Prolonged stretching in the late-late pregnant rat uterus inhibits contractions through potassium channel TREK-1

Prolonged mid- of and through potassium channel TREK-1. in the pregnant uterus close to unknown. Uterine tissues from late-late pregnant rats (day 20–21 of pregnancy) were isolated, and myometrial strips were prepared for isometric contraction measurements. We compared the oxytocin-induced contractions of myometrial strips treated with different stretch times and doses. Then, we used the potassium channel TREK-1 inhibitor L-methionine and TREK-1 agonist arachidonic acid to determine whether the changes caused by prolonged stretching involved changes in TREK-1 activity.


Background
During pregnancy, the uterus dramatically expands in volume to provide enough space for the growing fetus [1]. To guarantee su cient time for the development of the fetus before labor, mechanical quiescence of the myometrium is needed to decrease the muscle excitability caused by hypertrophy and distension of the myometrium during pregnancy [2]. Once the myometrium is stimulated at term, it transitions into an excitable state and becomes one of the strongest muscles to facilitate birth [3]. The mechanisms that facilitate uterine quiescence during pregnancy and enhance contractility at term are not fully understood.
Previous studies have shown that during pregnancy, uterine contractility dramatically decreases [4].
Prolonged stretching of the nonpregnant and mid-term pregnant rat uterus inhibits myometrial contractions. Similarly, during the late stage of a term pregnancy, human uterine contractility decreases compared to that of a preterm pregnancy. Additionally, prolonged stretching of the preterm pregnant uterus can inhibit contractions to a level that is similar to that of the term pregnant uterus [5]. All of these data show that stretching can inhibit myometrial contractions in the nonpregnant and mid-and late-term pregnant uteri of rats and humans. Whether the myometrium remains sensitive to stretching close to parturition is unclear.
Increases in intracellular calcium (Ca 2+ ) can trigger uterine contractions, especially through voltage-gated channels. The decline in intracellular Ca 2+ caused by membrane hyperpolarization and activation of different types of potassium (K + ) channels maintains smooth muscle relaxation [6,7]. A previous study has shown that potassium channels play a vital role in uterine functions, and several of these channels have been proposed to maintain uterine quiescence during pregnancy up to full term [8]. The TREK-1 channel, a stretch-activated tetraethyl ammonium-insensitive potassium current in smooth muscles that has been isolated from human and rat myometrial tissues, decreases myometrial smooth muscle excitability through its contributions toward returning the depolarized cell to a more negative resting potential and maintaining the resting cell near the K + equilibrium potential [9,10].
In the human pregnant uterus, higher TREK-1 expression is observed in term and twin pregnancies.
Prolonged stretching of the preterm human uterus increases TREK-1 expression [5]. The sex hormone progesterone decreases uterus contractions in late pregnant rats partly through TREK-1 channels [11].
Whether prolonged stretching of the rat uterus close to parturition can still stimulate TREK-1 function remains unknown.
In the present research, we used the rat uterus at 20-21 days of pregnancy to investigate (1) the contractility differences of the pregnant uterus close to parturition (late-late pregnancy) under prolonged stretching and (2) the TREK-1 function in processing prolonged stretching with late-late pregnant uterine contractions.

Tissue preparation
The study was conducted on late-late pregnant Sprague-Dawley rats purchased from Animal Laboratory Center of Anhui Medical University (aged 12 weeks, weighing 300 to 350 gram (g) when pregnant). Because the last day of pregnancy in rats is day 21 or day 22, we de ned late-late pregnancy as 20-21 days of gestation in rats, which is close to parturition but without the start of uterine contractions. The rats were housed in the animal facility and maintained on standard rat chow and tap water ad libitum in a 12:12-h light-dark cycle. 10 rats were used in stretch group, 10 rats were used in arachidonic acid group and 10 rats were used in L-methionine. The animals were euthanized by inhalation of 100% carbon dioxide (CO 2 ) in a closed chamber, and death was indicated by cessation of breathing and heartbeats.
The pregnant rat uterus was rapidly excised, and connected tissues were cleaned under microscopic visualization followed by the removal of the fetus and placenta. The uterus was immediately immersed in Krebs solution for further dissection. Because oxytocin is also synthesized within the decidual tissues immediately adjacent to the myometrium, the decidua was removed from the myometrial strips prior to myographic assessment. From each rat, longitudinal uterine strips (3 mm × 7 mm, width × length) were isolated and suspended vertically in four individual, temperature-controlled organ bath chambers containing 5 milliliters (ml) of Krebs solution bubbled with 95% O 2 /5% CO 2 at 37℃. The end of each uterine strip was attached to a xed glass hook at the bottom of the organ bath chamber, and the other end was hooked to an isometric force transducer (6240 biological treatment systems). Changes in isometric contractions were recorded.
All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Ethics Committee at the First A liated Hospital of Anhui Medical University, Hefei, China (20170210).

Isometric contraction measurement
The uterine strips of pregnant rats were stretched under the control of 2 g basal tension to equilibrate for 1 h with constant Krebs solution and stimulated twice with 96 mM potassium chloride (KCl) to obtain a maximal response. Each control KCl contraction was followed by three washes in Krebs solution, 10 min each. To demonstrate the effect of prolonged stretching on myometrial contractions, the uterine strips were subjected to either control 2 g basal tension or high 8 g tension for 18 h in tissue culture medium. On the next day, the bathing solution was changed to Krebs solution, and the oxytocin contraction was measured.
To determine whether TREK-1 was involved in the uterine contraction changes associated with prolonged stretching, contraction experiments were repeated in the presence of TRKE-1 inhibitor L-methionine (Sigma-Aldrich) and activator arachidonic acid (Sigma-Aldrich). The area under the curve (AUC) was measured at time 0 and was subtracted from the AUC measured after 5 min of application of each oxytocin concentration by the R1640 data analysis system (Chengdu, China). The AUC representing total contractions was also normalized to the weight of the myometrial strip and is presented as the AUC per gram of tissue. The oxytocin-induced contraction response was calculated for 20, 40, and 60 min.

Solutions and drugs
Normal Krebs solution contained (in mM): 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose, 2.5 CaCl2, and 1.2 MgCl2 (Sigma). The pH of the Krebs solution was 7.3-7.4 when bubbled with 95% O2 5% CO 2 at 37.0 ± 0.5 °C. A high KCl solution (96 mM) was prepared similarly to normal Krebs but with equimolar substitution of NaCl with KCl. Oxytocin (Shanghai Hefeng Pharmaceutical Company, China) was dissolved in deionized water. A stock solution of L-methionine (Sigma, 10 − 1 M) was prepared in deionized water. A stock solution of arachidonic acid (Sigma, 10 − 3 M) was prepared in ethanol. The nal concentration of ethanol was less than 0.1% and had no effect on uterine contractions. The tissue culture medium used for prolonged incubation of the uterine strips for 16 h comprised Minimum Essential Medium supplemented with penicillin, streptomycin, and amphotericin B (Gibco/Invitrogen, Grand Island, NY). All other chemicals were of reagent grade or better.

Statistical analysis
Data were analyzed and presented as the mean ± SEM, with "n" representing the number of subjects per group. For uterine contraction experiments, individual concentration-contraction curves were constructed. Data were rst analyzed using ANOVA of tissue stretching (control 2 g basal tension vs 8 g stretching) and tissue treatment (treated with arachidonic acid or L-methionine vs nontreated control tissues). When a signi cant difference was observed, the data were further analyzed using Bonferroni's post hoc test for multiple comparisons. Student's unpaired t-test was used for comparisons of two means. Differences were considered statistically signi cant if P < 0.05.

Results
Oxytocin-induced contractions in the late-late pregnant rat uterus under prolonged stretching In the isolated late-late pregnant rat uterine strips, oxytocin caused an increase in the frequency and amplitude of uterine contractions that did not return to baseline, leaving a measurable maintained response above baseline. Oxytocin caused a concentration-dependent increase in the maintained contractions, and the contractions of the rat uterine strips peaked at 10 − 7 M oxytocin, as previously described. The oxytocin-induced contractions decreased in the uterine strips under prolonged 8 g stretching compared to those in the control tissues under control 2 g basal tension (Fig. 1a). The cumulative data suggested that the oxytocin-induced maintained contractions in the AUC/g tissue were signi cantly reduced in tissues under 8 g stretching compared to control tissues under 2 g basal tension for 20 min, 40 min and 60 min, respectively (Fig. 1b).

Effect of TREK-1 activator on oxytocin-induced uterine contractions
For the uterine strips from the late-late pregnant rats and those that were precontracted with oxytocin (10 − 7 M), treatment with the vehicle was associated with a slight time-dependent decline in the AUC/g tissue, representing total uterine contractions, and treatment with activator of TREK-1 appeared to further modulate oxytocin contractions (Fig. 2a). Cumulative data revealed that the TREK-1 activator arachidonic acid (10 − 5 M) caused a further decline in the AUC/g tissue, representing total contractions of the uterine strips without prolonged stretching for 20 min, 40 min and 60 min, respectively. However, arachidonic acid caused no further decline in contractions compared to the uterine strips stretched under 8 g stretching for 18 h (Fig. 2b, c, d, e).

Effect of TREK-1 inhibitor on oxytocin-induced uterine contractions
For the uterine strips from the late-late pregnant rats and those that were precontracted with oxytocin (10 − 7 M), treatment with the TREK-1 blocker L-methionine (1 mM) minimized the decline in uterine contractions (Fig. 3a). Treatment with TREK-1 blocker L-methionine signi cantly enhanced the AUC/g tissue, representing total contractions of the uterine strips with prolonged 18 h stretching for 20 min, 40 min and 60 min, respectively (Fig. 3b, c, d, e).

Discussion
The present study of the late-late pregnant rat uteri showed that (1)  The regulation of uterine contractions is critical for normal healthy pregnancy and preterm labor [12]. During pregnancy, the uterus expands progressively and proportionately to increases in fetal size. In fullterm pregnancy, the uterine stretching imposed by the fully developed fetus reaches a threshold level to initiate labor. We evaluated late-late pregnant uterus contractions and found that prolonged stretching of the uterus could signi cantly inhibit uterine contractions. The results showed that the myometrial relaxation system continued to function late in gestation, when parturition was very close, at which point spontaneous contractions would start. The quiescent uctuation of the myometrium could be induced in the parturient woman. These observations are consistent with the ndings that parturition is triggered at a speci c point.
Previous studies have shown that threshold increases in uterine wall stretching can increase myometrial oxytocin receptor expression and in turn increase oxytocin-induced uterine contractions during normal labor [13]. Other studies have suggested that during the nal stages of pregnancy, which are close to parturition, threshold increases in uterine stretching cause the upregulation of contraction-associated proteins (CAPs) that can also play a role in the induction of labor [14,15].
Potassium channel TREK-1 is a type of CAP, and have been found to be expressed in the human and rat myometrium, particularly during pregnancy [16]. Based on the topology of the K + channel subunits, these TREK K2P channels are thought to maintain background outward K + current and resting membrane potential and counterbalance membrane depolarization and muscle contractions during pregnancy [17].
The activity of the TREK-1 channel has been shown to be regulated by numerous factors, including arachidonic acid (AA), L-methionine (L-M), pH, stretching, phosphorylation, nitric oxide, and temperature [18]. The present study indicates that the TREK-1 channel activator AA causes a dramatic enhancement in myometrial contractility, while its inhibitor L-M does not signi cantly increase contractility.
Our study demonstrated that prolonged stretch treatment decreased the myometrial contractility of the late-late pregnant rat uterus, which was similar to its myometrial relaxation in the early stage of pregnancy. It is unclear whether the pregnancy-associated and stretching-related reductions in the uterine contractions observed in late-late pregnancy, which is very close to parturition, re ect changes in TREK-1 expression/activity.
To determine whether prolonged stretching inhibits the myometrial contractility of the late-late pregnant rat uterus via the TREK-1 channel, isometric contraction measurements were performed to demonstrate that the reduced myometrial contractions caused by prolonged stretching were reversed under treatment with the TREK-1 inhibitor L-M, while there was no further contraction decrease under treatment with the TREK-1 activator AA.
The changes in the myometrial contractility of late-late pregnant uterus treated with L-M or AA after prolonged stretching suggest that the TREK-1 channel plays a vital role in the process of stretchingmediated regulation of uterine contractions before labor. The reversed contraction caused by L-M suggests that prolonged stretching decreases uterine contractions by regulating TREK-1 activity, while the myometrial relaxation caused by AA reaches saturation, possibly due to functional limitations in the TREK-1 channel.

Conclusion
A certain degree of stretching can maintain the balance of myometrial contraction and relaxation in latelate pregnant rats. The expression dynamics of potassium channel TREK-1 may be key to maintaining myometrial contraction/relaxation alterations. The limitation of the present study is that all of the data were collected from rats, which may differ from human beings in terms of these effects.

Not applicable
Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Effects of prolonged stretching on oxytocin-induced contractions. In isolated late-late pregnant rat uterine strips, oxytocin caused a concentration-dependent increase in the maintained contraction, reaching a peak at 10-7 M in the rat uterine strips, as previously described. The AUC/g tissue of the oxytocin-induced contraction was calculated for 20, 40, and 60 min with prolonged stretching (8 g) or basal stretching (2 g) treatment. The data are presented as the mean ± SEM; 2 g, n =10; 8 g, n=7. *Signi cantly different (P <0.05) Figure 2 Effect of TREK-1 activation on late-late uterine contractions Uterine strips from late-late pregnancy were either not treated (2 g stretch) or pretreated with TREK-1 activator arachidonic acid (