Ascorbic acid augments the adenylyl cyclase-cAMP system mediated POMC mRNA expression and beta-endorphin secretion...

Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidylglycine-alpha-amidating monooxygenase for the post-translational modification of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion of immunoreactive beta-endorphin (ir-beta EP) and mRNA expression of proopiomelanocortin (POMC) following the activation of cAMP-dependent protein kinase A pathway in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum-free media and incubated with vehicle or various doses of AA in the presence or absence of forskolin, 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryladenosine 3'5'-(cyclic)monophosphate [(Bu)2cAMP]. Whereas the basal ir-beta EP release was 22.0 +/- 0.4 pg/well (mean +/- S.E.; n = 3), 10 microM of forskolin treatment increased ir-beta EP release approximately 4.2-fold. Co-incubation with AA enhanced forskolin induced ir-beta EP release and that this enhancing effect of AA was both time related and dose-dependent, with an ED50 of approximately 10 microM and an Emax of 100 microM. At the concentration of 10 microM, AA augmented ir-beta EP release approximately 6.1-fold that of cultures treated with forskolin alone. A similar potentiating effect of AA was also seen in cultures co-treated with IBMX or with (Bu)2cAMP. These enhancing effects of AA were similarly found in the abundance of total cAMP and of POMC mRNA of cultures which received identical treatments. However, it is important to point out that AA alone did not modulate ir-beta 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 pathway-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 adenylyl cyclase-cAMP system.