Endocrine Abstracts

Introduction: Hypothalamic gonadotropin releasing hormone (GnRH) regulates mammalian reproductive system. It binds receptors (GnRHRs) expressed in gonadotrope cells of anterior pituitary, regulating the synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). GnRHR is a G protein-coupled receptor (GPCR) coupled to Gαq/11 protein and, to a lesser extent, to Gαi/Gαs proteins, simultaneously activating Ca²⁺, β-catenin signaling and mitogen-activated protein (MAP)-kinases. GnRHR ligands were developed to be used as GnRH agonists and antagonists.

Aim: The aim of this study is to compare three GnRH antagonists, Cetrorelix, Ganirelix and Teverelix, in modulating GnRH-induced intracellular signaling in vitro. To this purpose, dose-finding and kinetics were evaluated.

Methods: Experiments were performed in GnRHR-transfected HEK293 cells, and in the human derived neuroblastoma (SH-SY5Y) cell line, naturally expressing GnRHR. GnRH-induced intracellular Ca²⁺ increase and cAMP production were evaluated by BRET, while CREB and ERK1/2 phosphorylation by Western blotting, in the presence or in the absence of GnRH antagonists.

Results: Upon stimulation by increasing doses of GnRH (pM-μM range), intracellular cAMP and Ca²⁺ accumulation occurred in HEK293 cells transiently over-expressing GnRHR (cAMP EC₅₀=11.58±0.29 nM, n=3; Ca²⁺ EC₅₀=25.97±0.15 nM, n=3). Moreover, 1 μM GnRH treatment produced a rise in intracellular cAMP sustained over 50 min (n=3) in transfected HEK293, while it resulted in no significant cAMP accumulation in SH-SY5Y cells expressing endogenous GnRHR (P<0.05, one-way ANOVA; n=3). In GnRHR-transfected HEK293 cells, Ca²⁺ increase induced by treatment using concentration of 3-fold the EC₅₀ GnRH was suppressed by 1 μM of antagonists (P<0.05, one-way ANOVA; n=2), while no significant inhibition was detected at lower doses of Ganirelix and Teverelix (pM-nM range). Only 100 nM Cetrorelix prevented GnRH-induced intracellular Ca²⁺ increase (P<0.05, one-way ANOVA; n=2). nM concentrations of GnRH failed to induce CREB and ERK1/2 phosphorylation in transfected HEK293 cells and in SH-SY5Y cells.

Discussion: Cetrorelix, Ganirelix and Teverelix have different in vitro activity, evaluated in terms of GnRH-induced intracellular Ca²⁺ increase, suggesting that these drugs may act differently at the molecular level.

Conclusions: Since GnRH antagonists may be not equivalent in vitro, drug-specific effects in vivo should not to be excluded.