Obstet Gynecol Sci.  2018 Jan;61(1):14-22. 10.5468/ogs.2018.61.1.14.

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

Affiliations
  • 1Department of Obstetrics and Gynecology, Eulji General Hospital, Eulji University, Seoul, Korea. obdrseo@eulji.ac.kr
  • 2Department of Obstetrics and Gynecology, Eulji Medi-Bio Research Institute, Eulji University, Daejeon, Korea.

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 KATP 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, KATP 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.

Keyword

Corticotropin-releasing hormone; Pregnancy; KATP channels; Human myometrium

MeSH Terms

Adenosine Triphosphate
Adenosine*
Animals
Blotting, Western
Corticotropin-Releasing Hormone*
Female
Humans*
In Vitro Techniques
KATP Channels
Mice
Myocytes, Smooth Muscle
Myometrium*
Oxytocin
Parturition
Potassium Channels*
Potassium*
Pregnancy
Receptors, Corticotropin-Releasing Hormone
RNA, Messenger
Adenosine
Adenosine Triphosphate
Corticotropin-Releasing Hormone
KATP Channels
Oxytocin
Potassium
Potassium Channels
RNA, Messenger
Receptors, Corticotropin-Releasing Hormone

Figure

  • 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.


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