MOLECULAR CONSEQUENCES OF TOLERANCE AND DEPENDENCE

本申请的长期目标是1)确定 特定的基因和相应的蛋白质参与了 建立和维持对阿片类药物的耐受性和依赖性 药物，2)了解这些基因是如何被慢性阿片类药物调节的 滥用，3)阿片肽和阿片受体在脑内的作用 内分泌和免疫系统，以及4)前脑啡肽的调节 (Pe)基因和阿片受体在内分泌、免疫和中枢 慢性阿片类药物滥用导致的神经系统。基因差异 慢性阿片类药物治疗导致的表达将通过 这种方法被称为差异菌落杂交。Poly(A+)mRNA 将从吗啡处理和未处理的大鼠的大脑中分离出来，并 将制备放射性标记的cDNAs。这些cdna探针将与 吗啡处理大鼠脑内cDNA文库的构建。殖民地 含有因接触吗啡而受到调控的cDNA 被选中并排序。蛋白质的氨基酸序列将是 从cDNA中推导出来的。它将确定基因是如何调节的， 即转录或信使核糖核酸稳定性的变化。一项详细的研究 脑中特定的mRNA将通过原位杂交进行 确定表达该基因的细胞类型，并给出定量 关于其监管的信息。体育运动中大小差异的本质 睾丸中的mRNA将通过分子克隆和cdna进行测定。 测序。表达PE基因的细胞类型在内分泌和 免疫系统将被识别，其个体发育将 用原位杂交法进行测定。组织特异性产品 PE加工过程中产生的产物将通过高效液相色谱法和放射免疫法进行鉴定。 影响PE信使核糖核酸翻译的因素有 用体外翻译来表征。脑组织中的阿片受体 内分泌和免疫系统将被描述为MU， Delta或kappa亚型与亚型特异性的体外结合分析 配基。慢性吗啡对PE基因的调节作用 暴露将在内分泌、免疫和中枢神经系统进行调查。 使用印迹杂交和原位杂交的系统。对阿片类药物的监管 由耐受和依赖产生的受体将在这些 纸巾。关于药物滥用的分子后果的知识 将为治疗提供基础，并将对 我们社会中考虑娱乐性吸毒的年轻男女。 此外，希望这些信息将有助于 不产生耐受性和生理反应的止痛药的设计 依赖。

The long-term objectives of this application are 1) the identification of specific genes and corresponding proteins which are involved in the establishment and maintenance of tolerance to and dependence on opiate drugs, 2) understanding how these genes are modulated by chronic opiate abuse, 3) the function of opioid peptides and opiate receptors in the endocrine and immune systems, and 4) the regulation of the proenkephalin (PE) gene and opiate receptors in the endocrine, immune, and central nervous systems resulting from chronic opiate abuse. Differences in gene expression resulting from chronic opiate treatment will be examined using the methodology known as differential colony hybridization. Poly(A+)mRNA will be isolated from the brains of morphine-treated and untreated rats and radiolabeled cDNA will be prepared. The cDNA probes will be hybridized to a brain cDNA library generated from morphine-treated rats. Colonies which contain cDNAs which are regulated as a result of exposure to morphine will be selected and sequenced. The amino acid sequence of the protein will be deduced from the cDNA. It will be established how the gene is modulated, i.e., changes in transcription or mRNA stability. A detailed study of the specific mRNA in brain will be conducted using in situ hybridization to identify the cell type which expresses the gene and give quantitative information on its regulation. The nature of the size difference in PE mRNA in the testis will be determined by molecular cloning and cDNA sequencing. The cell types which express the PE gene in the endocrine and immune systems will be identified and their ontogenetic development will be determined using in situ hybridization. The tissue specific products resulting from processing of PE will be identified by HPLC and RIA. Factors which influence the translation of the PE mRNA will be characterized using in vitro translation. The opiate receptors in the endocrine and immune systems will be characterized as being of the mu, delta, or kappa subtype using in vitro binding assays with subtype-specific ligands. The regulation of the PE gene as a result of chronic morphine exposure will be investigated in the endocrine, immune, and central nervous systems using blot and in situ hybridization. Regulation of opiate receptors resulting from tolerance and dependence will be examined in these tissues. Knowledge concerning the molecular consequences of drug abuse will provide a basis for treatment and will act as a deterrent for the young men and women of our society that contemplate recreational drug use. In addition, it is hoped that this information will be helpful in the design of analgesic drugs which do not produce tolerance and physical dependence.