CELLULAR MECHANISM OF ADENOSINE CARDIAC ACTIONS

腺苷(ADO)具有与发生有关的电生理效应。 以及一些心律失常的治疗。这项建议是一项直接的 我们正在进行的研究成果，以定义潜在的细胞 腺苷对心肌细胞的作用机制(S)。虽然主要关注的是 这个项目是关于A1-腺苷受体介导的作用，心脏的A2-腺苷 已知的刺激腺酰环化酶的受体也将被研究。 这项将在豚鼠心房和心室肌细胞上进行的研究 四个目标旨在阐明引起的电生理信号 A1-和A2-腺苷受体的激活及其跨膜偶联 在正常和改变细胞反应期间的机制。我们的目标是 确定：1)A1-Ado诱导细胞活化的偶联机制 心房内向整流钾电流(IKAdo)和异丙肾上腺素(ISO) 刺激钙内流(ICa)，2)时程和幅度 A1-Ado介导的反应脱敏及其潜在机制3) 测定激活A2-腺苷的电生理反应 受体，4)腺苷对基础ICA(心房)和异丙肾上腺素刺激的影响 延迟整流钾电流(Ik，心室)及其后果 对心房收缩功能和心室动作电位时程的影响 (Apd)。ADO对单个KADO通道活动的影响 宏观电流IKAdo、基础ICa、ISO刺激的ICa和Ik将 用膜片钳技术测量由内向外的膜片和 分别为全细胞。定义A1-腺苷受体偶联 抗α1单抗-4A的作用机制 ADO介导的膜电流效应将被确定。这个 百日咳毒素诱导的ADP核糖化的大小和时程 α1将与抑制ADO和Ado的活动相关 用不可逆A1-受体偶联效率测定受体-效应偶联效率 腺苷受体拮抗剂(M-DITC-XAC)。阐明潜在的机制 细胞对A1-腺苷反应的改变(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-效应器的偶联效率将被确定。 假设A2激动剂。腺苷对心房基础ICA的影响 细胞将在cAMP介导的效应的条件下进行研究 都被cAMP依赖的蛋白激酶抑制剂RP-cAMPS所废除。 伴随着ICA测量，心肌细胞的抽动缩短将 使用视频图像边缘探测器进行监测。标准电压钳位 将使用协议来确定ADO在ISO刺激的IK上的作用 以及该效应在β-半衰期对心室动作电位时程的调节程度。 肾上腺素能刺激。这种多学科和详细的方法 应该提供了对潜在的细胞机制的新的洞察 ADO的心脏作用，并进一步确定这一点的重要性 核苷作为心脏功能的调节剂。

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.