MOLECULAR CONSEQUENCES OF TOLERANCE AND DEPENDENCE

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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.