Phosphorylation of ciliary target proteins

描述(由申请人提供)：在呼吸道疾病恶化时，哮喘、慢性阻塞性肺疾病和支气管扩张患者呼出的呼吸冷凝物中的低pH值可测量到呼吸道酸化。在临床上，肾上腺素能激动剂通过刺激跨膜腺苷酸环化酶(Tmacs)来增加cAMP，目的是在疾病加重时增强粘液纤毛运输和支气管扩张。对于支气管扩张，吸入的药物需要穿过呼吸道上皮。目前使用的大多数支气管扩张剂在生理或酸性pH下都有正电荷。因此，这些药物不能自由地在呼吸道上皮细胞间扩散以达到它们的目标。我们已经证明，pH依赖的有机阳离子/肉碱转运体OCTN1和OCTN2在呼吸道上皮细胞中表达，它们以pH依赖的方式将包括沙丁胺醇在内的阳离子转运到呼吸道上皮细胞中。在酸性的呼吸道环境中，正电荷的支气管扩张剂的摄取量减少，这表明它们的支气管扩张剂的作用将会减弱。我们还表明，细胞内的酸化直接抑制纤毛跳动。作为一种应对措施，细胞内HCO3刺激纤毛的增加是通过一种新的胞质(或可溶性)腺苷环化酶(SAC)产生cAMP的途径来实现的。这些新的发现表明，Phi和HCO3-在调节人类呼吸道内稳态方面发挥了重要作用。我们假设，pH直接和HCO3-可能通过SAC在调节呼吸道上皮细胞功能方面发挥关键作用，包括对带电阳离子的摄取，如支气管扩张剂。这一假设将通过三个具体目标进行全面检验，其中包括评估吸入沙丁胺醇对正常人类志愿者呼吸道血管扩张能力的生物测定。目的1验证cAMP的产生是由tmacs和sac共同完成的假说，因此受到HCO3-、pH、Ca2+变化的影响。目的2将验证一种假设，即pH依赖的阳离子被呼吸道上皮细胞摄取将在酸化过程中使气道表面获得更高浓度的肾上腺素能激动剂。目的3验证呼吸道酸化抑制包括沙丁胺醇在内的跨上皮阳离子摄取进入人体呼吸道粘膜下组织的假设。因此，这项应用解决了一种新颖的临床相关的粘液纤毛清除调节机制，以及使用翻译和最先进的方法在上皮表面和上皮下组织中药物可用性的调节机制。与公共卫生相关。呼吸道疾病，特别是其恶化，在美国和世界范围内构成了重要的健康问题。在疾病恶化期间，呼吸道会酸化，这种pH的变化预计会减少支气管扩张剂通过呼吸道上皮到其靶点(即平滑肌)的摄取，并对粘液纤毛清除的某些方面产生不利影响。这项应用检查了支气管扩张剂进入呼吸道粘膜的机制，以及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其目标的方式，并防止粘液纤毛功能障碍或将清除量恢复到正常水平是最终的临床目标。因此，这项提议可能确定新的治疗目标，有助于减轻呼吸道疾病恶化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.