PSYCHOSTIMULANT ACTIONS /BIOGENIC AMINE RECEPTORS /TRANS

精神兴奋剂，包括可卡因以及该中心开发的新型可卡因类似物，通过阻断转运蛋白介导的生物胺（多巴胺，5-HT和去甲肾上腺素）的再摄取发挥作用。该项目利用新型托烷和哌甲酯类似物，沿着由其他中心项目开发的特定动物模型，研究转运蛋白结合位点，并检查慢性药物治疗对G蛋白偶联受体的影响。该项目的第一个目的是利用该中心开发的动物模型来研究精神兴奋剂（可卡因和新型可卡因类似物）的长期治疗如何影响生物胺受体与大脑中G蛋白的偶联。这些研究将利用[35 S] GTP γ S放射自显影，其提供G蛋白受体激活的神经解剖定位。由于该技术允许在同一动物的脑切片中同时测定许多不同的受体，因此它是有效分析中心衍生动物脑组织的理想方法。该项目将跟进先前的研究，这些研究表明，慢性阿片类药物治疗在特定的脑干核团中产生了μ阿片类药物激活的G蛋白的选择性衰减。将采用三种不同的药物治疗模式：使用高效托烷类似物WF-23的长期治疗，Roberts博士在子项目0011中开发的可卡因狂欢模型中的长期自我给药，以及Smith和Martin博士在子项目0005中开发的快速球（海洛因/可卡因组合）自我给药模型。第二个目标将描述新的不可逆可卡因类似物（托烷和哌甲酯）作为多巴胺转运蛋白结构和功能的探针的特性。由于这些化合物将用于行为研究的其他中心项目，我们的实验室将首先在体外和体内，这些类似物的基本不可逆结合特性的特点，使用转运放射性配体结合膜和放射自显影。将用[1251]标记最有效的类似物，并通过SDS-PAGE和纯化从多巴胺转运蛋白转染的细胞中分离的[1251]-标记的胰蛋白酶肽片段来检查托烷和哌甲酯之间结合的差异。

Psychostimulants, including cocaine as well as novel cocaine analogs developed by this Center, act by blocking transporter-mediated reuptake of biogenic amines (dopamine, 5-HT and norepinephrine). This project utilizes novel tropane and methylphenidate analogs, along with specific animals models developed by other Center projects, to study transporter binding sites, and to examine the consequences of chronic drug treatment on G-protein-coupled receptors. The first aim of this project utilizes animal models developed by the Center to examine how chronic treatment with psychostimulants (both cocaine and novel cocaine analogs) affect coupling of biogenic amine receptors to G-proteins in brain. These studies will utilize [35S]GTPgammaS autoradiography, which provides a neuroanatomical localization of the activation of G-protein receptors. Because this technique allows for an number of different receptors to be assayed simultaneously in brain sections from the same animal, it is an ideal method to efficiently analyze brain tissue from Center-derived animals. This project will follow up on previous studies showing that chronic opioid treatment produced selective attenuation of mu opioid-activated G-proteins in specific brainstem nuclei. Three different drug treatment paradigms will be employed: a chronic treatment with the highly potent tropane analog WF-23, chronic self-administration in a binge model of cocaine developed by Dr. Roberts in subproject 0011, and a speedball (heroin/cocaine combination) self-administration model developed by Drs. Smith and Martin in subproject 0005. The second aim will characterize the properties of novel irreversible cocaine analogs (both tropanes and methylphenidates) as probes of dopamine transporter structure and function. Since these compounds will be used in behavioral studies by other Center projects, our laboratory will first characterize the basic irreversible binding properties of these analogs both in vitro and in vivo, using transporter radioligand binding in membranes and autoradiography. The most potent analogs will be labeled with [1251] and differences in binding between tropanes and methylphenidates will be examined by SDS-PAGE and purification of [1251]-labeled tryptic peptide fragments isolated from cells transfected with dopamine transporters.