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受体调节信号的机制， 在正常和缺血的心室肌中的转导。虽然 腺苷A1受体是一种典型的G蛋白偶联受体， 似乎与其他GPCR不同，因为它对 心室心肌有重要证据表明，GPCR，异源三聚体 GTP结合（G）蛋白和相关的第二信使，如腺苷酸 环化酶和蛋白激酶C（PKC）亚型，富含离散的 细胞内区室，包括富含胆固醇的烧瓶形 质膜的内陷，称为小窝。基于 申请人的初步数据表明，心室肌细胞腺苷A1受体 富含小窝，假设A1受体介导的 正常和缺血心肌中的信号传导受亚细胞 分隔具体目标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中的A1受体和相关G蛋白。 具体目标2将检验腺苷A1受体 抗肾上腺素能作用与B-肾上腺素能受体的组分共定位。 受体第二信使系统具体目标3将确定是否 腺苷A1受体激活调节局部PKC的激活 丝氨酸-苏氨酸蛋白磷酸酶同工酶在正常和缺血性 肌细胞研究将在成年大鼠分离的心室肌细胞中进行 在常氧和模拟缺血条件下。亚细胞的作用 腺苷A1受体信号传导的区室化将通过 细胞裂解物的亚细胞分级分离。亚细胞区室化 腺苷A1和B-肾上腺素能受体，G蛋白亚单位，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受体调节相关， 通过测量肌细胞抽搐幅度研究兴奋-收缩偶联， 细胞内钙、总cAMP和颗粒cAMP水平，以及细胞溶质和 颗粒蛋白磷酸酶活性。腺苷A1受体调节 PKC同工型易位和蛋白磷酸酶将与 在模拟缺血-再灌注期间防止细胞死亡。有 有大量证据表明，腺苷具有许多有益作用， 缺血-再灌注心肌，但很少有信息 关于作用机制。拟议的研究将提供一个 更好地理解GPCR对正常和 缺血心肌细胞，这可能有助于开发新的治疗方法 缺血性心脏的治疗方案

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.