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.

哮喘是一种可逆性气道阻塞的病理状态，由肺部炎症和细支气管平滑肌过度收缩组成。诱导支气管平滑肌收缩的主要天然存在的物质是G蛋白偶联受体（GPCR）的配体，如过敏原蛋白酶、凝血酶和过敏原-IgE激活的肥大细胞颗粒中所含的物质（如组胺、半胱氨酰白三烯（LTD 4）、内皮素1、腺苷和缓激肽）。通常，这些激动剂诱导异源三聚体G蛋白G-α q的活化，其增加平滑肌细胞中细胞内钙的浓度，促进肌动蛋白-肌球蛋白相互作用和肌纤维缩短。 相反，作用于G-α-s-偶联受体的配体，如沙丁胺醇，增加细胞内cAMP（cAMP）水平，促进ASM松弛。虽然嗜酸性粒细胞炎症是过敏性哮喘的典型特征，但它不是AHR的先决条件，这表明结构细胞（如气道平滑肌（ASM））的潜在异常有助于哮喘素质。 平滑肌中促收缩信号、GPCR信号的调节异常可介导平滑肌收缩性增强。 G蛋白信号传导调节因子（Regulators of G protein signaling，RGS）蛋白的大家族通过保守的RGS结构域结合G蛋白α亚基Gi和Gq（但不结合Gs），并通过催化其内在的GT3活性和通过阻断下游效应物相互作用使它们失活。尽管它们通常被认为是GPCR信号通路的负调节剂，但RGS蛋白在肺中的生理功能大多是未知的。 我们鉴定了几种RGS蛋白（特别是RGS 4、RGS 5）在支气管平滑肌中的表达。 在2014财年，我们证明了RGS 5基因缺陷的小鼠由于ASM中GPCR诱导的Ca 2+稳态异常而发生自发性哮喘。来自Rgs 5-/-小鼠的精确切割肺切片（PCLS）在基线时收缩最大，与过敏原激发无关。RGS 5缺乏对变应性炎症的参数影响不大，包括支气管肺泡灌洗液（BALF）中的细胞计数、粘蛋白产生、ASM质量和上皮下胶原沉积。出乎意料的是，来自Rgs 5-/-小鼠的BALF中的IL-13水平相对于WT低得多。这些研究表明，RGS 5的缺乏在不存在过敏性炎症的情况下在小鼠中赋予自发性AHR。 在重度哮喘中，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，其特征在于肺ASM质量和硬度增加。RGS 4的表达仅限于ASM的一个亚群，并且被有丝分裂原特异性上调，这诱导了与在哮喘中观察到的类似的过度增殖和收缩减退的ASM表型。 我们目前正在研究急性和慢性过敏性气道炎症模型中的表型，包括整体和平滑肌特异性Rgs 4基因缺失，以及过表达Rgs 4的小鼠。与密歇根大学的Neubig博士合作，我们将在动物模型和细胞培养中研究RGS 4特异性抑制剂对哮喘表型和ASM增生和收缩发展的影响。 这是第一代RGS抑制化合物。