Compartmentation of Myocyte Adenosine Receptor Signaling

描述(由申请人提供)：大量用于治疗缺血性心脏病的治疗剂，靶向G蛋白偶联受体(GPCR)和蛋白激酶。腺苷受体GPCR4个亚型均可在哺乳动物心肌中表达，所有4个亚型均可调节蛋白激酶信号转导。已发表的和申请人实验室的初步发现表明，腺苷A2a受体调节腺苷A1受体刺激的心脏保护效应。另外的初步发现表明，腺苷A1受体和4-阿片受体之间的相互作用调节每个受体的心脏保护作用。腺苷A1和4-阿片受体的心脏保护作用依赖于多种蛋白激酶。这一建议的总体假设是，腺苷受体亚型之间以及腺苷和阿片受体之间在介导其心脏保护作用方面的相互作用是由于亚细胞蛋白激酶信号的调节。具体目的1将确定腺苷受体亚型和阿片受体之间的相互作用在减轻完整心脏和分离的心肌细胞缺血再灌注损伤中的作用。具体目的2将确定腺苷受体亚型和阿片受体相互作用的心脏保护作用是否源于对完整心肌和分离心肌细胞亚细胞蛋白激酶信号的调节。具体目标3将描述腺苷受体亚型和阿片受体之间的相互作用对心肌细胞收缩、细胞内钙处理和线粒体功能的调节作用。研究将在野生型小鼠和腺苷A1受体、A2a受体、4-阿片受体和：-阿片受体缺失的小鼠身上进行。通过PKC、MAPK和AKT通路的受体信号将在正常和缺血再灌流分离的小鼠心脏和分离的心室肌细胞产生的核、胞浆、线粒体和膜亚细胞部分中进行检测。受体诱导的完整心脏亚细胞蛋白激酶信号的调节将与心肌梗死范围和心功能相关。分离的心肌细胞中的亚细胞信号将与心肌细胞的收缩能力、细胞内钙稳态和线粒体功能相关。虽然缺血心肌的保护似乎是通过激活多种蛋白激酶来实现的，但心脏直视手术后的心肌肥大、心力衰竭和心室重构等情况也与蛋白激酶活性的升高有关。这些对GPCRs及其相互作用在正常和缺血心肌亚细胞蛋白激酶信号转导中的深入研究结果可能有助于开发新的治疗缺血性心脏的方法。

DESCRIPTION (provided by applicant): A significant number of therapeutic agents for the treatment of ischemic heart disease target G protein coupled receptors (GPCR) and protein kinases. Up to four subtypes of one GPCR, the adenosine receptor, may be expressed in mammalian myocardium and all four subtypes have been shown to modulate protein kinase signaling. Published and preliminary findings from the applicant's laboratory indicate that adenosine A2a receptors modulate the cardioprotective effects of adenosine A1 receptor stimulation. Additional preliminary findings indicate that interactions between the adenosine A1 receptor and the 4-opioid receptor modulate the cardioprotective effects of each receptor. The cardioprotective effects of both adenosine A1 and 4-opioid receptors are dependent on multiple protein kinases. The overall hypothesis of this proposal is that the interactions between adenosine receptor subtypes and between adenosine and opioid receptors in mediating their cardioprotective effects are due to the modulation of subcellular protein kinase signaling. Specific Aim 1 will determine the role of interactions among adenosine receptor subtypes and opioid receptors in reduction of myocardial ischemia-reperfusion injury in intact hearts and isolated myocytes. Specific Aim 2 will determine whether the cardioprotective effects of adenosine receptor subtype and opioid receptor interactions are due to modulation of subcellular protein kinase signaling in intact myocardium and isolated cardiomyocytes. Specific Aim 3 will delineate the effects of interactions among adenosine receptor subtypes and opioid receptors on the modulation of myocyte contractility, intracellular calcium handling and mitochondrial function in isolated cardiomyocytes. Studies will be conducted in wild-type mice and mice with deletions of adenosine A1 receptors, A2a receptors, 4-opioid, and :-opioid receptors. Receptor signaling via the PKC, MAPK and AKT pathways will be examined in nuclear, cytosolic, mitochondrial and membrane subcellular fractions generated from normal and ischemic-reperfused isolated mouse hearts and isolated ventricular myocytes. Receptor- induced modulation of subcellular protein kinase signaling in intact hearts will be correlated with myocardial infarct size and ventricular function. Subcellular signaling in isolated myocytes will be correlated with myocyte contractility, intracellular calcium homeostasis and mitochondrial function. Although protection of ischemic myocardium appears to be mediated via activation of multiple protein kinases, conditions such as myocardial hypertrophy, heart failure, and ventricular remodeling following open heart surgery are also associated with increased protein kinase activity. The results of these in-depth studies of GPCRs and their interactions on subcellular protein kinase signaling in normal and ischemic myocardium may facilitate the development of new therapies for the treatment of the ischemic heart.