Different calcium pools participate in the activation of ERK1/2 by estradiol in male rat hypothalamic neurons in vitro

CABRERA ZAPATA, L.E.; BOLLO, M.; CAMBIASSO, M.J.

Mendoza

Workshop; 2015 International Workshop in Neuroendocrinology; 2015

Estrogens generate awide diversity of rapid ?non-classical? effects which occur in a range fromsome seconds to few minutes, including the triggering of Ca2+signals and the activation of several signaling pathways such as the proteinkinase C (PKC) and the extracellular signal-regulated kinase 1 and 2 (ERK1/2)cascades. Previous studies from our laboratory have shown that 17β-estradiol(E2) induces axonal growth through ERK1/2 activation in hypothalamic neurons ofmale embryos of 16 days of gestation (E16). Moreover, both axogenesis andERK1/2 activation mediated by the hormone depend on Ca2+ and Ca2+-dependentPKC isoforms. In the present study we investigate the Ca2+ pools thatparticipate in the activation of ER1/2 by E2. Primary hypothalamic neuroncultures were established from E16 male rat embryos and fed withastroglia-conditioned media for 48 hr. After 2 days in vitro, hypothalamic neurons were pre-treated for 1 hr with anextracellular Ca2+ chelator (EGTA), an L-type voltage-gated Ca2+channel (L-VGCC) blocker (nifedipine), a ryanodine receptor (RyR) inhibitor(ryanodine), or an inositol-1,4,5-trisphosphate receptor (IP3R)inhibitor (2-APB); and then pulsed for 15 min with E2. Finally, cultures were scrapedand homogenized to analyze the ERK1/2 phosphorylation levels by Western blot. E2treatment rapidly induced ERK activation, which was completely abolished byryanodine and partially attenuated by nifedipine and 2-APB. Pre-treatment withEGTA slightly decreased E2-induced ERK1/2 phosphorylation butthis effect was not statistically significant. In summary, the results suggestthat Ca2+ mobilization from extracellular space and from endoplasmicreticulum are both necessary to E2-induced ERK1/2 activation. Whereas L-VGCCsand IP3Rs might participate in the Ca2+ signaling evoked bythe hormone, the release of Ca2+ through RyRs plays a predominantrole in E2-induced ERK1/2 activation.This research was financially supported by grants fromCONICET, ANPCyT and SECyT-UNC, Argentina. Lucas E. Cabrera Zapata has held a fellowship from ConsejoInteruniversitario Nacional.