Beta-Adrenergic Receptor Function in Atrial Myocytes

心房肌细胞中的β-肾上腺素能受体功能

• 批准号: 7437291
• 负责人: STEPHEN Lloyd LIPSIUS
• 金额: $ 35.2万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7437291
• 关键词: 3-Phosphoinositide Dependent Protein Kinase-1; Actins; Affect; Binding; CD29 Antigen; Calmodulin; Caveolins; Cell surface; Cells; Complex; Coupled; Coupling; Cyclic AMP; Cyclic GMP; Cytoskeleton; Depressed mood; Disease; Employee Strikes; Event; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; F-Actin; Fibrosis; Fluorescence; Focal Adhesion Kinase 1; GTP-Binding Proteins; Goals; Heart; Heart Atrium; Histocytochemistry; Homeostasis; Immunoblotting; Integrin beta Chains; Integrins; Laminin; Laminin Receptor; Lead; Measurement; Mediating; Methods; Microfilaments; Muscle; Muscle Cells; Nitric Oxide; Pathway interactions; Phospholipase A2; Phosphorylation; Phosphotransferases; Production; Proteins; Range; Receptor Signaling; Regulation; Rho-associated kinase; Signal Pathway; Signal Transduction; Signaling Protein; Site; Transfection; Tyrosine Phosphorylation; Viral; base; beta-adrenergic receptor; caveolin 1; insight; patch clamp; paxillin; phospholamban; radioligand; receptor; receptor binding; receptor function; research study; rho

DESCRIPTION (provided by applicant): My long range goal is to elucidate the mechanisms by which beta-adrenergic receptor (beta-AR) stimulation regulates Ca2+ homeostasis and how the extracellular matrix-integrin-cytoskeletal complex remodels a-AR function in atrial muscle. Atrial muscle contains 3 functionally different beta-AR signaling pathways that couple to various G-proteins: Pathway A: Beta1-AR/Gs, Pathway B: Beta2-AR/Gs and Pathway C: Beta2-AR/Gs/Gi. We hypothesize that Pathways A and B act via global mechanisms while Pathway C acts via local mechanisms to regulate Ca2+ homeostasis as defined by stimulation of L-type Ca2+ current, intracellular Ca2+ content and release, contraction, and phosphorylation of phospholamban. Pathway C acts via IP3-mediated NO signaling to exert local control of Ca2+ homeostasis. Alternatively, Pathway C may act via cytosolic Ca2+-dependent phospholipase A2 signaling to locally regulate Ca2+ homeostasis. Atrial disease is commonly associated with atrial fibrosis, i.e. structural remodeling. Structural remodeling is associated with increases in extracellular matrix (ECM) proteins, and changes in integrin receptor signaling. Our preliminary findings indicate that the ECM protein laminin acts via beta1-integrin receptors and the actin-based cytoskeleton to selectively alter each Beta-AR signaling pathway. These ECM-integrin-mediated changes in beta-AR function are striking similar to the remodeling of Beta-AR function exhibited by the failing heart. We therefore hypothesize that stimulation of Beta1- integrin receptors by laminin initiates a cascade of signaling events that lead to the local recruitment and autophosphorylation of focal adhesion kinase (FAK) which in turn transduces a signal via Src-mediated tyrosine phosphorylation of paxillin to produce local alterations in the myocyte cytoskeleton that directly remodels Beta-AR regulation of Ca2+ homeostasis. We propose to use whole cell patch clamp recordings, epi- and confocal fluorescence measurements of [Ca2+]i and NOi, radioligand receptor binding, immunoblots, immuno-histochemistry, and viral transfection methods to determine the mechanisms underlying Beta-AR regulation of Ca2+ homeostasis and how stimulation of Beta-integrin receptors by laminin remodels these Beta-AR regulatory mechanisms. These experiments should provide a comprehensive understanding of the mechanisms by which beta-ARs regulate Ca2+ homeostasis in atrial myocytes, as well as important insight into how ECM-integrin-cytoskeletal signaling contributes to remodeling of beta-AR function in the diseased heart.

描述（由申请人提供）：我的长期目标是阐明β-肾上腺素能受体（β-AR）刺激调节Ca 2+稳态的机制以及细胞外基质-整合素-细胞骨架复合物如何重塑心房肌中的α-AR功能。心房肌含有3种功能不同的β-AR信号通路，其与各种G蛋白偶联：通路A：β 1-AR/Gs，通路B：β 2-AR/Gs和通路C：β 2-AR/Gs/Gi。我们假设通路A和B通过全局机制起作用，而通路C通过局部机制起作用以调节Ca 2+稳态，如通过刺激L型Ca 2+电流、细胞内Ca 2+含量和受磷蛋白的释放、收缩和磷酸化所定义的。途径C通过IP 3介导的NO信号传导发挥局部控制Ca 2+稳态的作用。或者，途径C可以通过胞质Ca 2+依赖性磷脂酶A2信号传导来局部调节Ca 2+稳态。心房疾病通常与心房纤维化（即结构重构）相关。结构重塑与细胞外基质（ECM）蛋白的增加和整合素受体信号传导的变化有关。我们的初步研究结果表明，ECM蛋白层粘连蛋白通过β 1-整联蛋白受体和肌动蛋白为基础的细胞骨架，选择性地改变每个β-AR信号通路。这些ECM整合素介导的β-AR功能变化与衰竭心脏表现出的β-AR功能重塑惊人相似。因此，我们假设层粘连蛋白刺激β 1-整联蛋白受体启动了一系列信号传导事件，导致局部募集和粘着斑激酶（FAK）的自磷酸化，FAK又通过Src介导的桩蛋白酪氨酸磷酸化转导信号，从而在肌细胞骨架中产生局部改变，直接重塑β-AR对Ca 2+稳态的调节。我们建议使用全细胞膜片钳记录，[Ca 2 +]i和NOi的epi和共聚焦荧光测量，放射性配体受体结合，免疫印迹，免疫组织化学和病毒转染方法来确定β-AR调节Ca 2+稳态的机制以及层粘连蛋白对β-整合素受体的刺激如何重塑这些β-AR调节机制。这些实验应该提供一个全面的了解的机制，β-AR调节心房肌细胞中的Ca 2+稳态，以及重要的洞察如何ECM整合素细胞骨架信号有助于重塑β-AR功能在患病的心脏。