REGULATION OF PROSPHOINOSITIDE-LINKED CNS RECEPTORS

该计划项目合并了两个长期和积极进行中的 本实验室的兴趣：定量测定 大脑中的M受体，以及脑组织的生物化学 通过肌醇磷脂裂解途径激活受体。 这些理解胆碱能信号转导的方法 在中枢神经系统，第一个细胞外和第二个细胞内， 并在此结合探讨胆碱能的实验模型。 功能障碍。我们的大部分生化知识都是关于 到目前为止，受体的功能仅限于 体外标记配体与膜的受体结合， 急性或慢性后的突触神经体或脑切片 暴露在神经活性物质中。我们在这里研究一下 体内慢性情况，如去传入或暴露于 药物，不仅依赖于受体数量的调节，而且 亲和力，也是对PPI相关功能的调节 第二信使，受体激活的结果。这些 包括：二酰甘油激活的蛋白激酶C和 肌醇三磷酸释放后钙离子的动员。在……里面 慢性化的体外模型包括长期暴露于 神经母细胞瘤和原代神经元或神经胶质细胞培养 抗胆碱能药、抗抑郁药和对Li+。一项建议 发育模型是基于我们观察到的增强耦合 M受体在胎儿和新生儿细胞反应中的作用 大脑。毒扁豆碱受体的调节可能与 还将探索其他神经递质-效应器系统。 我们还将研究肌醇磷脂在体内的作用。 突触前功能，以及各种药物的作用 对乙酰胆碱释放的治疗。 将用于解决此问题的技术包括 ~(32)P标记的磷脂酰肌醇转换率 甘油二酯和甘油二酯的积累及化学测定 磷脂酰肌醇。肌醇磷酸代谢将是 调查，包括肌醇的形成和分解 三磷酸和四磷酸肌醇。蛋白 控制和受体调节条件下的磷酸化 将特别着重于蛋白激酶C- 在突触小泡和生长锥体中的中介反应。 受体数量和亲和力的变化将通过 定量受体放射自显影。

This program project merges two long-term and actively ongoing interests of this laboratory: the quantitative determination of muscarinic receptors in the brain, and the biochemistry of receptor activation via the phosphoinositide cleavage pathway. These approaches to understanding cholinergic signal transduction in the CNS, the first extracellular and the second intracellular, and here combined to explore experimental models of cholinergic dysfunction. Much of our biochemical knowledge regarding receptor function up to the present has been restricted to the receptor binding of labeled ligands in vitro to membranes, synaptoneurosomes or brain sections following acute or chronic exposure to a neuroactive agent. We here examine the effects of chronic in vivo conditions, such as deafferentation or exposure to drugs, not only upon the regulation of receptor number and affinity, but also on the regulation of the function of PPI-related second messengers, a consequence of receptor activation. These include: the diacylglycerol-activated protein kinase C and the mobilization of Ca2+ following inositol trisphosphate release. In vitro models of chronicity involve long-term exposure of neuroblastoma and primary neuronal or glial cultures to various anticholinergic agents, antidepressants and to Li+. A proposed developmental model is based on our observed enhanced coupling of muscarinic receptors to the cell response in fetal and neonatal brain. Possible interaction of muscarinic receptor regulation with other neurotransmitter-effector systems will also be explored. We will also investigate the role of phosphoinositides in presynaptic function, as well as the effects of the various drug treatments on acetylcholine release. Techniques that will be brought to bear upon this problem include 32P-labeled phosphoinositide turnover, (3H)inositol phosphate accumulation, and chemical determination of diacylglycerol and of the phosphoinositides. Inositol phosphate metabolism will be investigated, including the formation and breakdown of inositol trisphosphate and inositol tetraphosphate. Protein phosphorylation under control and receptor-regulated conditions will be examined with particular emphasis on protein kinase C- mediated reactions in synaptic vesicles and growth cones. Changes in receptor number and affinity will be localized via quantitative receptor autoradiography.