REGULATION OF NA/CL/K COTRANSPORT IN TRACHEAL EPITHELIUM

基底外侧Na-K-Cl共转运（NKCC）在衬里上皮细胞中的作用 肺对于最佳的粘膜纤毛清除至关重要，因为它提供 CI用于液体分泌。 破坏CI和体液的疾病状态 运动，如哮喘，慢性支气管炎和囊性纤维化（CF）， 以及暴露于外来刺激物会导致粘液过多， 异常的粘液清除。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 活性，从而阻止Cl分泌，并最终未能纠正 过多的粘液积聚。 因此，在本提案中，我们提出 问：NKCC是如何启动以支持跨上皮CI分泌的？ 该实验室的新证据指出蛋白激酶C（PKC）- 气管上皮细胞（TEC）中NKCC的依赖性激活 激素（α 1-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。 尽管在NKCC模型中描绘了细胞内CI水平（Cii）， 为了直接调节通过CI跨质膜的CI通量， TEC中的电化学梯度，预期增加的刺激 Cii启动NKCC。 初步研究强烈暗示 细胞内信号通路作为CI依赖性 激活NKCC。 下文将对此进行详细研究 具体目的：1）验证Cii调节NKCC的假说 通过alpha 1-AR激动剂或高渗应激激活。 变化 [CI]i将确定由两个刺激引起的。 Cii水平将 被操纵以研究对NKCC激活的后续影响， NKCC活动。将研究NKCC失活的动力学。 2)为了检验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 将使用特定底物研究酶活性， 包括来自人和肾的NKCC。所需的stPP同种型 将确定NKCC的停用。3)为了验证这个假设， Cii调节NKCC和PKC之间的蛋白质-蛋白质相互作用和/或 蛋白磷酸酶 蛋白质相互作用及其依赖性 [CI]和活化的酶将使用免疫纯化的 NKCC、PKC和蛋白磷酸酶。 体内定位的变化 具有刺激的酶将通过共聚焦显微镜测定。 一 该项目的长期成果是开发药理学 操纵蛋白质相互作用的工具， PKC和NKCC的联合调节NKCC活性， 病理生理状态。

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.