Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes)

• Aim: characterize the immediate GnRH response in medaka LH-producing gonadotropes.
• GnRH2 induces a transient increase in cytosolic Ca2+.
• The Ca2+ response is dependent on both intra- and extracellular sources.
• The extracellular contribution is partly through L-type Ca2+ channels.
• GnRH2 induces a membrane depolarization and increased action potential frequency.

We have characterized the response to gonadotropin-releasing hormone 2 (GnRH2) in luteinizing hormone producing cells from gfp-transgenic medaka. Teleosts have separate cells producing the two types of gonadotropins, enabling us for the first time to study the intracellular signaling that controls secretion of each gonadotropin separately. Pituitary cell cultures were prepared, and lhb-producing cells were selected by their GFP expression. Cytosolic Ca2+ imaging revealed three response patterns to GnRH2, one monophasic and two types of biphasic patterns. The Ca2+ sources were examined by depleting intracellular Ca2+ stores and preventing influx of extracellular Ca2+. Both treatments reduced response amplitude, and affected latency and time to peak. Blocking L-type Ca2+ channels reduced amplitude and time to peak, but did not remove extracellular Ca2+ contribution. Patch-clamp recordings showed spontaneous action potentials in several cells, and GnRH2 increased the firing frequency. Presence of Ca2+-activated K+ channels was revealed, BK channels being the most prominent.