Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action

摘要

We have shown recently that Mn²⁺ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn²⁺ induces luteinizing hormone-releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30-day-old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N-monomethyl-l-arginine (NMMA); 300 or 500 μm) or a soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 100 or 250 μm) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 μm) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 μm MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300–500 μm NMMA to the medium did not block Mn²⁺-stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 μm MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn²⁺ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn²⁺-stimulated secretion of LHRH in a dose-dependent manner, indicating that GC is the site of action of Mn²⁺. Additionally, we showed that Mn²⁺ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 μm) inhibited Mn²⁺-induced LHRH release. These data demonstrate that the principal action of Mn²⁺ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.