Beta adrenergic receptor resensitization in asthma

摘要 我们的建议是研究气道β2-肾上腺素能受体（β 2-AR）功能障碍的机制 哮喘的高反应性解释了频繁的哮喘控制观察到的反常的丧失， 使用β-激动剂。哮喘病理与一氧化氮（NO）升高有关， 具有较高呼出NO分数（FENO）的患者具有更大的高反应性和气流阻塞， 对β-激动剂激发和再激发的反常损失（Bonini等，AJRCCM，2013）。β-激动剂激活 βAR通过cAMP引起气道平滑肌松弛，同时导致βAR磷酸化， 脱敏和内化。βAR脱敏作用已被广泛了解，但其机制尚不清楚 使β 2 AR再敏感，恢复细胞表面活性β 2 AR。我们最近发现了 调节β2AR再活化途径，其中，通过蛋白质的去磷酸化/再活化β2AR， 磷酸肌醇3-激酶γ（PI 3 K γ）可阻断磷酸酶2A（PP 2A），从而抑制β2AR 限制细胞中βAR对β-激动剂[PI 3 K γ-PP 2A轴]反应的再敏感化（Vasudeven et al.，摩尔细胞， 2011年）。目前尚不清楚是否脱敏和/或再敏化机制的基础损失β- 激动剂反应与哮喘中NO升高的关系。与NO介导的β2AR通过S- 我们在原代小鼠气道平滑肌细胞（ASMCs）中的初步数据显示， NO不是通过脱敏途径引起β 2 AR功能障碍，而是通过再敏化途径引起的。NO激活 PI 3 K γ通过S-亚硝基化PI 3 K γ阻断PP 2A活性从而抑制β2AR再敏感。此外，本发明还 PP 2A活性由其甲基化决定，我们发现亚硝基化的PI 3 K γ抑制PP 2A活性。 部分通过去甲基化。一致地，来自哮喘患者的原代ASMCs具有明显更多的 细胞内磷酸化β 2AR，对β激动剂的cAMP反应较低，PI 3 K γ活性较高，PP 2A较低 与非哮喘患者相比，在质膜和核内体的活性。基于这些发现，我们 假设NO通过亚硝基化/激活PI 3 K γ抑制β 2 AR再敏化，PI 3 K γ抑制PP 2A， 限制了哮喘气道中高NO的β 2 AR再激活，结果是功能细胞的累积损失 表面β 2 AR，使细胞对支气管收缩剂更敏感，即气道高反应性 （AHR）。PI 3 K γ敲除（PI 3 K γ-/-）小鼠AHR最低，且AHR恢复，这有力地支持了我们的假设。 抑制PP 2A后，PI 3 K γ-/-的变化。为了解决我们的假设，我们建议全面确定 NO对β 2 AR再敏感（Aim 1）的影响及其在气道高反应性（Aim 2），并确定NO介导的非经典激活PI 3 K γ的机制，抑制PP 2A和 β 2 AR在哮喘中的再增敏作用（目的3）。

Abstract Our proposal investigates mechanisms of β2-adrenergic receptor (β2AR) dysfunction that underlies airway hyper-responsiveness in asthma accounting for paradoxical loss of asthma control observed with frequent use of β-agonist. Asthma pathology is associated with elevation of nitric oxide (NO) and indeed, patients with higher fraction of exhaled NO (FENO) have greater hyper-reactivity and airflow obstruction with paradoxical loss to β-agonist challenge and re-challenge (Bonini et al., AJRCCM, 2013). β-agonist activation of βAR causes airway smooth muscle relaxation via cAMP in parallel leading to βAR phosphorylation, desensitization and internalization. βAR desensitization is well understood but mechanistically, little is known about β2AR resensitization to recover active β2ARs on cell surface. We recently uncovered mechanisms regulating β2AR reactivation pathway wherein, dephosphorylation/reactivation of the β2AR by protein phosphatase 2A (PP2A) is blocked by phosphoinositide 3-kinase γ (PI3Kγ), which serves to inhibit β2AR resensitization limiting βAR response in the cell to β-agonist [PI3Kγ-PP2A axis] (Vasudeven et al., Mol Cell, 2011). It is not known whether desensitization and/or resensitization mechanisms underlie the loss in β- agonist response with elevated NO in asthma. Contrary to NO mediating β2AR desensitization via S- nitrosylation of proteins, our preliminary data in primary murine airway smooth muscle cells (ASMCs) reveal that NO causes β2AR dysfunction not via desensitization rather, via resensitization pathways. NO activates PI3Kγ by S-nitrosylation of PI3Kγ blocking PP2A activity thereby, inhibiting β2AR resensitization. Further, PP2A activity is determined by its methylation and we find that nitrosylated PI3Kγ inhibits PP2A activity in part by de-methylation. Consistently, primary ASMCs from asthmatic patients have markedly more intracellular phosphorylated-β2ARs, less cAMP response to β-agonist, greater PI3Kγ activity and lesser PP2A activity compared to non-asthmatics at plasma membrane and endosome. Based on these findings, we hypothesize that NO inhibits β2AR resensitization by nitrosylation/activation of PI3Kγ which inhibits PP2A and limits β2AR reactivation in asthmatic airways with high NO. The result is cumulative loss of functional cell surface β2ARs, pre-disposing cells to greater sensitivity for broncho-constrictors, i.e. airway hyper-reactivity (AHR). In strong support of our hypotheses, PI3Kγ null (PI3Kγ-/-) mice have minimal AHR, and AHR returns in the PI3Kγ-/- upon PP2A inhibition. To address our hypothesis, we propose to comprehensively determine the effects of elevated NO on β2AR resensitization (Aim1) and its role in airway hyper-responsiveness (Aim 2), and to identify mechanisms of NO-mediated non-canonical activation of PI3Kγ that inhibits PP2A and β2AR resensitization in asthma (Aim 3).