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

1. Juan Pablo González Urzola Universidad Pontificia Bolivariana
2018 - 1

2. Introduction
• Corticotropin-releasing
hormone (CRH):
 neuropeptide hormone
 Its main function is the stimulation
of the pituitary synthesis of ACTH,
as part of the HPA Axis.
 Can suppress appetite, increase
subjective feelings of anxiety, and
perform other functions like
boosting attention
 The distal action of CRH is
immunosuppression via the action
of cortisol
• ATP-sensitive K+ channel:
 Type of potassium channel that is
gated by intracellular nucleotides,
ATP and ADP
 Are heteromultimers consisting of
Kir and SUR subunits.
SUR subunits (SUR1, SUR2A, and
SUR2B)
Kir subunits (Kir6.1 and Kir6.2)
 result in the expression of
different subtypes in various
tissues

3. • Pregnancy
 Potassium ion channels play an important role in the
activation of uterine muscles
 Changes in potassium ion channel expression or activity
can lead to changes in myometrial excitability and
contractility
 CRH is produced in the placenta and fetal membranes
during human pregnancy
 Concentration of CRH in the maternal plasma begins to
increase rapidly during the third trimester of pregnancy,
reaching a peak during labor.
Introduction

4. Corticotropin-releasing hormone (CRH) is a
crucial regulator of human pregnancy and
parturition. Adenosine triphosphate (ATP)-
sensitive potassium 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
General objective

5. Métodos - Cultivo
Mujeres parto a
termino
Muestras
1 x 1 x 1 cm
Lavado en Hanks
Muestras
3 x 3 mm
Cultivo
16 horas
37 °C
DMEM
Filtración y
centrifugacion
Citometria de
flujo
Cultivo
DMEM
37 °C
5% CO2
Ambiente
humidificado al
95%

6. Métodos - Tratamiento
Células del
músculo liso del
miometrio
humano
subcultivadas
(HSMC)
Pretratamiento
Antalarmina
Astressin 2b
1 hora
10^7 mmol/L
Tratamiento
CRH
6,12,24,48 horas
CRH
24 horas
CRH
0,1,10,10^2,10^3
10^4
pmol/L

7. Métodos - PCR
Reaccion en cadena de la polimerasa.
Técnica cuyo objetivo principal es la amplificación
directa de un gen o un fragmento de DNA o
indirecta de un RNA, presentes en mezclas de
muy diversas fuentes. Es decir permite obtener
muchas copia de una secuencia de DNA mediante
la replicación de una hebra sencilla se DNA por la
DNA polimerasa.
Se usó RT-PCR para evaluar la expresión de ARNm
para las subunidades Kir6.1 y SUR2B del canal de
potasio sensible al ATP de acuerdo con las
concentraciones de CRH. Este experimento se
realizó 5 veces, utilizando HSMC de diferentes
pacientes

8. Métodos - Western Blot
Técnica que permite la identificación de la
reacción entre un antígeno y un anticuerpo.
Primero se realiza una electroforesis para para
separar el componente proteico que contiene la
muestra, posteriormente se identifica el antígeno
con un anticuerpo.
Se realizo la identificación con:
• Kir6.1 antihumano de cabra
• SUR2B antihumano de cabra
• Anti-β-actina de cabra

9. Resultados

10. Resultados

11. Resultados

12. Resultados

13. Discussion
Author What they proposed Yes or not
Sasaki
The CRH concentrations used in the
present study were based on the
results of Sasaki
Yes
Campbell
There is a considerable change in the
maternal plasma CRH concentrations
throughout pregnancy Yes
Zhang
CRH also increases the contractile
effects of oxytocin in pregnant
myometrium, but there are
conflicting reports indicating that it
can show relaxant effects
Yes
Hillhouse and Grammatopoulos
they determined the mechanisms
underlying CRH dual action during
pregnancy.
Yes

14. 1. According to the results, it can be concluded
that changes in CRH concentration during
pregnancy have an effect on uterine
contractions, however it can determine the
onset of labor too, because in high
concentrations stimulates the onset of uterine
contractions.
2. The article shows the strongly influence of the
CRH concentrations in the expression of ATP-
Sensitive K+ channels , how as we know and
the results show it , there is a reduction of the
expression of the channels in high
concentrations of CRH.
Conclusions