Ethanol-mediated cilia motility dysfunction

乙醇介导的纤毛运动功能障碍

• 批准号: 8672565
• 负责人: Joseph H Sisson
• 金额: $ 41.77万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8672565
• 关键词: Address; Adult Respiratory Distress Syndrome; Affect; Air; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcoholic beverage heavy drinker; Alcohols; Antioxidants; Arginine; Breathing; Bronchitis; Cells; Chlamydomonas; Choking; Cilia; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Dynein ATPase; Electron Spin Resonance Spectroscopy; Enzymes; Ethanol; Event; Fingers; Food; Functional disorder; Generations; Genetic; Genetic Models; Heat-Shock Proteins 90; Host Defense; Human; Impairment; Individual; Infection; Infectious Agent; Intoxication; Knowledge; Laboratories; Lead; Legal; Life; Link; Lung; Measurement; Mediating; Modeling; Molecular Chaperones; Motion; Motor; Mucociliary Clearance; Mucous body substance; Mus; Nitric Oxide; Nitric Oxide Synthase; Organelles; Organism; Orthologous Gene; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Pneumonia; Prevention strategy; Production; Protein Dephosphorylation; Protein phosphatase; Proteins; Publishing; Reactive Oxygen Species; Regulation; Research; Risk; Role; Signal Transduction; Slice; Stress; Supplementation; System; Testing; Tube; alcohol effect; alcohol exposure; alcohol use disorder; arm; cell motility; cilium motility; cofactor; desensitization; design; feeding; gene discovery; human NOS3 protein; injured airway; innovation; insight; kinetosome; lung basal segment; motor control; novel; oxidant stress; particle; prevent; protein activation; research study; tetrahydrobiopterin; treatment strategy

DESCRIPTION (provided by applicant): Alcohol abuse has long been known to impair lung host defenses increasing the risk in heavy drinkers for bronchitis, pneumonia and acute respiratory distress syndrome. Our laboratory has focused on alcohol's impact on mucociliary clearance in the conducting airways of the lung, which provides the first line of defense of the lung against inhaled infectious agents, particles and debris. We have established two key observations that drive this proposal: 1) brief modest alcohol intake stimulates airway ciliary motility; and 2) sustained heavy alcohol intake impairs mucociliary clearance increasing the risk for airway injury and pneumonia. We have termed this impairment "Alcohol-Induced Ciliary Dysfunction" (AICD). Our mechanistic studies have demonstrated that both the brief alcohol stimulation and sustained AICD involve ciliated cell mechanisms dependent on the regulation of nitric oxide (NO), cilia-associated cyclases and cyclic nucleotide-dependent kinases (PKG & PKA). Recently, we published that these alcohol-triggered ciliary regulation pathways are distinctly localized to the basal body of each airway cilium in what we have called the alcohol-responsive "ciliary metabolon". This pathway is exquisitely sensitive to very low concentrations of alcohol (1-10 mM, which is less than half of the legal intoxication limit) and is fully functionl in isolated airway cilia organelles. This leads us to hypothesize that: Alcohol causes reversible airway ciliary dysfunction due to modified nitric oxide signaling and altered regulation of key cila proteins, including the dynein motors and the mechanisms that regulate dyneins. In this proposal, we outline experiments designed to answer four new questions: 1) How does brief alcohol exposure stimulate NO production in airway cilia? We explore this question in Aim #1 by examining the role alcohol plays in activating nitric oxide synthase (eNOS) by enhancing the chaperone function of eNOS by heat shock protein 90 (HSP90). 2) Why does sustained alcohol exposure deplete ciliary NO levels? We address this question in Aim #2 by defining how eNOS becomes uncoupled by sustained alcohol exposure due to L- arginine depletion and the generation of reactive oxygen species (ROS). We think increased ciliary ROS leads to depletion of the eNOS cofactor tetrahydrobiopterin (BH4) resulting in cilia desensitization. 3) How do cilia regulatory enzymes interact to modify motility? In Aim #3 we will determine how sustained alcohol exposure activates protein phosphatase 1 (PP1), which leads to dephosphorylation of HSP90 and other key cilia activation proteins such as PKA. 4) What downstream cilia motor molecules are modified by alcohol exposure? Using the model genetic system of the alcohol-sensitive motile organism, Chlamydomonas, Aim #4 will focus on how sustained alcohol modifies ciliary outer dynein arms, which are the motor proteins that make cilia beat. The studies we propose will greatly extend our knowledge of how to prevent and treat AICD and will expand our insight into how alcohol alters cilia molecules critical for airway cilia function. 1

描述(由申请人提供)：长期以来，酒精滥用一直被认为会损害肺部宿主防御系统，增加酗酒者患支气管炎、肺炎和急性呼吸窘迫综合征的风险。我们的实验室专注于酒精对肺传导气道粘液纤毛清除的影响，这为肺提供了抵御吸入感染物、颗粒和碎片的第一道防线。我们已经建立了两个关键的观察结果来支持这一建议：1)短暂的适度酒精摄入刺激了呼吸道纤毛运动；2)持续大量酒精摄入损害了粘液纤毛清除，增加了呼吸道损伤和肺炎的风险。我们将这种损害称为“酒精诱导的睫毛功能障碍”(AICD)。我们的机制研究表明，短暂的酒精刺激和持续的AICD都涉及纤毛细胞机制，依赖于一氧化氮(NO)、纤毛相关环化酶和环核苷酸依赖的蛋白激酶(PKG&PKA)的调节。最近，我们发表了这些由酒精触发的纤毛调节通路，明显定位于每个呼吸道纤毛的基底体，我们称之为酒精反应的纤毛代谢。这条通路对极低浓度的酒精(1-10 mM，不到法定中毒限度的一半)非常敏感，并且在分离的呼吸道纤毛细胞器中完全起作用。这导致了我们的假设：酒精导致可逆性的气道纤毛功能障碍，这是由于一氧化氮信号的改变和关键的cila蛋白的调节的改变，包括动力蛋白马达和调节动力蛋白的机制。在这项提案中，我们概述了旨在回答四个新问题的实验：1)短暂的酒精暴露如何刺激呼吸道纤毛中NO的产生？在目标1中，我们通过研究酒精在通过热休克蛋白90(HSP90)增强eNOS的伴侣功能来激活eNOS中所起的作用来探索这个问题。2)为什么持续的酒精暴露会消耗睫状体NO水平？我们在目标2中解决了这个问题，定义了由于L精氨酸耗尽导致的持续酒精暴露和活性氧物种(ROS)的产生是如何使eNOS解偶联的。我们认为纤毛ROS的增加导致eNOS辅因子四氢生物蝶呤(BH4)的耗竭，从而导致纤毛脱敏。3)纤毛调节酶如何相互作用来调节运动性？在目标3中，我们将确定持续酒精暴露如何激活蛋白磷酸酶1(PP1)，从而导致HSP90和其他关键纤毛激活蛋白(如PKA)的去磷酸化。4)酒精暴露会改变哪些下游纤毛马达分子？利用对酒精敏感的运动有机体衣藻的模型遗传系统，目标#4将专注于持续酒精如何改变纤毛动力蛋白外臂，这是使纤毛跳动的马达蛋白。我们提出的研究将极大地扩展我们对如何预防和治疗AICD的知识，并将扩大我们对酒精如何改变对呼吸道纤毛功能至关重要的纤毛分子的洞察。1