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The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.

The receptors and actions of gonadotropin-releasing hormone (GnRH) were analyzed in cultured testicular cells from 20.5-day fetal rats, in which treatment with luteinizing hormone (LH) maintained Leydig cell steroidogenesis and gonadotropic responses for up to 2 weeks. Testicular GnRH receptors were present on the 5th postnatal day, but were not demonstrable in fetal testes or 2-day cultures thereof. However, GnRH receptors were readily detectable in 4-day cultured fetal testes and were increased by exposure to GnRH agonists. In LH-treated cultures, GnRH sites were reduced by about 50% and did not increase during incubation with GnRH agonists. In such cultures, GnRH agonists inhibited LH-dependent steroid production and abolished the acute testosterone response to human chorionic gonadotropin. The half-maximal inhibitory concentration of [D-Ala6]des-Gly10-GnRH-N-ethylamide (3 X 10(-10)M) was commensurate with its binding affinity for testis receptors (Kd = 1.4 X 10(-10)M). In contrast, GnRH agonists had no inhibitory effects in 2-day cultures prior to the detection of GnRH receptors. The expression of functional GnRH receptors during culture in the absence of gonadotropin and their suppression in LH-treated cultures suggest that pituitary gonadotropins exert a tonic inhibitory effect upon testicular GnRH receptors. The demonstrated inhibitory actions of GnRH on steroidogenesis, with the expression of GnRH receptors in cultured fetal testes and 5-day postnatal testes, indicate that GnRH agonists could influence the actions of gonadotropins upon Leydig cell function in the neonatal testis.

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.