SALT TRANSPORT IN SURGICAL DIARRHEAL DISEASE

在肠道中，产电性Cl-分泌形成了多种 以分泌性腹泻为特征的病理状态，包括 结肠炎相关性结肠炎，细菌毒素介导的腹泻，造口术 腹泻，某些神经内分泌综合征，和许多炎症 肠道疾病。 电流性Cl分泌的当前模型 强调在根尖水平调节产电性Cl分泌 膜氯离子通道，因为最近认识到的关系， 囊性纤维化的发病机制。 相比之下， 已知基底外侧膜转运蛋白的调节 功能 为了在高速率下维持细胞组成 分泌，基底外侧Cl入口和顶端Cl出口必须精确 匹配，但机制，协调顶端和 基底外侧转运事件（“串扰”）的完成是 不完全理解。 这项建议的总体目标是从根本上审查 Na+/K+/2Cl协同转运蛋白的调节、基底外侧氯吸收 分泌Cl的上皮细胞的机制。Na+/K+/2Cl协同转运蛋白 是一种高度保守的膜转运蛋白， 在分泌和分泌细胞中盐和水运输中的不可或缺的作用， 吸收上皮细胞，可能参与细胞体积的调节，在这两个 上皮细胞和非上皮细胞。 越来越多的证据表明， Na+/K+/2Cl协同转运蛋白的调节是一个复杂的过程 涉及蛋白质磷酸化和改变的数量， 质膜中的转运蛋白。 本项目的初步数据 提出了一种新的可能性，即F-肌动蛋白细胞骨架可能 动态地影响这些事件。 拟议的研究将使用已建立的培养人 肠细胞系作为模型系统。 具体目标是 项目是1）在功能上表征监管的 基底外侧膜Na+/K/2Cl共转运蛋白对cAMP介导的 激动剂、非cAMP促分泌剂和渗透扰动，2） 协同转运蛋白功能的调节性变化， 基底外侧膜上的活性协同转运蛋白位点，3）定义 F-肌动蛋白微丝系统在调节细胞凋亡中的可能作用 协同转运蛋白的数量和活性，以及，4）识别一些 参与协同转运蛋白调节的细胞内信号通路 功能 这些研究有助于提高我们对产电氯离子的认识 通过阐明Na+/K+/2Cl的调节机制， 肠上皮细胞的共转运。 通过确定 离子转运整合膜蛋白（离子转运整合膜蛋白）之间的相互作用 协同转运蛋白）和F-肌动蛋白微丝系统，这些实验 可能证明对其他重要的离子传输有更广泛的影响 过程，并应产生洞察的功能，细胞骨架 在上皮细胞中。 希望能对基本的 机制潜在的产电氯分泌将建立一个合理的 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.