REGULATION OF NA+ & K+ TRANSPORT ACROSS EPITHELIA

这个项目的长期目标是了解钠如何 和钾跨上皮细胞运输。其中最多的两个 调节血浆钾和钠水平的重要部位有 肾脏集合管系统和结肠。目前的研究 建议使用2个模型上皮系统来研究 钠钾转运的调节：1)蟾蜍A6细胞系 2)兔远端小管原代细胞培养。这些 两种上皮细胞均通过对盐皮质激素敏感的方式吸收钠 机制，类似于其他所谓致密上皮中的机制。在……里面 此外，兔远端肾小管上皮细胞具有已被充分研究过的K- 传输机制。该项目的重点是监管 环醛固酮对心尖部和基底侧部离子通道的影响 核苷酸和其他因素。电生理学方法 包括传统的和离子敏感的微电极方法， 阻抗分析、电流波动分析和膜片钳 将使用各种技术。从这些方法中获得新的信息 将获得关于膜电位、电导率 和区域以及单通道属性。使用这个 信息，就有可能确定这些渠道是如何 被调节，例如受通道动力学的改变、 明渠电导或膜面积的变化 传导通道的数量。第一个具体目标是 表征膜电导、面积和钠通道 培养的A6上皮细胞的特性。以下问题将 被问到：1)醛固酮和抗利尿激素是否激活 钠离子通道的人群相同吗？2)哪些因素调节 基侧膜钾电导？3)什么是 醛固酮对膜电导的长期影响 和区域？4)去除激素刺激后，情况如何 上皮细胞通道“失活”？第二个目标是 肾远端小管顶膜钾通道的特征 细胞。具体地说，将提出以下问题：1)是否 醛固酮激活一组不同的K通道或 它通过修改通道选通或其他方式来增加K电导 属性。2)荷尔蒙刺激的时间进程是什么 这个K电导是下调还是上调 与钠吸收有什么关系？这一结果将提供重要的 在单膜和通道上的定量信息 关于慢性激素刺激对健康影响的水平 上皮膜特性。

The long term goal of this project is to understand how sodium and potassium are transported across epithelia. Two of the most important sites for the regulation of plasma K+ and Na+ levels are the renal collecting duct system and the colon. The present study proposes to use 2 model epithelial systems to investigate the regulation of Na+ and K+ transport: 1) the A6 cell line from toad kidney, and 2) primary cell cultures of rabbit distal tubule. These epithelia both absorb Na+ by mineralocorticoid-sensitive mechanisms, similar to those in other so-called tight epithelia. In addition rabbit distal tubule cells possess well-studied K+- transport mechanisms. The focus of the project is the regulation of apical and basolateral ionic channels by aldosterone, cyclic nucleotides, and other factors. Electrophysiological methods including conventional and ion-sensitive microelectrode methods, impedance analysis, current fluctuation analysis and patch clamp techniques will be used. From these methods new information will be obtained concerning membrane potentials, conductances and areas as well as single channel properties. Using this information, it will be possible to determine how these channels are regulated, e.g. by changes in channel kinetics, alteration of open-channel conductance or changes in membrane area and the number of conducting channels. The first specific aim is to characterize membrane conductances, areas, and Na+ channel properties in cultured A6 epithelia. The following questions will be asked: 1) Do aldosterone and anti-diuretic hormone activate the same population of Na+ channels? 2) What factors regulate the basolateral membrane potassium conductance? 3)What are the long term effects of aldosterone on membrane conductances and areas? 4) After removal of hormonal stimulation how are epithelial channels "deactivated"? The second objective is to characterize apical membrane K+ channels in renal distal tubule cells. Specifically, the following questions will be asked: 1) Does aldosterone activate a distinct population of K+ channels or does it increase K+ conductance by modifying channel-gating or other properties. 2) What is the time course of hormonal stimulation of this K+ conductance and is it downregulated or upregulated in relation to Na+ absorption? The results will provide important quantitative information at the single-membrane and channel level concerning the effects of chronic hormonal stimulation on epithelial membrane properties.