ASCORBIC-ACID AUGMENTS THE ADENYLYL CYCLASE-CAMP SYSTEM MEDIATED POMCMESSENGER-RNA EXPRESSION AND BETA-ENDORPHIN SECRETION FROM HYPOTHALAMIC NEURONS IN CULTURE

Besides acting as an important cofactor in the biosynthesis of catecho lamine, ascorbic acid (AA) also modulates the activity of peptidylglyc ine-alpha-amidating monooxygenase for the post-translational modificat ion of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion of immunoreactive beta-endor phin (ir-beta EP) and mRNA expression pf proopiomelanocortin (POMC) fo llowing the activation of cAMP-dependent protein kinase A pathway in r at hypothalamic neurons. Primary cultures of hypothalamic neurons from ;neonatal rats as previously described were employed in the present st udies. Six days after plating, cultures were replenished with serum-fr ee media and incubated with vehicle or various doses of AA in the pres ence or absence of forskolin, 3-isobutyl-1-methylxanthine (IBMX), N-6, 2'-O-dibutyryladenosine 3'5'-(cyclic)monophosphate [(Bu)(2)cAMP]. Wher eas the basal ir-beta EP release was 22.0 +/- 0.4 pg/well (mean +/- S. E n = 3), 10 mu M of forskolin treatment increased ir-beta EP release approximately 4.2-fold. Co-incubation with AA enhanced forskolin induc ed ir-beta EP release and that this enhancing effect of AA was both ti me related and dose-dependent, with an ED(50) of approximately 10 mu M and an E(max) of 100 mu M. At the concentration of 10 mu M, AA augmen ted ir-beta EP release approximately 6.1-fold that of cultures treated with forskolin alone. A similar potentiating effect of AA was also se en in cultures co-treated with IBMX or with (Bu)(2)cAMP. These enhanci ng effects of AA were similarly found in the abundance of total cAMP a nd of POMC mRNA of cultures which received identical treatments. Howev er, it is important to point out that AA alone did not modulate ir-bet a EP release or the abundance of POMC mRNA or total cAMP levels of the hypothalamic cultures when protein kinase A pathway was not activated . We thus conclude that AA augments cAMP-dependent protein kinase A pa thway-induced production and release of beta EP from rat hypothalamic neurons: in culture. Furthermore, this biological effect of AA is, at least in part, mediated through enhancing the responsiveness of the ad enylyl cyclase-cAMP system.