Beta adrenergic receptor resensitization in asthma

哮喘中的β肾上腺素能受体再敏化

• 批准号: 9205534
• 负责人: Sathyamangla V Prasad
• 金额: $ 39.63万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9205534
• 关键词: Accounting; Acute; Address; Adenylate Cyclase; Adrenergic beta-Agonists; Agonist; Air; Asthma; Binding; Biochemical; Bronchoconstrictor Agents; Bronchodilation; Cell membrane; Cell surface; Cells; Cessation of life; Chronic; Coculture Techniques; Coupling; Cyclic AMP; Cyclic GMP-Dependent Protein Kinases; Data; Development; Endosomes; Epithelial; Epithelial Cells; Exhalation; Functional disorder; GRK6 gene; Goals; Human; Inflammation; Knock-out; Knockout Mice; Link; Liquid substance; Lung; Maps; Mediating; Methylation; Modeling; Mus; Muscle Cells; Muscle relaxation phase; Nitric Oxide; Nitric Oxide Donors; Obstruction; Ovalbumin; Pathology; Pathway interactions; Patients; Phosphatidylinositols; Phosphorylation; Protein Dephosphorylation; Protein Inhibition; Protein Methylation; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Recycling; Regulation; Role; Signal Transduction; Site; Testing; Transferase; airway hyperresponsiveness; asthmatic; asthmatic airway; asthmatic patient; base; beta-2 Adrenergic Receptors; beta-adrenergic receptor; beta-arrestin; cGMP-dependent protein kinase Ibeta; desensitization; functional loss; in vivo; insight; novel; novel therapeutics; respiratory smooth muscle; response

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).

抽象的 我们的建议调查了β2-肾上腺素能受体（β2AR）功能障碍的机制，该功能障碍是气道的基础 在哮喘中的高反应性，该哮喘控制的悖论丧失频率是频率的 使用β-激动剂。哮喘病理与一氧化氮（NO）的升高有关，实际上是患者 呼出的NO（FENO）的较高分数具有更大的反应性和气流反对性 β-激动人心的挑战和重新挑战（Bonini等人，AJRCCM，2013年）的矛盾损失。 β激活的激活 βAR通过cAMP并行导致气道平滑肌松弛，导致βAR磷酸化， 脱敏和内在化。 βAR脱敏是有充分理解的，但从机械上讲，知之甚少 大约β2AR汇总化以在细胞表面恢复活性β2AR。我们最近发现了机制 调节β2AR重新激活途径，其中蛋白质对β2AR的去磷酸化/重新激活 磷酸酶2a（PP2A）被磷酸肌醇3-激酶γ（PI3Kγ）阻塞，这有抑制β2AR 敏化限制细胞中对β-激动剂的βAR反应[PI3Kγ-PP2A轴]（Vasudeven等，Mol Cell，，Mol Cell， 2011）。尚不清楚脱敏和/或脱敏机制是否是β-损失的基础 激动剂的反应与哮喘中的NO升高。与没有通过s-介导的β2AR脱敏相反 蛋白质的硝基化，我们在原代鼠气道平滑肌细胞（ASMC）中的初步数据显示 没有通过脱敏而导致β2AR功能障碍，而是通过脱敏途径。没有激活 PI3Kγ通过pI3Kγ阻断PP2A活性的S-亚硝基化，从而抑制β2AR汇总化。更远， PP2A活性取决于其甲基化，我们发现亚硝基化的PI3Kγ抑制了PP2A活性 部分通过脱甲基化。一致地，来自哮喘患者的初级ASMS明显更明显 细胞内磷酸化-β2AR，对β-激动剂的cAMP反应较小，PI3Kγ活性较大，PP2A较少 与质膜和内体上的非asthmatics相比，活性。基于这些发现，我们 假设NO通过硝基化/激活PI3Kγ抑制β2AR均质化，从而抑制PP2A和 限制了高NO的哮喘气道中的β2AR重新激活。结果是功能单元的累积丧失 表面β2AR，对支气管收缩剂的敏感性更高的细胞，即气道高反应性 （啊）。为了大力支持我们的假设，PI3KγNULL（PI3Kγ-/ - ）小鼠的AHR最小，AHR返回 在pp2a抑制后的pi3kγ - / - 中。为了解决我们的假设，我们建议全面确定 NO升高对β2AR脱敏的影响（AIM1）及其在气道高反应性中的作用（AIM） 2），并确定抑制PP2A和PI3Kγ的无介导的非传统激活的机制 哮喘中的β2AR脱敏（AIM 3）。