L-TYPE CA2+ CHANNELS MEDIATE JOINT MODULATION BY GAMMA-AMINOBUTYRIC-ACID AND GLUTAMATE OF [CA2+], AND NEUROPEPTIDE SECRETION IN IMMORTALIZED GONADODROPIN-RELEASING HORMONE NEURONS

To examine the role of calcium signaling in the joint modulation of gonadotropin-releasing hormone (GnRH) secretion by gamma-aminobutyric acid (GABA) and glutamate, cytoplasmic calcium ([Ca2+](i)) responses to the two transmitters were analyzed in monolayer networks of the GT1-7 line of immortalized GnRH neurons. [Ca2+](i) was increased by GABA and the GABA(A) receptor agonist, muscimol, and these responses were inhibited by the GABA(A) receptor antagonist, bicuculline. In contrast, the GABA(B) receptor agonist, baclofen, and the GABA(B) receptor antagonist, phaclofen, had no effect on basal and GABA- and glutamate-induced Ca2+ levels in GT1-7 neurons. The GABA-and muscimol-induced responses consisted of a spike increase in [Ca2+](i) followed by a decrease to a plateau; both the increase and the subsequent decrease in [Ca2+](i) depended on agonist concentration. Glutamate, N-methyl-D-aspartate (NMDA), and kainate also increased [Ca2+](i), but were less effective than GABA. GABA-, glutamate-, NMDA-, and kainate-induced [Ca2+](i) responses were almost abolished in Ca2+-free medium and were markedly attenuated by nifedipine. The [Ca2+](i) response to GABA was unaffected by prior application of glutamate, and vice versa. This additive effect of glutamate on the GABA-induced [Ca2+](i) response was mimicked by prior or simultaneous application of low (10 mM) KCl. The [Ca2+](i) response to simultaneous application of GABA and glutamate was also equal to the sum of the individual responses, whereas the GnRH secretory response was larger. However, the secretory responses to GABA and glutamate applied individually or together, were markedly attenuated by nifedipine. These results indicate that in GT1-7 neurons the combined actions of GABA and glutamate on [Ca2+](i) are additive of the actions of each transmitter, which are nifedipine-sensitive. However, the combined actions of the two transmitters on GnRH release are more than additive, possibly reflecting the non-linear coupling between [Ca2+](i) and exocytosis in neuroendocrine cells.