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

哮喘是一种可逆性气道阻塞的病理状态，包括肺部炎症以及细支气管平滑肌的过度收缩。 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，然而，这表明在这种疾病中可能存在气道平滑肌（ASM）收缩性的原发性异常。 引起支气管平滑肌收缩的主要物质是G蛋白偶联受体（GPCRs）的天然配体。 过敏反应是由IgE致敏的肺肥大细胞上的高亲和力IgE受体的过敏原交联引发的，并且这种过敏机制被认为是哮喘病理生理级联反应中最常见的诱因。 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GPCR以诱导支气管收缩。 实例包括组胺、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，促进肌动球蛋白相互作用。 相反，作用于G-α-s-偶联受体的配体，如异丙肾上腺素，增加细胞内cAMP（cAMP）水平，促进ASM松弛。 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），并通过增强其内在的GT3活性和通过阻断下游效应物相互作用使其失活。RGS蛋白在肺中的生理功能是未知的。 该项目的主要目的是确定哪些RGS蛋白在肺中的特定细胞类型中表达，并列举它们在该器官中的功能。 第一个目的主要是通过免疫组织化学和免疫印迹使用特异性抗体。 通过PCR和免疫印迹显示RGS 5在人和小鼠支气管平滑肌细胞中表达。在人类培养的气道平滑肌（ASM）细胞中，RGS 5的表达似乎受到白细胞介素-13（IL-13）的调节，IL-13是哮喘中发现的AHR的核心细胞因子。 我们正在使用Rgs 5敲除小鼠和RNA干扰来敲低培养的人ASM细胞中的基因表达，以检查Rgs 5在ASM中的功能。为了阐明RGS 5如何调节小鼠ASM收缩，我们使用切片收缩技术。切除小鼠的小直径支气管，制备肺薄切片并在组织培养皿中培养。 几天后，通过肺泡张力获得拴系到培养皿的支气管肺泡单元，在激动剂刺激之前和之后，可以通过显微镜评估气道直径（收缩）。 初步数据表明，与野生型相比，来自这些小鼠的培养的气管平滑肌细胞中，敲除细胞中的钙向毒蕈碱受体激动剂卡巴胆碱和前列腺素E2（PGE 2）的动员减少。未来的研究将在过敏性气道炎症模型中检查过敏原致敏和激发后这些小鼠的支气管收缩性。在过敏原暴露前后，通过体积描记法测量整个动物的气道反应性。 我们还建立了siRNA双链体，其有效地降低培养的人ASM细胞中内源性RGS 5的表达。我们目前正在研究siRNA转染的人ASM细胞和野生型和基因敲除小鼠培养的小鼠ASM中的细胞内钙浓度。 我们还计划直接研究培养的ASM细胞的增殖和合成功能（例如细胞因子分泌）。

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 human cultured airway smooth muscle (ASM) cells, RGS5 expression appears to be regulated by interleukin-13 (IL-13), which is a cytokine central to the AHR found in asthma. We are examining the function of RGS5 in ASM using Rgs5 knockout mice and RNA interference to knock down gene expression in cultured human ASM cells. To elucidate how RGS5 regulates ASM contraction in mice, we are using the slice contraction technique. The small diameter bronchi of the mice are excised, and thin lung sections are prepared and cultured in a tissue culture dish. A bronchoalveolar unit tethered to the dish by alveolar tension is obtained after several days, from which airway diameter (contraction) can be evaluated by microscopy before and after agonist stimulation. Preliminary data indicate cultured tracheal smooth muscle cells from these mice, the mobilization of calcium in knockout cells to the muscarinic receptor agonist carbachol and prostaglandin E2 (PGE2) were reduced compared to wild-type. Future studies will examine bronchial contractility in these mice after allergen sensitization and challenge in a model of allergic airway inflammation. Airway responsiveness of whole animals will be measured by plethysmography before and after allergen exposure. We have also established siRNA duplexes that effectively reduce expression of endogenous RGS5 in cultured human ASM cells. We are currently examining intracellular calcium concentration in siRNA-transfected human ASM cells and cultured mouse ASM from wild-type and knockout mice. We also plan to study the proliferation and synthetic function (e.g. cytokine secretion) of cultured ASM cells directly.