Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. II. Extracellular calcium dependence and dopaminergic inhibition of gonadotropin responses.

Primary static cultures of dispersed goldfish pituitary cells obtained by controlled trypsinization released gonadotropin (GTH) in response to 2-hr stimulations of 0.1 nM to 1 microM [Trp7,Leu8]-gonadotropin-releasing hormone (sGnRH), [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa), and [His5,Trp7,Tyr8]-GnRH (cGnRH-II) in a dose-dependent manner. Coincubation with 10 to 1000 nM of a dopamine agonist, apomorphine, dose dependently reduced the GTH response to increasing concentrations of sGnRH. Apomorphine at 1 microM completely abolished the dose-dependent GTH response to sGnRHa and cGnRH-II, but only partially inhibited the GTH-releasing action of high concentrations of sGnRH. Addition of calcium ionophores, 1 to 100 microM A23187 and 10 to 100 microM ionomycin, significantly increased GTH release. The ED50S of the GTH response to A23187 and ionomycin were 0.88 +/- 0.15 and 13.67 +/- 2.76 microM, respectively. Incubation with Ca2(+)-deficient media (media prepared without the addition of Ca2+ salts) did not significantly affect basal GTH release, but severely decreased the hormone response to increasing concentrations of sGnRH, A23187, and ionomycin. These results confirm the direct inhibitory dopaminergic influence on GTH release in goldfish and further suggest that extracellular Ca2+ plays a role in mediating GnRH action on gonadotropes in fish.