ATRIAL MYOCYTE FUNCTION--STRETCH AND THE CAVEOLAE

心房肌细胞功能——伸展和小窝

• 批准号: 6030697
• 负责人: ERNEST PAGE
• 金额: $ 34.96万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6030697
• 关键词: animal tissue; atrium; biological signal transduction; cadherins; cardiac myocytes; caveolas; cell component structure /function; dystrophin; electrospray ionization mass spectrometry; heart function; immunoelectron microscopy; laboratory rat; phosphorylation; protein structure; stretch receptors; tissue /cell culture; water channel

In the heart and its constituent muscle cells stretch is a major determinant of both the strength of cardiac contraction and the rate of secretion of protein hormones. We have shown that caveolae, abundant small vesicular invaginations of the plasma membrane, change their shape during stretch and what may be an analogous process, hyperosmotic shrinkage. These organelles may thus be part of the apparatus that transduces stretch signals in cardiomyocytes. During the previous grant period we have made significant progress on identification and purification of cardiac caveolae and their constituent proteins. Recently, we have made a breakthrough in this area by using an immunopurification protocol with antibodies directed against the muscle- specific caveolin-3 isoform. This improvement now allows us to ask a series of specific questions about the structure and function of cardiac caveolae and their relationship to perturbations such as stretch and osmolarity. There are three specific aims: Specific Aim 1 will (a) use immunoelectron microscopy to track the internalization of aquaporin1 from caveolae to an as yet unidentified locus in the atrial cytoplasm or elsewhere, (b) identify and localize the vesicles and microtubules involved, (c) look for and study the associated phosphorylations and signaling mechanism, and (d) determine if steps (a - c) manifest stretch dependence. Specific Aim 2 will (a) use a newly made rabbit polyclonal antibody to the muscle specific caveolin isoform, cav3 to optimize caveolar isolation from sheep and rat hearts; (b) use immuno-electron microscopy to identify immuno-purified caveolae, and standard analytic methods to identify specific lipid and protein components; (c) use microscale (e.g., electrospray) methods to define the protein composition of caveolae (d) study the signaling events in immunopurifed caveolae, and look for reversible phosphorylation, ADP-ribosylation, and effects of stretch. Specific Aim 3 examines the interaction of caveolae with the recently discovered extracellular matrix protein T-cadherin and the medically interesting caveolae-associated cytoskeletal protein dystrophin and its membrane anchor dystroglycan.

在心脏及其组成的肌肉细胞中，拉伸是一个主要因素 心脏收缩的强度和收缩率的决定因素 蛋白质激素的分泌。 我们已经表明，小窝，丰富 质膜的小囊泡内陷，改变其形状 在拉伸期间以及可能是类似的过程，高渗 收缩。 因此，这些细胞器可能是该装置的一部分 转导心肌细胞中的拉伸信号。 在上一次补助期间 在此期间，我们在识别和识别方面取得了重大进展 纯化心脏小窝及其组成蛋白。 最近，我们在这方面取得了突破，通过使用 使用针对肌肉的抗体的免疫纯化方案 特定的 Caveolin-3 亚型。 这一改进现在允许我们询问 关于心脏结构和功能的一系列具体问题 小凹及其与拉伸和等扰动的关系 渗透压。 共有三个具体目标： 具体目标 1 将 (a) 使用 免疫电子显微镜追踪水通道蛋白1的内化 从小窝到心房细胞质中尚未识别的位点 或其他地方，(b) 识别并定位囊泡和微管 涉及，（c）寻找并研究相关的磷酸化和 信号机制，以及 (d) 确定步骤 (a - c) 是否明显拉伸 依赖性。 具体目标 2 将 (a) 使用新制作的兔多克隆抗体 肌肉特异性小窝蛋白亚型抗体，cav3 进行优化 与绵羊和大鼠心脏的小窝隔离； (b) 使用免疫电子 显微镜识别免疫纯化的小凹，以及标准分析 鉴定特定脂质和蛋白质成分的方法； (三) 使用 微尺度（例如电喷雾）方法来定义蛋白质 小凹的组成 (d) 研究免疫纯化中的信号传导事件 小凹，并寻找可逆磷酸化、ADP-核糖基化和 拉伸的效果。 具体目标 3 检查小窝的相互作用 与最近发现的细胞外基质蛋白 T-钙粘蛋白和 医学上有趣的小窝相关细胞骨架蛋白 肌营养不良蛋白及其膜锚定肌营养不良聚糖。