Effect of Adenosine A2 receptor activation on the mitochondrial death pathway

描述（由申请人提供）：最近大量研究表明，再灌注时线粒体死亡途径与坏死和细胞凋亡有关。因此，阻止线粒体死亡途径是再灌注时成功保护心脏的先决条件。虽然在再灌注时腺苷A2受体激活已被提出以保护心脏，但其机制尚不清楚。本提案的目的是确定A2受体激活对线粒体死亡途径的影响，并阐明A2受体激活影响的信号传导机制。我们假设在再灌注时通过NECA刺激A2受体激活，通过涉及eNOS、NO、PKG、锌和GSK-3的信号级联阻断线粒体死亡途径。我们将通过使用细胞生物学、分子生物学、生理学、药理学和基因靶向等多种技术来检验这些假设。目的1将通过测量线粒体功能、梗死面积和凋亡指标，确定再灌注时A2受体激活对线粒体死亡途径的影响。目的2将通过定义PKA、酪氨酸激酶和HSP90在eNOS激活中的作用来确定A2受体激活产生NO的机制。目的3将确定PKG在A2受体激活的保护作用中的作用。我们将通过过表达或敲除PKG基因来确定PKG的作用。Aim 4将确定PKG激活的下游靶点，这些靶点导致A2受体激活对线粒体死亡途径的预防作用。为了实现这一目标，我们将描述细胞内游离锌、线粒体KATP通道和GSK-3的作用。该建议的成功实施，将对A2受体在再灌注时活化对缺血/再灌注损伤保护作用的信号机制提供重要的新见解，对治疗急性心肌梗死患者具有重要的临床意义。

DESCRIPTION (provided by applicant): A large number of recent studies have associated a mitochondrial death pathway at reperfusion with both necrosis and apoptosis. Preventing the mitochondrial death pathway, therefore, is a prerequisite for successful cardioprotection at reperfusion. Although adenosine A2 receptor activation at reperfusion has been proposed to protect the heart, its mechanisms are still not clear. The objective of this proposal is to determine the effects of A2 receptor activation on the mitochondrial death pathway and to elucidate the signaling mechanisms underling the effects of A2 receptor activation. We hypothesize that stimulation of A2 receptor activation by NECA at reperfusion interdicts the mitochondrial death pathway through a signal cascade involving eNOS, NO, PKG, zinc, and GSK-3. We will test these hypotheses by using diverse techniques of cell biology, molecular biology, physiology, pharmacology, and gene targeting. Aim 1 will determine the effects of A2 receptor activation at reperfusion on the mitochondrial death pathway by measuring mitochondrial function, infarct size, and indexes of apoptosis. Aim 2 will determine the mechanisms by which A2 receptor activation produces NO by defining the roles of PKA, tyrosine kinase, and HSP90 in activation of eNOS. Aim 3 will determine the role of PKG in the protective action of A2 receptor activation. We will define the role of PKG by overexpressing or knocking out the PKG gene. Aim 4 will ascertain the downstream targets of PKG activation that lead to the preventive effects of A2 receptor activation on the mitochondrial death pathway. Towards this goal we will characterize the roles of intracellular free zinc, mitochondrial KATP channels, and GSK-3. The successful execution of this proposal should produce important new insights into the signaling mechanisms underlying the protective effects of A2 receptor activation at reperfusion against ischemia/reperfusion injury, and have great clinical significance for treating patients with acute myocardial infarction.