The goal of the proposed study is to understand the long-term effects of nicotine and staurosporine on the release of neurotransmitter and [Met5]enkephalin (ME) and the expression of the proenkephalin A (proENK) gene in bovine adrenal medullary (BAM) cells. The specific goal of this project was to elucidate the mechanisms for (1) the release of ME induced by nicotine and staurosporine, and (2) the regulation of the expression of the proENK mRNA. Using primary cultured bovine adrenal chromaffin cells, we found that nicotine increased the release of ME. The pattern of ME release induced by nicotine was biphasic, short-term secretion followed by long-term secretion. The post-treatment study with cholinergic receptor antagonists, hexamethonium and atropine, revealed that long-term (6-9 hrs) stimulation of nicotinic receptors are required for the long-term secretion of ME and the expression of proENK gene. Nuclear run-on experiments showed that nicotine increased transcriptional rate for proENK gene about 2.5-fold 0.5, 1, 3, 5, and 9 hr after drug treatment, indicating that an increase of proENK gene expression induced by nicotine was mediated by an increase of transcription activity. Currently we are studying the mechanisms involved in short-term and long-term secretion of ME. In addition to the opioid system, we are examining the effects of nicotine on catecholamines secretion, tyrosine hydroxylase (TH) gene expression and TH transcription activity. Long-term treatment of BAM cells with staurosporine, a PKC inhibitor, caused neurite-like growth similar to PMA, a PKC activator. Staurosporine decreased the secretion of ME into the media while it increased intracellular content of ME, its precursor, proenkephalin, and proENK mRNA. Like PMA, staurosporine increased AP-1 DNA binding activity. However, the effects of different PKC inhibitors, such as K252a, H7 and sphingosine, were different from staurosporine's responses. The results indicate that staurosporine produced these long-term effects by mimicking a PKC activator, rather than inhibiting PKC activity. This study will reveal the mechanism underlying the regulation of adrenal medullary function of sympathetic neurotransmission.
