SALT TRANSPORT IN SURGICAL DIARRHEAL DISEASE

在肠道中，产生电的氯离子分泌形成了各种 以分泌性腹泻为特征的病理状态，包括 抗生素相关性结肠炎、细菌毒素介导的腹泻、造口 腹泻，某些神经内分泌综合征，以及大量炎症 肠道疾病。电生氯分泌的现有模型 强调在根尖水平上对生电氯分泌的调节 膜氯通道是由于最近认识到的 这一途径在囊性纤维化的发病机制中起重要作用。相比之下，几乎没有 已知基底侧膜转运蛋白的调节 功能。为了在高速率下保持细胞组成 分泌物，基侧氯进入和根尖氯出口必须精确 相匹配，然而顶端和顶端的协调 完成了基侧传输事件(“串扰”) 不完全理解。 这项建议的总体目标是在根本层面上审查 Na~+/K~+/2Cl共转运蛋白的调节 上皮细胞分泌氯离子的机制。Na~+/K~+/2Cl共转运蛋白 是一种高度保守的膜转运蛋白，除了其 在盐分和水分运输中的不可或缺的作用 吸收上皮可能参与两者的细胞体积调节。 上皮细胞和非上皮细胞。越来越多的证据表明 Na+/K+/2Cl共转运蛋白的调控是一个复杂的过程 涉及蛋白质的磷酸化和数量的改变 质膜上的转运蛋白。本项目的初步数据 提示了F-肌动蛋白细胞骨架可能 动态地影响这些事件。 建议的研究将使用已建立的培养的人进行。 肠道细胞系作为模型系统。这一行动的具体目标 项目有1)从功能上描述对 基底侧膜Na~+/K/2Cl共转运体对cAMP介导的反应 激动剂、非cAMP促分泌剂和渗透压扰动，2)相互关联 协同转运体功能的调节变化与数量的变化 基底膜上活性共转运体的位置，3)以确定 F-肌动蛋白微丝系统在细胞周期调控中的可能作用 共转运体的数量和活性，以及，4)识别一些 参与共转运蛋白调控的细胞内信号通路 功能。 拟议的研究应提高我们对电生氯的理解 阐明Na~+/K~+/2Cl调节细胞分泌的机制 肠上皮细胞的共转运。通过确定 离子转运的完整膜蛋白(The 共转运体)和F-肌动蛋白微丝系统，这些实验 可能会对其他重要的离子传输产生更广泛的影响 过程，并应深入了解细胞骨架的功能 在上皮细胞中。希望通过对基本原理的理解 电源性氯分泌的机制将建立一个合理的 设计新的治疗方法的基础 分泌性腹泻。

In the intestine, electrogenic Cl-secretion forms the basis for a variety of pathologic states characterized by secretory diarrhea, including antibiotic-associated colitis, bacterial toxin-mediated diarrhea, ostomy diarrhea, certain neuroendocrine syndromes, and numerous inflammatory diseases of the intestine. Current models of electrogenic Cl secretion emphasize regulation of electrogenic Cl secretion at the level of apical membrane Cl channels because of the recently recognized relationship of this pathway to the pathogenesis of cystic fibrosis. In contrast, little is known about the regulation of basolateral membrane transporter function. In order to maintain cellular composition during high rates of secretion, basolateral Cl entry and apical Cl exit must be precisely matched, yet the mechanism by which coordination of apical and basolateral transport events ("crosstalk") is accomplished is incompletely understood. The general goal of this proposal is to examine at a fundamental level the regulation of the Na+/K+/2Cl cotransporter, the basolateral Cl uptake mechanism of Cl secreting epithelial cells. The Na+/K+/2Cl cotransporter is a highly conserved membrane transporter which, in addition to its integral role in salt and water transport in both secretory and absorptive epithelia, may be involved in cell volume regulation in both epithelial and non-epithelial cells. Accumulating evidence suggests that the regulation of the Na+/K+/2Cl cotransporter is a complex process involving protein phosphorylation and alteration in numbers of transporters in the plasmalemma. Preliminary data for this project suggest the novel possibility that the F-actin cytoskeleton may dynamically influence these events. The studies proposed will be carried out using established cultured human intestinal cell lines as model systems. The specific goals of this project are 1) to characterize functionally the regulation of the basolateral membrane Na+/K/2Cl cotransporter in response to cAMP-mediated agonist, non-cAMP secretagogue, and osmotic perturbation, 2) to correlate regulated changes in cotransporter function with changes in the number of active cotransporter sites on the basolateral membrane, 3) to define the possible role of the F-actin microfilament system in regulation of cotransporter number and activity, and, 4) to identify some of the intracellular signalling pathways involved in modulation of cotransporter function. The propose studies should improve our understanding of electrogenic Cl secretion by elucidating the mechanism for regulation of Na+/K+/2Cl cotransport in intestinal epithelial cells. By determining the interaction between an ion-transporting integral membrane protein (the cotransporter) and the F-actin microfilament system, these experiments may prove to have broader implications for other important ion transport processes and should yield insight into the function of the cytoskeleton in epithelial cells. It is hoped that an understanding of fundamental mechanisms underlying electrogenic Cl secretion will establish a rational basis for designing new therapeutic approaches to the problem of secretory diarrhea.