This project consists of several studies on the molecular genetics of protein precursors of neuropeptides in the mammalian nervous system. A major study concerns regulation by hormones and neurotransmitters of the gene for proenkephalin (pEnk), the precursor of the opioid peptides methionine- and leucine-enkephalin, in clonal cell lines of neural origin, as well as in rat brain. NG108-15 neuroblastoma-glioma hybrid cells and C6 rat glioma cells contain pEnk mRNA, quantitated by blot hybridization. C6 cells contain a much higher abundance (3-6 pg/ug RNA) but lower enkephalin content than NG108-15 cells. Treatment of C6 cells with compounds that raise the cyclic AMP concentration by activation of adenylate cyclase (e.g. beta-adrenergic receptor agonists such as (-)-norepinephrine or forskolin) elevate the pEnk mRNA abundance. Glucocorticoid hormones such as dexamethasone or cortisol, while having no effect alone on the pEnk mRNA level, potentiate the effect of cyclic AMP elevation, producing maximum elevations of 8-fold. C6 cells contain proenkephalin but do not process this precursor significantly. Treatment with norepinephrine + dexamethasone raises the content of proenkephalin 11-fold. Treatment of cells with glucocorticoid + forskolin for 1-6 hr increases pEnk gene transcription at least 4-7-fold. These results suggest that glucocorticoids and neurotransmitters that elevate cyclic AMP transcriptionally regulate enkephalin biosynthesis in enkephalinergic cells. Studies have been initiated on the regulation of expression of the gene for proneuropeptide Y (pNPY), the precursor of neuropeptide Y, a putative regulator in the autonomic nervous system. pNPY mRNA is relatively abundant in NG108-15 hybrid cells. Treatment of these cells with glucocorticoids elevate pNPY mRNA 2-fold. Probable rat pNPY cDNA clones are being characterized. In collaboration with Dr. H.-Y. Yang (NIMH) efforts have continued to clone cDNA for precursor(s) to two anti-analgesic neuropeptides isolated by her group.
