PURINERGIC SIGNALING IN CULTURED HEART CELLS

腺苷和腺苷5‘-三磷酸(ATP)的心脏作用是 由存在于心肌细胞上的嘌呤能受体介导。这些 受体分为三磷酸腺苷受体(P2嘌呤能)和 腺苷受体(P1能)，它进一步细分为 腺苷A1和A2受体。腺苷A2受体，偶联 对腺苷环化酶活性和心肌细胞收缩功能的刺激作用 心肌细胞是一种新的刺激性嘌呤能受体 心脏的潜在生理学和病理生理学意义。 虽然已经确定了心脏腺苷A1受体的特征，但很少 已知腺苷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，由于ATP 具有显著的正性肌力作用，且易被水解。 对于腺苷，出现了关于是否激活 腺苷A2受体参与正性变力作用 三磷酸腺苷。本提案的总体目标是研究 腺苷A2受体亚型的功能与调控 嘌呤能受体亚型之间的相互作用。具体来说， 以14日龄鸡胚培养的心肌细胞为模型 它们同时表达A1和A2腺苷受体，如下 假说将会得到检验。1)A2亚型表现出差异性 与A1亚型相比，腺苷激动剂的反应调节。 2)A2亚型的激活能够对抗A1亚型- 对基础收缩幅度的中介作用。3)刺激 A2亚型参与了ATP的正性变力作用，其机制是通过 涉及ATP诱导、腺苷A2受体介导的机制 刺激腺苷环化酶活性和环磷酸腺苷的积累。 4)A2亚型的激活能够对抗A1亚型- 异丙肾上腺素对心肌细胞增殖的介导拮抗作用 收缩能力，即A1亚型介导的抗肾上腺素能反应 到腺苷。A1-选择性激动剂和拮抗剂，选择性 腺苷A2受体途径的脱敏及其拮抗作用 将使用A2选择性拮抗剂来确定潜在的 A2亚型的收缩功能，如2)至4)所述。 从14日龄胚胎培养的心房肌细胞表达A1，但不表达 A2亚型并表现出基础收缩幅度的降低 对腺苷的反应以及正性变力反应减弱 为ATP干杯。因此，心房肌细胞是一种有用的心脏模型。 测试A2a和A2b亚型在中介中的相对重要性 2)、4)中列出的各种收缩功能。编码基因的c DNA A2a或A2b受体的速率(插入到质粒pcDNA1中， 由CMV启动子驱动)将单独导入培养的 腺苷和三磷酸腺苷对心肌细胞的收缩作用 选择性地表达A2a或A2b亚型将被比较。 这些研究应该提供对功能和功能的重要见解 腺苷A2受体亚型的调控及其机制研究 三磷酸腺苷和腺苷在心脏中的心脏作用。

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.