DEXAMETHASONE AND FORSKOLIN SYNERGISTICALLY INCREASE [MET(5)]ENKEPHALIN ACCUMULATION IN MIXED BRAIN-CELL CULTURES

Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10(-7) M) and the adenylate cyclase agonist forskolin (FSK, 10(-5) M) on [Met(5)]enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures. In enriched glia l cultures, DEX and FSK each stimulated the accumulation of ME 2-3-fol d over basal media levels, but there was little additional stimulation when these agonists were combined. In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combi nation of these agonists produced a pronounced synergistic effect on m edia ME accumulation (6-10-fold over basal levels). The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was poten t (acting over a concentration range of 10(-11)-10(-7) M), mimicked by corticosterone (10(-6) M), and blocked by the glucocorticoid receptor antagonist RU486. There was a pronounced time lag (2 days) for the sy nergistic effects of DEX + FSK to develop. In situ hybridization and i mmunocytochemical studies suggested that astrocytes were the major sou rce for the increased ME production in all mixed neuronal/glial cultur es examined. Creating a mixed culture by plating fetal neurons onto co nfluent, enriched P7 glial cultures inhibited accumulation of ME in th e media. DEX + FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures. The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial culture s relative to a 2.5-fold increase in the enriched glial cultures. Neur onal inhibition of basal glial ME production could explain the similar synergistic effects of DEX + FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain.