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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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况，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物质是G蛋白偶联受体（GPCR）的配体，例如过敏原蛋白酶、凝血酶以及过敏原IgE激活的肥大细胞颗粒中所含的物质（例如组胺、半胱氨酰白三烯（LTD4） )、内皮素 1、腺苷和缓激肽)。一般来说，这些激动剂会诱导异源三聚体 G 蛋白 G-α q 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（例如沙丁胺醇）会增加细胞内环 AMP (cAMP) 的水平，从而促进 ASM 松弛。尽管嗜酸性粒细胞炎症是过敏性哮喘的典型表现，但它并不是 AHR 的先决条件，这表明气道平滑肌 (ASM) 等结构细胞的潜在异常会导致哮喘素质。 ASM 中的前收缩 GPCR 信号传导失调可能会介导收缩性增强。 G 蛋白信号调节蛋白 (RGS) 蛋白大家族通过保守的 RGS 结构域与 G 蛋白 α 亚基 Gi 和 Gq（但不是 Gs）结合，并通过催化其内在 GTP 酶活性和阻断下游效应子相互作用来灭活它们。尽管它们通常被认为是 GPCR 信号通路的负调节因子，但 RGS 蛋白在肺部的生理功能大多未知。我们鉴定了支气管平滑肌中几种 RGS 蛋白（特别是 RGS4、RGS5）的表达。在 2014 财年，我们证明了 RGS5 基因缺陷的小鼠由于 ASM 中 GPCR 诱导的 Ca2+ 稳态异常而发生自发性哮喘。幼稚 Rgs5-/- 小鼠的精密切割肺切片 (PCLS) 在基线时收缩最大，与过敏原激发无关。 RGS5 缺乏对过敏性炎症参数影响不大，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，Rgs5-/- 小鼠 BALF 中的 IL-13 水平比 WT 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 AHR。在严重哮喘中，支气管扩张剂和类固醇不敏感的气流阻塞是通过未知机制形成的，其特征是肺 ASM 质量和硬度增加。 RGS4 表达仅限于 ASM 亚群，并被有丝分裂原特异性上调，从而诱导与顽固性哮喘中观察到的类似的过度增殖和低收缩 ASM 表型。我们目前正在急性和慢性过敏性气道炎症模型中检查整体和平滑肌特异性 Rgs4 基因缺失的表型，以及过度表达 RGS4 的小鼠。我们将与密歇根大学的 Neubig 博士合作，在动物模型和细胞培养物中研究 RGS4 特异性抑制剂对哮喘表型发展以及 ASM 增生和收缩的影响。这是第一代 RGS 抑制化合物。