Korean J Physiol Pharmacol.  2016 Sep;20(5):547-556. 10.4196/kjpp.2016.20.5.547.

Myometrial relaxation of mice via expression of two pore domain acid sensitive K⁺ (TASK-2) channels

• Kyeong KS
• Hong SH
• Kim YC
• Cho W
• Myung SC
• Lee MY
• You RY
• Kim CH
• Kwon SY
• Suzuki H
• Park YJ
• Jeong EH
• Kim HS
• Kim H
• Lim SW
• Xu WX
• Lee SJ
• Ji IW

• KMID: 2350512
• DOI: http://doi.org/10.4196/kjpp.2016.20.5.547

Abstract

Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K⁺ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K⁺ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K⁺ channels (TASK-2). NIOK in the presence of K⁺ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

Keyword

Longitudinal muscle; Myometrium; Relaxation; TASK-2

MeSH Terms

Acidosis
Animals
Blotting, Western
Bupivacaine
Estrogens
Female
Isometric Contraction
Lidocaine
Methionine
Mice*
Microscopy, Confocal
Muscle, Smooth
Myometrium
Oxytocin
Pregnancy
Quinidine
Relaxation*
Tea
Uterine Contraction
Uterus
Bupivacaine
Estrogens
Lidocaine
Methionine
Oxytocin
Quinidine
Tea

Figure

• 

  Fig. 1 Effect of quinidine on the isometric contraction of uterine longitudinal muscle in mice.(A) Oxytocin (OXT) produced initial and tonic contractions; however, tonic contractions were not sustained in non-pregnant myometrium (NP, non-pregnant; TP, term-pregnant). (B) Quinidine (10, 20, and 30 µM) produced concentration-dependent phasic contractions. Tonic contractions were also produced at high concentrations of quinidine in pregnant myometrium. (C) Quinidine was applied to myometrium in the presence of TEA (10 mM). TEA (10 mM) produced strong phasic contractions in non-pregnant myometrium (1.2±0.15 g) but not in pregnant myometrium. Quinidine (10~30 µM), even in the presence of TEA, increased contraction frequency in non-pregnant myometrium and produced stronger contractions in pregnant myometrium.

• 

  Fig. 2 Regulation of pregnant mouse myometrial contractility by TASK-2 channel inhibitors in the presence of TEA and 4-AP.The TASK-2 inhibitors produced contractions in pregnant myometrium in the presence of TEA and 4-AP. (A) Extracellular acidosiso (pHo=6.4) produced contractions in pregnant myometrium. (B) Bupivacaine (100 nM) also produced robust contractions even in the presence of nerve blockers. (C) L-methionine (1 mM), which inhibits stretch-dependent K2P channels (TREK-1), produced contractions that spontaneously decayed to near baseline values within 10~15 min. However, lidocaine produced robust contraction in the presence of L-methionine. (D~E) Data are summarized.

• 

  Fig. 3 Regulation of TEA and 4-AP insensitive non-inactivating outward current (NIOK) in single mouse myometrial cells.(A, B) In the presence of TEA (10 mM) and 4-AP (5 mM), NIOK were observed under whole-cell voltage clamp. Quinidine (50 µM) inhibited NIOK currents more significantly in pregnant cells. (C) NIOK was inhibited by pHo=6.4, bupivacaine, oxytocin (OXT), and estrogen in pregnant myometrial cells. (D) Representative I/V relationships are shown. (E) Data are summarized. NIOK was inhibited to 126, 86, 87, 85, 81, 68 and 78% of the control by pHo=8.4, pHo=6.4, OXT (10 and 50 nM), bupivacaine (500 nM), estrogen (1 µM), and Ba2+ (3 mM), respectively (n= 5, 5, 4, 3, 6, 4 and 3, respectively; *p<0.05). Such as TASK-2 is very promising for managing labor at term and developing target medicines. As shown in Fig. 3C~3E, OXT (10 and 50 nM) inhibited the NIOK current to 87±1.94% and 85±2.93% in pregnant cells in a reversible manner, respectively (p<0.05). Estrogen (1 µM) also inhibited the NIOK current to 68±7.65% (n=4; p<0.05).

• 

  Fig. 4 Identification of NIOK by reversal potentials (Erev).The reversal potentials (Erev) of NIOK were evaluated by changing the external K+ concentration from 4.5 to 30 mM. Erev shifted to new expected values at 30 mM of external K+. Instantaneous tail currents in the NIOK of non-pregnant and pregnant myometrial cells were reversed at –37 mV (n=3) and –41 mV (n=5) by 30 mM K+.

• 

  Fig. 5 Identification of TASK-2 by immunohistochemistry and Western blot in mouse myometrium.(A, B) Laser scanning confocal micrograph shows TASK-2 immunoreactivity on the membrane of a single pregnant cell. Confocal images at 2 µm intervals showed localization of the TASK-2 channel on the cell membrane of pregnant myometrial cells. Highly increased immunoreactivity for the TASK-2 antibody in single pregnant myometrial cells ws observed compared to that in non-pregnant cells. (C) TASK-2 was increased in pregnancy compared to the control under Western blot (595 vs 100, p<0.05; n=3 and 4).

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