CELLULAR MECHANISM OF ADENOSINE'S CARDIAC ACTIONS

腺苷（Adenosine，Ado）具有与肿瘤发生相关的电生理效应 和治疗多种心律失常。 这是一个直接的建议。 我们正在进行的研究的结果，以确定潜在的细胞 Ado对心肌细胞作用的机制。 虽然主要焦点 本项目的重点是A1-Ado受体介导的作用，心脏A2-Ado 受体，已知刺激腺苷酸环化酶，也将进行研究。 在豚鼠心房和心室肌细胞中进行的研究 四个目标旨在阐明电生理信号引起的 通过激活A1-和A2-Ado受体及其跨膜偶联 在正常和改变的细胞反应机制。 其宗旨是 为了确定：1）A1-Ado诱导的激活的偶联机制， 心房内向整流钾电流（IKAdo）和异丙肾上腺素（ISO）- 刺激性钙内向电流（伊卡）; 2）时程和幅度 A1-Ado介导的反应的脱敏和潜在机制3） 确定对A2-Ado激活的电生理反应 4）Ado对基础伊卡（心房）和ISO刺激的ICa（心房）的作用 延迟整流钾电流（IK，心室）和这些结果 心房收缩力和心室动作电位时程的作用 (APD)，分别。 Ado对单钾通道活性的影响， 宏观电流IKAdo、基础伊卡、ISO刺激的伊卡和IK将是 用膜片钳技术在由内而外的膜膜片中测量， 全细胞，分别。 定义A1-Ado受体偶联 机制，单克隆抗体-4A（抗α 1）对 将确定对膜电流的α介导效应。 的 百日咳毒素诱导的ADP-核糖基化的幅度和时间过程 α 1将与抑制Ado的作用相关， 用不可逆A1- Ado受体拮抗剂（M-DITC-XAC）。 阐明潜在的机制 细胞对A1-Ado的反应性改变（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-效应物偶联效率将被确定。 假设A2-激动剂。 腺苷对心房肌基础伊卡的影响 细胞将在cAMP介导的作用 被cAMP依赖性蛋白激酶抑制剂Rp-cAMPS消除。 伴随着伊卡测量，肌细胞的收缩缩短将 使用视频图像边缘检测器进行监控。 标准电压钳 将使用协议来确定Ado对ISO刺激的IK的作用 以及该效应在β- 肾上腺素能刺激 这种多学科和详细的方法 应该提供新的洞察细胞机制的基础上， Ado的心脏作用，并进一步确定其重要性， 核苷作为心脏功能调节剂。

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.