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 microchambers. 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 microM of forskolin (an activator of adenylate cyclase) for 5 minutes showed a 2.6-fold increase in GnRH release. Likewise, the addition of 100 microM 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 microM 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.
Chen
Eileen C
EC
Department of Obstetrics and Gynecology, Southern California Permanente Medical Group, Los Angeles, California, USA.
Javors
Martin A
MA
Norris
Catherine
C
Siler-Khodr
Theresa
T
Schenken
Robert S
RS
King
Thomas S
TS
D000262:Adenylyl Cyclases / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics*; D020746:Calcium Channels, L-Type / Q000502:physiology*; D018929:Cell Culture Techniques; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry; D007476:Ionophores / Q000494:pharmacology; D009474:Neurons
D020746:Calcium Channels, L-Type; D007476:Ionophores; D005576:Colforsin; D007987:Gonadotropin-Releasing Hormone; D000001:Calcimycin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium