CARDIAC A1-ADENOSINE RECEPTOR RESERVE

描述：(改编自申请者的摘要)最近在 申请者的实验室显示，腺苷抑制的效力 异丙肾上腺素刺激的大鼠心肌细胞L型钙内流(β-ICa，L) 心房肌细胞比激活内向整流高12倍 K+电流(IKAdo)。本申请中提出的研究是设计的 为了检验以下与细胞分化潜能相关的假设 腺苷抑制心房肌细胞β-ICA、L和激活IKAdo： 目的1-不同的A1受体亚型(A1a和A1b)抑制 β-ICA，L和IKAdo的激活；目的#2-单个受体偶联到 这两种反应，但有更大的受体储备，以抑制 β-ICA，L为激活IKAdo；目标#3-不同的A1ADOR激动剂 会有不同程度的受体储备。重要的是 受体储备作为心肌细胞对A1ADO受体反应性的决定因素 激动剂将被分解。一种新合成的不可逆A1ADOR 将使用拮抗剂灭活A1 ADORs以进行受体分析 保留。将使用药理学方法定义A1ADOR亚型和 要确定两者的受体占用-响应关系 A1ADOR介导的反应(激活IKAdo/抑制β-Ica，L) 心房肌细胞与心房单相的直接缩短 A1ADOR激动剂引起的动作电位(MAP)。研究将会是 用新鲜分离的豚鼠心房肌细胞和分离的 灌流的心脏。激活IKAdo和抑制β-ICA，L 腺苷和A1ADOR激动剂将通过使用整个细胞来记录 膜片钳技术。腺苷和阿司匹林引起的房图缩短 A1ADOR激动剂将通过标准电生理学进行定量 方法：研究方法。虽然这个项目的主要重点是受体储备， 他们还将调查(目标4)更高的效力和 潜在的更大的抗β-肾上腺素能作用储备可能会赋予 内源性腺苷对交感神经兴奋的紧张性抑制 麻醉豚鼠心脏。内生性假说 腺苷对心脏刺激作用的紧张性抑制作用 交感神经活性的增加将通过测量 硝普钠低血压引起的反射性心动过速的程度， 在A1ADOR拮抗剂和变构存在的情况下 激动剂与A1ADOR结合的增强剂。这些研究将具有重要的意义 A1ADOR激动剂的设计和使用在高血压中的临床意义 心脏病的治疗，以及腺苷在调节中的作用 心脏功能的变化。这些研究的长期目标是了解 对A1ADOR激动剂的器官和/或反应选择性的机制 是可以实现的。

DESCRIPTION: (Adapted from the Applicant's Abstract) Recent studies in the applicants' laboratory revealed that the potency of adenosine to inhibit isoproterenol-stimulated L-type calcium inward current (beta-ICa,L) of atrial myocytes is 12-fold higher than to activate the inwardly rectifying K+ current (IKAdo). The studies proposed in this application are designed to test the following hypotheses related to the differential potency of adenosine to inhibit beta-ICa,L and to activate IKAdo in atrial myocytes: Aim #1 - distinct A1 receptor subtypes (A1a and A1b) subserve inhibition of beta-ICa,L and activation of IKAdo; Aim #2 - a single receptor is coupled to both responses, but there is a greater receptor reserve for inhibition of beta-ICa,L than for activation of IKAdo; Aim #3 - different A1AdoR agonists will have different magnitudes of receptor reserve. The importance of receptor reserve as a determinant of cardiomyocyte responsiveness to A1AdoR agonists will be resolved. A newly synthesized irreversible A1AdoR antagonist will be used to inactivate A1AdoRs for the analysis of receptor reserve. Pharmacological methods will be used to define A1AdoR subtypes and to determine the receptor occupancy-response relationships for both A1AdoR-mediated responses (activation of IKAdo/inhibition of beta-Ica,L) in atrial myocytes and for the direct shortening of the atrial monophasic action potential (MAP) caused by various A1AdoR agonists. Studies will be carried out with guinea pig freshly isolated atrial myocytes and isolated perfused hearts. Activation of IKAdo and inhibition of beta-ICa,L by adenosine and A1AdoR agonists will be recorded by use of the whole cell patch-clamp technique. Shortening of the atrial MAP caused by adenosine and by A1AdoR agonists will be quantitated by standard electrophysiological methods. Although the main focus of this project is on receptor reserve, they will also investigate (Aim #4) whether the higher potency and potentially greater reserve for the anti beta-adrenergic action may confer tonic inhibition by endogenous adenosine of sympathetic activation of the heart in the anesthetized guinea pig. The hypothesis that endogenous adenosine exerts tonic inhibition of the cardiostimulatory effects of increased sympathetic neural activity will be tested by measuring the magnitude of reflex tachycardia caused by nitroprusside-induced hypotension, in the absence and presence of A1AdoR antagonists and of an allosteric enhancer of agonist binding to the A1AdoR. The studies will have important clinical implications for the design and use of A1AdoR agonists in the treatment of cardiac disease, and for the role for adenosine in regulation of cardiac function. The long-term goal of the studies is to understand mechanisms by which organ and/or response selectivity to A1AdoR agonists is achievable.