Dependence of 3',5'-cyclic adenosine monophosphate--stimulated gonadotropin-releasing hormone release on intracellular calcium levels and L-type calcium channels in superfused GT1-7 neurons.

Objective:Immortalized GT1-7 neurons were used to characterize the interactive roles of adenylate cyclase-3′,5′-cyclic adenosine monophosphate (cAMP) and L-type calcium channels on gonadotropin-releasing hormone (GnRH) release.Methods:Dibutyryl (db)-cAMP was used as an active analog of endogenous cAMP, and forskolin was used to activate adenylate cyclase. Extracellular calcium was chelated using EGTA and L-type calcium channels were blocked using nimodipine. The selective Ca2+ ionophore A23187 was employed to increase intracellular calcium levels. GT1-7 neurons were grown on Cytodex-3 beads (Pharmacia Biotech, Uppsala, Sweden) and placed in special superfusion microhambers. The cells were superfused at a rate of 6.2 mL/h with media 199 (M-199; Gibco, Grand Island, NY; pH 7.35, 37C); effluent fractions were collected at 5-minute intervals for analysis of GnRH concentrations by radioimmunoassay.Results:Basal GnRH release from superfused GT1-7 neurons ranged from 10 to 62 pg · min-1. mL-1. Coexposure of the ...