Compartmentation of Myocyte Adenosine Receptor Signaling

描述(由申请人提供)：拟议研究的总体目标 目的是确定腺苷A1受体对信号的调控机制 正常和缺血心肌中的信号转导。尽管 腺苷A1受体是一种典型的G蛋白偶联受体，它 似乎与其他gpr不同，因为它不会对 室壁心肌。有重要证据表明，GPCR异三聚体 GTP结合(G)蛋白和相关的第二信使，如腺苷 环化酶和蛋白激酶C(PKC)亚型，在不连续的 细胞内间隔，包括富含胆固醇的烧瓶形 质膜内陷，称为小凹。基于 申请人的初步数据显示，心室肌细胞腺苷A1受体 都富含小窝，推测A1受体介导的 正常和缺血心肌的信号转导是由亚细胞调控的 车厢分隔。特异性靶点1将决定受体的作用 激动剂和内源性腺苷对腺苷亚细胞定位的影响 正常和缺血心肌细胞的A1受体和相关G蛋白。 特定目标2将检验腺苷A1受体的假设 抗肾上腺素能作用与B-肾上腺素能成分共同定位 受体第二信使系统。具体目标3将决定是否 腺苷酶A1受体激活调节局部PKC的激活 正常和缺血时丝氨酸-苏氨酸蛋白磷酸酶的异构体 肌细胞。这项研究将在成年大鼠分离的心肌细胞中进行 在常氧和模拟缺血条件下。亚细胞的作用 腺苷A1受体信号的区隔将由以下因素决定 细胞裂解产物的亚细胞分级。亚细胞区划 腺苷A1和B-肾上腺素能受体、G蛋白亚基、PKC亚型和 蛋白磷酸酶，用免疫印迹、免疫沉淀和 免疫荧光将与腺苷A1受体调控相关 通过测量心肌细胞的收缩幅度来研究兴奋-收缩耦合， 细胞内钙、总cAMP和颗粒cAMP水平，以及胞浆和 颗粒蛋白磷酸酶活性。腺苷A1受体对血管生成的调节作用 PKC异构体易位和蛋白磷酸酶将与 对模拟缺血再灌流过程中细胞死亡的保护。的确有 大量证据表明腺苷对人体有多种有益作用 缺血再灌流的心室肌，但信息很少 关于行动机制的可用信息。拟议的研究将提供一个 更好地理解GPCR对正常和正常人群信号转导的调节 缺血心肌细胞可能有助于开发新的治疗方法 治疗缺血型心脏的方案。

DESCRIPTION (provided by applicant): The overall aim of the proposed research is to determine the mechanisms of adenosine A1 receptor modulation of signal transduction in normal and ischemic ventricular myocardium. Although the adenosine A1 receptor is a typical G protein coupled receptor (GPCR), it appears to differ from other GPCR in that it exerts no direct effects in ventricular myocardium. There is significant evidence that GPCR, heterotrimeric GTP binding (G) proteins, and associated second messengers, such as adenylyl cyclase and protein kinase C (PKC) isoforms, are enriched in discrete intracellular compartments, including cholesterol-enriched, flask-shaped invaginations of the plasma membrane, referred to as caveolae. Based on the applicant's preliminary data, that ventricular myocyte adenosine A1 receptors are enriched in caveolae, it is hypothesized that A1 receptor-mediated signaling in normal and ischemic myocardium is regulated by subcellular compartmentation. Specific Aim 1 will determine the effects of receptor agonists and endogenous adenosine on the subcellular localization of adenosine A1 receptors and associated G proteins in normal and ischemic myocytes. Specific Aim 2 will test the hypothesis that the adenosine A1 receptor anti-adrenergic effect is co-localized with the components of the B-adrenergic receptor second messenger system. Specific Aim 3 will determine whether adenosme A1 receptor activation modulates the activation of localized PKC isoforms or serine-threonine protein phosphatases in normal and ischemic myocytes. Studies will be conducted in adult rat isolated ventricular myocytes under normoxic and simulated ischemia conditions. The role of subcellular compartmentalization of adenosine A1 receptor signaling will be determined by subcellular fractionation of cell lysates. Subcellular compartmentation of adenosine A1 and B-adrenergic receptors, G protein subunits, PKC isoforms, and protein phosphatases, assessed with immunoblotting, immunoprecipitation, and immunofluorescence will be correlated with adenosine A1 receptor modulation of excitation-contraction coupling studied by measuring myocyte twitch amplitude, intracellular calcium, total and particulate cAMP levels, and cytosolic and particulate protein phosphatase activities. Adenosine A1 receptor modulation of PKC isoform translocation and protein phosphatases will be correlated with protection against cell death during simulated ischemia-reperfusion. There is substantial evidence that adenosine has numerous beneficial effects in ischemic-reperfused ventricular myocardium, but there is little information available regarding mechanism of action. The proposed studies will provide a better understanding of GPCR regulation of signal transduction in normal and ischemic cardiac myocytes which may aid in the development of new treatment regimens for the ischemic heart.