Abstract

Incubation of primary cultures of chromaffin cells from bovine adrenal medulla with 8-bromo-adenosine 3',5'-monophosphate (8-Br-cyclic AMP) 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-cyclic AMP treatment was apparent in 12 hr and continued over 2 days. Corresponding changes were detected in enkephalin-like immunoreactivity but with a 24-hr 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-cyclic AMP increased both the high molecular weight fraction and the low molecular weight 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 cyclic AMP, with a similar time course. These results therefore suggest the possibility of coordinate regulation by cyclic AMP of the expression of the cotransmitters, catecholamines and enkephalin peptides, in the adrenal medulla.