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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015002&req=5

Figure 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).

Mentions: We studied outward currents from single isolated murine longitudinal myometrial cells. Non-inactivating outward K+ currents (NIOK) in pregnant cells using a step depolarizing pulse (see Methods) were inhibited to 43.6±3.50% by TEA (n=5) and to 41.8±5.17% by 4-AP plus TEA (n=5) at +80 mV (data not shown). The remaining current, which coincides with TEA and 4-AP resistant current (TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK)), is shown in the left panel of Fig. 3A. The reversal potentials (Erev) of TEA and 4-AP resistant currents were briefly evaluated by changing the external K+ concentration from 5 to 30 mM (see Methods ; Fig. 4). Erev shifted to new expected values at 30 mM of external K+. Using these protocols, instantaneous tail currents of outward current in non-pregnant and pregnant myometrial cells were reversed at –36.8±7.04 mV (from –60.6±2.17 mV; n=3) and –40.6±4.50 mV (from –64.6±4.14 mV; n=5) by 30 mM K+. Therefore, this result suggests that outward current remained after treatment with K+ channel blockers and was a Ca2+-insensitive NIOK current. The characteristic of Ca2+-insensitivity of NIOK is responsible for independence of intra- and extracellular Ca2+ including the block of KCa channels by TEA in experimental condition (see methods).


Myometrial relaxation of mice via expression of two pore domain acid sensitive K + (TASK-2) channels
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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015002&req=5

Figure 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).
Mentions: We studied outward currents from single isolated murine longitudinal myometrial cells. Non-inactivating outward K+ currents (NIOK) in pregnant cells using a step depolarizing pulse (see Methods) were inhibited to 43.6±3.50% by TEA (n=5) and to 41.8±5.17% by 4-AP plus TEA (n=5) at +80 mV (data not shown). The remaining current, which coincides with TEA and 4-AP resistant current (TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK)), is shown in the left panel of Fig. 3A. The reversal potentials (Erev) of TEA and 4-AP resistant currents were briefly evaluated by changing the external K+ concentration from 5 to 30 mM (see Methods ; Fig. 4). Erev shifted to new expected values at 30 mM of external K+. Using these protocols, instantaneous tail currents of outward current in non-pregnant and pregnant myometrial cells were reversed at –36.8±7.04 mV (from –60.6±2.17 mV; n=3) and –40.6±4.50 mV (from –64.6±4.14 mV; n=5) by 30 mM K+. Therefore, this result suggests that outward current remained after treatment with K+ channel blockers and was a Ca2+-insensitive NIOK current. The characteristic of Ca2+-insensitivity of NIOK is responsible for independence of intra- and extracellular Ca2+ including the block of KCa channels by TEA in experimental condition (see methods).

View Article: PubMed Central - PubMed

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.

No MeSH data available.