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.

哮喘是可逆气道阻塞的病理状况，由肺部炎症和支气管平滑肌的超收缩性组成。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，凝血酶和那些在过敏原激活的肥大细胞颗粒中包含的配体（例如Bradykinin）。通常，这些激动剂会诱导异三聚体G蛋白G-Alpha Q的激活，从而增加平滑肌细胞中细胞内钙的浓度，从而促进肌动蛋白 - 膜肌球蛋白相互作用和肌肉纤维缩短。相比之下，作用于G-Alpha-S偶联受体（例如沙丁胺醇）增加细胞内循环AMP（CAMP）的配体，促进ASM松弛。尽管嗜酸性炎性炎症代表过敏性哮喘，但它并不是AHR的先决条件，这表明在结构细胞（例如气道平滑肌（ASM））中的潜在异常有助于哮喘的素质。 ASM中procctallige，GPCR信号的失调可能介导增强的收缩力。 G蛋白信号传导（RGS）蛋白的一大批调节剂与G蛋白α亚基GI和GQ（但不是GS）结合，通过保守的RGS结构域结合，并通过催化其内在的GTPase活性和阻止下游效应的效应器相互作用来催化它们。尽管通常认为它们是GPCR信号通路的负调节剂，但肺中RGS蛋白的生理功能主要是未知的。我们确定了支气管平滑肌中几种RGS蛋白（尤其是RGS4，RGS5）的表达。在2014财年，我们证明了RGS5遗传缺陷的小鼠由于ASM中GPCR诱导的Ca2+稳态异常而产生了自发性哮喘。从基线上最大收缩的Nave RGS5 - / - 小鼠的精密切割肺切片（PCL），与过敏原挑战无关。 RGS5缺乏对过敏性炎症的参数几乎没有影响，包括支气管肺泡灌洗液（BALF），粘蛋白产生，ASM质量和上皮下胶原蛋白沉积的细胞计数。出乎意料的是，相对于WT，RGS5 - / - 小鼠的BALF中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的缺乏赋予小鼠自发的AHR。在严重的哮喘中，支气管扩张剂和对类固醇不敏感的气流阻塞的发展是通过未知的机制发展出来的，其特征是肺ASM质量和刚度增加。 RGS4表达仅限于ASM的亚群，并被有丝分裂原特异性上调，这诱导了类似于在顽固性哮喘中观察到的高增殖性和低取消质量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，使用全球和平滑肌特异性RGS4基因缺失以及过表达RGS4的小鼠的表型。与密歇根大学的Neubig博士合作，我们将研究RGS4特异性抑制剂对动物模型和细胞培养中哮喘表型和ASM增生的发展以及ASM增生以及收缩的影响。这是第一代RGS抑制化合物。