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：
7964504
负责人：
Kirk m Druey
金额：
$ 28.5万
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/7964504
关键词：
Acetylcholine Actomyosin Adenosine Adrenergic beta-Agonists Affinity Agonist Allergens Allergic Allergic Reaction Animals Antibodies Asthma Backcrossings Binding Bradykinin Bronchoconstriction Calcium Calcium-Sensing Receptors Cells Collaborations Cyclic AMP Cytoplasmic Granules Disease Drug Delivery Systems Endothelin-1 Extinction (Psychology)Family Future 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 Genetic Transcription Goals Guanosine Triphosphate Phosphohydrolases Heterotrimeric GTP-Binding Proteins Histamine Human IgE IgE Receptors Immunoblotting Immunohistochemistry Inflammatory Infiltrate Isoproterenol Knockout Mice Leukotriene Antagonists Leukotriene D4 Ligands Lung Lung Inflammation Measures Modeling Mus Muscle Contraction Muscle relaxation phase Myosin Light Chains Normal tissue morphology Organ Parathyroid Adenoma Parathyroid gland Phenotype Phosphorylation Physiological Plasma Plethysmography Production Protein Binding RGS Domain RGS Proteins RNA Interference Regulation Relaxation Respiratory physiology Role Serum Signal Pathway Slice Small Interfering RNA Smooth Muscle Smooth Muscle Myocytes Subfamily lentivirinae Therapeutic Agents airway hyperresponsiveness airway obstruction allergic airway inflammation cell type crosslink cysteinyl-leukotriene depressed in vivo interest mast cell receptor coupling release of sequestered calcium ion into cytoplasm respiratory smooth muscle response small hairpin RNA

项目摘要

Asthma, a pathological condition of reversible airways obstruction, comprises both inflammation of the lung as well as hyper-contractility of the bronchiolar smooth muscle. Such airway hyperresponsiveness (AHR) can exist in the absence of frank inflammatory infiltrates, however, suggesting that primary abnormalities in airway smooth muscle (ASM) contractility may exist in this disease. The major substances that induce bronchial smooth muscle contraction are natural ligands of G protein coupled receptors (GPCRs). Allergic reactions are initiated by allergen crosslinking of of high affinity IgE receptors on lung mast cells sensitized by IgE, and this allergic mechanism is considered the most common inciter of the pathophysiological cascade in asthma. Many of the compounds contained in mast cell granules or synthesized by mast cells act on procontractile GPCRs to induce bronchoconstriction. Examples include 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 actomyosin interactions. In contrast, ligands acting on G-alpha-s-coupled receptors, such as isoproterenol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. 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 accentuating their intrinsic GTPase activity and by blocking downstream effector interactions. The physiological function of RGS proteins in the lung is unknown. The principal objective of this project is to determine which RGS proteins are expressed in specific cell types in the lung and to enumerate their functions in this organ. The first objective is accomplished primarily by immunohistochemistry and immunoblotting using specific antibodies. RGS5 was shown to be expressed by PCR and immunoblotting in human and mouse bronchial smooth muscle cells. In 2009, we found that beta adrenergic agonists, a primary therapeutic agent for asthma, induced a marked reduction in transcription of several RGS mRNAs in human ASM, including RGS5. Human ASM with siRNA-induced extinction of RGS5 expression showed enhanced activity of signaling pathways leading to contraction like intracellular calcium flux and myosin light chain phosphorylation in response to physiological ligands such as bradykinin and acetylcholine. Contraction of precision-cut lung slices (PCLS) treated with RGS5-encoding lentivirus was reduced compared to control while the responses of slices infected with RGS5 shRNA lentivirus were increased. Contraction of PCLS from Rgs5 knockout mice in response to acetylcholine were markedly enhanced. These studies indicate that RGS5 is a physiological regulator of ASM contractility. Future studies will examine bronchial contraction in vivo using Rgs5 knockout mice after allergen sensitization and challenge in a model of allergic airway inflammation. Airway responsiveness of whole animals will be measured by invasive plethysmography before and after allergen exposure. An outgrowth of this project was to examine the function of RGS5 in parathyroid in collaboration with Dr. Olson. We found that (1)RGS5 is highly expressed in parathyroid cells; (2) parathyroid adenomas express elevated levels of RGS5 compared to matched pair normal tissue; (3) RGS5 can inhibit calcium-induced IP3 production in response to Calcium sensing receptor (CaSR) stimulation; (4) mice nullizygous for RGS5 have abnormally depressed plasma PTH levels but normal serum calcium. Future studies will examine the phenotype of Rgs5-/- mice backcrossed onto strains mimicking primary hyper- and hypo-parathyroidism.

哮喘是一种可逆性气道阻塞的病理状况，包括肺部炎症以及细支气管平滑肌的过度收缩。然而，这种气道高反应性（AHR）可以在没有明显炎症浸润的情况下存在，这表明这种疾病可能存在气道平滑肌（ASM）收缩性的主要异常。诱导支气管平滑肌收缩的主要物质是G蛋白偶联受体（GPCR）的天然配体。过敏反应是由 IgE 致敏的肺肥大细胞上高亲和力 IgE 受体的过敏原交联引发的，这种过敏机制被认为是哮喘病理生理级联反应的最常见诱因。肥大细胞颗粒中含有的或由肥大细胞合成的许多化合物作用于促收缩性 GPCR，从而诱导支气管收缩。例子包括组胺、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，促进肌动球蛋白的相互作用。相反，作用于 G-α-s 偶联受体的配体（例如异丙肾上腺素）会增加细胞内环 AMP (cAMP) 的水平，从而促进 ASM 松弛。 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，并通过增强其内在 GTP 酶活性和阻断下游效应子相互作用来灭活它们。 RGS 蛋白在肺中的生理功能尚不清楚。该项目的主要目标是确定哪些 RGS 蛋白在肺部特定细胞类型中表达，并枚举它们在该器官中的功能。第一个目标主要通过使用特异性抗体的免疫组织化学和免疫印迹来实现。通过 PCR 和免疫印迹显示 RGS5 在人和小鼠支气管平滑肌细胞中表达。 2009年，我们发现β肾上腺素能激动剂（哮喘的主要治疗药物）可诱导人类ASM中几种RGS mRNA（包括RGS5）的转录显着减少。具有 siRNA 诱导的 RGS5 表达消除的人 ASM 显示信号通路活性增强，导致细胞内钙流和肌球蛋白轻链磷酸化等收缩，以响应生理配体（如缓激肽和乙酰胆碱）。与对照相比，用RGS5编码慢病毒处理的精密切割肺切片（PCLS）的收缩减少，而用RGS5 shRNA慢病毒感染的切片的反应增加。 Rgs5 敲除小鼠的 PCLS 对乙酰胆碱的反应显着增强。这些研究表明 RGS5 是 ASM 收缩性的生理调节剂。未来的研究将使用 Rgs5 敲除小鼠在过敏原致敏和过敏性气道炎症模型中激发后检查体内支气管收缩情况。在过敏原暴露之前和之后，将通过侵入性体积描记法测量整个动物的气道反应性。该项目的一个成果是与 Olson 博士合作检查 RGS5 在甲状旁腺中的功能。我们发现（1）RGS5在甲状旁腺细胞中高表达； (2) 与匹配的正常组织相比，甲状旁腺腺瘤表达 RGS5 水平升高； (3) RGS5 可以抑制钙感应受体 (CaSR) 刺激后钙诱导的 IP3 产生； (4)RGS5无效合子的小鼠血浆PTH水平异常降低，但血清钙正常。未来的研究将检查与模仿原发性甲状旁腺功能亢进和低下的品系回交的 Rgs5-/- 小鼠的表型。