DOPAMINE D2-RECEPTOR MEDIATED SIGNAL TRANSDUCTION

这个项目的总体目标是探索信号转导。 多巴胺通过D2受体的作用机制，相信 这可能会阐明这些受体在调节 神经元活动。因此，目前的提案需要两个 方法-首先，建立一个模型细胞系统，在这个系统中，多巴胺 细胞内第二信使cAMP和[Ca~(2+)]_i的调节作用 可以描绘，其次，要研究电生理 D2受体介导的信号发生生化扰动的后果 纹状体中的转导。要评估的模型系统将是 神经母细胞瘤x中国仓鼠胚胎脑外植体杂交细胞系 (NCB-20)，它表达多巴胺、5-羟色胺、缓激肽、 鸦片和乙酰胆碱。第一组实验将确定 D2受体是否抑制NCB-20细胞的腺苷环化酶 这些受体是否也可能扰乱这些细胞的[Ca~(2+)]i动员 细胞，通过测量D2受体对磷脂酰肌醇的影响 代谢和[Ca~(2+)]i动员。在第二组实验中， 将优化纹状体内注射百日咳的条件 毒素，使很大一部分百日咳毒素底物 是ADP核糖化的；将对其有效性进行确定 这种治疗消除了受体介导的抑制 腺苷环化酶；随后，受体介导的改变 将对这些动物的电生理特性进行检测 与假注射对照组相比。

The overall goal of this project is to explore signal transduction mechanisms for dopamine acting through D2-receptors, in the belief that this may elucidate the role played by these receptors in regulating neuronal activity. Consequently, the present proposal takes two approaches - first, to establish a model cell system in which dopamine's role in modulating the intracellular second messengers, cAMP and [Ca2+]i can be delineated and secondly, to investigate the electrophysiological consequences of a biochemical perturbation of D2-receptor mediated signal transduction in striatum. The model system to be evaluated will be a neuroblastoma x Chinese hamster embryonic brain explant hybrid cell line (NCB-20), which expresses receptors for dopamine, serotonin, bradykinin, opiates and acetylcholine. The first group of experiments will determine whether D2-receptors inhibit adenylate cyclase in NCB-20 cells and whether these receptors also may perturb [Ca2+]i mobilization in these cells, by measuring D2-receptor effects on phosphatidylinositol metabolism and [Ca2+]i mobilization. In the second group of experiments, conditions will be optimized for the intrastriatal injection of pertussis toxin, so that a significant portion of the pertussis toxin substrates are ADP-ribosylated; a determination will be made on the effectiveness with which this treatment eliminates receptor-mediated inhibition of adenylate cyclase; subsequently, receptor-mediated alterations in electrophysiological properties will be examined in these animals compared to sham-injected controls.