MEMBRANE PROCESSES MEDIATING NACL TRANSPORT

这个项目的总体目标是通过以下方式了解这些机制 肾脏对尿液中盐分的重吸收 受控，特别是在远端肾单位，荷尔蒙信号 根据身体需要调节钠的重吸收。这个 这个应用程序的具体目标可以概括为两个 类别。首先是对监管的详细描述，由 肾上腺皮质离子转运蛋白的矿化皮质激素 收集大鼠肾小管。二是对中国传统文化的探索 醛固酮调节上皮钠活性的机制 频道。 为了实现第一个目标，我们将使用膜片钳技术来 监测整个细胞的通道和泵介导的电流 膜(全细胞记录)以及通过单独的通道 (单声道录制)。时间进程和程度 心尖钠通道、心尖钾通道和基底外侧通道的激活 Na/K泵将在体外进行评估，在操作设计为 对动物体内矿化皮质醇状态的影响。这些 操作包括改变饮食(低钠、高钾)、钠耗竭 使用利尿剂，如速尿，并直接输注类固醇。 需要解决的具体问题包括(1)渠道变更 活动是由血浆醛固酮水平变化引起的吗？(2) 整体运输活动的变化是由 传导通道的数量？(3)通道的表达是否均匀 是膜上的还是簇状的？(4)单个细胞在 分级还是全有或全无的时尚？和(5)是频道的变化 伴随着NA/K泵活动的平行变化？ 第二组目标将测试几个具体的假设，这些假设涉及 钠离子通道的调控机制。这些是(1) 醛固酮激活预先存在的通道而不刺激通道 信使核糖核酸合成，(2)醛固酮促进脂肪或蛋白质甲基化 (3)醛固酮激活Na/H 导致细胞质碱化的交换，以及(4)ADH，起作用 通过cAMP，促进含有醛固酮的囊泡融合- 可激活的通道与顶膜。这些假设将是 使用膜片钳电生理学测试，荧光测量 细胞Ph，并通过注射mRNA在卵母细胞中表达通道。 拟议的工作应加强我们对以下过程的理解 调节身体健康中可能存在缺陷的氯化钠含量 囊性纤维化和高血压等疾病状态。

The overall goal of this project is to understand the mechanisms by which the reabsorption of salt from the urine by the kidney is controlled, particularly in the distal nephron where hormonal signals regulate Na reabsorption according to the needs of the body. The specific goals of this application can be put into two general categories. The first is a detailed description of the regulation by the adrenal mineralocorticoids of ion transporters in the cortical collecting tubule of the rat kidney. The second is an exploration of the mechanisms by which aldosterone controls the activity of epithelial Na channels. To achieve the first goal, we will use the patch clamp technique to monitor channel and pump-mediated currents across the entire cell membrane (whole-cell recordings) as well as through individual channels (single-channel recordings). The time course and the extent of activation of apical Na channels, apical K channels, and basolateral Na/K pumps will be evaluated in vitro after manipulations designed to effect the mineralocorticocoid status of the animals in vivo. These manipulations include changes in diet (low Na, high K), Na depletion using diuretics such as furosemide, and direct infusion of steroids. Specific issues to be addressed include (1) Are changes in channel activity accounted for by changes in levels of plasma aldosterone? (2) changes in overall transport activity accounted for by changes in the number of conducting channels? (3) Are channels expressed evenly over the membrane or in clusters? (4) Are individual cells stimulated in a graded or and all-or-none fashion? and (5) Are changes in channel activity accompanied by parallel changes in activity of the NA/K pumps? The second set of aims will test several specific hypotheses concerning the mechanisms through which Na channels are regulated. These are (1) aldosterone activates pre-existing channels without stimulating channel MRNA synthesis, (2) aldosterone promotes lipid or protein methylation leading to activation of channels, (3) aldosterone activates Na/H exchange leading to alkalinization of the cytoplasm, and (4) ADH, acting through CAMP, promotes the fusion of vesicles containing aldosterone- activatable channels with the apical membrane. These hypotheses will be tested using patch-clamp electrophysiology, fluorescence measurements of cell Ph, and expression of channels in oocytes by injection of MRNA. The proposed work should enhance our understanding of processes that regulate body NaCl content in health and which may be defective in disease states such as cystic fibrosis and hypertension.