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J Clin Endocrinol Metab. Jun 2010; 95(6): 2902–2908.
Published online Apr 9, 2010. doi:  10.1210/jc.2009-2137
PMCID: PMC2902072

Melatonin Sensitizes Human Myometrial Cells to Oxytocin in a Protein Kinase Cα/Extracellular-Signal Regulated Kinase-Dependent Manner


Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night.

Objective: The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells.

Design: To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP3)/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation.

Results: I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1.

Conclusions: MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway, resulting in specific activation of PKCα and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.

The initiation of parturition is a complex multifactorial process. Our previous work investigating the role of melatonin (MEL) in human myometrial physiology (1,2) indicated that MEL signaling plays a significant role in the inhibition of oxytocin (OT) receptor (OTR) mRNA transcription, increases intercellular communication via gap junctions, and synergistically enhances OT-induced contractility. In all processes, MEL signaling occurred via the MEL type 2 receptor (MT2R) and protein kinase C (PKC).

MT2R and OTR are both G protein-coupled receptors that have been shown to couple with the Gq/11 α-subunit (3,4). In the myometrium, OT has been shown to activate phospholipase C (PLC) leading to the generation of inositol trisphosphate (IP3), which mediates intracellular calcium release. This results in calmodulin-dependent phosphorylation of the myosin light chain (MLC) by MLC kinase (MLCK) and activation of PKC (4). OT has also been shown to activate the Rho kinase signaling cascade, leading to inhibition of MLC phosphatase (MLCP) (5). The similarities between the MEL and OT signaling pathways provide multiple points for potential cross talk and potentiation that could lead to increased MLC phosphorylation and increased contractility.

In addition to modulation of the phosphorylation of MLC, the availability of actin for myosin binding is regulated by caldesmon and calponin (6). The phosphorylation of caldesmon by ERK1/2 reduces the affinity of caldesmon for actin, which makes actin more readily available for myosin binding during the cross-bridge cycle. This effect is modulated by PKC activity and presents a mechanism for sensitization of the myometrial cells to procontractile signals.

In the present studies, we sought to obtain a better understanding of the specific details of the signaling pathway that facilitates MEL effects on human myometrial smooth muscle cells. We focused on similarities between the MT2R and OTR signaling pathways because these appear to converge on the regulation of OTR transcription in human myometrial smooth muscle cells (1) and may underlie the synergy of MEL and OT action on human myometrial contractions in vitro (2). Our experiments identify the specific PKC isoform responsible for MEL effects on contractility in the human myometrium and show that MEL increases the availability of myometrial actin for the myosin cross-bridge cycle through modulation of caldesmon phosphorylation.

Materials and Methods

Cell culture

Telomerase immortalized myometrial cells (hTERT) were maintained in DMEM/F12 (Mediatech, Manassas, VA) medium with 10% Fetal II Plus serum (Valley Biomedical, Winchester, VA) with penicillin/streptomycin and gentamicin at 37 C and 5% CO2. Cells were trypsinized at 90% confluency and plated in T175 cell culture flasks at a 1:5 dilution or six-well plates at 20,000 cells per well. For pharmacological experiments, cells were treated with iodo-MEL (I-MEL; 1 nm) (Tocris Bioscience, Ellisville, MO) or OT (1 nm) (Sigma-Aldrich, St. Louis, MO) or cotreated as described in Results. The pharmacological inhibitors, MT2R-specific antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT; 10 nm), specific MAPK kinase (MEK) inhibitor PD 98059 (10 μm), Rho kinase inhibitor Y27632 (10 μm), MLCP inhibitor microcystin-LW (1 μm), PKC inhibitor C1 (10 μm), or the PLC inhibitor U73122 (1 μm) (all obtained from Tocris) were applied as a pretreatment 1 h before application of I-MEL or OT. I-MEL was used due to its increased stability in culture. After treatment, the cells were trypsinized, pelleted, washed in PBS, and frozen at −20 C until further analysis.

Immunoblotting and immunoprecipitation

For in vitro investigations, cultured hTERT cells were collected by trypsinization and gentle scraping. Cells were suspended in PBS and pelleted by centrifugation. Protein extraction was performed according to the method of Shearman and colleagues (7). After electrophoretic separation on a 10% SDS-polyacrylamide gel, proteins were semidry blotted in buffer onto a Whatman PROTRAN nitrocellulose membrane (Whatman, Dassel, Germany). Molecular size markers (Bio-Rad, Hercules, CA) were included. The membrane was incubated for 60 min at 20 C in blocking buffer containing 5% milk powder before overnight incubation at 4 C with phospho-PKCα/β (Cell Signaling Technologies, Beverly, MA) or anti-actin (Sigma) at a dilution of 1:1000 in blocking buffer. After washing in buffer [20 mm Tris (pH 7.6), 137 m NaCl, 0.05% Tween 20], the membrane was incubated at 20 C for 1 h with a peroxidase-conjugated affinity-purified goat antirabbit Ig (Sigma) in a 1:2000 dilution. Chemiluminescent signals were then detected with the Pierce ECL Western blotting substrate (Pierce, Rockford, IL) using CL-XPosure film (Pierce).

Western blotting for caldesmon or phosphocaldesmon (both Abcam, Cambridge, MA); p42/44 MAPK (phospho-ERK1/2), ERK1/2, phospho-PKCα/β II (Ser638/641), -δ (Thr505), -δ/θ (Ser643/676), θ- (Thr538), and -ζ/λ (Thr410/403); phospho-PKD/PKCμ (Ser916); and phosphomyosin light chain 2 (Ser19) and myosin light chain 2 (all antibodies from Cell Signaling) was performed in accordance with the manufacturer's protocol at a dilution of 1:500. The goat antirabbit Ig was diluted 1:2000 in a 5% milk/Tris-buffered saline/Tween 20 solution. Densitometric analysis was performed using AIS Image Analysis Software (Ontario, Canada) of images acquired with a digital camera. Western blots were repeated a minimum of three times to ensure reproducibility.

Immunoprecipitation was performed as follows. Protein G-Sepharose beads were equilibrated in protein extraction buffer for 1 h at room temperature. Beads were then washed three times for 20 min in 500 μl protein extraction buffer. Rabbit anti-MLC2 antibody (Cell Signaling) was linked to protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) at a 1:50 dilution for 1 h at room temperature. Sample protein extracts were precleared with 10 μl of equilibrated protein G-Sepharose beads for 20 min. Samples were then spun down for 10 sec at 3000 rpm. Samples were transferred to antibody-linked beads and incubated overnight at 4 C. Samples were spun at 3000 rpm for 10 sec and supernatant transferred to tubes as immunodepleted samples. Beads were then washed six times in 1 ml protein extraction buffer (10 sec spin at 3000 rpm). Protein loading buffer was then added to samples for Western blotting, and the samples were denatured at 95 C for 5 min and then rocked at room temperature for 5 min. Samples were spun for 1 min at 12,000 rpm, and supernatant was used in Western blot analysis for phospho-MLC or MLC.

PKC activity assay

hTERT cells were cultured and treated in T75 plates, harvested, extracted for protein, and immunoprecipitated for PKCα as described for Western blotting with the following exception. Samples for PKC activity were assayed after the final 1-ml wash and were not denatured. PKC activity assay was performed in accordance with manufacturer's protocol (Millipore, Bedford, MA), but the PKC activator cocktail was not added. [32P]ATPγ (3000 Ci/μmol) was purchased from PerkinElmer (Norwalk, CT).

Myometrial cell contractility assay

Myometrial cell contractility was assayed using a collagen disk retraction assay as described by Devost and Zingg (8) plating 10,000 hTERT cells per well. This method assesses contraction amplitude (not frequency) over the course of stimulation and, hence, reflects modulation of the myometrial contractile machinery (9). Samples were pretreated with inhibitors for 1 h before overnight treatment with OT and/or MEL as described in Results, and each treatment was performed in triplicate. Myometrial cell contractility was quantified by capturing images of the fixed collagen disks with a digital camera and analyzing for total area using AIS Image Analysis Software. The results were normalized to the cell-free control sample areas and expressed as a percentage of untreated control area.

IP3 turnover assay

Cells were cultured for at least 24 h. After a brief wash in fresh medium, the cells were then incubated overnight with myo-[2-3H]inositol (10 μCi/ml) (PerkinElmer) to label cell inositol phospholipids (IPs). The cells were rinsed three times with physiological saline to remove the unincorporated radioactivity and equilibrated in medium for 60 min. The cells were treated for 15 min with 10 mm lithium chloride (final concentration) before stimulation to inhibit inositol-1-monophosphatase activity. The cells were then treated for 30 min with I-MEL, OT, or both in LiCl at 37 C. The cells were lysed and cellular proteins precipitated by addition of 1 ml 95 C water, followed by freezing, thawing, and scraping. The extracts were then centrifuged 2 min at high speed. The supernatant was used for IP determination and pellet for 3H incorporation into membrane lipids. The 3H-labeled IPs in the supernatant were purified by anion-exchange column chromatography on Dowex-1 columns (AG 1-X8, formate form; Bio-Rad). The cell extracts were applied to the top of columns without disturbing resin, and flow-through was discarded. Sample tubes were rinsed three times with 0.5 ml distilled water, and the rinses were added to the columns. The columns were washed with 8 ml room temperature 60 mm ammonium formate/5 mm Na2B4O7. The sample fractions were collected into scintillation vials as follows. The columns were eluted with two successive aliquots of each elution buffer and then washed with 8 ml of the previous elution buffer before application of successive elution buffers. The buffer compositions were inositol monophosphate (IP1), 200 mm ammonium formate/100 mm formic acid; inositol bisphosphate (IP2), 400 mm ammonium formate/100 mm formic acid; and IP3, 1 m ammonium formate/100 mm formic acid.

The cell pellets were processed by adding 0.5 ml lipid clearing solution (500 mm KCl/5 mm myoinositol) and 0.5 ml chloroform. The pellets were then vortexed heavily, shaken vigorously for 10 min at room temperature, and centrifuged for 20 sec at 5000 × g. Two hundred microliters of the organic phase were transferred to scintillation vials and evaporated to dryness at room temperature. The eluted radioactivity in each sample was quantified by adding 5 ml scintillation fluid (Fisher Scientific, Pittsburgh, PA) and then counting in a liquid scintillation counter. The data are reported as counts per minute per well. Results are expressed as IP3 turnover (10) for IP3 and total IP (IP1 + IP2 + IP3) because IP1 and IP2 are dependent on, and highly correlated with, IP3 formation (10,11).

Statistical analyses

Experiments were repeated a minimum of three times. Replicate values for each data point were averaged, and differences were statistically analyzed using a one-way ANOVA followed by the Bonferroni post hoc test (Prism; GraphPad, San Diego,CA) with P < 0.05 as the criterion level for significance. All error bars shown represent sem.


MEL effects on contractility and MLC phosphorylation do not involve Rho kinase signaling or inhibition of MLCP

Rho kinase activation has been shown to increase myometrial contractility through inhibition of MLCP (12). We hypothesized that MEL could act to inhibit MLCP activity via the Rho kinase signaling pathway promoting contractility. To determine MEL's effect on the Rho kinase pathway, we performed collagen retraction assays (2). Cells were treated with I-MEL, OT, and the Rho kinase inhibitor Y27632. Treatment with Y27632 reduced basal and OT-induced contractility but had no significant effect on MEL-induced contractility. Y27632 treatment of samples cotreated with OT and I-MEL showed a modest inhibition of contractility down to levels seen in samples treated with I-MEL alone, I-MEL and Y27632, and OT and Y27632 (Fig. 1A1A).). This effect suggests that contractility due to I-MEL does not involve Rho kinase signaling.

Figure 1
MEL actions on the Rho kinase signaling pathway and MLCP activity. A, Gel contraction assay using human telomerase-immortalized hTERT myometrial smooth muscle cells. Results are presented as collagen disk surface area (square millimeters) normalized to ...

We next performed Western blots for phospho-MLC20. MLC is phosphorylated by MLCK at two sites, Thr18 and Ser19, which are both detected by our phospho-MLC antibody. To confirm that I-MEL-induced contractility is not due to inhibition of MLCP, cells were pretreated with the MLCP inhibitor microcystin-LW and then treated with I-MEL. Lanes containing extract from cells treated with both I-MEL and microcystin showed an increase in phospho-MLC levels, specifically a second band corresponding to Ser19- and Thr18-phosphorylated MLC, which is absent in all other treatment groups. Neither inhibition of the phosphatase with microcystin nor treatment with I-MEL resulted in the multiple phosphorylation of the MLC. This suggests that rather than inhibiting MLCP, I-MEL's action is via kinase activity. The absence of the second band in samples treated with I-MEL alone indicates that MLCP activity remains unaffected (Fig. 1B1B).

MEL activates PKCα in human myometrial smooth muscle cells

Next we explored the IP3/DAG signaling mechanisms that are used by MEL. Our previous work has shown that MEL signaling in the human myometrium is mediated by PLC and PKC (1,2). To specifically dissect this signaling mechanism, we used IP3 turnover assays, immunoprecipitation, and Western blotting to determine the activation levels of the pathway and identify the specific PKC isoform. Cells were treated with I-MEL, OT, and the inhibitors 4P-PDOT and U73162 (see Materials and Methods). IP3 turnover was significantly increased in samples treated with I-MEL (179% of control levels) and OT (238% of control levels) and after I-MEL/OT cotreatment (360% of control levels). Pretreatment with 4P-PDOT reduced IP3 turnover in I-MEL/OT-cotreated cells to OT levels, and pretreatment with U73122 reduced levels to those of control (Fig. 2A2A).). Total IP turnover displayed the same trend confirming that I-MEL treatment augments PLC activity (Fig. 2B2B).). Subsequent standard fura-2 calcium imaging experiments showed no detectable increase in intracellular calcium due to I-MEL treatment, whereas treatment with OT showed a dramatic and rapid increase in intracellular calcium (data not shown), suggesting that I-MEL acts to sensitize myometrial cells to subsequent procontractile signals.

Figure 2
MEL actions on IP3 and total IP turnover. A, IP3 turnover after treatment with I-MEL (1 nm), OT (1 nm), the MT2R antagonist 4P-PDOT (10 nm), and the PLC inhibitor U73122 (1 μm). a, P < 0.05 vs. control; b, P < 0.05 vs. I-MEL-treated ...

Western blots for a panel of phospho-PKC isoforms (see Materials and Methods) were performed to define those activated by treatment with I-MEL. I-MEL treatment resulted in an increase in phospho-PKCα/β in a Western blot screening of multiple PKC isoforms (see Materials and Methods). To identify which isoform was activated, samples were immunoprecipitated using an antibody against PKCα and Western blotted with the phospho-PKCα/β antibody. This produced a band in the immunoprecipitated samples. Treatment with the MT2R antagonist 4P-PDOT reduced phospho-PKC levels to control levels (Fig. 3A3A).). Western blots on the immunodepleted supernatant showed no detectable phospho-PKCα/β (data not shown). To further confirm this result, we performed PKC enzyme assays. 32P incorporation into the PKC substrate was increased in I-MEL-treated samples (Fig. 3B3B)) and in samples immunoprecipitated for PKCα. Activity was absent in immunodepleted samples, confirming PKCα activation by I-MEL.

Figure 3
MEL activates PKCα. A, Treated samples were immunoprecipitated with anti-PKCα, and then Western blots were performed for phospho-PKCα/β. hTert cells were treated with 1 nm I-MEL with or without 10 nm 4P-PDOT. B, PKC activity ...

MEL treatment increases ERK1/2 and caldesmon phosphorylation

Another regulator of smooth muscle contractility is caldesmon. Phosphorylation of caldesmon results in a decrease in its affinity for actin, which increases actin availability for myosin binding (6). We hypothesized that PKCα activation by I-MEL would result in caldesmon phosphorylation in an ERK1/2-dependent manner. Western blots for phospho-caldesmon and p42/44 MAPK (ERK1/2) confirmed that after 15 min, I-MEL increases both ERK1/2 and caldesmon phosphorylation. This action could be abolished by pretreatment of cells with the MEK inhibitor PD98059 and by the general PKC inhibitor C1 (Fig. 44).

Figure 4
MEL activates ERK1/2-dependent phosphorylation of caldesmon. Representative Western blots are shown for phospho- (P-)ERK1/2 and phospho- (P-)caldesmon for samples collected after 15-min treatments with I-MEL (1 nm) or OT (1 nm) or samples pretreated with ...


Continuous monitoring of normal uterine contractile activity during late-term pregnancy in humans has shown increased frequency between 2030 and 0200 h (13). Studies on the timing of human labor onset and deliveries show that the initiation of labor peaks between 2400 and 0500 h, regardless of gestational age (14). Little is known regarding the physiological mechanisms underlying the timing of human parturition to the night phase. MEL, the molecular messenger of circadian night, peaks at night, and levels in late-term pregnancy are significantly elevated (15). Our previous work revealed that the myometria from nonlaboring term pregnant women generally have low levels of both OT and MEL receptor protein, whereas myometria from laboring term pregnant women have high levels (2). Furthermore, our earlier work pointed to PKC as a probable mediator of MEL signaling in the context of human myometrial contractility and gap junction regulation (1,2).

The purpose of this investigation was to further elucidate the MEL signaling pathway in human myometrial cells that accounts for its effects on contractility. The immortalized cell line that we use has been shown to be an excellent model of human myometrial smooth muscle cells, and depending on the culture conditions (e.g. the present of steroids) these cells are reflective of either nonpregnant or pregnant myometrial tissues (1,2,8,16). Our previous work suggested involvement of the PLC/IP3 signaling pathway and involvement of PKC, resulting in a synergistic effect on OT-induced contractility. This synergistic effect could be accomplished by multiple mechanisms that regulate the activities of the MLCK, MLCP, and actin availability for myosin cross-bridge cycling (6,17,18). Herein we follow the MEL signaling cascade from IP3/DAG generation by activation of PLC through the signaling cascade to phosphorylation of caldesmon. I-MEL treatment resulted in an increase in IP3 and total IP turnover in samples treated with I-MEL alone and in samples cotreated with OT (Fig. 22,, A and B). As expected, the increase in I-MEL/OT-cotreated samples was abolished by treatment with the MT2R-specific antagonist 4P-PDOT, which agrees with our previous data indicating that I-MEL effects on contractility are mediated through MT2R. Somewhat unexpectedly, we were not able to detect an increase in intracellular Ca2+ in cells treated with I-MEL alone. Treatment with OT did, however, produce a strong increase in intracellular calcium (data not shown). MEL has been shown to synergistically augment intracellular calcium signaling but has no detectable effect on calcium alone in MCF-7 cells (19). Also, MEL has been shown to augment norepinephrine-induced vasoconstriction in a calcium-independent manner in the rat (20).

PKC has long been implicated as an important factor in the uterine contractile machinery. PKCα has been reported to be expressed in the human myometrium at term (21) and to have a direct role in myometrial contractility (22). Our data show that I-MEL increases PKCα phosphorylation as well as PKC enzyme activity. The lack of PKC activity in the immunodepleted fraction of the samples (Fig. 3B3B)) points to a very specific activation of PKCα by I-MEL. PKCα has been shown to phosphorylate Raf (23), starting the MAPK signaling cascade through MEK leading to ERK1/2 phosphorylation, which can subsequently phosphorylate targets such as caldesmon and direct phosphorylation of MLCK (12). We thus followed the PKC cascade to ERK1/2 and caldesmon. I-MEL alone clearly increased phosphorylation of caldesmon and markedly activated ERK1/2 (Fig. 44).). This effect was completely blocked by the MEL receptor antagonist 4P-PDOT and largely abolished by inhibition of ERK1/2 and PKC signaling (Fig. 44).). Interestingly, an inverse relationship between caldesmon and phospho-caldesmon was noted, suggesting a nearly complete phosphorylation of the former upon stimulation with I-MEL or OT. Samples treated with OT alone showed an increase in caldesmon phosphorylation with only a weak elevation in phosphorylated ERK1/2. Whereas the PKC inhibitor C1 blocked the OT-induced increase of both caldesmon and ERK1/2 phosphorylation, the MEK inhibitor PD98059 blocked only the latter. This may be explained by the recent results of Devost and colleagues (24), who have reported rapid activation and inactivation of ERK1/2 as well as activation of p38MAPK by OT in the hTERT cell line. Both ERK1/2 and p38MAPK have been reported to phosphorylate caldesmon (25,26). The combination of OT plus I-MEL did not appear to enhance the already strong phosphorylation of caldesmon or ERK1/2 achieved by I-MEL (Fig. 44).). Thus, the synergistic response of myometrial smooth muscle cells to this hormonal combination likely reflects the contribution of additional intracellular mechanisms, such as IP3 turnover (Fig. 22),), phosphorylation of MLC (2), increased gap junction activity (2), etc.

In conclusion, the present studies expand on our previous model for procontractile MEL signaling in human myocytes (Fig. 55)) by confirming increased IP3 turnover due to MEL activation of MT2R. Additionally, we identify PKCα as the mediator of MEL downstream effects. PKCα then activates the ERK1/2 signaling cascade. ERK1/2 activation has been shown to phosphorylate caldesmon. Caldesmon has been shown to be a negative regulator of smooth muscle contractility by binding actin, thus preventing myosin binding. Phosphorylation of caldesmon greatly reduces its affinity for actin, thus making actin more readily available. This mechanism has been proposed as a mechanism of sensitization in smooth muscle (12). Combined with our previous observations showing MEL enhancement of OT-induced contractility (2), the present data provide a mechanism by which MEL can sensitize the human myometrium to procontractile OT signaling. This mechanism explains our previous observation that MEL acts permissively through sensitization of myometrial cells to the effects of OT and thereby increasing myometrial contractility. Sensitization of the myometrium to subsequent procontractile signals in vivo would provide a mechanism underlying the observed increase in nocturnal uterine contractility and parturition in late-term pregnancy. These data provide new insights into the mechanisms underlying the timing of birth and regulation of the contractile machinery in the myometrium and reveal a novel physiological mechanism for MEL actions whose further characterization may serve in the development of new pharmacological strategies for the management of preterm and/or delayed parturition.

Figure 5
Proposed model for myometrial sensitization by MEL. MEL activates PLC, which generates DAG, resulting in PKCα activation. PKCα initiates the Raf/MEK/ERK1/2 signaling cascade leading to phosphorylation of caldesmon (hCaD), which increases ...


We thank Dr. Ann Word for graciously providing the telomerase immortalized hTERT human myometrial cell line. We also thank Dr. Joel Tabak for his assistance with calcium imaging.


These studies were supported by intramural grants from the Florida State University COFRS program and the Florida State University Research Foundation. C.C. was recipient of a summer research fellowship from Florida State University College of Medicine.

Current address for C.C.: Howard Hughes Medical Institute, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.

This contribution represents a portion of the doctoral studies of J.S.

Disclosure Summary: J.T.S. and C.C. have nothing to disclose. J.O. has received a provisional patent U.S. 60991866.

First Published Online April 9, 2010

Abbreviations: DAG, Diacylglycerol; IP, inositol phosphate; IP1, inositol monophosphate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; MEL, melatonin; MEK, MAPK kinase; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; MT2R, MEL type 2 receptor; OT, oxytocin, OT receptor; PKC, protein kinase C; PLC, phospholipase C; 40-PDOT, 4-phenyl-2-propionamidotetralin.


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