Ethanol-mediated cilia motility dysfunction

描述（由申请人提供）：长期以来，人们一直认为酒精滥用会损害肺宿主的防御能力，增加重度饮酒者患支气管炎、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导气管粘膜纤毛清除的影响，这是肺抵御吸入感染性病原体、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推动这一建议：1)短暂适度的酒精摄入刺激气道纤毛运动；2)持续大量饮酒损害纤毛粘膜清除率，增加气道损伤和肺炎的风险。我们将这种损伤称为“酒精性纤毛功能障碍”（AICD）。我们的机制研究表明，短暂的酒精刺激和持续的AICD都涉及依赖于一氧化氮（NO）、纤毛相关环化酶和环核苷酸依赖性激酶（PKG和PKA）调节的纤毛细胞机制。最近，我们发表了这些酒精触发的纤毛调节通路明显定位于每个气道纤毛的基底，我们称之为酒精反应性的“纤毛代谢”。这一途径对极低浓度的酒精（1-10毫米，不到法定中毒限度的一半）非常敏感，并在孤立的气道纤毛细胞器中完全起作用。这导致我们假设：由于一氧化氮信号的改变和关键纤毛蛋白（包括动力蛋白马达和调节动力蛋白的机制）的调节改变，酒精导致可逆性气道纤毛功能障碍。在本提案中，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气道纤毛中NO的产生？我们在Aim #1中探讨了这个问题，通过研究酒精在激活一氧化氮合酶（eNOS）中的作用，通过热休克蛋白90 （HSP90）增强eNOS的伴侣功能。2)为什么持续酒精暴露会降低纤毛NO水平？我们在Aim #2中通过定义由于L-精氨酸耗竭和活性氧（ROS）的产生而导致的持续酒精暴露如何使eNOS解耦来解决这个问题。我们认为纤毛活性氧增加导致eNOS辅助因子四氢生物蝶呤（BH4）的耗竭，导致纤毛脱敏。3)纤毛调节酶如何相互作用改变纤毛运动？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)哪些下游纤毛运动分子会因酒精暴露而改变？利用对酒精敏感的运动生物——衣藻的模型遗传系统，Aim #4将关注持续的酒精如何改变纤毛外动力蛋白臂，这是使纤毛跳动的运动蛋白。我们提出的研究将极大地扩展我们对如何预防和治疗AICD的认识，并将扩展我们对酒精如何改变对气道纤毛功能至关重要的纤毛分子的见解。1

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 PUBLIC HEALTH RELEVANCE: Cilia, the wave-producing finger-like projections of the bronchial tubes that clear mucus and inhaled particles from the lungs, are normally activated by stress events to clear mucus faster. Catching a cold, choking on food or breathing in dirty air cause cilia to produce nitric oxide, which makes them beat faster to clear out the lungs. After heavy alcohol drinking, lung cilia become unresponsive to stress, because they can no longer make nitric oxide, which leads to mucus congestion and infections such as bronchitis & pneumonia. Our research is focused on how alcohol shuts off nitric oxide production by lung cilia with an emphasis on discovering ways to reverse and/or prevent alcohol-related cilia damage to avoid lung infections. 1