PROTEIN(S) INVOLVED IN NEUROTRANSMISSION

5-羟色胺(5-羟色胺)是中枢神经系统的重要神经递质 系统和突触后5-羟色胺受体与5-羟色胺摄取系统 与人类的病因学和/或治疗有关 精神抑郁。该项目将使用两个芳基叠氮化合物作为光亲和力 从分子水平表征5-羟色胺-1a受体和 5-羟色胺摄取载体。这些信息将极大地帮助 对精神抑郁的理解。两个探测器，它们是开发出来的 在这个实验室里， 1-(2-(4-azidophenyl)ethyl)-4-(3-trifluoromethylphenyl)piperazine (P-叠氮基-TFMPP)和5-羟色胺叠氮苯甲胺(SABA)。 P-叠氮基-TFMPP与5-羟色胺-1A的结合具有很高的亲和力 用氚化合物进行的实验将在大鼠脑中进行 以评估标记的特异性。一旦这是 证明，[~3H]p-叠氮基-TFMPP将被用来共价标记 光解实验中的5-羟色胺-1A受体。标记的蛋白质将是 SDS-聚丙烯酰胺凝胶电泳法和5-羟色胺受体分离 激动剂和拮抗剂将用于鉴定多肽 与5-羟色胺-1A受体有关。药理程序将是 用来显示多肽的突触位置。一名活跃分子 5-羟色胺-1A受体将用洗涤剂增溶，并通过 以对叠氮基-TFMPP的对氨基类似物为亲和层析 柱状配体。将使用p-叠氮基-TFMPP进行实验以评估 5-羟色胺-1A受体与5-羟色胺刺激的关系 大鼠海马腺苷环化酶。光解实验 未标记的p-叠氮基-TFMPP将证明5-羟色胺是否刺激 腺苷环化酶可在抑制的同时发生不可逆转的抑制 [~3H]p-叠氮基-TFMPP与5-HT-1A受体结合。 5-羟色胺摄取位点光亲和标记的特异性 将建立大鼠脑粘液体和[~3H]SABA人血小板 SABA标记的多肽将通过以下方法分离和鉴定 光解组织样品的SDS-聚丙烯酰胺凝胶电泳法。 三环类抗抑郁药对标记物的保护作用 将对载体多肽进行评估以确定这些 药物通过与含有5-羟色胺的相同多肽结合来抑制5-羟色胺的摄取 底物结合部位。特异性蛋白质的比较 用[~3H]SABA和[~H]叠氮丙咪胺标记将进一步提供 关于载体大分子和分子间关系的信息 三环结合部位。

Serotonin (5-HT) is an important neurotransmitter in the central nervous system and postsynaptic serotonin receptors and the serotonin uptake system have been implicated in the etiology and/or therapeutic treatment of human mental depression. This project will use two aryl azides as photoaffinity probes to characterize, in molecular terms, the 5-HT-1A receptor and the serotonin uptake carrier. This information should greatly aid in the understanding of mental depression. The two probes, which were developed in this laboratory, are 1-(2-(4-azidophenyl)ethyl)-4-(3-trifluoromethylphenyl)piperazine (p-azido-TFMPP) and serotonin azidobenzamidine (SABA). p-Azido-TFMPP has high affinity for the 5-HT-1A site and binding experiments with the tritiated compound will be performed with rat brain membranes to assess the specificity of the labeling. Once this is demonstrated, [3H]p-azido-TFMPP will be used to covalently label the 5-HT-1A receptor in photolysis experiments. The labeled proteins will be separated by SDS-polyacrylamide gel electrophoresis and serotonin receptor agonists and antagonists will be used to identify the polypeptide associated with the 5-HT-1A receptor. Pharmacological procedures will be used to demonstrate the synaptic location of the polypeptide. An active 5-HT-1A receptor will be solubilized with detergents and purified by affinity chromatography using the p-amino analogue of p-azido-TFMPP as the column ligand. Experiments will be performed with p-azido-TFMPP to assess the relationship between the 5-HT-1A receptor and the serotonin stimulated adenylate cyclase in rat hippocampus. Photolysis experiments with unlabeled p-azido-TFMPP will demonstrate whether the serotonin stimulated adenylate cyclase can be irreversibly inhibited concomitant with inhibition of [3H]p-azido-TFMPP binding to 5-HT-1A receptors. The specificity of the photoaffinity labeling of the 5-HT uptake site in rat brain syanptosomes and human platelets by [3H]SABA will be established [3H]SABA labeled polypeptides will be separated and identified by SDS-polyacrylamide gel electrophoresis of the photolyzed tissue samples. The ability of tricyclic antidepressants to protect against labeling of the carrier polypeptide will be evaluated to determine whether or not these drugs inhibit 5-HT uptake by binding to the same polypeptide that contains the substrate binding site. Comparison of the proteins specifically labeled by [3H]SABA and [3H]azidoimipramine will provide further information concerning the relationship of the carrier macromolecule and the tricyclic binding site.