The inhibitory regulation of PRL secretion by dopamine (DA) or the dopaminergic agonists bromergocryptine (CB-154) and apomorphine was studied in cultured GH3 cells, an established rat anterior pituitary cell line which produces both PRL and GH. The basal release of PRL from GH3 cells was unaffected when incubated for 6 h with DA concentrations as high as 10(-4) M. The inability of DA to suppress PRL secretion could not be explained by the catabolism of DA or the presence of unknown inhibitors (e.g. estradiol) in the fetal calf serum present in the incubation media. Apomorphine and CB-154 were only partially effective in suppressing PRL release at high concentrations of 10(-4) and 10(-5) M, respectively. Various concentrations of the dopaminergic antagonist d-butaclamol did not reverse the inhibitory action of 10(-5) M CB-154, while equal concentrations (10(-5) M) of both d- and l-butaclamol significantly suppressed PRL release. The greatly lowered responsiveness of GH3 cells to dopaminergic inhibition of PRL is suggestive of some impairment of DA receptors. This hypothesis is supported by radioligand binding studies in which high affinity dopaminergic binding sites are absent in the same cell line used in this study.

Dopamine (DA) and DA agonists bind with high affinity to anterior pituitary receptors which mediate the inhibition of PRL release. Spiperone (SPIP), a DA antagonist, has also been successfully used to characterize pituitary DA receptors with a dissociation constant (Kd) of less than 1 nM. We studied the binding of SPIP to GH3D6 cells which secrete only PRL and GH. This clone was derived from a radiation-induced tumor of the rat anterior pituitary. Equilibrium binding of [3H]SPIP to living GH3 cells showed no high affinity receptors, but a low affinity (Kd = 0.83 microM) and saturable (0.06 fmol/cell) population of sites was observed. In addition, saturable binding with a similar affinity (Kd = 0.57 microM) was noted in broken GH3 cells. The interaction was completely reversible and temperature dependent. The concentration of various ligands required to compete for half of the [3H]SPIP binding to whole cells were: chlorpromazine, 0.17 microM; haloperidol, 0.68 microM; pimozide, 0.77 microM; d-butaclamol, 1.16 microM; 1-butaclamol, 1.30 microM; SPIP, 1.49 microM; bromergocryptine, 4.98 microM; apomorphine, 13.9 microM; and DA, 100 microM. The absence of a high affinity site in GH3 cells is consistent with the decreased effectiveness of various agonists and antagonists on PRL secretion. It is possible that the low affinity interactions observed in GH3 cells are normally present in the anterior pituitary and brain and do not simply represent an alteration of receptor affinity.