Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.
Exposure of bovine chromaffin cells in primary culture to 5 microM reserpine or 25 microM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 microM forskolin (or cholera toxin at 50 micrograms/ml) causes a 1.5- to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 microgram/ml). Reserpine (5 microM) elicits a 1.5- to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Met]enkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Met]enkephalin pentapeptide and a concomitant decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAenk) by RNA blot hybridization with a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (less than 17 hr) 3- to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr period. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004744:Enkephalin, Methionine / Q000235:genetics; D004745:Enkephalins / Q000235:genetics*; D007700:Kinetics; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D012110:Reserpine / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D004745:Enkephalins; D012333:RNA, Messenger; D004744:Enkephalin, Methionine; D012110:Reserpine; D003513:Cycloheximide; D000242:Cyclic AMP