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MOLECULAR MECHANISMS OF DIAZEPAM TOLERANCE

地西泮耐受性的分子机制

基本信息

批准号：
2858049
负责人：
ERMINIO COSTA
金额：
$ 9.22万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-01-01 至 2000-12-31
项目状态：
已结题

项目摘要

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（改编自申请人的摘要）：耐受性苯二氮卓类药物（BZ）限制了其抗惊厥药的临床使用，抗焦虑剂和抗恐慌剂。在临床实践中要保持药物疗效水平增加 BZ 剂量以克服耐受性和这常常导致身体依赖。我们必须了解其中的机制 BZ耐受性的研究，以克服其临床使用中的问题我们相信这也会带来一个初步的了解 GABA 能传输受到调节。 BZ 可分为 FULL 或 GABA-a 受体（FAM 和分别为 PAM）。两种药物均作用于 GABA-a 受体，包括其结构中有一个 alpha1-5 和一个 gamma2-3 亚基，但它们放大 GABA 作用的内在活性不同。地西泮（FAM），但咪达西尼（PAM）不会在啮齿动物中诱导耐受性。在初步研究表明，在耐受性大鼠中，编码 alpha1、gamma2 和 alpha5 GABA-a 受体的 mRNA 表达当耐受性逆转时，亚基也会逆转。这样的改变不存在于接受咪达西尼的大鼠中，其剂量与地西泮可诱导耐受。这两种药物将用作研究探索地西泮耐受机制的工具。我们假设对地西泮的耐受性是由 GABA-a 受体的改变引发的亚基表达仅限于功能专用的大脑区域。我们提出对地西泮耐受机制进行系统研究具有以下目标的大鼠：（1）确定地西泮的起效和持续时间对其抗惊厥药、抗冲突药和认知干扰药的耐受性使用咪达西米作为对照的作用，（2）PCR分析14中的变化开始和结束时28个脑区的亚基mRNA含量地西泮耐受性并测试咪达西尼是否产生类似的变化或拮抗地西泮的变化，(3) 用脑中金免疫标记进行测量切片相关的 8 个不同 GABA-a 受体亚基的含量使用咪达西尼作为对照和/或对地西泮耐受的大鼠的大脑区域作为拮抗剂和（4）研究神经元亚基表达的变化从相关大脑区域制备的分离培养物的膜耐受的老鼠；确定相关亚基的受体共表达不同大小的金颗粒的双重免疫标记。