Dependence of 3′,5′-Cyclic Adenosine Monophosphate—Stimulated Gonadotropin-Releasing Hormone Release on Intracellular Calcium Channels in Superfused GT1-7 Neurons

Abstract

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 cells to forskolin and A23187 produced an additive effect on stimulated release of GnRH. Cells exposed to 1 μM of forskolin (an activator of adenylate cyclase) for 5 minutes showed a 2.6-fold increase in GnRH release. Likewise, the addition of 100 μM of db-cAMP to the superfusion for 5 minutes demonstrated a 2.3-fold increase in the amplitude of GnRH secretion. Maintaining the superfused cells in medium containing 5 mM EGTA had no obvious effect on basal GnRH release but blocked the effect of db-cAMP to increase GnRH release. Similarly, the addition of 10 μM nimodipine to the superfusion medium blocked db-cAMP-stimulated GnRH release.

Conclusions: These findings provide additional evidence that cAMP-mediated GnRH release from GT1-7 neurons is dependent on influx of extracellular calcium via L-type Ca²⁺ channels.

Keywords

GT1-7 neurons GnRH cAMP L-type calcium channels

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