GNRH INTERACTION WITH ANTERIOR-PITUITARY .2. CYCLIC-AMP AS AN INTRACELLULAR MEDIATOR IN THE GNRH ACTIVATED GONADOTROPH

Suspensions of ovine anterior pituitary cells were prepared by sequential incubation with hyaluronidase-collagenase and trypsin. After an 18 h culture in suspension, cells were used 1) as a bioassay system to evaluate the LH releasing potency of GnRH and analogs of GnRH and 2) to investigate the role of cAMP as an intracellular second messenger for GnRH-induced release of LH. GnRH stimulated release of LH from ovine anterior pituitary cells was dose-dependent, with an ED50 of 0.35 nM GnRH. Release of cAMP was below the limits of detection at all concentrations of GnRH. Therefore, a phosphodiesterase inhibitor (0.5 mM MIX) and dopamine (0.5 μM) were added to the incubation medium to inhibit degradation of cAMP and to reduce secretion of cAMP by lactotrophs, respectively. Under these conditions, both basal and GnRH stimulated release of LH were potentiated. GnRH-induced release of LH and cAMP was dose-dependent and the log-linear portion of the response curves was parallel. The minimum effective dose was 0.032 nM GnRH for release of both cAMP and LH. Maximal release of LH was effected by 2 nM GnRH, whereas 125 nM GnRH was required to induce maximal release of cAMP. D-Ala6-GnRH ethylamide also induced release of LH and cAMP in a dose-dependent manner. A GnRH-induced increase in total LH (medium plus cellular) was not demonstrable during the 120 min incubation. GnRH specific release of cAMP was noted within 5 min and was maximal by 180 min, while GnRH specific release of LH was first apparent at 10 min and was maximal by 180 min. Although total LH increased with time of incubation, no GnRH specific changes in total LH were observed at incubation times to 240 min. Dibutyryl-cAMP (1 mM) stimulated release of LH to an extent greater than 200 nM GnRH but had no effect on total LH. The results suggest that cAMP may be an intracellular second messenger for release of LH in the GnRH activated gonadotroph.