KININS AND EPITHELIAL ION TRANSPORT MECHANISMS

这项建议中的工作将促进对 激肽释放酶-激动素系统及其在上皮细胞调控中的作用(S) 离子转运--维持正常细胞的重要过程 功能。囊性纤维化现在被认为是由上皮性肿瘤引起的 氯化物转运缺陷，例如， 对同一过程进行不适当的监管。离子传输现象 也有助于控制血压和糖尿病，以及 激肽释放酶-激动素系统中的异常与这两种情况有关 这些疾病。 这项提案的具体目标将确定是否 激肽释放酶-激肽系统以自分泌或旁分泌的形式参与 人结肠上皮细胞T84的氯离子转运调节因子 来源于腺癌的细胞系。这些单元中的系统将 被激活或被抑制，以及对氯离子运动的影响 在Ussing密室里被监控。这将通过(1)以下方式实现 用适当的试剂激活或抑制蛋白激肽释放酶， 并通过阻止动作来确定结果的特异性 在其受体上释放的激肽，以及(2)通过增加 使用生理信号的系统活动，例如生长 细胞作为裸鼠体内的肿瘤，或增加细胞外 钾浓度，增加激肽释放酶产量的处理 和激动素敏感性。 生理诱导的分子机制研究进展 激肽释放酶-激肽系统的变化也将被调查。 125I-缓激肽结合研究将测量激动素受体的变化 数量和/或亲和力，以及钙动员和 肌醇磷酸合成将测量第二信使的变化 制作。激肽释放酶(或类似激肽释放酶)基因的表达 研究通过评估mRNA转录本，以及Na+-K+-Cl-的作用 共转运系统在IV中的作用将通过抑制 在钾挑战前与布美他尼共转运。 上皮细胞离子转运的调节是复杂的， 涉及多种神经和荷尔蒙的影响。澄清： 激肽释放酶-激肽系统对这些过程的贡献将 降低这种复杂性。这些发现不仅与 对基本细胞过程的定义，也是对 对人类疾病的理解。

The work in this proposal will advance the understanding of the kallikrein-kinin system and its role(s) in the regulation of epithelial ion transport, a process important to the maintenance of normal cell function. Cystic fibrosis is now thought to result from an epithelial defect in chloride transport, e.g., and secretory diarrhea from inappropriate regulation of the same process. Ion-transporting phenomena also contribute to aspects of blood pressure control and diabetes, and aberrancies in the kallikrein-kinin system have been associated with both of these diseases. The specific aims of this proposal will establish whether the kallikrein-kinin system participates as an autocrine or paracrine regulator of chloride transport in T84 cells, a human colonic epithelial cell line derived from an adenocarcinoma. The system in these cells will be activated or inhibited, and the effects on chloride movements monitored in the Ussing chamber. This will be accomplished (1) by activating or inhibiting the protease kallikrein with appropriate agents, and establishing the specificity of the results by blocking the actions of the released kinin peptide at its receptors, and (2) by increasing the activity of the system using physiological signals such as growing the cells as a tumor in the nude mouse, or increasing the extracellular potassium concentration, treatments that increase kallikrein production and kinin sensitivity. The molecular mechanisms underlying the physiologically-induced changes in the kallikrein-kinin system will also be investigated. 125I-bradykinin binding studies will measure changes in kinin receptor number and/or affinity, and determinations of calcium mobilization and inositol phosphate synthesis will measure changes in second messenger production. Kallikrein (or kallikrein like) gene expression will be studied by evaluating mRNA transcripts, and the role of the Na+-K+-Cl- cotransport system in the IV effect will be examined by inhibiting the cotransport with bumetanide prior to a potassium challenge. The regulation of ion transport in epithelia is complex, involving multiple neural and hormonal influences. Elucidation of the contribution of the kallikrein-kinin system to these processes will reduce this complexity. These findings will be relevant not only to the definition of fundamental cellular processes, but also to the understanding of human disease.