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靶G蛋白偶联受体（GPCR）和蛋白激酶的治疗剂。多达四种亚型的GPCR，腺苷受体，可能在哺乳动物心肌中表达，所有四种亚型已被证明调节蛋白激酶信号传导。申请人实验室发表的初步研究结果表明，腺苷A2a受体调节腺苷A1受体刺激的心脏保护作用。其他初步研究结果表明，腺苷A1受体和4-阿片受体之间的相互作用调节了每种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都依赖于多种蛋白激酶。该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。特异性目的1将确定腺苷受体亚型和阿片受体之间的相互作用在减少完整心脏和离体心肌细胞心肌缺血再灌注损伤中的作用。特异性目的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由于完整心肌和分离心肌细胞中亚细胞蛋白激酶信号的调节。特异性目标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性、细胞内钙处理和线粒体功能的调节的影响。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中进行。通过PKC， MAPK和AKT通路的受体信号将在正常和缺血再灌注的离体小鼠心脏和离体心室肌细胞产生的核，细胞质，线粒体和膜亚细胞部分进行检测。完整心脏中受体诱导的亚细胞蛋白激酶信号调节与心肌梗死面积和心室功能相关。在分离的肌细胞亚细胞信号将与肌细胞收缩力，细胞内钙稳态和线粒体功能相关。尽管缺血心肌的保护似乎是通过多种蛋白激酶的激活介导的，但心内直视手术后心肌肥大、心力衰竭和心室重构等情况也与蛋白激酶活性增加有关。这些深入研究gpcr及其在正常和缺血心肌中对亚细胞蛋白激酶信号传导的相互作用的结果可能有助于开发治疗缺血性心脏的新疗法。

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.