AI ADENOSINE RECEPTOR IN CULTURED HEART CELLS

本研究的主要目的是研究这一进程， 心肌腺苷A1的脱敏和增敏机制 受体（A1 AR）介导的生理反应，以确定是否改变 在A1 AR和/或A1 AR调节的高亲和力形式的水平中， 心脏对A1 AR激动剂刺激的生理反应性，以及 确定A1 AR激活导致 A1 AR介导的收缩反应。 单层心房细胞培养自 将14天鸡胚用作模型系统。 具体而言，我们将： a）表征由A1 AR激动剂引起的功能反应 刺激; B）进行放射性配体结合的生物化学研究 这些培养细胞的膜中A1 AR的拮抗剂和激动剂。 我们将进一步确定1）A1 AR是否与各种 潜在的效应物如腺苷酸环化酶，磷酸二酯酶， 鸟苷酸环化酶或钾通道，我们将研究的作用， 这些潜在的效应因子介导A1 AR的收缩效应， 激动剂; 2）百日咳毒素敏感性GTP结合蛋白参与 A1 AR与各种效应物之间的偶联; 3） 培养的心房细胞对A1 AR激动剂的作用导致A1 AR的衰减。 介导的功能反应，以及这种反应性降低是否是 与高亲和力A1 AR向低亲和力A1 AR的转化相关， 形式，A1 AR的下调，或两者; 4）慢性治疗 用腺苷受体拮抗剂或腺苷脱氨酶培养 诱导A1 AR介导的功能反应的敏化， 低亲和力A1 AR转化为高亲和力形式， 受体的上调或通过两种机制; 5） A1 AR伴随着刺激性神经递质水平的代偿性增加。 G蛋白（Gs）或β-肾上腺素能受体（β AR）伴随 对β-肾上腺素能刺激的反应性增加; 6）另一方面 另一方面，这些抑制性受体的致敏作用与 Gs或β AR水平代偿性降低，伴随 β-肾上腺素能反应性降低。 这些研究应该有助于 阐明A1 AR的增敏和脱敏机制 系统以及A1 AR的调节作用及其高亲和力 在调节心房肌细胞对A1 AR激动剂敏感性中的作用 刺激. 它们还应提供有关机制的见解， 负责腺苷的细胞作用。

The main objectives of the present study are to study the process and mechanism of desensitization and sensitization of the cardiac A1 adenosine receptor (A1AR)-mediated physiologic response, to determine whether changes in the levels of A1AR and/or the high-affinity form of the A1AR regulate physiologic responsiveness of the heart to A1AR agonist stimulation, and to determine the molecular mechanism(s) by which activation of A1AR causes the A1AR-mediated contractile response. Monolayer atrial cells cultured from 14 day chick embryo will be used as a model system. Specifically, we will: a) characterize the functional responses elicited by A1AR agonist stimulation; b) carry out biochemical studies of the binding of radioligand antagonist and agonist to the A1AR in membranes of these cultures cells. We will further determine whether 1) A1AR are coupled to the various potential effectors such as the adenylate cyclase, phosphodiesterase, guanylate cyclase or the potassium channel, and we will study the role of these potential effectors in mediating the contractile effects of A1AR agonist; 2) pertussis toxin-sensitive GTP-binding protein(s) is involved in the coupling between A1AR and the various effectors; 3) chronic exposure of cultured atrial cells to A1AR agonist causes attenuation of the A1AR- mediated functional responses and whether such decreased responsiveness is associated with a conversion of the high-affinity A1AR to a low-affinity form, a downregulation of the A1AR, or both; 4) chronic treatment of the culture with an adenosine receptor antagonist or adenosine deaminase induces sensitization of the A1AR-mediated functional response by causing the conversion of low-affinity A1AR to a high-affinity form, an upregulation of the receptor or by both mechanisms; 5) desensitization of A1AR is accompanied by a compensatory increase in the level of stimulatory G protein (Gs) or beta-adrenergic receptors (beta AR) with a concomitant increase in responsiveness to beta-adrenergic stimulation; 6) on the other hand, sensitization of these inhibitory receptors is associated with a compensatory decrease in the level of Gs or beta AR with concomitant decrease in beta-adrenergic responsiveness. These studies should help elucidate the mechanism of sensitization and desensitization of the A1AR system as well as the role of the regulation of A1AR and its high-affinity form in modulating the sensitivity of atrial myocytes to A1AR agonist stimulation. They should also provide insights into the mechanisms responsible for the cellular action of adenosine.