Coordinate and differential regulation of phenylethanolamine N-methyltransferase, tyrosine hydroxylase and proenkephalin mRNAs by neural and hormonal mechanisms in cultured bovine adrenal medullary cells

Primary cultures of bovine adrenal medullary cells (AM) in a chemically defined media were used to examine the role of neural and hormonal factors in the expression of proenkephalin A (pEK), phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) genes. Acetylcholine or nicotine reduced cellular content of catecholamines by 30% and increased the relative abundance of pEK, TH, and PNMT mRNAs. The increases produced by acetylcholine were +129%, +147%, and +43% for pEK, TH, and PNMT mRNA, respectively. The kinetics of increases produced by nicotine were different for the 3 mRNAs, with pEK and TH showing enhanced levels over 48 h incubation, while PNMT showed increase during the initial 18 h (+90%) followed by decline to control levels at 48 h. 8-Br cAMP and forskolin elicited a similar pattern of changes as nicotine, suggesting that cyclic AMP may be involved in the mediation of the nicotinic effects. To examine the role of depletion of cellular catecholamines in the regulation of mRNA levels, cells were exposed to tetrabenazine or reserpine. Decreases in cellular catecholamine contents were accompanied by increases in TH and pEK mRNA levels, while the expression of PNMT gene exhibited a transient 4-fold increase and then profound inhibition (60-95%) over a 48-h period. The tetrabenazine effect on TH and pEK mRNA was reduced by alpha-amanitin, suggesting transcriptionally-mediated regulation. Inductions of pEK but not TH or PNMT mRNAs were inhibited by cycloheximide. Hormonal regulation of TH, PNMT, and pEK mRNAs was examined by incubation of cells with dexamethasone. Low concentrations of dexamethasone (0.1, 10 nM) were effective to increase PNMT (+35%, +90%) and pEK (+27%, 45%) mRNA levels. TH mRNA was not affected by similar concentrations of dexamethasone, however, there was a 45% increase at 1 microM. Dexamethasone-elicited increases in PNMT mRNA levels were observed at 48 h and persisted up to 7 days, suggesting that hormonal mechanisms may be distinct from those mediating effects of nicotine, cAMP or tetrabenazine. Taken together, these results indicate that (1) the level of TH, PNMT, and pEK mRNAs are regulated by direct neural (acetylcholine) and hormonal (glucocorticoid) inputs to adrenal medullary cells; (2) effects of acetylcholine could be mediated by cyclic AMP and alterations in catecholamine content; and (3) expression of individual genes is regulated differentially. Such differential regulation of TH, PNMT, and pEK mRNAs may contribute to the long-term selective control of hormonal output from adrenomedullary cells.