Regulation of Normal and Asthmatic Lung Function by G-Protein-Coupled Receptors

G 蛋白偶联受体对正常和哮喘肺功能的调节

• 批准号: 8946374
• 负责人: Kirk m Druey
• 金额: $ 43万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946374
• 关键词: Actins; Acute; Adenosine; Adrenergic beta-Agonists; Agonist; Albuterol; Allergens; Allergic inflammation; Animal Model; Asthma; Binding; Bradykinin; Bronchoalveolar Lavage Fluid; Bronchodilator Agents; Calcium; Cell Count; Cell Culture Techniques; Cells; Chronic; Collaborations; Collagen; Contracts; Cyclic AMP; Cytoplasmic Granules; Deposition; Development; Disease susceptibility; Drug Targeting; Endothelin-1; Extrinsic asthma; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Deletion; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Heterotrimeric GTP-Binding Proteins; Histamine; Homeostasis; Human; Hyperplasia; IgE; Interleukin-13; Leukotriene Antagonists; Leukotriene D4; Ligands; Lung; Lung Inflammation; Mediating; Michigan; Mitogens; Modeling; Mucins; Mus; Muscle Contraction; Muscle Fibers; Muscle function; Muscle relaxation phase; Myosin ATPase; Obstruction; Peptide Hydrolases; Phenotype; Physiological; Production; Protein Binding; RGS Domain; RGS Proteins; Regulation; Relative (related person); Relaxation; Respiratory physiology; Role; Signal Pathway; Slice; Smooth Muscle; Smooth Muscle Myocytes; Specimen; Steroids; Thrombin; Tissues; Universities; airway obstruction; allergic airway inflammation; cysteinyl-leukotriene; eosinophilic inflammation; inhibitor/antagonist; interest; mast cell; muscle form; overexpression; receptor coupling; repository; respiratory smooth muscle

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchiolar smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for AHR, suggesting that underlying abnormalities in structural cells such as airway smooth muscle (ASM) contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. A large family of Regulators of G protein signaling (RGS) proteins binds to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (particularly RGS4, RGS5) in bronchial smooth muscle. In FY14, we demonstrated that the mice genetically deficient in RGS5 developed spontaneous asthma due to abnormal GPCR-induced Ca2+ homeostasis in ASM. Precision-cut lung slices (PCLS) from nave Rgs5-/- mice contracted maximally at baseline, independent of allergen challenge. RGS5 deficiency had little effect on parameters of allergic inflammation including cell counts in bronchoalveolar lavage fluid (BALF), mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, IL-13 levels were much lower in BALF from Rgs5-/- mice relative to WT. These studies showed that deficiency of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung ASM mass and stiffness. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. We are currently examining the phenotype of with global and smooth muscle-specifi Rgs4 gene deletion, as well as mice that overexpress RGS4,in models of acute and chronic allergic airway inflammation. In collaboration with Dr. Neubig at the University of Michigan, we will examine the effect of an RGS4-specific inhibitor on the development of the asthma phenotype and ASM hyperplasia and contraction in animal models and cell culture. This is a first-generation RGS inhibitory compound.

哮喘是一种可逆性呼吸道阻塞的病理状态，包括肺部炎症和细支气管平滑肌的过度收缩。引起支气管平滑肌收缩的主要天然物质是G蛋白偶联受体(GPCRs)的配体，如变应原蛋白水解酶、凝血酶，以及那些包含在过敏原-IgE激活的肥大细胞颗粒中的物质(如组胺、半胱氨基白三烯(LTD4)、内皮素1、腺苷和缓激肽)。一般来说，这些激动剂诱导异源三聚体G蛋白G-αQ的激活，从而增加平滑肌细胞内钙的浓度，促进肌动蛋白-肌球蛋白的相互作用和肌肉纤维的缩短。相反，作用于G-α-S偶联受体的配体，如沙丁胺醇，可以增加细胞内环磷酸腺苷(CAMP)的水平，从而促进ASM的松弛。虽然嗜酸性炎症是过敏性哮喘的典型表现，但它不是AHR的先决条件，这表明潜在的结构细胞异常，如气道平滑肌(ASM)，参与了哮喘的素质。ASM中前收缩、GPCR信号的失调可能介导了收缩能力的增强。 大量的G蛋白信号转导调节蛋白通过保守的RGS结构域与G蛋白α亚单位Gi和Gq(而不是Gs)结合，并通过催化其固有的GTPase活性和阻断下游效应器的相互作用来使其失活。虽然它们通常被认为是GPCR信号通路的负调控因子，但RGS蛋白在肺中的生理功能大多尚不清楚。我们鉴定了几种RGS蛋白(特别是RGS4、RGS5)在支气管平滑肌中的表达。 在2014财年，我们证明了RGS5基因缺陷的小鼠发生了自发性哮喘，这是由于GPCR诱导ASM内钙稳态异常所致。来自NAVE RGS5-/-小鼠的精密切割肺切片(PCL)在基线时收缩最大，与过敏原攻击无关。RGS5缺乏对过敏性炎症指标影响不大，包括支气管肺泡灌洗液(BALF)中细胞计数、粘蛋白产生、ASM质量和上皮下胶原沉积。出乎意料的是，与WT相比，RGS5-/-小鼠BALF中的IL-13水平要低得多。这些研究表明，在没有过敏性炎症的情况下，RGS5的缺乏会导致小鼠自发的AHR。 在严重哮喘中，对支气管扩张剂和类固醇不敏感的气流阻塞通过未知的机制发展，其特征是肺ASM质量和僵硬增加。RGS4的表达仅限于ASM的一个亚群，并被有丝分裂原特异性上调，这导致了与顽固性哮喘相似的ASM过度增殖和收缩减慢的表型。我们目前正在急性和慢性过敏性气道炎症模型中检测具有全局和平滑肌特异性RGS4基因缺失的小鼠以及过度表达RGS4的小鼠的表型。我们将与密歇根大学的纽比格博士合作，在动物模型和细胞培养中研究RGS4特异性抑制剂对哮喘表型和ASM增殖和收缩的影响。这是一种第一代RGS抑制化合物。