IMMORTALIZED GONADOTROPIN-RELEASING-HORMONE NEURONS SECRETE GAMMA-AMINOBUTYRIC-ACID - EVIDENCE FOR AN AUTOCRINE REGULATION

The immortalized hypothalamic neuronal cell lines GT1-1 and GT1-7 repr esent unique model systems to investigate the physiological control of gonadotropin-releasing hormone (GnRH) secretion. Using immunofluoresc ence microscopy, key proteins of regulated exocytosis, e.g. synaptotag min, synaptobrevin and SNAP-25 (synaptosomal associated protein of 25 kDa) were found in GT1 neurons. In addition, GT1 neurons contained syn aptophysin, a marker protein for small synaptic vesicles (SSVs) which are responsible for the storage of neurotransmitters such as gamma-ami nobutyric acid (GABA). Upon subcellular fractionation, a righter vesic le population characterized by synaptophysin separated from a denser v esicle population containing GnRH. Both vesicle populations contained synaptobrevin and synaptotagmin. Besides GnRH, GT1 neurons expressed g lutamic acid decarboxylase at the mRNA-level and synthesized GABA. Mor e importantly, GT1 neurons took up and stored H-3-GABA. The stored GAB A was released after stimulation with increasing K+ concentrations and by alpha-latrotoxin. Reducing the extracellular Ca2+-concentration ab olished stimulated secretion, indicating that GABA was released by reg ulated exocytosis. Hormone secretion from GT1 neurons is controlled by GABA via GABA(A) and GABA(B) receptors reflecting the situation in vi vo. Our data provide the first evidence that GT1 neurons possess a sec ond regulated secretory pathway sustained by SSVs storing and releasin g GABA. The released GABA influences GnRH secretion by an auto- or par acrine loop.