REGULATION OF NA+ & K+ TRANSPORT ACROSS EPITHELIA

该项目的长期目标是了解钠如何 钾离子通过上皮细胞运输 两个最 调节血浆K+和Na+水平的重要位点是 肾集合管系统和结肠。 本研究 建议使用2个模型上皮系统来研究 Na ~+、K ~+转运的调控：1）蟾蜍A_6细胞系 肾组织; 2）兔远曲小管原代细胞培养。 这些 上皮细胞均通过盐皮质激素敏感性吸收Na+ 机制，类似于其他所谓的紧密上皮细胞。 在 此外，兔远端小管细胞具有研究充分的K+- 运输机制。 工程的重点是规范 顶端和基底侧离子通道的醛固酮，环 核苷酸和其他因素。 电生理学方法 包括常规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 技术将被使用。 通过这些方法， 将获得关于膜电位，电导 和面积以及单通道特性。 使用此 信息，将有可能确定这些渠道如何 受到调节，例如通过通道动力学的变化、 开放通道电导或膜面积的变化， 通道数。 第一个具体目标是 表征膜电导、面积和Na+通道 培养的A6上皮细胞中的性质。 以下问题将 问：1）醛固酮和抗利尿激素是否激活 相同的Na+通道数量 2)哪些因素调节 基底外侧膜钾传导率 3)是什么 醛固酮对细胞膜电导的长期影响 和地区？ 4)去除激素刺激后， 上皮通道“失效”了吗 第二个目标是 肾远曲小管顶膜钾通道的研究 细胞 具体而言，将提出以下问题：1）是否 醛固酮激活不同的K+通道， 它通过改变通道门控或其它方式增加K+电导， 特性. 2)激素刺激的时间过程是什么 这种K+电导，它是下调还是上调， 与Na+吸收有关？ 结果将提供重要的 单膜和通道的定量信息 关于慢性激素刺激对 上皮细胞膜性质。

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.