REGULATION OF NA/CL/K COTRANSPORT IN TRACHEAL EPITHELIUM

气管上皮NA/CL/K协同转运的调控

• 批准号: 6389700
• 负责人: CAROLE M LIEDTKE
• 金额: $ 29.52万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389700
• 关键词: alpha adrenergic receptor; body fluid osmolarity; chloride ion; confocal scanning microscopy; enzyme activity; gel electrophoresis; hormone regulation /control mechanism; intracellular transport; ion transport; phosphoprotein phosphatase; potassium ion; protein kinase C; protein localization; protein protein interaction; radiotracer; respiratory epithelium; sodium ion; tissue /cell culture; trachea

Basolateral Na-K-CI cotransport (NKCC) in lining epithelial cells of the lung is crucial for optimal mucociliary clearance because it supplies CI for fluid secretion. Disease states that disrupt CI and fluid movement, such as asthma, chronic bronchitis, and cystic fibrosis (CF), and exposure to foreign irritants contribute to excess mucus and abnormal mucocillary clearance. Treatments aimed at correcting deficient CI secretion might fail to activate NKCC or might compromise NKCC activity thus preventing CI secretion and ultimately failing to correct excess mucus accumulation. Hence, in this proposal, we pose the question: how is NKCC turned on to support transepithelial CI secretion? New evidence from this laboratory points to protein kinase C (PKC)- dependent activation of NKCC in tracheal epithelial cells (TEC) by hormone (alpha1-adrenergic (AR)) stimulation and by hyperosmotic stress. However, modulation of NKCC is more complicated than activation of PKC. Although, intracellular CI levels (CIi) are depicted in models of NKCC to directly modulate CI flux across a plasma membrane through a CI electrochemical gradient, in TEC, stimuli that are expected to increase CIi activate NKCC. Preliminary studies strongly implicate an intracellular signalling pathway as a focal point for CI-dependent activation of NKCC. This will be studied in detail in the following specific aims: 1) To test the hypothesis that CIi modulates NKCC activation by alpha1-AR agonist or by hyperosmotic stress. Changes in [CI]i induced by the two stimuli will be determined. CIi levels will be manipulated to study subsequent effects on activation of NKCC and on NKCC activity. The kinetics of NKCC deactivation will be investigated. 2) To test the hypothesis that CIi modulates activity of protein kinases and phosphatases required for modulation of NKCC activity. Altered activity of protein kinases and phosphatases and the CI-dependence of the enzyme activities will be studied using specific substrates, including NKCC from human and kidney. stPP isotype(s) required for deactivation of NKCC will be identified. 3) To test the hypothesis that CIi modulates protein-protein interactions between NKCC and PKC and/or protein phosphatases. Protein-protein interaction and its dependence on [CI] and activated enzyme will be investigated using immunopurified NKCC, PKC, and protein phosphatases. Shifts in in vivo localization of enzymes with stimulation will be determined by confocal microscopy. A long term outcome of this project is the development of pharmacological tools to manipulate protein-protein interactions that facilitate association of PKC and NKCC to modulate NKCC activity in pathophysiological states.

基底外侧的 Na-K-CI 协同转运 (NKCC) 位于基底膜上皮细胞中 肺对于最佳粘液纤毛清除至关重要，因为它提供 CI 用于液体分泌。 破坏 CI 和液体的疾病状态 运动，例如哮喘、慢性支气管炎和囊性纤维化（CF）， 接触外来刺激物会导致粘液过多， 粘膜纤毛清除异常。旨在纠正缺陷的治疗 CI 分泌可能无法激活 NKCC 或可能损害 NKCC 从而阻止 CI 分泌并最终无法纠正 过多的粘液积聚。 因此，在本提案中，我们提出 问题：NKCC 如何启动以支持跨上皮 CI 分泌？ 该实验室的新证据指向蛋白激酶 C (PKC) - 气管上皮细胞 (TEC) 中 NKCC 的依赖性激活 激素（α1-肾上腺素能 (AR)）刺激和高渗应激。 然而，NKCC 的调节比 PKC 的激活更复杂。 尽管 NKCC 模型中描绘了细胞内 CI 水平 (CIi) 通过 CI 直接调节跨质膜的 CI 通量 电化学梯度，在 TEC 中，预计会增加的刺激 CIi 激活 NKCC。 初步研究强烈暗示 细胞内信号通路作为 CI 依赖性的焦点 NKCC 的激活。 下面将对此进行详细研究 具体目标：1) 检验 CIi 调节 NKCC 的假设 由 alpha1-AR 激动剂或高渗应激激活。 变化 将测定由两种刺激诱导的[CI]i。 CIi 水平将 被操纵以研究对 NKCC 激活的后续影响以及 NKCC 活动。将研究 NKCC 失活的动力学。 2) 检验 CIi 调节蛋白激酶活性的假设 和调节 NKCC 活性所需的磷酸酶。 改变 蛋白激酶和磷酸酶的活性以及 CI 依赖性 将使用特定底物研究酶活性， 包括来自人类和肾脏的 NKCC。所需的 stPP 同种型 NKCC 失活将被识别。 3）检验假设 CIi 调节 NKCC 和 PKC 之间的蛋白质-蛋白质相互作用和/或 蛋白质磷酸酶。 蛋白质-蛋白质相互作用及其依赖性 [CI] 和活化酶将使用免疫纯化进行研究 NKCC、PKC 和蛋白磷酸酶。 体内定位的转变 受刺激的酶将通过共聚焦显微镜测定。 一个 该项目的长期成果是药理学的发展 操纵蛋白质-蛋白质相互作用的工具，促进 PKC 和 NKCC 的关联调节 NKCC 活性 病理生理状态。