LOCALIZATION OF RECEPTOR ACITIVITY FOR DRUGS OF ABUSE

滥用药物受体活性的定位

• 批准号: 2897612
• 负责人: Laura J Sim-Selley
• 金额: $ 11.55万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2897612
• 关键词: G protein; autoradiography; cannabinoid receptor; guanosine triphosphate; laboratory rat; neuropeptide receptor; neuropeptides; neuropharmacology; receptor binding; receptor expression

DESCRIPTION: (Applicant's Abstract) A novel technique has been developed using [35S]GTPgS autoradiography to localize receptor-activated G-proteins. This technique is based upon previous studies using agonist-stimulated [35S]GTPgS binding, in the presence of excess GDP, to measure receptor-activated G-proteins in membranes. The advantage of [35S]GTPgS autoradiography over other techniques is the ability to localize functionally active G-proteins. The technique has been used to demonstrate G-proteins activated by several receptors: opioid, cannabinoid and GABA-B. [35S]GTPgS autoradiography has also been used to identify ORL1 peptide-stimulated [35S]GTPg binding. This demonstrates another advantage of the technique; since [35S]GTPgS is the radioligand used, any unlabeled agonist can be used in this technique. [35S]GTPgS autoradiography has also been used to examine G-protein activity following chronic drug treatment. In these studies, chronic morphine treatment decreased mu opioid-stimulated [35S]GTPgS binding in specific brainstem nuclei: the dorsal raphe nucleus, locus, coeruleus, parabrachial nucleus and commissural nucleus tractus solitarius. The proposed studies will begin with experiments to develop [35S]GTPgS autoradiography for additional receptor systems. Studies will then be performed to examine the effects of chronic cannabinoid or cocaine administration on receptor activation of G-proteins. Initially, studies will examine cannabinoid and dopamine-stimulated [35S]GTPgS binding, however, subsequent studies will examine other receptor systems as well. The third series of experiments will use a novel anatomical approach in which specific regions are lesioned, then [35S]GTPgS autoradiography is performed to 1) identify the origin of afferents to the nucleus and 2) determine whether the receptors on those fibers are pre- or post-synaptic. These studies will focus on the ORL1 and cannabinoid systems, since little anatomical information is available regarding these systems. This project will provide an excellent opportunity for Dr. Sim to utilize her neuroanatomical training, while developing training in the areas of signal transduction and pharmacology. Dr. Sim will conduct this research under the mentorship of Dr. Childers, an established investigator in the field of signal transduction mechanisms of drug of abuse. In addition collaborations have been established with members of the Neuroscience of Drug Abuse Research Center, with faculty specializing in behavior, pharmacology, physiology and molecular biology. This project will allow Dr. Sim to develop into an independent investigator trained in anatomy, pharmacology and signal transduction.

描述：（申请人摘要） 使用 [35S]GTPgS 放射自显影技术开发了一种新技术 定位受体激活的 G 蛋白。 该技术基于 先前的研究使用激动剂刺激的 [35S]GTPgS 结合， 存在过量 GDP，以测量受体激活的 G 蛋白 膜。 [35S]GTPgS 放射自显影相对于其他方法的优势 技术是定位功能活性 G 蛋白的能力。 这 技术已被用来证明G蛋白被多种物质激活 受体：阿片类药物、大麻素和 GABA-B。 [35S]GTPgS放射自显影有 也被用于鉴定 ORL1 肽刺激的 [35S]GTPg 结合。 这 展示了该技术的另一个优点；因为 [35S]GTPgS 是 使用放射性配体，任何未标记的激动剂都可以用于该技术。 [35S]GTPgS 放射自显影术也已用于检查 G 蛋白活性 长期药物治疗后。 在这些研究中，慢性吗啡 治疗减少了 mu 阿片类药物刺激的 [35S]GTPgS 特异性结合 脑干核：中缝背核、位核、蓝核、臂旁核 孤束核和连合核。 拟议的研究 将开始实验开发 [35S]GTPgS 放射自显影术 额外的受体系统。 然后将进行研究以检验 长期服用大麻素或可卡因对受体的影响 G蛋白的激活。 最初，研究将检查大麻素和 多巴胺刺激的 [35S]GTPgS 结合，然而，后续研究将 也检查其他受体系统。 第三系列实验 将使用一种新颖的解剖方法，其中特定区域受到损害， 然后进行 [35S]GTPgS 放射自显影以 1) 确定 传入细胞核的信号，2) 确定这些信号上的受体是否 纤维位于突触前或突触后。 这些研究将集中于 ORL1 和 大麻素系统，因为可用的解剖学信息很少 关于这些系统。 该项目将提供一个绝佳的机会 让 Sim 博士利用她的神经解剖学训练，同时发展 信号转导和药理学领域的培训。 沉博士将会 在 Childers 博士的指导下进行这项研究，Childers 博士是一位知名的 药物信号转导机制领域研究者 虐待。 此外，还与以下组织的成员建立了合作关系： 药物滥用神经科学研究中心，其教员专门从事 行为、药理学、生理学和分子生物学。 该项目将 让 Sim 博士发展成为一名受过培训的独立调查员 解剖学、药理学和信号转导。