MOLECULAR CONSEQUENCES OF TOLERANCE AND DEPENDENCE

本申请的长期目标是：1）识别 特定的基因和相应的蛋白质参与了 阿片类药物耐受性和依赖性建立和维持 药物，2）了解这些基因如何被慢性阿片类药物调节 滥用，3）阿片肽和阿片受体的功能， 内分泌和免疫系统，以及4）前脑啡肽的调节 (PE)内分泌、免疫和中枢神经系统中的基因和阿片受体 由于长期滥用鸦片而导致的神经系统损伤。 基因差异 慢性阿片类药物治疗引起的表达将使用 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大鼠的大脑中分离， 将制备放射性标记的cDNA。 cDNA探针将与 从吗啡处理的大鼠产生的脑cDNA文库。 的菌落 含有由于暴露于吗啡而受到调节的cDNA， 选择和排序。 蛋白质的氨基酸序列将是 从cDNA中推导出来的。 将确定基因是如何被调节的， 也就是说，转录或mRNA稳定性的变化。 详细研究了 将使用原位杂交进行脑中特定mRNA， 鉴定表达基因的细胞类型，并给出定量的 关于其监管的信息。 PE中尺寸差异的性质 睾丸中的mRNA将通过分子克隆和cDNA 测序 在内分泌系统中表达PE基因的细胞类型， 免疫系统将被识别，它们的个体发育将 使用原位杂交测定。 组织特异性产物 将通过HPLC和RIA鉴别PE加工产生的残留物。 影响PE mRNA翻译的因素将是 使用体外翻译表征。 大脑中的阿片受体 内分泌和免疫系统将被表征为属于μ， δ或κ亚型，使用亚型特异性的体外结合试验 配体。 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中进行研究。 系统使用印迹和原位杂交。 鸦片制剂的调节 受体产生的耐受性和依赖性将在这些检查 组织中 关于药物滥用的分子后果的知识 将提供治疗的基础，并将作为一种威慑， 我们社会中考虑使用娱乐性毒品的年轻男女。 此外，希望这一资料将有助于 设计不产生耐受性和物理性的镇痛药物 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.