Partial characterization of K +-induced increase in [Ca 2+] cyt and GnRH release in GT1-7 neurons

Abstract

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca 2+ and GnRH release in response to K +-induced depolarization of GT1-7 neurons. Our results showed that K + concentrations from 7.5 to 60 mM increased [Ca 2+] cyt in a concentration-dependent manner. Resting [Ca 2+] cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; n = 69). K +-induced increases in [Ca 2+] cyt ranged from 58.2 nM at 7.5 mM [K +] to 347 nM at 60 mM [K +]. K +-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K +] to about 60 pg/ml at 45 mM [K +]. K +-induced increases in [Ca 2+] cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca 2+ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca 2+ channel antagonist. Nimodipine (1 μM) alone partially inhibited K +-induced increases in [Ca 2+] cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K +-induced GnRH release or [Ca 2+] cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K +-induced increase in [Ca 2+] cyt significantly more ( p < 0.02) than nimodipine alone, suggesting that N-type Ca 2+ channels exist in GT1-7 neurons and may be part of the response to K +. The response of [Ca 2+] cyt to K + was linear with increasing [K +] whereas the response of GnRH release to increasing [K +] appeared to be saturable. K +-induced increase in [Ca 2+] cyt and GnRH release required extracellular [Ca 2+]. These experiments suggest that voltage dependent N- and L-type Ca 2+ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.