SELECTIVE ACTIVATION OF DOPAMINE RECEPTOR SUBPOPULATIONS

描述（改编自申请人的摘要）：本研究的基础 第一奖是对一个名为“功能性”的新概念的测试 选择性假说“它假定“非典型”药物的相互作用 单一G蛋白受体亚型可能会导致功能性影响， 作为激动剂与拮抗剂的极端（取决于所涉及的性质） G蛋白与药物-受体诱导的构象变化类型 互动）。 虽然这一假设被提出来推广到所有 G蛋白偶联受体系统，我建议测试和完善这一点， 通过研究D2样多巴胺（DA）受体假说。 中的第一数据 支持这一观点结果是， 六氢苯并[a]菲啶多巴胺能配体[即，dihydrexidine (DHX)及其类似物]在突触后激活，但不在突触前， D2样受体。 目前的应用将集中在机械 可以为潜在的“功能”提供坚实基础的研究 选择性假说“工作假设是，DHX及其 N-正丙基类似物对这些D2样受体具有完全的激动剂作用 与腺苷酸环化酶偶联，而由于这些药物是拮抗剂（或低浓度）， 效力部分激动剂）在与钾通道连接的D2受体上。 DHX和N-p-DHX都能强烈抑制几种细胞中的腺苷酸环化酶。 模型（例如，抑制毛喉素刺激或D1介导的cCAMP 纹状体、垂体催乳细胞或D2转染的C-6中的合成/外排 和MN 9D克隆细胞）。 然而，令人惊讶的是，这些药物几乎没有 或对已知（或推测）与钾离子偶联的D2受体无影响 （K+）通道（例如，它们不会抑制多巴胺细胞的放电或多巴胺 释放，并且它们仅诱导垂体中K+通道的弱激活 催乳素）。 药物的开发（如 六氢苯并[a]菲啶）可能导致 通过提供机会， 靶向受体相关事件的子集，从而避免不期望的 由于受体功能的广泛激活或阻断而引起的副作用。 这类药物将提供“药理学手术刀”， 方面 为了开始阐明功能选择性的机制，我 本实验主要研究DHX对大鼠D2受体功能的影响 纹状体 第一个目标是收集浓度/响应数据， 测定DHX和类似物对D2受体的效力和功效 与腺苷酸环化酶或K+通道相连。 超灌注纹状体切片将 用于比较对Da和ACh释放的影响（反射离子通道 激活），对cAMP流出（腺苷酸环化酶的指标）有影响 激活）。 自身受体对腺苷酸环化酶连接的多巴胺的作用 通过酪氨酸羟化酶的动力学分析来评估合成。 目的 2将结果从体外研究扩展到两个体内范例， 脑微透析（测量DA，ACh和cAMP溢出）和 γ-丁内酯（GBL）模型（测量对DA合成的影响）。 目标3中的实验将比较DHX激活G蛋白的模式 和典型的D2激动剂。 激动剂诱导的[α-32 P]GTP与 纹状体膜和MN 9D细胞中的G蛋白亚型将用作 G蛋白激活的标志物。

DESCRIPTION (Adapted from applicant's abstract): The foundation of this FIRST Award is the testing of a novel concept termed the "functional selectivity hypothesis." It posits that the interaction of "atypical" drugs with a single G-protein receptor isoform may cause functional effects as extreme as agonist vs. antagonist (depending on the nature of the involved G-proteins and the type of conformational changes induced by drug-receptor interaction). While this hypothesis is proposed to generalize to all G-protein coupled receptor systems, I propose to test and refine this hypothesis by studying D2-like dopamine (DA) receptors. The first data in support of this notion were result showing that novel hexahydrobenzo.a.phenanthridine dopaminergic ligands [i.e., dihydrexidine (DHX) and its analogs] activated post-synaptic, but not pre-synaptic, D2-like receptors. The current application will focus on mechanistic studies that can provide a firm underpinning for the underlying "functional selectivity hypothesis." The working hypothesis is that DHX and its N-n-propyl analog have full agonist actions at those D2-like receptors coupled to adenylate cyclase, whereas as these drugs are antagonists (or low efficacy partial agonists) at D2 receptors linked to potassium channels. Both DHX and N-p-DHX cause robust inhibition of adenylate cyclase in several models (e.g., inhibition of forskolin-stimulated or D1-mediated cCAMP synthesis/efflux in striatum, pituitary lactotrophs, or D2-transfected C-6 and MN9D clonal cells). Surprisingly, however, these same drugs have little or no effect on D2 receptors known (or presumed) to be coupled to potassium (K+) channels (e.g., they do not inhibit dopamine cell firing or dopamine release, and they induce only weak activation of K+ channels in pituitary lactotrophs). The development of drugs (such as the hexahydrobenzo[a]phenathridines) with functional selectivity may lead to dramatically improved pharmacotherapies by providing opportunities for targeting a subset of receptor-linked events, thus avoiding the undesirable side effects due to widespread activation or blockade of receptor functions. Such drugs would provide "pharmacological scalpels" for perturbing selected aspects. To begin to elucidate the mechanisms of functional selectivity, my experiments will focus on the actions of DHX on D2 receptor functions in rat striatum. The first aim is to collect concentration/response data to determine the potencies and efficacies of DHX and analogs at D2 receptors linked to adenylate cyclase or K+ channels. Superfused striatal slices will be used to compare effects on Da and ACh release (reflecting ion channel activation) with effects on cAMP efflux (an index of adenylate cyclase activation). Autoreceptor-mediated actions on adenylate-cyclase linked DA synthesis will be assessed by kinetic analysis of tyrosine hydroxylase. Aim 2 will extend results from in vitro studies to two in vivo paradigms, cerebral microdialysis (to measure DA, ACh and cAMP overflow) and the gamma-butyrolactone (GBL) model (to measure effects on DA synthesis). Experiments in Aim 3 will compare the pattern of G-protein activation by DHX and typical D2 agonists. Agonist-induced binding of [alpha-32P]GTP to G-proteins isoforms in striatal membranes and MN9D cells will be used as a marker of G-protein activation.