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 本实验室的利益：定量测定 大脑中的毒蕈碱受体，以及 通过磷酸肌醇裂解途径激活受体。 这些理解胆碱能信号转导的方法 在中枢神经系统中，第一个细胞外和第二个细胞内， 并在这里结合探索胆碱能的实验模型， 功能障碍 我们的大部分生物化学知识 到目前为止，受体功能仅限于 体外标记配体与膜的受体结合， 突触神经体或脑切片， 暴露于神经活性剂。 我们在这里研究的影响， 慢性体内状况，如传入神经阻滞或暴露于 药物，不仅对受体数量的调节， 亲和力，而且还对PPI相关的调节功能 第二信使，受体激活的结果。 这些 包括：二酰基甘油激活的蛋白激酶C和 三磷酸肌醇释放后Ca 2+的移动。 在 慢性病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物 抗胆碱能药、抗抑郁药和Li+。 一项拟议 发展模型是基于我们观察到的增强耦合 毒蕈碱受体对胎儿和新生儿细胞反应的影响 个脑袋 毒蕈碱受体调节与 还将探索其他神经递质效应系统。 我们还将研究磷酸肌醇在 突触前功能，以及各种药物的作用 乙酰胆碱释放的治疗。 将用于解决这一问题的技术包括 32 P标记的磷酸肌醇周转，（3 H）磷酸肌醇 积累和化学测定甘油二酯， 磷脂酰肌醇。 肌醇磷酸代谢将是 研究，包括肌醇的形成和分解 三磷酸和肌醇四磷酸。 蛋白 在控制和受体调节条件下的磷酸化 将特别强调蛋白激酶C- 在突触囊泡和生长锥介导的反应。 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.