MOLECULAR MECHANISMS OF DIAZEPAM TOLERANCE

地西泮耐受性的分子机制

• 批准号: 2635537
• 负责人: ERMINIO COSTA
• 金额: $ 8.95万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2635537
• 关键词: GABA receptor; anticonvulsants; antipsychotic agents; behavior test; benzodiazepines; brain mapping; cognition; diazepam; drug administration rate /duration; drug tolerance; immunocytochemistry; laboratory rat; messenger RNA; neurons; neuropharmacology; polymerase chain reaction; protein structure; receptor expression; tissue /cell culture

DESCRIPTION (Adapted from applicant's abstract): Tolerance to benzodiazepines (BZ) limits their clinical use of anticonvulsants, anxiolytic and antipanic agents. In the clinical practice to maintain the level of drug efficacy the BZ doses are increased to overcome tolerance and this often leads to physical dependence. We must understand the mechanisms of BZ tolerance in order to overcome the problems in their clinical use and we believe that this will also bring about an initial understanding of how GABAergic transmission is regulated. BZ can be classified as FULL or PARTIAL allosteric modulators of GABA action at GABA-a receptors (FAM and PAM, respectively). Both drug classes act on GABA-a receptors that include one alpha1-5 and one gamma2-3 subunit in their structure, but their intrinsic activity in amplifying GABA action differs. Diazepam, (a FAM), but not imidazenil, (a PAM) induces tolerance in rodents. In preliminary studies, we have shown that in tolerance rats there is a change in the expression of mRNAs encoding for alpha1, gamma2 and alpha5 GABA-a receptor subunits which reverses when tolerance is reversed. Such changes are not present in rats receiving imidazenil in doses equipotent to those of diazepam that induce tolerance. These two drugs will be used as research tools to explore the mechanism of diazepam tolerance. We hypothesize that tolerance to diazepam is triggered by an alteration of GABA-a receptor subunit expression restricted to functionally dedicated brain areas. We propose a systematic investigation of the mechanism of diazepam tolerance in rats with the following aims: (1) Determine onset and duration of diazepam tolerance to its anticonvulsant, anticonflict and cognition disrupting action using as a contrast imidazemil, (2) PCR analysis changes in 14 subunit mRNA content in 28 brain areas at the onset and termination of diazepam tolerance and test whether imidazenil produces similar changes or antagonizes diazepam changes, (3) Measure with gold immunolabeling in brain slices the content of 8 different GABA-a receptor subunits in pertinent brain areas of diazepam tolerant rats using imidazenil as a contrast and/or as antagonist and (4) Study changes of subunit expression in neuronal membranes of dissociated cultures prepared from pertinent brain areas of tolerant rats; determine receptor coexpression of pertinent subunits with double-immunolabelling with gold particles of different size.

描述（改编自申请人摘要）： 苯并二氮杂（BZ）限制了它们作为抗惊厥药的临床应用， 抗焦虑药和抗恐慌药。 在临床实践中， BZ剂量增加以克服耐受性， 这往往会导致身体依赖。 我们必须了解 BZ耐受性，以克服其临床使用中的问题， 我们相信，这也将使我们初步了解， γ-氨基丁酸能的传输受到调节。 BZ可以被分类为完整或 GABA作用于GABA-a受体的部分变构调节剂（FAM和 PAM）。 这两类药物都作用于GABA-a受体，包括 一个α 1 -5和一个γ 2 -3亚基，但它们的 增强GABA作用的内在活性不同。 地西泮（一种FAM）， 而不是咪唑菌腈（PAM）诱导啮齿类动物的耐受性。 初步 研究表明，在耐受大鼠中， 编码α 1、γ 2和α 5 GABA-α受体的mRNA的表达 当耐受性逆转时逆转的亚基。 这种变化不是 存在于接受咪唑菌胺的大鼠中，其剂量相当于 安定诱导耐受性 这两种药物将被用作研究 探讨安定耐受机制的工具。 我们假设 对地西泮的耐受性是由GABA-α受体的改变触发的 亚基表达仅限于功能专用的大脑区域。 我们 建议系统研究地西泮耐受的机制， 大鼠，目的如下：（1）确定地西泮的起效时间和持续时间 抗惊厥、抗冲突和认知障碍的耐受性 以咪唑安定为对照，（2）PCR分析14例 28个脑区亚单位mRNA含量在发病和终止 地西泮耐受性，并测试咪唑是否产生类似的变化， （3）金免疫标记法测定脑内 将8种不同的GABA-a受体亚单位的含量切片， 使用咪唑菌胺作为对照的地西泮耐受大鼠的脑区和/或 作为拮抗剂;（4）研究神经元亚基表达的变化 分离培养物的膜，从相关的脑区制备， 耐受大鼠;确定相关亚单位的受体共表达， 用不同大小的金颗粒进行双重免疫标记。