CELLULAR BASIS OF AIRWAY SECRETION CLEARANCE COUPLING

气管上皮细胞在肺部宿主防御中起着重要作用 通过清除气道中的异物， 粘膜纤毛清除率尽管这一关键作用，我们的理解 负责调节的细胞和分子机制 粘膜纤毛清除的过程还远未完成。广泛、长期 该项目的假设是，有效清除取决于 纤毛细胞的协同作用和协调反应， 粘液分泌细胞。我们称之为“清除分泌物 耦合。副交感神经胆碱能输入气道，通过 毒蕈碱受体在调节粘膜纤毛中起重要作用 清除和这些受体都发现在纤毛细胞和 粘膜下腺体在这一建议中，我们试图理解 负责调节纤毛跳动频率的机制 (CBF)乙酰胆碱（acetylcholine，ACh） 我们假设ACh通过m3毒蕈碱受体作用于 纤毛细胞，对CBF具有双重相反的作用，而且， 这些作用是由细胞质游离Ca 2+的变化介导的。 ACh激活该G蛋白产生的[Ca2+]i浓度 偶联受体这些对立行为的最终结果是， 给予纤毛细胞一种特殊程度的精细控制， 调节CBF反应。为了验证这个假设，我们将 使用高分辨率数字视频显微镜检查机制， ACh可升高和降低[Ca2+]i， Ca2+和CBF之间的耦合，并开始识别信号传导 介导这种耦合的分子。具体目标是： I）确定负责调节[Ca2+]i的机制， 纤毛细胞对乙酰胆碱的反应。 II）表征[Ca 2 +]i与CBF之间的偶联动力学。 III）表征Ca 2+作用的分子位点。 增加对正常机制的理解， 纤毛跳动将有助于确定有缺陷的粘膜纤毛的基础 在哮喘和慢性支气管炎等常见疾病中清除。 目前治疗粘膜纤毛功能障碍是不令人满意的。的 拟议的实验将有助于确定可能 作为这种气道的新治疗策略的基础 疾病

Tracheal epithelial cells play a vital role in pulmonary host defense by clearing foreign materials from the airways through the process of mucociliary clearance. Despite this critical role, our understanding of the cellular and molecular mechanisms responsible for the regulation of mucociliary clearance is far from complete. The broad, long-term hypothesis of this project is that effective clearance depends on cooperative actions and coordinated responses of ciliated cells and mucus-secreting cells. We refer to this as 'secretion-clearance coupling'. Parasympathetic cholinergic input to the airway, acting via muscarinic receptors, plays an important role in regulating mucociliary clearance and these receptors are found on both ciliated cells and submucosal glands. In this proposal, we seek to understand the mechanisms responsible for the regulation of ciliary beating frequency (CBF) by acetylcholine (ACh). We hypothesize that ACh, acting through m3 muscarinic receptors on ciliated cells, has dual, opposing actions on CBF and, further, that these actions are mediated by changes in cytoplasmic free Ca2+ concentration [Ca2+]i resulting from ACh activation of this G-protein coupled receptor. The net result of these opposing actions serves to give the ciliated cell an extraordinary degree of fine control in regulating CBF responses. In order to test this hypothesis, we will examine, using high resolution digital video microscopy, mechanisms by which ACh can raise and lower [Ca2+]i, measure the tightness of coupling between Ca2+ and CBF, and begin to identify signaling molecules that mediate such coupling. The specific aims are: I) To define the mechanisms responsible for regulating [Ca2+]i in ciliated cells in response to ACh. II) To characterize the kinetics of coupling between [Ca2+]i and CBF. Ill) To characterize the molecular site of Ca2+ action. An increased understanding of the normal mechanisms that regulate ciliary beating will help to define the basis for defective mucociliary clearance in such common diseases as asthma and chronic bronchitis. Present day therapy for mucociliary dysfunction is unsatisfactory. The proposed experiments will help to identify molecular targets that might serve as the basis for novel treatment strategies for such airway diseases.