Non-genomic actions of sex hormones on pregnant uterine contractility in rats: An in vitro study at term

Abstract

The non-genomic (prompt) actions of sex steroids on pregnant uterine contractility are not fully explored yet, the aim of our study was to clarify such effects of 17-β estradiol (E2), progesterone (P4) and testosterone (T) on late (22-day) pregnant uterine contractions together with the signaling pathways in rats in vitro. Methods: The uterine effects of sex steroids on KCl-stimulated contractions were examined in the presence of genomic pathway blocker actinomycin D and cycloheximide, sex hormone receptor antagonists (flutamide, fulvestrant, mifepristone) and also after removing the endometrium. The modifications in uterine G-protein activation and cAMP levels were also detected. Results: T and E2 both relaxed the uterine contractions in the concentration range of 10 −8-10 −3 M with an increase in the activated G-protein and cAMP levels of the uterus, while P4 was ineffective. Cycloheximide, actinomycin D, antagonist for T and E2 were not able to modify the responses along with the endothelium removal. Mifepristone blocked the relaxing effects of T and E2 and reduced the activation of G-protein and the formation of cAMP. Significance: T and E2 can inhibit KCl-stimulated contractions in the late pregnant uterus in high concentrations and in a non-genomic manner. Their actions are mediated by a G-protein coupled receptor that can be blocked by mifepristone. A single and high dose of T or E2 might be considered in premature contractions, however, further preclinical and clinical studies are required for the approval of such a therapeutic intervention.

Non-genomic uterorelaxant actions of corticosteroid hormones in rats: An in vitro and in vivo study

“…In addition, endometrium removal did not alter the relaxing action of DEX of FLD, therefore, the measured effects are linked to myometrial steroid receptors. This finding is similar to our previous study about sex steroids (Mirdamadi et al, 2020), but is in contrast with earlier studies of airway smooth muscles, where the prompt relaxing action of CSs is endothelium-dependent (Horvath, 2006). This suggests the different functions of the endometrium and the endothelium in the uterus and lungs, respectively.…”

“…Since each experiment lasted a maximum of 30 min, genomic action was ruled out. As a further proof for non-genomic action, pre-treatment with genomic pathway blocker actinomycin D (DNA synthesis blocker) did not modify the action of steroid drugs, similarly to earlier studies (Gutiérrez et al, 1994;Mirdamadi et al, 2020;Panettieri et al, 2019). In addition, endometrium removal did not alter the relaxing action of DEX of FLD, therefore, the measured effects are linked to myometrial steroid receptors.…”

“…To investigate the role of classical steroid receptors, the tissues were pre-treated with the following receptor antagonists for 10 min before KCl stimulation: spironolactone in10 −6 M for FLD (Mihailidou and Funder, 2005), mifepristone (MIF) in 10 −8 M for GR (Attardi et al, 2004), and in 10 −6 M (Mirdamadi et al, 2020) for both steroids mentioned. In another set of experiments, uterine tissues were pre-treated with the transcription inhibitor actinomycin D (10 −6 M) (Pérez et al, 2013), for 30 min before adding DEX or FLD.…”

“…To investigate the efficacy of steroids on G-protein-coupled receptors (GPCRs), especially G αi/o in a non-genomic manner, the changes of GDP to GTP were measured with radiolabeled, non-hydrolysable GTP. The experiment of [ 35 S]GTPγS was performed as described in our previous study (Mirdamadi et al, 2020). Uterine tissue samples of both pregnant and non-pregnant animals (n = 10) were grounded and homogenized with Ultra-Turrax® (IKA-Werke GmbH & Co. KG, Staufen in Breisgau, Germany) in an ice bath for 2 × 30 s with 20 vol (W/V) of ice-cold Tris-EDTA buffer (composed of 10 mm Tris-HCl, 1 mM EDTA, 0.6 mM MgCl 2 , and 0.25 M sucrose, pH 7.4), and afterwards suspended by a 4-layer gauze filter.…”

“…In our previous work, we reported the non-genomic actions of sex steroids on pregnant uterine smooth muscles in rats (Mirdamadi et al, 2020). In the present study, we aimed to investigate the fast, nongenomic action of MC fludrocortisone (FLD) and GC dexamethasone (DEX) on uterine contractions and signaling pathways both in pregnant and non-pregnant rats.…”