LONG-TERM STIMULATION OF NICOTINIC RECEPTORS IS REQUIRED TO INCREASE PROENKEPHALIN-A MESSENGER-RNA LEVELS AND THE DELAYED SECRETION OF [MET(5)]-ENKEPHALIN IN BOVINE ADRENAL-MEDULLARY CHROMAFFIN CELLS

The effects of nicotine on the transcriptional activity of the proENK gene, proenkephalin A (proENK) mRNA levels, and the secretion of [Met( 5)]-enkephalin (ME) were studied in bovine adrenal medullary chromaffi n (BAMC) cells. Nicotine (10 mu M) caused an immediate secretion (with in 1 hr) of ME followed by a delayed secretion (12-24 hr after treatme nt) into the medium. Posttreatment with the cholinergic antagonists, h examethonium (1 mM) and atropine (1 mu M), up to 6 hr after the nicoti ne treatment significantly inhibited the delayed secretion of ME induc ed by nicotine. However, nicotine-induced long-term secretion of ME wa s not affected when cholinergic antagonists were added 9 or 12 hr afte r the nicotine treatment. Long-term (24 hr) stimulation of BAMC cells with nicotine also increased proENK mRNA level. This nicotine-induced response was inhibited by posttreatment with cholinergic antagonists 0 .5, 1, 3 and 6 hr after the nicotine treatment. As with the secretion experiments, these cholinergic antagonists did not affect the nicotine -induced responses when they were added at 9 and 12 hr. Posttreatment with nimodipine(1 mu M), calmidazolium (1 mu M) or KN-62 (5 mu M) up t o 6 hr after the nicotine treatment significantly inhibited the increa ses of the long-term secretion of ME and proENK mRNA level induced by nicotine. However, these agents were ineffective in blocking the long- term secretion of ME and proENK mRNA level induced by nicotine when BA MC cells were posttreated after 9 and 12 hr. Nuclear run-on assays sho wed that nicotine increases the transcriptional rate for the proENK ge ne after 30 min and the response continues for up to 9 hr with a maxim al increase of 2- to 2.5-fold at 1 and 3 hr. Our results suggest that the long-term stimulation (for at least 6 hr) of nicotinic receptors i s required for the increases in proENK mRNA levels and the delayed sec retion of ME in BAMC cells. The nicotine-induced delayed secretion of ME followed an increased biosynthesis of ME during the first 6 hr whic h appeared to result from increased transcription of the proENK gene.