INTRACELLULAR MEDIATION OF GNRH ACTION ON GTH RELEASE IN TILAPIA

The objective of the present study was to confirm previous results on the mediation of GnRH signal in tilapia by providing evidence from exp eriments in cultured pituitary cells and from perifusion experiments u sing a GnRH-antagonist. After 4 days in culture under identical condit ions, cells taken from pituitaries of fish maintained at 26-degrees-C were more sensitive to GnRHa ([D-Ala6, Pro9-NEt]-LHRH) than those take n from fish maintained at 19-degrees-C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture w as augmented by Ca2+ ionophore (A23187; 1-100 muM) or ionomycin (0.02- 10 muM). The response of perifused pituitary to GnRH was reduced by ni modipine (1-10 muM) indicating that Ca2+ influx via voltage-sensitive Ca2+ channels is involved in the stimulation of GTH release. Activatio n of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 muM) or TPA (1-O-tetra-decanoyl phorbol-13-acetate; 1.25-125 nM) resulted in a dose-dependent stimulation of taGTH release from cultured cells. Arachidonic acid (0.33-330 muM) also augmented the release of taGTH fr om the culture. Four sequential pulses of sGnRH (100 nM) at 2h interva ls resulted in surges of taGTH release from perifused pituitary fragme nts; the surges were similar in magnitude with no signs of desensitiza tion. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 m uM) in the presence of GnRH-antagonist ([Pro2,6, Trp3]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did n ot alter the pattern of forskolin-stimulated GTH release, indicating t hat forskolin stimulation is exerted at the level of the adenohypophys eal cells. It is concluded that, as in other vertebrates, the transduc tion of GnRH stimulation of GTH release involves Ca2+ influx through v oltage-sensitive Ca2 + channels, mobilization of the ion from intracel lular sources, arachidonic acid and activation of PKC. Adenylate cycla se-cAMP system is also involved in the mediation but its relationship with other transduction cascades requires further investigations.