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Misoprostol, prostaglandin E1 analogue, used for labour induction. However, one-third of patients who have labour induced with prostaglandins do not reach vaginal delivery. The differential expression of prostaglandin receptors in myometrial cells could account for this differential response. Since delivery physiology also involves modulation of oxidative metabolism that can be potentially affected by pharmacological drugs, in the present investigation the role of misoprostol on expression of prostaglandin receptors, and oxidative markers of myometrial cells was evaluated. Samples of myometrial tissues procured from women with spontaneous (SL) and nonspontaneous (NSL) labours were cultured in vitro and exposed to different concentrations of misoprostol. Gene expression was evaluated by qRT-PCR and oxidative biomarkers were evaluated by spectrophotometric and fluorometric analysis. Cells from SL women presented greater responsiveness to misoprostol, since an upregulation of genes related to increased muscle contraction was observed. Otherwise, cells from NSL women had low responsiveness to misoprostol exposure or even a suppressive effect on the expression of these genes. Oxidative biomarkers that previously have been related to labour physiology were affected by misoprostol treatment: lipoperoxidation and protein carbonylation (PC). However, a decrease in lipoperoxidation was observed only in SL cells treated with low concentrations of misoprostol, whereas a decrease of PC occurred in all samples treated with different misoprostol concentrations. The results suggest a pharmacogenetic effect of misoprostol in labour induction involving differential regulation of EP receptor genes, as well as some minor differential modulation of oxidative metabolism in myometrial cells., (Copyright © 2016. Published by Elsevier Inc.)

Elucidation of cellular mechanisms regulating myometrial contractility is crucial for improvement in management of many obstetric abnormalities, such as premature delivery, uterine dystocia and post-partum haemorrhage. Myometrial contractions are triggered by periodic synchronous rises in intracellular calcium concentration ([Ca(2+)]i) elicited by spontaneously generated action potentials propagating throughout the entire myometrium. During labour, hormones like oxytocin and prostaglandins potentiate uterine contractions by increasing their duration, strength and frequency. The most informative approach to studying the mechanisms underlying hormonal modulation of uterine contractility is to record [Ca(2+)]i responses to hormones in intact myometrial samples that have not been subjected to enzymatic treatment for cell isolation or cell culture conditions. However, the spatio-temporal resolution of such recording is limited due to the motion artifacts occurring in contracting tissue. Here we describe the application of our newly developed motion correction algorithm to investigate the [Ca(2+)]i dynamics in control and oxytocin stimulated slices of human myometrium on a cellular level. We present evidence that oxytocin induces asynchronous [Ca(2+)]i oscillations in individual myocytes within intact myometrium which are similar to those observed in cultured cells. The oscillations occur between synchronous action potential-driven [Ca(2+)]i transients but appear to be unrelated to contractions. Furthermore, the oxytocin-triggered [Ca(2+)]i oscillations wane within 30-50min of hormone application, while the action potential induced [Ca(2+)]i transients remain augmented. We conclude that oxytocin-induced [Ca(2+)]i oscillations are not relevant to the acute regulation of myometrial contractility but may play a role in longer-term regulatory processes, for example, by triggering gene expression., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study examined the hypothesis that the force generated by myometrial strips from pregnant women is influenced by the smooth muscle content and fibre orientation of the strips and that correcting for these structural variables will provide a more accurate measure of contractility. Myometrial strips (n=72) were contracted by exposure to KCl, oxytocin, U44619 and phenylephrine and maximum responses were recorded. Morphological techniques were used to determine the cross-sectional area of the strips, the area occupied by smooth muscle and the area occupied by smooth muscle longitudinal in the strip. Maximum responses to contractile agents were expressed in terms of these three variables. The mean cross sectional area of strips was 2.01 ± 0.06 mm(2), of which 50% was smooth muscle, and 18% was smooth muscle longitudinal in the strip (n=72). There was much heterogeneity in responses, smooth muscle content and fibre orientation. Correction for morphological variability did not improve the heterogeneity in responses where coefficients of variation among strips from the same donor ranged from 43% to 63% when expressed in relation to longitudinal smooth muscle cross-sectional area. The standard method of preparation of myometrial strips for in vitro recording results in samples that are not structurally uniform. Correcting for the known structural variables does not provide a more accurate measure of maximum contractile responses. Because of the heterogeneity shown here, experiments that are dependent upon accurate estimation of maximum contractile responses require a large number of replicates to reach meaningful conclusions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim was to study whether bovine myometrial cells have an enzymatic system able to produce prostaglandins (PGs) and whether PGs synthesis is regulated by steroids in a similar manner as endometrial cells. In Experiment 1, immunohistochemical studies localized proteins for cyclooxygenase 2 (COX2), PGE synthase (PGES) and PGF2alpha synthase (PGFS) in myometrial and endometrial (as positive control) slices from days 14 to 16 of the estrous cycle. Enzymatically isolated myometrial cells (2.5 x 10(5)/ml) were cultured for 96 h to attach them to the bottom of the culture well. In Experiment 2, cells were preincubated for 30 min with progesterone (P4; 10(-5) M), and thereafter incubated for 4 or 6h with arachidonic acid (AA; 10(-5) M, as positive control), oxytocin (OT; 10(-7) M), and OT+P4. In medium, PGE and PGFM (PGF2alpha metabolite) were increased (P<0.05) by AA treatment after 4 and 6h, but by OT only after 6h of incubation. Progesterone did not affect (P>0.05) basal secretion of both PGs, but it diminished (P<0.05) the stimulatory effect of OT on PGF2alpha and PGE secretion after 6h of incubation. The amount of enzyme protein for COX2, PGES and PGFS analyzed by Western Blot was affected (P>0.05) by any of the factors added to the culture medium. In Experiment 3, myometrial cells were preincubated with P4 (10(-5)M) and pregnenolone (P5; 10(-5)M) for 30 min, and then incubated for 6h with OT (10(-7) M) and OT plus each of these steroids used. Expression of mRNA for COX2, but not PGFS and PGES, was found in the cells stimulated with OT. Neither P4 nor P5 affected expression of the studied genes; however, both steroids diminished (P<0.05) OT-stimulated mRNA expression of COX2. The data suggest that: (a) myometrial cells are able to synthesize both PGF2alpha and PGE and (b) synthesis of these PGs may be regulated by steroids through a transcription-independent manner, which modulated the effect of OT on COX2 mRNA expression.

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

Unlabelled: Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished., In Conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.

Scutellaria barbata D. Don (SB) is one of the herbs belonging to perennial plants, which is known in traditional Korean medicine as 'Ban-Ji-Ryun,' and has been used as an anti-inflammatory and anti-tumor agents against human uterine leiomyoma, mammalian and ovarian cancers. Although the difference between uterine smooth muscle cell (SMC) and leiomyomal SMCs has not been clearly established, the action of SB water extract was investigated using SMCs from normal myometrium and leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by SB treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under SB treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by treatment with SB in myometrial and leiomyomal SMCs. In contrast, cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not affected. Taken together, these results indicate that SB inhibits the proliferation of myometrial and leiomyomals SMC through the induction of alpha-SMA, calponin h1 and p27. It is suggested that SB may induce differentiation in uterine SMC and may influence tissue remodeling and reconstruction during physiological and pathophysiological events.

We measured the effects of stable thromboxane A2 (TXA2) analogues on signalling in cultured human myometrial cells. U46619 and/or IBOP stimulated total inositol phosphates (IPs) and cAMP production, RhoA-associated protein kinase (ROK) activity and elevated intracellular calcium [Ca2+]i. Pretreatment of the cells with pertussis toxin did not inhibit IPs or [Ca2+]i production but the thromboxane receptor (TP) antagonist SQ-29548 did inhibit IPs and cAMP production, the elevation of [Ca2+]i, and the increase in ROK activity. Pretreatment with thapsigargin inhibited [Ca2+]i elevation. TP receptor-stimulated ROK activity was inhibited by the ROK inhibitor Y27632 while ROK activity was enhanced by the caspase 3 inhibitor, Z-DEVD-FMK. TP receptor-stimulated IPs production is additive to prostaglandin F2alpha (FP) or prostaglandin E (EP) receptor-stimulated IPs production and neither FP nor EP receptor-stimulated IPs production is inhibited by SQ29548. Thus cultured human myometrial cells express at least two functional TP receptor subtypes; TPalpha-like (cAMP-stimulating) and TPbeta-like (IPs, [Ca2+] and ROK-stimulating).

Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.

The purpose of this study was to investigate the actions of 17beta-estradiol on the electrical activity of pregnant rat myometrium. The longitudinal layer of the myometrium was dissected from pregnant rats (17 to 19 days of gestation), and single cells were isolated by enzymatic digestion. Calcium currents and potassium currents were recorded by the whole-cell voltage-clamp method, and the single calcium-dependent potassium current was recorded by the outside-out patch-clamp method. The effects of 17beta-estradiol on these currents were investigated. When a myometrial cell was held at -50 mV, depolarization to a potential more positive than -30 mV produced an inward current followed by a slowly developing outward current. Application of tetraethylammonium inhibited the outward current while the inward current was completely abolished in a calcium-free solution. Estradiol at high concentrations (> 3 microM) inhibited both inward and outward currents in a voltage-dependent manner. Removal of estradiol restored the amplitude of the outward but not of the inward current. Estradiol (30 microM) also inhibited the activity of single calcium-dependent potassium channels without changing single channel conductance. In conclusion, estradiol at high concentrations inhibited: (1) voltage-dependent calcium, (2) calcium-dependent potassium and (3) voltage-dependent potassium currents. These actions of estradiol would prevent action potential generation and after-hyperpolarizations. Suppression of the after-hyperpolarization might further prevent spike generation due to slowing of the calcium channel's recovery from the inactivated state.

The synthesis of prostaglandins in the uterus at term are modulated by two isoforms of the enzyme cyclooxygenase (COX): constitutive COX-1 and inducible COX-2. This study aims to characterize the expression of the protein for COX-1 and -2 in the rat uterus and cervix during the estrous cycle, pregnancy, and labor, and in cultured myometrial cells. Western immunoblotting of proteins was performed and quantitation of protein was obtained densitometrically. Results indicate: 1) the rat uteri, cervix, and isolated myometrial cells express both COX-1 and COX-2 proteins, 2) during pregnancy, both COX-1 and -2 increase, with a dramatic increase at parturition (250%-280%), 3) a 2-fold increase of cervical COX-2 is seen at spontaneous labor, 4) during proestrus and estrus, uterine expression of COX-2 is elevated, 5) both COX-1 and -2 were expressed by rat myometrial cells and treatment with IL-1 beta (10 ng/mL) produced a significant increase in COX-2, and 6) immunocytochemical studies show that both COX-1 and -2 were primarily localized to the epithelial cells of the endometrium and smooth muscle cells in the circular layers of the myometrium in the uterus and to the epithelial cells and smooth muscle cells in the cervix. Thus, we propose that increased expression of COX-2 may be involved at term in increased uterine contractility and cervical ripening.

Estriol, estradiol, progesterone and diethylstilbestrol in the concentration range of 0.2-40 microM inhibited the spontaneous contractions of the myometrium in a dose-dependent manner but the differences in IC50 values obtained with different hormones were not statistically significant. All these hormones caused a concentration-dependent inhibition of the K(+)-induced contraction. The IC50 values were lowest for diethylstilbestrol and highest for estriol. Vasopressin at concentrations of 1.5 x 10(-6) - 1.8 x 10(-3) U/ml stimulated myometrial contractions. These responses were also inhibited by ovarian steroids and diethylstilbestrol. The IC50 values for estriol and progesterone were significantly higher than for estradiol or diethylstilbestrol. The values for estriol and progesterone did not differ significantly. In the uterine arteries, which lacked spontaneous activity, ovarian steroids and diethylstilbestrol inhibited contractions induced by K+ depolarization. As with myometrium, the lowest effect was observed with estriol and the highest with diethylstilbestrol. A dose-dependent inhibition by all four hormones (0.2-40 microM) of vasopressin-induced contractile responses of the uterine arteries was observed. With the lowest concentration of progesterone, however, the arterial response to vasopressin was enhanced. The increases by progesterone (0.02 and 0.2 microM) of responses induced by vasopressin were statistically significant (P < 0.05). The present data strongly suggest that, in human myometrium and uterine arteries, ovarian steroids and diethylstilbestrol cause a more pronounced inhibition of receptor-mediated than of voltage-dependent Ca2+ channels. The increase by a very low (physiological) concentration of progesterone of vasopressin-induced responses in both myometrium and arteries may be of significance in the pathophysiology of dysmenorrhea.

We have recently demonstrated that corticotropin releasing hormone (CRH) potentiates the contractile response to oxytocin of human gestational myometrium, using a high flow microsuperfusion system and electrical field stimulation. We now report this potentiation to be equivalent to that of 1 nM prostaglandin F2 alpha (PGF2 alpha), while 10 nM PGF2 alpha did not potentiate the response to oxytocin. Prostaglandin E2 (PGE2) also showed no augmentation of the contraction force of the myometrium in response to oxytocin. The CRH potentiated response was inhibited by the lipoxygenase and cyclooxygenase inhibitor BW755C (1 microM) and by indomethacin (0.1 microM), but not by the lipoxygenase inhibitor BW4C (1 microM). Measurements of prostaglandins in the superfusate showed no significant trends. It is concluded that the potentiation of contraction force to oxytocin by CRH is dependent on prostaglandins, probably PGF2 alpha and that leukotrienes, generated via the lipoxygenase pathway are not involved.

Catecholamine stimulation of adenyl cyclase in plasma membranes from rat myometrium was typical of beta-adrenoceptor stimulation. Thus, isoproterenol was the most potent followed by epinephrine and norepinephrine; phenylephrine, a pure alpha-agonist, did not activate the enzyme. The catecholamine-induced activation was potently inhibited by propranolol, whereas phenozybenzamine was without effect. The ED50 for 1-isoproterenol activation was 10- minus 7 M. Binding of [3H]-d-isoproterenol to the same preparations bore little resemblance to the pattern of 1-isoproterenol activation of adenyl cyclase. Propranolol, even at high concentrations, was without significant effect. The degree of binding was inversely rflated to the osmolarity suggesting that diffusion may have contributed to the binding. This contention was further supported by the time course of binding which was biphasic- the first component (smaller than 4 min) being less affected by osmolarity than the second (greater than 4 min). The optimum pH for binding was 7.4.