Airway muscle tone, RhoA & Cytoskeletal remodeling

气道肌张力，RhoA

• 批准号: 7104256
• 负责人: CAROL A HIRSHMAN
• 金额: $ 31.93万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104256
• 关键词: ADP ribosylation; G protein; actins; antisense nucleic acid; beta adrenergic agent; beta adrenergic receptor; biological signal transduction; bronchomotion; calcium flux; cytoskeleton; fluorescence microscopy; guanine nucleotide binding protein; guinea pigs; immunofluorescence technique; muscle contraction; paxillin; polymerase chain reaction; protein kinase A; protein tyrosine kinase; receptor coupling; receptor expression; respiratory pharmacology; smooth muscle; stimulant /agonist; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Although B2-adrenergic agonists are widely used for the acute treatment for asthma, these therapeutic agents are not ideal. They are not always effective in severe asthma, must be redosed frequently, are often inadequate as sole therapy and are associated with increased mortality. B2 agonists are thought to reduce symptoms of asthma by directly relaxing airway smooth muscle via the Gs/cAMP- PKA pathway but the events downstream from PKA are not known. Although PKA-independent mechanisms have been suggested, the signaling intermediates in a PKA-independent pathway have never been identified. Exciting preliminary data demonstrate B-adrenergic agonist-mediated relaxation of intact airway smooth muscle and actin deploymerization of primary cultures of human airway smooth muscle cells via a novel PKA-independent signaling pathway involving Src tyrosine kinases as well as by PKA-dependent inhibition of RhoA. RhoA is a monomeric G proteins that plays a central role in the dynamic regulation of the actin cytoskeleton in airway smooth muscle cells and in maintaining contraction in intact smooth muscle. We now seek to identify the intermediates in the PKA-independent pathway and to determine whether the PKA-dependent and/or PKA-independent signaling involve inhibition of RhoA. We hypothesize that B-adrenergic agonists induce airway smooth muscle relaxation and actin depolymerization by a novel PKA-independent pathway that involves Gs activation of Lck tyrosine kinase with subsequent activation of P190 RhoGAP leading to RhoA inhibition as well as by PKA-dependent inhibition of RhoA and dephosphorylation of cofilin. We propose: to confirm preliminary data demonstrating that B-adrenergic agonists inhibit contraction of intact airway smooth muscle by PKA-independent as well as by PKA-dependent mechanisms and to identify intermediates involved (Aim 1); to identify the Src tyrosine kinase involved in the PKA-independent pathway and explore the upstream (Gs protein) and downstream (P190 RhoGAP) intermediates (Aim 2); and to determine if B-adrenergic agonists induce actin depolymerization by PKA-induced phosphorylation and inhibition of RhoA; (Aim 3). Identification of intermediates of a signaling pathway that relaxes airway smooth muscle in a PKA-independent manner would provide new selective targets for asthma drug therapy and would be a significant advance in the field. Identifying novel pathways in airway smooth muscle that mediate relaxation will lead to new therapeutic intervention strategies that may be employed to impair the ability of airway smooth muscle to contract and cause acute airway narrowing in asthma.

描述（由申请人提供）：虽然B2-肾上腺素能激动剂广泛用于哮喘的急性治疗，但这些治疗剂并不理想。它们对严重哮喘并不总是有效，必须经常重新给药，作为单一疗法通常是不够的，并且与死亡率增加有关。 B2 激动剂被认为可通过 Gs/cAMP-PKA 途径直接放松气道平滑肌来减轻哮喘症状，但 PKA 下游的事件尚不清楚。尽管已经提出了不依赖于 PKA 的机制，但不依赖于 PKA 的途径中的信号传导中间体从未被鉴定过。令人兴奋的初步数据表明，通过涉及 Src 酪氨酸激酶的新型 PKA 独立信号通路以及 PKA 依赖性 RhoA 抑制，B 肾上腺素能激动剂介导完整气道平滑肌的松弛和人气道平滑肌细胞原代培养物的肌动蛋白部署。 。 RhoA 是一种单体 G 蛋白，在气道平滑肌细胞肌动蛋白细胞骨架的动态调节以及维持完整平滑肌的收缩中发挥核心作用。我们现在寻求鉴定 PKA 独立通路中的中间体，并确定 PKA 依赖性和/或 PKA 独立信号传导是否涉及 RhoA 的抑制。我们假设 B-肾上腺素能激动剂通过一种新的不依赖于 PKA 的途径诱导气道平滑肌松弛和肌动蛋白解聚，该途径涉及 Gs 激活 Lck 酪氨酸激酶，随后激活 P190 RhoGAP 导致 RhoA 抑制，以及 PKA 依赖性 RhoA 抑制和丝切蛋白的去磷酸化。我们建议：确认初步数据，证明 B-肾上腺素能激动剂通过 PKA 独立和 PKA 依赖性机制抑制完整气道平滑肌的收缩，并确定相关中间体（目标 1）；鉴定参与 PKA 独立通路的 Src 酪氨酸激酶，并探索上游（Gs 蛋白）和下游（P190 RhoGAP）中间体（目标 2）；并确定 B-肾上腺素能激动剂是否通过 PKA 诱导的磷酸化和 RhoA 抑制来诱导肌动蛋白解聚； （目标 3）。鉴定以不依赖PKA的方式放松气道平滑肌的信号通路中间体将为哮喘药物治疗提供新的选择性靶点，并且将是该领域的重大进步。识别气道平滑肌中介导放松的新途径将导致新的治疗干预策略，这些策略可用于削弱气道平滑肌收缩的能力并导致哮喘急性气道狭窄。