The major goal of this study is to understand the mechanism(s) underlying the activation of catecholamine enzymes [Tyrosine Hydroxylase (TH), Phenylethyl-N-methyltransferase (PNMT)] and opioid neuropeptide [Proenkephalin (PENK)] gene expression in response to a variety of effector stimuli in bovine adrenal medullary chromaffin (BAM) cells in vitro. When BAM cells were exposed to nicotine and TPA separately or together, the secretion of Met-ENK, the processed products of preproenkephalin was increased. A bimodal Met-ENK release mechanism was observed. For short-term effect the increase of Met-ENK secretion in modulator treated BAM cells was independent of new protein synthesis. On the other hand, the long-term effect of Met-ENK release required new protein synthesis because the presence of cycloheximide, a protein synthesis inhibitor effectively blocked the Met-ENK exocytosis. We observed at least an additive effect of Met-ENK secretion by nicotine and TPA together in BAM cell culture for short-term and long-term release. Furthermore, the PENK transcription was also elevated in drug-treated cells, indicating a transcription-secretion coupling mechanism in these studies. Based on the known genomic upstream sequence from the transcription site of human and rat ENK, we first used the AP-1 consensus responsive element to study the AP-1 related transcription factor(s) involved in the activation of ENK expression. The data of our DNA-protein interaction and gel shift studies demonstrated a good correlation between the elevation of PENK transcription and increase of the AP-1 transactivator. In summary, we have observed a transcription and secretion coupling effect of Met-ENK by nicotine-, TPA-, and nicotine and TPA-treated BAM cells. The new protein synthesis was only required for the long-term effect of secretion. An additive effect was seen with Nicotine in combination with TPA in BAM cell culture. Furthermore, at least a transcription factor, namely AP-1 protein was also stimulated in drug -treated cells, implying that the AP-1 trans-activating factor might be involved in PENK transcription activation.
