Phosphorylation of ciliary target proteins

描述（由申请人提供）：在气道疾病加重时，通过哮喘、COPD和支气管扩张患者呼出冷凝水中的低pH值来测量气道酸化。临床上，肾上腺素能激动剂通过刺激跨膜腺苷酸环化酶（tmac）来增加cAMP，目的是在疾病加重期间增强纤毛粘膜运输和支气管扩张。对于支气管扩张，吸入的药物需要穿过气道上皮。目前使用的大多数支气管扩张剂在生理或酸性ph下都带正电荷。因此，这些药物不能自由地穿过气道上皮到达它们的靶点。我们已经证明，ph依赖性有机阳离子/肉碱转运体OCTN1和OCTN2在气道上皮中表达，并且它们以ph依赖性的方式将阳离子（包括沙丁胺醇）转运到气道上皮细胞中。在酸性气道环境中，带正电荷的支气管扩张剂的摄取减少，表明其支气管扩张剂的作用将降低。我们还表明，细胞内酸化直接抑制纤毛跳动。作为应对措施，细胞内HCO3-刺激纤毛的增加通过一种新的细胞质（或可溶性）腺苷酸环化酶（sAC）产生cAMP的途径。这些新发现提示了pHi和HCO3-在调节人体气道内稳态中的重要作用。我们假设pH直接和HCO3-（可能通过sAC）在调节气道上皮细胞功能（包括支气管扩张剂等带电阳离子的摄取）中发挥关键作用。这一假设将以三个具体目标进行全面测试，包括评估吸入沙丁胺醇扩张正常人类志愿者气道血管的能力的生物测定。目的1将验证cAMP的产生是由tmac和sAC共同完成的假设，因此受到HCO3-、pH、Ca2+变化的影响。目的2将验证一个假设，即在酸化过程中，气道上皮细胞对ph依赖性阳离子的摄取将使气道表面有更高浓度的肾上腺素能激动剂。目的3将验证气道酸化抑制经上皮阳离子摄取的假设，包括沙丁胺醇进入人类气道粘膜下组织。因此，该应用解决了一种新的和临床相关的粘膜纤毛清除和上皮表面以及上皮下组织药物可用性的调节机制，使用了翻译和最先进的方法。公共卫生相关性。气道疾病及其恶化在美国和世界范围内构成了重要的健康问题。疾病加重时气道酸化，预计这种pH变化会减少支气管扩张剂通过气道上皮到达靶点（即平滑肌）的摄取，并对纤毛粘膜清除产生不利影响。本应用研究了支气管扩张剂进入气道粘膜的机制和粘膜纤毛清除调节的新方面。改善支气管扩张剂在疾病恶化期间达到目标的方法，防止纤毛粘膜功能障碍或恢复清除率至正常水平是最终的临床目标。因此，这一建议可能确定新的治疗靶点，有助于减轻气道疾病加重对个人和社会的负担。

DESCRIPTION (provided by applicant): In airway disease exacerbations, the airway acidifies as measured by low pH in exhaled breath condensate in asthma, COPD and bronchiectasis. Clinically, ¿-adrenergic agonists are used to increase cAMP via stimulation of transmembrane adenylyl cyclases (tmACs), with the intent to enhance mucociliary transport and bronchodilation during disease exacerbations. For bronchodilation, inhaled drugs need to cross the airway epithelium. The majority of the currently used bronchodilators have a positive charge at physiological or acidic pH. Thus, these drugs cannot freely diffuse across the airway epithelium to reach their targets. We have shown that the pH-dependent organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in airway epithelia and that they transport cations, including albuterol, into airway epithelial cells in a pH-dependent manner. Uptake of positively charged bronchodilators is decreased in an acidic airway environment, suggesting that their bronchodilatory effects will be reduced. We have also shown that intracellular acidification directly inhibits ciliary beating. As a counter measure, increases in intracellular HCO3- stimulate cilia via a pathway involving cAMP production by a novel, cytosolic (or soluble) adenylyl cyclase (sAC). These new findings suggest a significant role for pHi and HCO3- in the regulation of human airway homeostasis. We hypothesize that pH directly and HCO3-, likely through sAC, play critical roles in regulating airway epithelial cell functions, including uptake of charged cations such as bronchodilators. This hypothesis will be comprehensively tested with three specific aims, including a bioassay that assesses the ability of inhaled albuterol to dilate airway blood vessels in normal human volunteers. Aim 1 will test the hypothesis that cAMP production is accomplished by both tmACs and sAC and thus influenced by changes in HCO3-, pH, Ca2+. Aim 2 will test the hypothesis that pH-dependent cation uptake into airway epithelial cells will make ¿-adrenergic agonists available in higher concentrations on the airway surface during acidification. Aim 3 will test the hypothesis that airway acidification inhibits transepithelial cation uptake, including albuterol, into airway submucosal tissues in human beings. This application thus addresses a novel and clinically relevant regulation mechanism of mucociliary clearance and of drug availability on the epithelial surface as well as in the subepithelial tissue using a translational and state-of-the-art approach. PUBLIC HEALTH RELEVANCE. Airway diseases and specifically their exacerbations pose important health problems in the USA and worldwide. The airway acidifies during disease exacerbations and this change in pH is predicted to decrease uptake of bronchodilators across the airway epithelium to their target (namely smooth muscle) and adversely affect aspects of mucociliary clearance. This application examines mechanisms by which bronchodilators are taken up into the airway mucosa and novel aspects of mucociliary clearance regulation. Improving ways that bronchodilators reach their target during disease exacerbations and preventing mucociliary dysfunction or restoring clearance to normal levels is the ultimate clinical goal. This proposal thus may identify new therapeutic targets that could help to decrease the burden from airway disease exacerbations on individuals and on society.