Morphine and other opiates bind to specific cell-surface receptors to alter various biosynthetic and electrophysiological properties on opiate- sensitive neurons in the brain. At least three subtypes of opiate receptors (mu, delta and k) linked to differential biological functions have been identified and localized in various regions of the brain. The most widespread mu-receptors have high affinity for morphine-like compounds and may be involved in modulation primary perception of stimuli and sensory and motor functions. Little is known about the post-receptor mechanism(s) of morphine action although accumulated evidence has suggested a coupling to G proteins. The proposed study represent an effort aiming toward understanding the coupling mechanism(s) of extra cellular opiate stimulation to regulation of gene expression. Three classes of molecules will be studied: (1) the principle opioid peptide precursors, proenkephalin (PE) and pro- opiomelanocortin (POMC), (2) the rate-limiting catecholaminergic biosynthetic enzyme, tyrosine hydroxylase (TH) and (3) the immediate response gene products, especially the Fos protein encoded by proto- oncogene c-fos, which is implicated in the signal transduction process.
Four specific aims are proposed as follows: (1) Determine the effects of opiate treatment on expression of PE, POMC, TH and c-fos genes and corresponding protein/-peptide levels in rat brain by Slot-blotting, Northern blotting, Western blotting analysis and radioimmunoassay. 2) Determine the effects of opiate treatment on expression of PE, POMC, TH and c-fos genes and corresponding protein/peptide levels in a human neuroblastoma cell line, SH-SY5Y, by the same techniques as in AIM 1. 3) Determine the distribution of opiate-inducible protein (Fos) by immunocytochemistry, and c-fos mRNA by in situ hybridization in rat brain, 4) Examine the relationship of c-fos expression to morphine- induced alterations of PE, POMC and TH genes on a functional level in SH- SY5Y cells by transfection techniques. Accomplishment of this proposed study will provide new insights into opiate tolerance and dependance at the level of gene regulation, as well as a new understanding of the anatomical distribution of neurons that have opiate receptors.
