G PROTEIN REGULATION--ADENYLYL CYCLASE IN AIRWAY MUSCLE

G蛋白调节--气道肌中的腺苷酸环化酶

• 批准号: 2210389
• 负责人: CHARLES W EMALA
• 金额: $ 9.14万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-28 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2210389
• 关键词: G protein; SDS polyacrylamide gel electrophoresis; adenylate cyclase; antibody specificity; asthma; beta adrenergic receptor; biological signal transduction; cyclic AMP; dogs; enzyme activity; laboratory rabbit; leukocytes; muscarinic receptor; muscle tone; radionuclides; receptor binding; respiratory muscles; scintillation counter; second messengers; smooth muscle; western blottings

Since Szentivanyi proposed in 1968 that asthma might be due to an inherited or acquired deficit in beta-adrenergic function, much research has focused on the beta-adrenergic receptor (beta2AR) and the beta- adrenergic cascade. However, 22 years later the question is still unsettled, as research has focused largely on the leukocyte, which is poor model of human asthma. Studies of bronchial muscle tissue from animal models of asthma and from human asthmatics obtained either at surgery or at autopsy show a very definite deficit in beta-adrenergic function. However, the intracellular events underlying this beta- adrenergic deficit are unknown. We have a well characterized animal model of airway hyperresponsiveness that clearly demonstrates decreased sensitivity to beta-adrenergic agonists in vivo and in vitro. We hypothesize that the decreases sensitivity to beta-adrenergic agonists in vivo and in vitro. We hypothesize that the decreased sensitivity to beta-adrenergic agonists relates to a specific defect either in generation or degradation of intracellular cAMP. To identify directly the intracellular lesion we plan to measure numbers and affinities of beta2AR and muscarinic receptor subtypes, second messengers, adenylyl cyclase and their regulation by the signal transducing G proteins in the airway smooth muscle of the basenji-greyhound dog. We are currently measuring adenylyl cyclase activity in the white blood cell and airway smooth muscle. We have identified the presence of Gs/alpha, Gi/alpha, and beta gamma using human polyclonal antibodies in Western blots. Preliminary studies in the white blood cell reveals decreased adenylyl cyclase activity in response to beta agonists in the BG dog despite normal quantities of G proteins. The reduced in vitro sensitivity to beta agonists in our animal model provides us with the unique opportunity of easy accessibility to large quantities of airway smooth muscle for our in vitro studies from tho allergic lines of animals - one hyperresponsive and one normoresponsive, allowing us to focus on the deficit leading to hyperresponsiveness, not confounded by allergy or drug therapy. We also have the unique ability to control and manipulate environmental and genetic influences. Results from the proposed work will provide new information about the intracellular mechanisms which regulate airway smooth muscle tone in this model.

自 1968 年 Szentivanyi 提出哮喘可能是由于 遗传性或获得性β-肾上腺素能功能缺陷，大量研究 专注于β-肾上腺素能受体（β2AR）和β- 肾上腺素能级联。 然而22年后，问题仍然是 尚未解决，因为研究主要集中在白细胞上， 人类哮喘的不良模型。 支气管肌肉组织的研究 哮喘动物模型和人类哮喘患者获得的 手术或尸检显示β-肾上腺素能非常明显的缺陷 功能。 然而，这种β-的细胞内事件 肾上腺素能缺乏尚不清楚。 我们有一种特征明确的动物 气道高反应性模型清楚地表明气道高反应性下降 体内和体外对β-肾上腺素能激动剂的敏感性。 我们 假设对β-肾上腺素能激动剂的敏感性降低 体内和体外。 我们假设敏感性降低 β-肾上腺素能激动剂与特定缺陷有关 细胞内cAMP的产生或降解。 直接识别 我们计划测量细胞内病变的数量和亲和力 beta2AR 和毒蕈碱受体亚型，第二信使，腺苷酸 环化酶及其信号转导 G 蛋白的调节 巴森吉灵缇犬的气道平滑肌。 我们目前 测量白细胞和气道中的腺苷酸环化酶活性 平滑肌。 我们已经确定了 Gs/alpha、Gi/alpha 的存在， 和 β γ 在蛋白质印迹中使用人多克隆抗体。 白细胞的初步研究显示腺苷酸减少 尽管 BG 狗对 β 激动剂有反应，但环化酶活性 正常量的 G 蛋白。 体外敏感性降低 我们的动物模型中的β激动剂为我们提供了独特的 轻松获得大量气道畅通的机会 用于我们体外研究的动物过敏系肌肉 - 之一 过度反应和正常反应，使我们能够专注于 缺陷导致高反应性，不与过敏或 药物治疗。 我们还拥有独特的控制和操纵能力 环境和遗传的影响。 拟议工作的结果 将提供有关细胞内机制的新信息 在此模型中调节气道平滑肌张力。