Effects of corticotropin-releasing hormone on the expression of adenosine triphosphate-sensitive potassium channels (Kir6.1/SUR2B) in human term pregnant myometrium

Abstract

Objective: Corticotropin-releasing hormone (CRH) is a crucial regulator of human pregnancy and parturition. Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels are important for regulating myometrial quiescence during pregnancy. We investigated regulatory effects of different concentrations of CRH on KATP channel expression in human myometrial smooth muscle cells (HSMCs) in in vitro conditions.

Methods: After treating HSMCs with different concentrations of CRH (1, 10, 10², 10³, 10⁴ pmol/L), mRNA and protein expression of K_ATP channel subunits (Kir6.1 and SUR2B) was analyzed by reverse transcription-polymerase chain reaction and western blot. We investigated which CRH receptor was involved in the reaction and measured the effects of CRH on intracellular Ca²⁺ concentration when oxytocin was administered in HSMCs using Fluo-8 AM ester.

Results: When HSMCs were treated with low (1 pmol/L) and high (10³, 10⁴ pmol/L) CRH concentrations, K_ATP channel expression significantly increased and decreased, respectively. SUR2B mRNA expression at low and high CRH concentrations was significantly antagonized by antalarmin (CRH receptor-1 antagonist) and astressin 2b (CRH receptor-2 antagonist), respectively; however, Kir6.1 mRNA expression was not affected. After oxytocin treatment, the intracellular Ca²⁺ concentration in CRH-treated HSMCs was significantly lowered in low concentration of CRH (1 pmol/L), but not in high concentration of CRH (10³ pmol/L), compared to control.

Conclusion: Our data demonstrated the regulatory effect was different when HSMCs were treated with low (early pregnancy-like) and high (labor-like) CRH concentrations and the KATP channel expression showed significant increase and decrease. This could cause inhibition and activation, respectively, of uterine muscle contraction, demonstrating opposite dual actions of CRH.

Keywords: Corticotropin-releasing hormone; Human myometrium; KATP channels; Pregnancy.

Fig. 1

Molecular expression of Kir6.1 and SUR2B with 1–10⁴ pmol/L corticotropin-releasing hormone (CRH) in human myometrial smooth muscle cells. Blots are representative of 5 experiments. The expression level is presented as the ratio of the radioactivity of the band to that of the corresponding β-actin band. Values are presented as mean±standard error for 5 cultures from 5 patients. (A) mRNA expression of Kir6.1 and SUR2B was measured by reverse transcription-polymerase chain reaction. (B) Protein expression of Kir6.1 and SUR2B was measured by western blot analysis. ^a)P<0.05 compared with control.

Fig. 2

Effects of corticotropin-releasing hormone (CRH)-R1/-R2 antagonists on mRNA expression of Kir6.1 and SUR2B at low (1 pmol/L) (A) and high (10⁴ pmol/L) (B) concentrations of CRH. mRNA expression of Kir6.1 and SUR2B was measured by reverse transcription-polymerase chain reaction. Cells were pretreated with CRH-R1 antagonist (antalarmin) and CRH-R2 antagonist (astressin 2B) for 1 hour before treatment with CRH. Thereafter, cells were harvested for mRNA analysis. Representative bands are presented. The expression level is presented as the ratio of the radioactivity of the band to that of the corresponding β-actin band. Values are presented as mean±standard error for 5 cultures from 5 patients. ^a)P<0.05.

Fig. 3

Effects of corticotropin-releasing hormone (CRH) on oxytocin-induced increases in intracellular Ca²⁺ concentrations in human myometrial smooth muscle cells (n=3). CRH treatment was performed 24 hours before treating cells with oxytocin (OXY) and measuring intracellular Ca²⁺ using Fluo-8 AM ester. The Ca²⁺ traces were obtained by connecting the experimental value measured every 5 seconds. The representative traces are shown. Each value represents the mean measured for 3 replicate wells from one 96-well plate. ^a)P<0.05.