Ethanol-mediated cilia motility dysfunction

描述(由申请人提供)：众所周知，大量饮酒会损害肺部保护自己免受肺炎和支气管炎等感染的能力。肺部对这种感染的第一道防线是捕获吸入的颗粒，并使用粘液纤毛器将它们赶出肺外。在一个健康的人身上，这个装置产生粘液，粘液捕获吸入的颗粒物，然后被纤毛推出肺外，纤毛是肺部衬里的指状突起。纤毛受损会导致肺炎和肺功能丧失。我们已经证实，酒精通过改变称为激酶的关键蛋白质，对这个脆弱的粘液纤毛器官造成深刻的伤害。其中两个激酶，PKA和PKG，是人们所熟知的调节细胞功能的分子，包括肺纤毛。我们在酒精喂养的小鼠身上的实验证明，酒精严重损害了这些激酶的功能，导致粘液纤毛器的正常“战斗或逃跑”反应丧失，需要产生一氧化氮。虽然我们已经确定酒精会导致这个问题，但我们不知道如果酒精被去除，这个问题会持续多久。我们也不知道酒精是如何改变纤毛中这些重要的依赖于激酶的功能的。这导致了我们的假设：慢性酒精暴露通过改变呼吸道上皮纤毛中的特定调节蛋白而导致时间依赖性和可逆性的粘液纤毛功能损害。在这项建议中，我们提出了旨在回答两个问题的实验：1.酒精引起的粘液纤毛功能损害是可预防的还是可逆的？酒精对纤毛蛋白有哪些影响？我们建议通过四个特定目标的实验来回答这些问题：1.表征慢性酒精损伤粘膜纤毛功能的时间进程和可逆性；2.明确氧化剂应激在酒精介导的一氧化氮依赖的纤毛运动调节中所起的信号通路和作用；3.确定存在于酒精暴露改变的纤毛上的激酶磷酸化靶点；以及4.使用分离的纤毛模型鉴定酒精介导的纤毛损伤的分子决定因素。虽然我们已经了解了很多关于酒精损伤对粘液纤毛功能的影响和机制，但我们提出的研究将扩展我们的知识，使我们了解如何预防或治疗这种损伤，以及酒精究竟如何改变对纤毛功能和肺健康至关重要的分子。

DESCRIPTION (provided by applicant): Heavy alcohol intake is known to damage the ability of the lung to protect itself from infections such as pneumonia and bronchitis. The lung's first line of defense against such infection is to trap inhaled particles and propel them out of the lung using the mucociliary apparatus. In a healthy individual, this apparatus produces mucus, which traps inhaled particles, and is then propelled out of the lung by cilia, the fingerlike projections of the lung lining. Damage to cilia results in pneumonia and loss of lung function. We have established that alcohol profoundly injures this delicate mucociliary apparatus by altering critical proteins called kinases. Two of these kinases, PKA and PKG, are well-described molecules that regulate cell functions including lung cilia. Our experiments in alcohol-fed mice have demonstrated that alcohol profoundly impairs the function of these kinases, resulting in a loss of the normal "fight or flight" responsiveness of the mucociliary apparatus in a manner that requires the production of nitric oxide. While we have established that alcohol causes this problem, we do not know how long it persists if alcohol is removed. We also do not know exactly how alcohol alters these important kinase-dependent functions in cilia. This leads us to hypothesize that: Chronic alcohol exposure causes time-dependent and reversible impairment of mucociliary function by altering specific regulatory proteins in airway epithelial cilia. In this proposal, we propose experiments designed to answer two questions: 1. Is alcohol-driven impairment of mucociliary function preventable or reversible? and 2. Which cilia proteins are affected by alcohol? We propose to answer these questions through experiments proposed in four specific aims: 1. Characterize the time course and reversibility of chronic alcohol impairment of mucociliary function; 2. Define the signaling pathways and role that oxidant stress plays in alcohol-mediated nitric oxide-dependent regulation of ciliary motility, 3. Determine the kinase phosphorylation targets present on cilia that are altered by alcohol exposure; and 4. Identify the molecular determinants of alcohol-mediated cilia injury using isolated cilia models. Although we have learned much about the impact and mechanisms of alcohol injury to mucociliary function, the studies we propose will extend our knowledge into how this may be prevented or treated and exactly how alcohol alters molecules critical for cilia function and lung health.