协调和动态相互作用来确定，这不仅调节 NHE3，还影响彼此的关联和调节NHE3以协调的方式。目标 I 研究在极化肠 Na 吸收细胞 (Caco-2) 中表达的每种蛋白质，并使用与生化方法相关的转运测定，包括 siRNA 和具有分子修饰的 NHE3 或相关蛋白质的药理学和分子（显性阴性）抑制剂，以确定NHE3 与这些支架蛋白的物理关联对 NHE3 基础活性、刺激活性和抑制活性以及参与其调节的 NHE3 复合物的影响。 Aim II 测试了这样的假设：NHE3 C 末端的一个共同结构域与 Aim I 中的所有蛋白质相关联，代表“开关”的一部分，该开关使 NHE3 在完全刺激状态、基础活性和抑制状态之间来回移动，它将 NHE3 功能定义为消化的一部分。这将通过确定 Aim I 中研究的蛋白质与 NHE3 的关联是否随着 NHE3 活性调节的变化而以可重复和时间依赖性的方式变化来进行检查；这些 NHE3 相关蛋白的敲低和药理学/分子抑制剂是否会改变 NHE3 与其他相互作用蛋白的关联；以及消除与单个或多个这些关联蛋白的相互作用（包括通过诱变方法）的后果。公共健康相关性：该提案的基础是迫切需要治疗急性和慢性腹泻疾病的新药，这可以通过确定刺激特定肠道钠转运蛋白（刷状缘 Na/H）的方法来实现。交换器 NHE3，处于基础状态和抑制状态，如大多数腹泻疾病中所发生的那样。本研究探讨了与 NHE3 细胞质部分结合的几种蛋白质在基础条件下以及当 NHE3 受到刺激或抑制以模拟消化和腹泻疾病期间发生的变化时影响其转运活性的机制。此外，将检查这些蛋白质与 NHE3 的协调关联，这些蛋白质都与 NHE3 的小区域结合，以确定它们是否共同解释了 NHE3 在消化和腹泻过程中如何改变其活性。