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 experiments in cultured pituitary cells and from perifusion experiments using a GnRH-antagonist. After 4 days in culture under identical conditions, cells taken from pituitaries of fish maintained at 26°C were more sensitive to GnRHa ([D-Ala(6), Pro(9)-NEt]-LHRH) than those taken from fish maintained at 19°C. Cells from female pituitaries were more responsive than those from males. taGTH release in culture was augmented by Ca(2+) ionophore (A23187; 1-100 μM) or ionomycin (0.02-10 μM). The response of perifused pituitary to GnRH was reduced by nimodipine (1-10 μM) indicating that Ca(2+) influx via voltage-sensitive Ca(2+) channels is involved in the stimulation of GTH release. Activation of protein kinase C by OAG (1-oleyl-2-acetyl glycerol; 0.16-160 μM) 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 μM) also augmented the release of taGTH from the culture. Four sequential pulses of sGnRH (100 nM) at 2h intervals resulted in surges of taGTH release from perifused pituitary fragments; the surges were similar in magnitude with no signs of desensitization. Sequential stimulation with graded doses of sGnRH (0.1 nM to 1 μM) in the presence of GnRH-antagonist ([Pro(2,6), Trp(3)]-GnRH) resulted in an attenuation of taGTH release. However, the GnRH-antagonist did not alter the pattern of forskolin-stimulated GTH release, indicating that forskolin stimulation is exerted at the level of the adenohypophyseal cells. It is concluded that, as in other vertebrates, the transduction of GnRH stimulation of GTH release involves Ca(2+) influx through voltage-sensitive Ca(2+) channels, mobilization of the ion from intracellular sources, arachidonic acid and activation of PKC. Adenylate cyclase-cAMP system us also involved in the mediation but its relationship with other transduction cascades requires further investigations.