ALLOSTERIC ENHANCEMENT OF THE CARDIAC ADENOSINE RECEPTOR

最近，一系列2-氨基-3-苯甲酰基噻吩被证明是 腺苷（Ado）对A1- 大鼠脑和FRTL-5甲状腺细胞中的Ado受体 线 心脏A1-β R系统的正变构作用尚未被证实。 以前调查过。 本研究旨在：（1） 表征并确定由以下因素引起的增强的特异性 外源性心脏活动的原型AE，PD 81，723（PD）， 内源性Ado，2）定义PD对A1-ADIR的选择性， 亚型，并对变构增强进行数学建模，3） 研究对PD反应的物种和受体亚型差异， 并使用重组miR确定PD结合结构域，和4） 合成和筛选具有改进效力和选择性的新Aes BXR亚型。 为了实现这些广泛的目标，多学科 （药理学、分子生物学、化学）方法。 研究将使用豚鼠离体心脏和新鲜的 分离的心肌细胞、培养的细胞系和稳定的细胞 用天然的和突变的重组WXR亚型转染。 的 帕金森病对房室结传导阻滞的影响 热量和膜电流（例如，IKAdo）单心房和 心室肌细胞将使用电生理学方法定量 技术. 帕金森病对心肌细胞膜结合的影响 对具有与Ado类似的效果的药物的反应（例如， 卡巴胆碱）将被确定，以评估PD作为增强剂的特异性 阿多的行为 研究Aes对心肌细胞的增强作用， 对内源性Ado、缺氧、缺血和Ado抑制剂的反应 代谢将被用来提高心肌间质 在PD存在和不存在下的Ado浓度。 确定 PD对给定AXR亚型的选择性，PD对A1介导的 房室结传导减慢和A2介导的冠状血管舒张 将进行比较，并将放射性配体结合的BXR激动剂的重组 A1、A2 a、A2 b和A3-miR在CHO细胞中永久表达， DDT 1 MF-2和PC-12细胞系中的内源性A1-和A2-miR将被 定量。 PD和细胞间的别构相互作用的性质 将通过研究Aes对a）AesR激动剂的作用来评估AesR， 和拮抗剂放射性配体结合参数，B） A1-A1 R，和c）A1 R与G蛋白的偶联。 PD对 BXR激动剂与克隆和表达的BXR亚型的结合， 各种物种（包括人）和嵌合受体构建体 将用于指导定点诱变工作，以确定 对AE活动重要的BXR区域。 的最终目标 这一建议是为了证明， 的Ado是增加对局部释放的Ado的反应的有用试剂 在心脏中，从而作为部位=特异性药物。 这些化合物可以 证明是一种理想的治疗方法， 增强内源性腺苷的作用。

Recently a series of 2-amino-3 benzoylthiophenes were shown to be allosteric enhancers (AEs) of the actions of adenosine (Ado) on the A1- Ado receptor (A1-AdoR) in the rat brain and in the FRTL-5 thyroid cell line. Positive allosterism of the cardiac A1-AdoR system has not been previously investigated. This research proposal is designed to 1) characterize and establish the specificity of the enhancement caused by the prototype AE, PD 81,723 (PD), of the cardiac actions of exogenous and endogenous Ado, 2) define the selectivity of PD for the A1-AdoR vs other AdoR subtypes, and mathematically model the allosteric enhancement, 3) investigate species and receptor-subtype differences in response to PD, and determine the PD binding domain(s) using recombinant AdoRs, and 4) synthesize and screen new Aes with improved potency an selectivity for AdoR subtypes. To accomplish these broad objectives, a multidisciplinary (pharmacology, molecular biology, chemistry) approach is proposed. Studies will be carried out with guinea pig isolated hearts and freshly isolated cardiomyocytes, cultured cell lines, and cells stably transfected with native and mutated recombinant AdoR subtypes. The effect of PD on AdoR-mediated slowing of AV nodal conduction in isolated heats and on membrane currents (e.g., IKAdo) in single atrial and ventricular myocytes will be quantitated using electrophysiological techniques. The effects of PD on membrane binding of, and cardiac responses to, drugs that have effects similar to those of Ado (e.g., carbachol) will be determined to assess the specificity of PD as enhancer of the actions of Ado. To investigate potentiation by Aes of cardiac responses to endogenous Ado, hypoxia, ischemia, and inhibitors of Ado metabolism will be used to elevate the myocardial interstitial concentration of Ado in the presence and absence of PD. To determine the selectivity of PD for a given AdoR subtype, PD's effects on A1-mediated slowing of AV nodal conduction and on A2-mediated coronary vasodilation will be compared, and radioligand binding of AdoR agonists to recombinant A1,A2a,A2b and A3-AdoRs permanently expressed in CHO cells and to endogenous A1- and A2-AdoRs in DDT1MF-2 and PC-12 cell lines will be quantitated. The nature of the allosteric interaction between PD and the AdoR will be assessed by studying the effect of Aes on a) AdoR agonist, and antagonist radioligand binding parameters, b) affinity states of the A1-AdoR, and c) coupling of AdoRs to G proteins. The effects of PD on binding of AdoR agonists to cloned and expressed AdoR subtypes from various species (including human) and to chimeric receptor constructs will be used to guide site-directed mutagenesis efforts to identify the region(s) of the AdoR important for AE activity. The ultimate goal of this proposal is to demonstrate that allosteric enhancers of the actions of Ado are useful agents to increase the response to locally released Ado in the heart and thereby act as site=specific drugs. These compounds may prove to be an ideal therapeutic approach to selectively and specifically amplify the actions of endogenous adenosine.