ALLOSTERIC ENHANCEMENT OF THE CARDIAC ADENOSINE RECEPTOR

最近，一系列2-氨基-3-苯甲酰基噻吩类化合物被证明是 腺苷(ADO)对A_1~+作用的变构增强剂 大鼠脑和FRTL-5甲状腺细胞的腺苷受体(A1-ADAR) 排队。心脏A1-ADAR系统的阳性变构尚未得到 之前被调查过。本研究方案设计为1) 描述并确定由以下原因引起的增强的特异性 外源性心脏活动的原型声发射，PD 81,723(Pd) 内源性ADO，2)确定A1-ADOR对PD的选择性 ADAR亚型，并对变构增强进行数学建模，3) 研究PD反应的物种和受体亚型的差异， 和用重组ADOR确定PD结合结构域(S)，和4) 合成和筛选具有更高效价和选择性的新AES ADAR亚型。为了实现这些广泛的目标，多学科的 (药理学、分子生物学、化学)方法。 研究将在豚鼠离体心和新鲜心脏上进行 分离的心肌细胞、培养的细胞系和稳定的细胞 转染天然和突变的重组ADAR亚型。这个 PD对ADAR介导的房室结传导减慢的影响 温度和膜电流(如IKAdo)在单个心房和 将使用电生理学方法对心室肌细胞进行定量 技巧。钯对心肌细胞膜结合的影响 对具有与腺苷类似效果的药物的反应(例如， 卡巴胆碱)将用于评估PD作为增强剂的特异性 阿多的行为。原子发射光谱分析对心脏的增强作用 对内源性ADO、缺氧、缺血和ADO抑制剂的反应 代谢将被用来提升心肌间质 在存在和不存在PD的情况下，ADO浓度。要确定 Pd对给定的ADAR亚型的选择性，Pd对A1介导型的影响 房室结传导减慢和A2介导的冠状动脉扩张 将比较和放射性配基结合的ADAR激动剂到重组 A1、A2a、A2b和A3-ADORs在CHO细胞中永久表达 DDT1MF-2和PC-12细胞内源性A1-和A2-ADO受体 量化的。Pd和Pd之间变构相互作用的性质 将通过研究AES对ADAR激动剂的影响来评估ADAR， 和拮抗剂放射性配基结合参数，b)亲和状态 A1-ADOR；c)ADORs与G蛋白的偶联。帕金森氏病对人体健康的影响 ADAR激动剂与克隆和表达的ADAR亚型的结合 各种物种(包括人类)和嵌合受体结构 将用于指导定点突变工作，以识别 对声发射活动具有重要意义的区域(S)。的最终目标是 这项建议是为了证明变构增强剂的作用 是提高对当地释放的ADO的反应的有用的药物 在心脏中，从而起到部位=特异性药物的作用。这些化合物可能 事实证明，这是选择性和特异性治疗的理想方法 放大内源性腺苷的作用。

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.