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: 2702490
• 负责人: CAROLE M LIEDTKE
• 金额: $ 25.53万
• 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/2702490
• 关键词: alpha adrenergic receptor; body fluid osmolarity; chloride ion; confocal scanning microscopy; enzyme activity; gel electrophoresis; hormone regulation /control mechanism; intermolecular interaction; intracellular transport; ion transport; phosphoprotein phosphatase; potassium ion; protein kinase C; protein localization; 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Na-K-CI共转运(NKCC)在兔眼衬里上皮细胞中的表达 肺对于最佳的粘液纤毛清除至关重要，因为它提供 用于液体分泌的CI。扰乱CI和液体的疾病状态 运动，如哮喘、慢性支气管炎和囊性纤维化(CF)， 暴露在外来刺激物中会导致粘液过量和 粘液毛细血管清除异常。以补虚为主的治法 CI分泌可能无法激活NKCC或可能损害NKCC 从而阻止CI的分泌并最终无法纠正 粘液堆积过多。因此，在这项提案中，我们提出了 问：NKCC是如何支持跨上皮CI分泌的？ 来自该实验室的新证据表明，蛋白激酶C(PKC)- NKCC在气管上皮细胞(TEC)中的依赖激活 激素(α1-肾上腺素能(AR))刺激和高渗应激。 然而，对NKCC的调控比对PKC的激活更为复杂。 尽管细胞内CI水平(CII)在NKCC模型中被描述 通过CI直接调制跨质膜的CI通量 电化学梯度，在TEC中，预计将增加的刺激 CII激活NKCC。初步研究强烈表明 细胞内信号通路是CI依赖的焦点 激活NKCC。这将在以下内容中详细研究 具体目的：1)检验CII调控NKCC的假说 α1-AR激动剂或高渗应激激活。中的更改 [CI]由这两个刺激诱导的I将被确定。CII水平将 被操纵以研究对NKCC激活的后续影响 NKCC活动。对NKCC的失活动力学进行了研究。 2)检验CII调节蛋白激酶活性的假说 以及调节NKCC活性所需的磷酸酶。更改后的 蛋白激酶和磷酸酶的活性及其CI依赖性 将使用特定的底物来研究酶的活性， 包括来自人和肾脏的NKCC。需要STPP同型(S) 将确定NKCC的停用。3)检验假设 CII调节NKCC与PKC和/或 蛋白磷酸酶。蛋白质-蛋白质相互作用及其依赖性 [CI]和激活的酶将用免疫提纯进行研究 NKCC、PKC和蛋白磷酸酶。在体定位的研究进展 有刺激作用的酶将通过共聚焦显微镜进行测定。一个 这一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联合作用调节NKCC活性 病理生理状态。