NA+,K+,CL-COTRANSPORT IN A KIDNEY EPITHELIAL CELL LINE

这项建议的长期目标是了解该机制， 布美他尼敏感离子的调节及其生理意义 联运系统。在分化的肾上皮细胞系中， MDCK，在严格控制的条件下，布美他尼(和速尿 敏感体系催化1-Na+，1-紧耦合共转运 K+和2Cl-。在各种各样的组织中，这种运输系统 与细胞体积的调节有关。在许多上皮细胞中， 布美他尼敏感的转运也在氯的再吸收中起作用。 MDCK细胞系作为研究离子的系统具有许多优点 运输，包括三个独立突变体的可用性 布美他尼敏感的运输系统有缺陷。这其中的一个 突变体的病毒含量不到正常水平的10%。 布美他尼敏感的Na+和K+通量，而另一个突变体有 失去了50%的K+通量活性，但保留了近100% Na+通量活性的变化。第三个突变体有多个缺陷， 很可能会发生监管突变。这一运输系统在 跨上皮运输和细胞体积调节将是 利用这三个突变体进行了研究。此外，两国关系 我们将研究细胞体积调节和生长调节之间的关系。 多种激素和其他物质在调节运输中的作用 也将接受调查。同时，还会显示 布美他尼在极化细胞单层中的结合位点将是 用放射性布美他尼测定。光亲和性衍生物 将合成布美他尼并进行测试，用于特定的标记 亲本和突变体转运系统的多肽成分(S) 细胞系。质膜囊泡将被用来证实和 扩展在整个细胞中所做的观察。我们将把注意力集中在 检测未偶联的氯化钠和氯化钾的转运并确定 支持布美他尼敏感转运的细胞内ATP。

The long term goals of this proposal are to understand the mechanism, regulation and physiological importance of the bumetanide-sensitive ion cotransport system. In the differentiated kidney epithelial cell line, MDCK, under carefully controlled conditions the bumetanide- (and furosemide sensitive system catalyzed the tightly coupled cotransport of 1 Na plus, 1 K plus and 2 Cl minus. In a wide variety of tissues, this transport system has been implicated in the regulation of cell volume. In many epithelia, bumetanide-sensitive transport also functions in chloride reabsorbtion. The MDCK cell line was many advantages as a system with which to study ion transport, including the availability of three independent mutants defective in the bumetanide-sensitive transport system. One of these mutants has less than 10 percent of the normal levels of bumetanide-sensitive Na plus and K plus fluxes, while another mutant has lost 50 percent of the K plus flux activity but retained nearly 100 percent of the Na+ flux activity. The third mutant has multiple defects and is likely to have a regulatory mutation. The role of this transport system in transepithelial transport and cellular volume regulation will be investigated using these three mutants. In addition, the relationship between cellular volume regulation and growth regulation will be examined. The role of a variety of hormones and other agents in regulating transport will also be investigated. Concurrently, the number and location of the bumetanide binding sites in polarized cellular monolayers will be determined using radioactive bumetanide. Photoaffinity derivatives of bumetanide will be synthesized and tested for specific labeling of the polypeptide component(s) of the transport system in the parental and mutant cell lines. Plasma membrane vesicles will be utilized to corroborate and extend the observations made in whole cells. Attention will be directed at detecting uncoupled NaCl and KCl transport and at determining the role of intracellular ATP in supporting bumetanide-sensitive transport.