NITRIC-OXIDE SYNTHESIZED BY GONADOTROPIN-RELEASING-HORMONE NEURONS ISA MEDIATOR OF N-METHYL-D-ASPARTATE (NMDA)-INDUCED GNRH SECRETION

N-Methyl-D-aspartate (NMDA) directly stimulates gonadotropin-releasing hormone (GnRH) neurons to secrete GnRH. It is not known if this stimu latory effect of NMDA is mediated by NO. Northern blot analysis of the immortalized hypothalamic GnRH neuronal cells (GT1-1) mRNA with a neu ronal NOS cDNA revealed this clonal cell line expressed neuronal NOS t ranscripts as a single 10.5-kb band. Immunoblot analysis of GT1-1 prot eins with anti-neuronal NOS serum showed that the GT1-1 cells contain neuronal NOS. GT1-1 cells were used to study the effects of NO and NMD A on GnRH release. L-Arginine (10(-2)M), a precursor of NO enhances ba sal GnRH secretion. Both oxyhemoglobin (Hb)(10(-6)-10(-4)M), a NO scav enger and N-omega-nitro-L-arginine (NNA)(10(-3),10(-2)M), a NOS inhibi tor and inactivator block basal as well as NMDA-induced GnRH release. Sodium nitroprusside (SNP) (10(-4), 10(-3)M), a NO donor stimulates Gn RH release, an effect inhibited by Hb. Incubation of GT1-1 cells in Ca 2+-free medium abolished the stimulatory effect of NMDA on GnRH releas e. In contrast, incubation in medium with increasing concentrations of Ca2+ enhances basal GnRH release as well as augments NMDA-mediated Gn RH release. The results demonstrate that L-arginine-NO pathway is func tional in the GT1-1 cells and an increase in intracellular Ca2+ [Ca2+] (i) following NMDA receptor activation activates NOS to generate NO. W e conclude that endogenous NO mediates, at least in part, basal as wel l as NMDA-stimulated GnRH release and may play a role as an intercellu lar messenger in synchronizing pulsatile GnRH release.