Inhibition of an apical cAMP/cGMP transporter(MRP4)in the gut induces diarrhea

描述(申请人提供)：这份持续支持的申请将专注于研究囊性纤维化跨膜传导调节因子(CFTR)、多药耐药蛋白4(MRP4)和诱导型一氧化氮合酶(INOS)之间的蛋白质-蛋白质相互作用如何导致溃疡性结肠炎(UC)的腹泻症状。我们的统一假设是，CFTR、NHERF2、MRP4和iNOS在UC的肠上皮细胞膜或质膜附近形成一个大分子复合体，这个大分子复合体是UC腹泻发病过程所必需的。具体地说，我们认为在UC中iNOS在肠上皮细胞的质膜上过度表达，导致持续高水平的一氧化氮(NO)的产生，从而触发NO-cGMP途径并产生区域化的cGMP。由于iNOS在质膜上或质膜附近与CFTR、NHERF2和MRP4形成大分子复合体，这种NO依赖的区段化cGMP触发CFTR氯通道的过度激活，从而导致腹泻表型。大分子复合体的破坏将取消iNOS依赖的cGMP生成和CFTRCl-通道功能的功能耦合。本研究的具体目的是：1.验证iNOS在UC患者的肠上皮细胞中高表达的假设，并验证其是否与CFTR和MRP4偶联并上调CFTRCl-通道功能；2.验证iNOS、CFTR和MRP4在肠上皮细胞质膜上或质膜附近形成大分子复合体的假设，以及该大分子复合体是UC腹泻发病过程所必需的。这些研究不仅有助于我们更好地了解UC常见腹泻症状的机制，而且可能为该病的治疗干预提供新的靶点和方法。因此，本申请中建议的研究将对许多UC患者和某些形式的分泌性腹泻患者具有临床意义。

DESCRIPTION (provided by applicant): This application for continued support will focus on studying how protein-protein interactions among the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance protein 4 (MRP4) and inducible nitric oxide synthase (iNOS) contribute to the diarrheal symptom observed in Ulcerative Colitis (UC). Our unifying hypothesis is that CFTR, NHERF2, MRP4 and iNOS form a macromolecular complex at or near the plasma membrane in gut epithelial in UC and this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. Specifically, we proposed that iNOS is overexpressed at the plasma membrane of gut epithelia in UC, resulting in the production of sustained high level of nitric oxide (NO) which triggers NO-cGMP pathway and generates compartmentalized cGMP. Since iNOS forms a macromolecular complex with CFTR, NHERF2 and MRP4 at or near the plasma membrane, this NO-dependent compartmentalized cGMP triggers the hyperactivation of CFTR chloride channels and thus causes diarrhea phenotype. Disruption of the macromolecular complex will abolish the functional coupling of iNOS-dependent cGMP generation and CFTR Cl- channel function. The specific aims of this proposal are: Specific Aim 1. To test the hypothesis that iNOS is overexpressed in UC-affected human gut epithelia and to test whether it is coupled to CFTR and MRP4 and upregulates CFTR Cl- channel function Specific Aim 2. To test the hypothesis that iNOS, CFTR, and MRP4 form a macromolecular complex at or near the plasma membrane of gut epithelial cells and that this macromolecular complex is required for the pathogenic process of diarrhea observed in UC. The proposed studies will not only help us better understand the mechanisms underlying the diarrhea symptom commonly observed in UC, but probably provide novel targets and methods for therapeutic interventions of the disease. Therefore, the studies proposed in this application will have clinical relevance for many individuals suffering from UC and also for those suffering from certain forms of secretory diarrheas.