ATRIAL MYOCYTE FUNCTION--STRETCH AND THE CAVEOLAE

心房肌细胞功能——伸展和小窝

• 批准号: 2704652
• 负责人: ERNEST PAGE
• 金额: $ 33.94万
• 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/2704652
• 关键词: animal tissue; atrium; biological signal transduction; cadherins; caveolas; cell component structure /function; dystrophin; electrospray ionization mass spectrometry; heart cell; heart function; immunoelectron microscopy; laboratory rat; muscle cells; 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.

在心脏及其成分肌肉细胞中伸展 既决定心脏收缩的强度和速率 蛋白质激素的分泌。 我们已经证明了小屋，丰富 质膜的小囊泡内陷，改变其形状 在拉伸期间，可能是一个类似过程，高渗 收缩。 因此，这些细胞器可能是仪器的一部分 在心肌细胞中传递拉伸信号。 在上一个赠款期间 时期我们在识别和 心脏小窝及其成分蛋白的纯化。 最近，我们通过使用一个 具有针对肌肉的抗体的免疫治疗方案 特定的小窝蛋白-3同工型。 现在，这种改进使我们能够问一个 有关心脏结构和功能的一系列特定问题 Caveolae及其与拉伸和扰动的关系 渗透性。 有三个特定的目标：特定目标1将（a）使用 免疫电子显微镜跟踪Aquaporin1的内在化 从Caveolae到心房细胞质中尚未确定的基因座 或其他地方，（b）识别和定位囊泡和微管 参与，（c）寻找并研究相关的磷酸化和 信号传导机制，（d）确定步骤（a -c）是否明显伸展 依赖。 特定目标2将（a）使用新制作的兔多克隆 肌肉特异性小窝蛋白同工型的抗体，CAV3以优化 从绵羊和老鼠的心中隔离洞穴； （b）使用免疫电子 显微镜检查以鉴定免疫纯化的小窝和标准分析 鉴定特定脂质和蛋白质成分的方法； （c）使用 微观（例如，电喷雾）定义蛋白质的方法 小窝的组成（d）研究免疫保健中的信号传导事件 小窝，寻找可逆的磷酸化，ADP-核糖基化和 拉伸的影响。 特定目标3检查了小窝的相互作用 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的细胞骨架蛋白 肌营养不良及其膜锚dystroglycan。