ENKEPHALIN BIOSYNTHESIS IN ADRENAL-MEDULLA - MODULATION OF PROENKEPHALIN MESSENGER-RNA CONTENT OF CULTURED CHROMAFFIN CELLS BY 8-BROMO-ADENOSINE 3',5'-MONOPHOSPHATE
Incubation of primary cultures of chromaffin cells from bovine adrenal medulla with 8-bromo-adenosine 3'',5''-monophosphate (8-Br-cAMP) resulted in an increase in proenkephalin mRNA content. The mRNA that increased was detected by hybridization analysis using a cDNA probe and migrated with an apparent size of approximately 1400 bases. The increase in proenkephalin mRNA following 8-Br-cAMP treatment was apparent in 12 h and continued over 2 days. Corresponding changes were detected in enkephalin-like immunoreactivity but with a 24-h lag: the cellular content increased significantly after 2 days of treatment and continued to rise over the next 2 days, whereas changes in the amount released to the medium followed the same time course. Dose-response curves for the increase in the content of proenkephalin mRNA and of enkephalin-containing peptides were essentially identical. Chromatographic characterization of the enkephalin-like peptides demonstrated that 8-Br-cAMP increased both the high MW fraction and the low MW fraction, which was shown by high-pressure liquid chromatography to contain Met5-enkephalin, Leu5-enkephalin and Met5-enkephalin-Arg6-Phe7. Previous results in chromaffin cells have demonstrated that the synthesis of tyrosine hydroxylase is also regulated by cAMP, with a similar time course. These results suggest the possibility of coordinate regulation by cAMP of the expression of the cotransmitters, catecholamines and enkephalin peptides, in the adrenal medulla.