ACTIVE POTASSIUM TRANSPORT ACROSS COLONIC EPITHELIUM

描述：这项提案的总体目标是确定 控制活性钾分泌和吸收的调控机制 在哺乳动物的结肠里。工作模式是空泡化的柱状结构 哺乳动物结肠隐窝细胞具有钾、氯和氢退出途径 跨越根尖膜以及基底外侧的钾和氯通道 薄膜。需要检验的假设是，协调的，尽管 不同激素/调节剂对这些过程的不同调节 对结肠表现出的一系列分泌行为负责 上皮组织。因此，肾上腺素和醛固酮引起低比率的液体 被认为是一种调节型调节的分泌，而 前列腺素E_2(PGE_2)导致液体流动速度加快，被认为是 更像是一种冲水式的分泌物。这两项中的任何一项都有功能障碍 空泡化的柱状细胞中的调节级联将形成 各种胃肠道疾病，从分泌性腹泻到 炎症性肠病。在前两个具体目标中，监管 钾离子通道和氯离子通道在冲洗和调制过程中的作用机制 我们将主要通过膜片钳来检查刺激类型 方法论。这些研究将允许在顶端和 基侧膜需单独研究。它们将得到补充 通过使用荧光探针测量细胞内氯，以及通过 形态(差分干涉衬度显微镜)和 跟踪液泡释放的生物物理(膜电容)研究。在 第三个具体目标，使用荧光探针，参与 细胞内钙和氢在离子通道中的调节作用 调查过了。初步数据显示，这两人参与了 前列腺素E_2、醛固酮和肾上腺素的作用途径。此外 将评估根尖膜H/K泵的活性，并观察是否存在 原位杂交法测定H/K-ATPase转录本。

DESCRIPTION: The overall goal of this proposal is to determine the regulatory mechanisms controlling active potassium secretion and absorption in the mammalian colon. The working model is that the vacuolated columnar cells of the mammalian colonic crypt possess pathways for K, Cl and H exit across the apical membrane as well as K and Cl channels on the basolateral membrane. The hypothesis to be tested is that the coordinated, albeit differential regulation, of these processes by different hormones/modulators is responsible for the range of secretory behavior exhibited by the colonic epithelium. Thus epinephrine and aldosterone cause a low rate of fluid secretion that is proposed to be a modulatory type of regulation whereas prostaglandin E2 (PGE2) causes a high rate of fluid flow that is proposed to be more of a flushing type of secretion. A dysfunction in either of these regulatory cascades in the vacuolated columnar cells would form the basis of a variety of gastrointestinal diseases ranging from secretory diarrhea to inflammatory bowel disease. In the first two specific aims the regulatory mechanisms acting on K and Cl channels during the flushing and modulatory type of stimulation will be examined, largely through patch clamp methodology. These studies will allow the events at the apical and basolateral membranes to be studied separately. They will be complemented by measurements of intracellular Cl using a fluorescent probe, and by morphological (differential interference contrast microscopy) and biophysical (membrane capacitance) studies to track vacuole release. In the third specific aim, using fluorescent probes, the involvement of intracellular Ca and H in the regulation of the ion channels will be investigated. Preliminary data suggest the involvement of these two pathways in the actions of PGE2, aldosterone and epinephrine. In addition apical membrane H/K pump activity will be assessed and the presence of an H/K ATPase transcript determined by in situ hybridization.