CELLULAR BASIS OF AIRWAY SECRETION CLEARANCE COUPLING

气管上皮细胞在肺宿主防御中的重要作用 通过以下过程清除呼吸道中的异物 粘液纤毛清除。尽管发挥了关键作用，但我们的理解是 负责调控的细胞和分子机制 粘液纤毛的清除还远未完成。广泛的、长期的 这个项目的假设是，有效的清除取决于 纤毛细胞和纤毛细胞的协同作用和协同反应 分泌粘液的细胞。我们把这称为‘分泌物清除’。 联结‘。副交感胆碱能传入呼吸道，通过 M受体在粘液纤毛调节中的重要作用 Clearance和这些受体都存在于纤毛细胞和 粘膜下腺。在这项建议中，我们试图了解 纤毛搏动频率的调节机制 (CBF)乙酰胆碱(ACh)。 我们假设ACh通过m3的M受体作用于 纤毛细胞对CBF有双重、相反的作用，而且 这些作用是通过细胞质内游离钙离子的变化来调节的。 ACh激活该G蛋白引起的[Ca~(2+)]i浓度 偶联受体。这些对立行为的最终结果是 对纤毛细胞进行特殊程度的精细控制 调节CBF反应。为了检验这一假设，我们将 使用高分辨率数字视频显微镜检查机械装置 ACh可升高和降低[Ca~(2+)]i，衡量血管紧张度 Ca~(2+)与CBF的偶联，并开始识别信号 调节这种耦合的分子。具体目标是： I)确定负责调节[Ca2+]i的机制 纤毛细胞对ACh的反应。 Ii)研究[Ca~(2+)]_i与脑血流量的偶联动力学。 来表征钙离子作用的分子部位。 对调控的正常机制有了更深入的了解 纤毛跳动有助于确定粘液纤毛缺陷的基础 清除哮喘、慢性支气管炎等常见病。 目前治疗粘液纤毛功能障碍的方法并不令人满意。这个 拟议中的实验将有助于识别可能 为这种呼吸道的新治疗策略奠定了基础 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.