PURINERGIC SIGNALING IN CULTURED HEART CELLS

腺苷和三磷酸腺苷（ATP）的心脏作用是 由心肌细胞上的嘌呤能受体介导。 这些 受体分为ATP受体（P2嘌呤能）和 腺苷受体（P1嘌呤能），进一步细分为 腺苷A1和A2受体。 腺苷A2受体， 腺苷酸环化酶活性和肌细胞收缩性的刺激， 心室肌细胞是一种新的刺激性嘌呤能受体 在心脏中具有潜在的生理和病理生理意义。 虽然心脏腺苷A1受体已被表征，但很少 已知腺苷A2受体及其亚型（A2 a和A2 b）， 或A1和A2受体之间的相互作用。 此外，由于ATP 发挥显著的正性肌力作用， 对于腺苷，出现了关于是否激活 腺苷A2受体有助于正性肌力作用 ATP 本提案的总体目标是研究 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。 具体地说， 以14天鸡胚心室肌细胞为模型， 其表达A1和A2腺苷受体， 将对假设进行检验。 1)A2亚型表现出不同的 与A1亚型相比，对腺苷激动剂的反应调节。 2)A2亚型的激活能够对抗A1亚型- 介导对基础收缩幅度的影响。 3)刺激 A2亚型通过以下方式促进ATP的正性肌力作用： ATP诱导的腺苷A2受体介导的 腺苷酸环化酶活性和环AMP积累的刺激。 4)A2亚型的激活能够对抗A1亚型- 异丙肾上腺素刺激的肌细胞增殖的介导拮抗作用 收缩性，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受体通路的脱敏和用 A2选择性拮抗剂将用于确定 A2亚型的收缩功能，如2）至4）所述。 从14天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降低， 对腺苷的反应以及减弱的正性肌力反应 到ATP。 因此，心房肌细胞代表了一种有用的心脏模型 检测A2 a和A2 b亚型在介导 2）thr 4）中列出的各种收缩功能。 的cdna RATE A2 a或A2 b受体（插入质粒pcDNA 1， 由CMV启动子驱动）将单独转染到培养的 心房肌细胞;腺苷和ATP的收缩作用 将比较选择性表达A2 a或A2 b亚型的细胞。 这些研究应该提供重要的见解的功能， 腺苷A2受体亚型的调节及其机制 心脏中ATP和腺苷的心脏作用。

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.