PURINERGIC SIGNALING IN CULTURED HEART CELLS

培养心脏细胞中的嘌呤能信号传导

• 批准号: 3367385
• 负责人: BRUCE T LIANG
• 金额: $ 21.22万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3367385
• 关键词: adenosine; adenosine triphosphate; adenylate cyclase; chick embryo; cyclic AMP; embryo /fetus tissue /cell culture; heart cell; heart contraction; heart pharmacology; heart ventricle; isoproterenol; purinergic receptor; receptor coupling; receptor expression; receptor sensitivity; transfection

The cardiac actions of adenosine and adenosine 5'-triphosphate (ATP) are mediated by purinergic receptors present on cardiac myocytes. These receptors are classified into receptors for ATP (P2 purinergic) and receptors for adenosine (P1 purinergic), which is further subdivided into the adenosine A1 and A2 receptors. The adenosine A2 receptor, coupled to stimulation of adenylyl cyclase activity and myocyte contractility on ventricular myocytes represents a novel stimulatory purinergic receptor of potential physiologic and pathophysiologic significance in the heart. While cardiac adenosine A1 receptors have been characterized, very little is known about the adenosine A2 receptor and its subtypes (A2a and A2b), or the interaction between the A1 and A2 receptors. Further, since ATP exerts a pronounced positive inotropic effect and is readily hydrolyzed to adenosine, questions arise regarding whether activation of the adenosine A2 receptor contributes to the positive inotropic effect of ATP. The overall objective of the present proposal is to study the function and regulation of subtypes of adenosine A2 receptor and the interaction between subtypes of purinergic receptors. Specifically, using ventricular myocytes cultured from 14-day chick embryos as a model which express both A1 and A2 adenosine receptors, the following hypotheses will be tested. 1) The A2 subtypes exhibits differential regulation in response to adenosine agonists compared to the A1 subtype. 2) activation of the A2 subtype is capable of opposing the A1 subtype- mediated effect on the basal contractile amplitude. 3) stimulation of the A2 subtype contributes to the positive inotropic effect of ATP by a mechanism that involves an ATP-induced, adenosine A2 receptor-mediated stimulation of adenylyl cyclase activity and cyclic AMP accumulation. 4) activation of the A2 subtype is capable of opposing the A1 subtype- mediated antagonism of isoproterenol-stimulated increase in myocyte contractility, that is, the A1 subtype-mediated anti-adrenergic response to adenosine. A1-selective agonist and antagonist, selective desensitization of the adenosine A2 receptor pathway and blocking with an A2-selective antagonist will be used to determine the potential contractile functions of the A2 subtype as outlined in 2) through 4). Atrial myocytes cultured from 14-day embryos express the A1 but not the A2 subtype and exhibit a decrease in basal contractile amplitude in response to adenosine as well as a diminished positive inotropic response to ATP. The atrial myocyte therefore represents a useful cardiac model to test the relative importance of the A2a and A2b subtypes in mediating the various contractile functions listed in 2) thr 4). The cDNA encoding either the rate A2a or A2b receptor (inserted in the plasmid pcDNA 1, driven by CMV promoter) will be transfected individually into cultured atrial myocytes; the contractile effects of adenosine and ATP in myocytes expressing selectively the A2a or the A2b subtypes will be compared. These studies should provide significant insights into the function and regulation of subtypes of adenosine A2 receptor and into the mechanisms of cardiac action of ATP and adenosine in the heart.

腺苷和5'-三磷酸腺苷（ATP）的心脏作用是 由心肌细胞上存在的嘌呤能受体介导。 这些 受体分为ATP的受体（P2嘌呤能）和 腺苷的受体（P1嘌呤能），进一步细分为 腺苷A1和A2受体。 腺苷A2受体，耦合 刺激腺苷酸环化合酶活性和心肌收缩力 心室心肌细胞代表一种新型的刺激性嘌呤能受体 心脏潜在的生理和病理生理意义。 虽然心脏腺苷A1受体的表征很少 了解腺苷A2受体及其亚型（A2A和A2B）， 或A1和A2受体之间的相互作用。 此外，自ATP 发挥明显的阳性性肌效应，并且很容易水解 对于腺苷，关于激活是否激活的问题 腺苷A2受体有助于阳性肌力作用 ATP。 本提案的总体目的是研究 腺苷A2受体亚型的功能和调节 嘌呤能受体亚型之间的相互作用。 具体来说， 使用从14天小鸡胚胎培养的心室心肌细胞作为模型 同时表达A1和A2腺苷受体，以下 假设将进行检验。 1）A2亚型表现出差异 与A1亚型相比，对腺苷激动剂的响应。 2）A2亚型的激活能够反对A1亚型 介导对基底收缩幅度的影响。 3）刺激 A2亚型有助于A ATP的阳性肌力作用 涉及ATP诱导的腺苷A2受体介导的机制 刺激腺苷酸环化酶活性和环状AMP积累。 4）A2亚型的激活能够反对A1亚型 异丙肾上腺素刺激的介导肌细胞的介导的拮抗作用 收缩力，即A1亚型介导的抗肾上腺素能反应 到腺苷。 A1选择性激动剂和对手，选择性 腺苷A2受体途径的脱敏并与 A2选择性拮抗剂将用于确定电势 A2亚型的收缩功能如2）至4）。 从14天胚胎培养的心肌细胞表达A1，但不能 A2亚型并显示出基底收缩幅度的减少 对腺苷的反应以及阳性正性反应减少 到ATP。 因此，心肌细胞代表有用的心脏模型 测试A2A和A2B亚型在中介中的相对重要性 2）thr 4）中列出的各种收缩功能。 cDNA编码 速率A2A或A2B受体（插入质粒pcDNA 1， 由CMV启动子驱动）将分别转染到培养中 心肌细胞；腺苷和ATP在肌细胞中的收缩作用 将比较有选择地表达A2A或A2B子类型。 这些研究应提供有关功能和 调节腺苷A2受体的亚型和机制 心脏中ATP和腺苷的心脏作用。