calcimycin and preproenkephalin

CV-1 cells were stably transfected with a preproenkephalin A (PPE) promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid containing -176 to +171 bp of the human PPE gene. Low levels of CAT were expressed constitutively. The reporter enzyme activity was induced by treatment of the cells for 6 h with drugs that increased intracellular cAMP (forskolin and 8-bromo-cAMP), intracellular calcium (A23187), or protein kinase C activity (tetradecanoyl phorbol-4-acetate, TPA) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Co-administration of dexamethasone reduced the magnitude of phorbol ester-stimulated CAT activity by about 50%, while there were smaller but not significant effects on forskolin- or A23187-stimulated expression of this reporter construct. In transient transfections which included the PPE-CAT reporter gene and a glucocorticoid receptor expression plasmid, dexamethasone significantly reduced stimulated expression of the reporter by TPA, forskolin, and A23187. The effect was observed with 10(-8)-10(-6) M dexamethasone and was blocked by the presence of the glucocorticoid antagonist RU486, suggesting that the effect of dexamethasone was mediated by the glucocorticoid receptor. The promoter region contained in this construct lacks a classical glucocorticoid response element or known negative elements; thus, dexamethasone may reduce stimulated expression of the PPE promoter via indirect effects.

Topics: Calcimycin; Cell Line; Chloramphenicol O-Acetyltransferase; Colforsin; Depression, Chemical; Dexamethasone; Enkephalins; Gene Expression Regulation, Enzymologic; Genes, Reporter; Glucocorticoids; Humans; Ionophores; Promoter Regions, Genetic; Protein Precursors; Receptors, Glucocorticoid; Tetradecanoylphorbol Acetate

Decreasing transsynaptic activity through surgical adrenal denervation or by medullary explantation, increases Leu-enkephalin immunoreactivity (Leu-Enk) and preproenkephalin mRNA (prepro-EK). Membrane depolarization prevents this rise. To determine whether depolarizing effects are mediated by intracellular movement of calcium ions, explanted medullae were depolarized in the presence of EGTA or the calcium ion 'channel' blockers D600 or verapamil. Inhibition of Ca2+ influx prevented the effects of KCl-induced depolarization on the rise in Leu-Enk and on prepro-EK. Increasing intracellular Ca2+ with the ionophore A23187, in the absence of depolarizing agents, reproduced the effects of depolarization. By contrast, medullae grown in the presence of A23187, but in Ca2+-free medium, showed similar increases in prepro-EK mRNA and Leu-Enk, indicating an absolute requirement for Ca2+. In addition, KCl-inhibitory effects could be partially blocked by the calmodulin and protein kinase-C antagonist, trifluoperazine. However, KCl effects were not antagonized by the preferential calmodulin inhibitors W7, W13 or calmidizolium even at doses 10-fold higher than required to prevent calmodulin-dependent effects. Thus, these data suggest that inhibitory effects of transsynaptic activity and membrane depolarization on adrenal enkephalin occurs through Ca2+ and perhaps through a protein kinase-C dependent pathway, mechanisms known to augment catecholamine biosynthesis. It appears then that the same or similar molecular mechanisms can result in differential regulation of these co-localized transmitter systems.

Topics: Adrenal Medulla; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Enkephalin, Leucine; Enkephalins; Male; Organ Culture Techniques; Protein Kinase C; Protein Precursors; Rats; Rats, Inbred Strains; RNA, Messenger; Transcription, Genetic; Trifluoperazine