CELLULAR MECHANISM OF ADENOSINE'S CARDIAC ACTIONS

腺苷心脏作用的细胞机制

• 批准号: 3349016
• 负责人: LUIZ BELARDINELLI
• 金额: $ 18.78万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3349016
• 关键词: G protein; adenosine; cardiotonic agents; cell type; coronary vasodilator; cyclic AMP; electrophysiology; guinea pigs; heart block; heart contraction; heart function; heart pharmacology; isoproterenol; molecular pathology; myocardial ischemia /hypoxia; receptor coupling; tachycardia; western blottings

Adenosine (Ado) has electrophysiological effects relevant to the genesis and therapy of a number of cardiac arrhythmias. This proposal is a direct outgrowth of our ongoing research to define the underlying cellular mechanism(s) of Ado actions on cardiac myocytes. Although the main focus of this project is on A1-Ado receptor mediated actions, the cardiac A2-Ado receptor, known to stimulate adenylyl cyclase, will also be investigated. The study to be performed in guinea pig atrial and ventricular myocytes has four objectives aimed to elucidate the electrophysiological signals caused by activation of A1- and A2-Ado receptors and their transmembrane coupling mechanisms during normal and altered cellular responsiveness. The aims are to determine: 1) the coupling mechanism of A1-Ado induced activation of atrial inwardly rectifying K+ current (IKAdo) and isoproterenol (ISO)- stimulated calcium inward current (ICa), 2) the time-course and magnitude of desensitization of A1-Ado mediated-responses and underlying mechanism 3) determine the electrophysiological response to activation of A2-Ado receptors, 4) the effect of Ado on basal ICa (atria) and ISO-stimulated delayed rectifier K+ current (IK, ventricle) and the consequences of these actions for atrial contractility and ventricular action potential duration (APD), respectively. Ado effects on single KAdo-channel activity and on macroscopic currents IKAdo, basal ICa, ISO-stimulated ICa and IK will be measured with the patch-clamp technique in inside-out membrane patches and whole-cells, respectively. To define the A1-Ado receptor coupling mechanism, the effect of the monoclonal antibody-4A (against alpha1) on Ado-mediated effects on membrane currents will be determined. The magnitude and time-course of pertussis toxin-induced ADP-ribosylation of alpha1 will be correlated with inhibition of the actions of Ado and receptor-effector coupling efficiency determined with the irreversible A1- Ado receptor antagonist (M-DITC-XAC). To elucidate potential mechanisms for the altered responsiveness of cells (desensitization) to A1-Ado mediated actions, changes in receptor number and affinity, G-protein subunits, and receptor-effector coupling efficiency will be determined. The hypothesis that A2-agonist. The effect of Ado on basal ICa in atrial cells will be investigated under conditions in which cAMP-mediated effects are abolished by the cAMP-dependent protein kinase inhibitor Rp-cAMPS. Concomitant with ICa measurements, twitch shortening of the myocytes will be monitored with a video-image edge detector. Standard voltage-clamp protocols will be used to determine the action of Ado on ISO-stimulated IK and the extent by which the effect modulates ventricular APD during beta- adrenergic stimulation. This multidisciplinary and detailed approach should provide new insight into the cellular mechanisms underlying the cardiac actions of Ado and further establish the importance of this nucleoside as a modulator of cardiac function.

腺苷 (Ado) 具有与起源相关的电生理效应 以及多种心律失常的治疗。 这个提议是直接的 我们正在进行的研究的成果，以定义潜在的细胞 Ado 对心肌细胞的作用机制。 虽然主要焦点 该项目的重点是 A1-Ado 受体介导的作用，心脏 A2-Ado 已知可刺激腺苷酸环化酶的受体也将受到研究。 在豚鼠心房和心室肌细胞中进行的研究 旨在阐明引起的电生理信号的四个目标 通过激活 A1- 和 A2-Ado 受体及其跨膜偶联 正常和改变的细胞反应期间的机制。 目标是 确定：1）A1-Ado诱导激活的偶联机制 心房内向整流 K+ 电流 (IKAdo) 和异丙肾上腺素 (ISO)- 受激钙内向电流（ICa），2）时程和幅度 A1-Ado 介导反应的脱敏及其潜在机制 3) 确定 A2-Ado 激活的电生理反应 受体，4) Ado 对基础 ICa（心房）和 ISO 刺激的影响 延迟整流 K+ 电流（IK，心室）以及这些的后果 心房收缩力和心室动作电位持续时间的动作 （APD），分别。 Ado 对单个 KAdo 通道活性的影响 宏观电流 IKAdo、基础 ICa、ISO 刺激 ICa 和 IK 将 使用膜片钳技术在从内到外的膜片上进行测量 分别是全细胞。 定义 A1-Ado 受体偶联 机制，单克隆抗体 4A（针对 alpha1）对 将确定 Ado 介导的对膜电流的影响。 这 百日咳毒素诱导的 ADP-核糖基化的强度和时间过程 alpha1 将与 Ado 和 Ado 的作用的抑制相关 用不可逆的 A1- 确定受体-效应器耦合效率 Ado 受体拮抗剂（M-DITC-XAC）。 阐明潜在机制 改变细胞对 A1-Ado 的反应性（脱敏） 介导的作用、受体数量和亲和力的变化、G 蛋白 亚基和受体-效应器偶联效率将被确定。 假设A2-激动剂。 Ado对心房基础ICa的影响 将在 cAMP 介导的效应条件下研究细胞 被 cAMP 依赖性蛋白激酶抑制剂 Rp-cAMPS 消除。 伴随着 ICa 测量，肌细胞的抽搐缩短将 使用视频图像边缘检测器进行监控。 标准电压钳 协议将用于确定 Ado 对 ISO 刺激的 IK 的作用 以及该效应在 β- 期间调节心室 APD 的程度 肾上腺素能刺激。 这种多学科和详细的方法 应该为潜在的细胞机制提供新的见解 Ado 的心脏作用并进一步确立了这一点的重要性 核苷作为心脏功能的调节剂。