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The levels of the secretion of [Met^(5)]-enkephalin (ME) in addition to proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin cells were studied in the presence of cycloheximide, an inhibitor of protein synthesis, and two modulators of ME secretion and proENK synthesis, nicotine and 12-0-tetradecanoylphorbol-13-acetate (TPA). Nicotine at a concentration of 10 μM caused a rapid secretion of ME followed by a long-term secretion (9-24 h after treatment) into the medium. However, the treatment with TPA (1 μM) augmented ME secretion rather slowly and the magnitude of increase in ME secretion continued to increase with time. Post-treatment with cycloheximide (20 μM) Hp to 5 h significantly inhibited the long-term secretion of ME induced by nicotine, TPA or a combined treatment of nicotine and TPA without affecting the short-term secretion. However, the long-term secretion of ME induced by nicotine, TPA, or a combined treatment of nicotine and TPA was not affected, when cycloheximide was added 9 h after the drug treatment. Long-term (24 h) stimulation of bovine adrenal medullary chromaffin cells with nicotine, TPA or a combined treatment of nicotine and TPA also increased the proENK mRNA level. The increase of proENK mRNA induced by nicotine, TPA, or a combined treatment of nicotine and TPA was inhibited by post-treatment with cycloheximide 1 and 5 h after the treatment of these drugs. However, cycloheximide did not affect the increased proENK mRNA level induced by nicotine, TPA, or a combined treatment of nicotine and TPA, when cycloheximide was given 9 h after treatments. The results indicate that de novo protein synthesis was necessary for proENK gene activation and the secretion of ME. This de novo protein synthesis should occur within at least 5 h after the treatment of bovine adrenal medullary chromafin cells with nicotine, TPA, or a combined treatment of nicotine and TPA.
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