The purpose of this project is to determine which second messenger pathways are involved in receptor regulation of rat adrenal medulla (and spleen, and ultimately brain) enkephalin levels. Previous work from our lab demonstrated that nicotine stimulates enkephalin gene expression in cultured bovine chromaffin cells. Nicotine increases intracellular CA2+ concentration (Cai) and protein kinase C (PKC) translocation in these cells, and these pathways stimulate the expression of AP-1 binding proteins (Fras and Juns), which bind to and stimulate transcription of the enkephalin gene in bovine cells. However, AP-1 binding proteins are elevated in rat adrenal medulla, and acute nicotine treatment of rats decreases the AP-1 binding (presumably by CA2+ increasing phosphatase activity); chronic bidaily nicotine, and also saline injections increased adrenal AP-1 binding. Adrenal enkephalin levels are increased over naive levels by all three treatments, but most markedly by acute, repeated nicotine. The enkephalin promotor contains two additional potential transcription factor sites, the cAMP (and CA2+) responsive element (CRE), and the NFKB site, which may be more important for the response to nicotine. Glial (and possibly neuronal) enkephalin levels are known to be increased by elevating cAMP, and we have demonstrated a glucocorticoid potentiation of this stimulation. Spleen enkephalin levels appear sensitive to NFKB regulation; we have also observed a surprising sex difference in enkephalin levels in the spleen, and cultured brain cell transcription factor levels and their responses to agents known to elevate enkephalin levels, to determine whether the enkephalin gene is regulated similarly or differently in each of these tissues.
