Oxytocin-induced Contractions Research Articles

Calix[4]arene С-956 as a selective inhibitor of Ca2+-pump of the plasma membrane and a modulator of the contractile function in the myometrium

Background. At present, creating and testing pharmacological instruments for selective inhibition of Са2+-pump of the plasma membrane, which would become the foundation for medical preparations, for instance, for the treatment of the impaired excitability of the cardiac and smooth muscles, remains critically significant. We have demon­strated in our previous experiments that calix[4]arene С-956 is effective in inhibi­ting Са2+, Mg2+-ATPase activity of the plasma membrane of myometrium cells. The aim of this study was to investigate the regularities and mechanisms of the impact of calix[4]arene С-956 on Са2+-transporting activity of Са2+, Mg2+-ATPase of the plasma membrane (PM) and the contractile function of rat myometrium. Materials and Methods. The experiments were conducted using outbred white non-pregnant rats. Ca2+-transporting activity of myocytes PM preparations loaded with Ca2+-sensitive fluorescent probe fluo-4 AM was investigated. The registration of the contractile activity in the preparations of longitudinal smooth muscles of uterine horns with preserved endothelium was done in the isometric mode. Results. It was determined that calix[4]arene C-956 causes blocking of the transport function of the calcium pump of preparations of plasma membranes of uterine myocytes. The C-956 compound causes an increase in the amplitude of spontaneous contractions and a change in their mechanokinetic parameters during a short-term effect on multicellular preparations of rat myometrium. Calix[4]arene C-956 also significantly affects the contractions caused by high-potassium depolarization of the PM and oxytocin, increa­sing their amplitude and decreasing the rate of relaxation. Blocking the synthesis of nitric oxide significantly enhances the effects of C-956 on spontaneous and high-potassium- and oxytocin-induced contractions of the myometrium. Conclusions. The results of our research indicate that the main target of the action of calix[4]arene C-956 on myocytes is the calcium pump of the PM. With the preliminary inhibition of nitric oxide synthases followed by the use of C-956, we were able to fully demonstrate the contribution of the calcium pump of the PM to the regulation of uterine contractions.

Interleukin-1β activates matrix metalloproteinase-2 to alter lacrimal gland myoepithelial cell structure and function.

The aim of the present study is to investigate the role of c-Jun N-terminal kinase (JNK) and matrix metalloproteinase-2 (MMP-2) in mediating the effects of interleukin-1β (IL-1β) on the function of lacrimal gland myoepithelial cells (MECs). MECs isolated from an α-smooth muscle actin-green fluorescent protein (SMA-GFP) transgenic mouse were treated with IL-1β alone or in the presence of SP600125, a JNK inhibitor, or ARP100, an MMP-2 inhibitor. The GFP intensity and the cell size/area were measured, and on day 7, the SMA, calponin, and pro-MMP-2 protein levels and the MEC contraction were assessed. At baseline, the control and treated cells showed no differences in GFP intensity or cell size. Starting on day 2 and continuing on days 4 and 7, the GFP intensity and cell size were significantly lower in the IL-1β-treated samples, and these effects were alleviated following inhibition of either JNK or MMP-2. Compared with the control, the levels of SMA and calponin were lower in the IL-1β-treated samples, and both the JNK and MMP-2 inhibitors reversed this trend. The pro-MMP-2 protein level was elevated in the IL-1β-treated samples, and this effect was abolished by the JNK inhibitor. Finally, oxytocin-induced MEC contraction was diminished in the IL-1β-treated samples, and both the JNK and MMP-2 inhibitors reversed this effect. Our data suggest that IL-1β uses the JNK/MMP-2 pathways to alter MEC functions, which might account for the diminished tears associated with aqueous-deficient dry eye disease.

The oxytocin antagonist cligosiban reduces human prostate contractility: Implications for the treatment of benign prostatic hyperplasia.

With increasing life expectancy, benign prostatic hyperplasia (BPH) consequently affects more ageing men, illustrating the urgent need for advancements in BPH therapy. One emerging possibility may be the use of oxytocin antagonists to relax smooth muscle cells in the prostate, similar to the currently used (although often associated with side effects) α1-adrenoceptor blockers. For the first time we used live-imaging, combined with a novel image analysis method, to investigate the multidirectional contractions of the human prostate and determine their changes in response to oxytocin and the oxytocin antagonists atosiban and cligosiban. Human prostate samples were obtained and compared from patients undergoing prostatectomy due to prostate cancer as well as from patients with transurethral resection of prostate tissue due to severe BPH. The two cohorts of tissue samples showed spontaneous multidirectional contractions, which significantly increased after the addition of oxytocin. Different to atosiban, which showed ambiguous effects of short duration, only long-acting cligosiban reliably prevented, as well as counteracted, any contractile oxytocin effect. Furthermore, cligosiban visibly reduced not only oxytocin-induced contractions, but also showed intrinsic activity to relax prostatic tissue. Thus, the oxytocin antagonist cligosiban could be an interesting candidate in the search for novel BPH treatment options.

In vitro inhibition of uterine contractions using electrospun nanofibers loaded with nifedipine and ML7.

To formulate a nanofiber-based controlled drug delivery system that could be effective in preventing uterine contractions and can be used for the treatment of preterm labor. We utilized uterine tissue samples obtained from ten pregnant women who underwent cesarean section at term to investigate the effect of nanofibers on spontaneous and induced myometrial contractions. We prepared nifedipine and ML7-loaded nanofibers using the electrospinning method with Poly(D,L-lactide-co-glycolide) (PLGA) polymer, resulted in seven groups of nanofibers, including a control group. Group I served as the control, Group II was non-drug loaded nanofiber, Group III was nifedipine (10-5 M) loaded nanofiber, Group IV was ML7 (3x10-5 M) loaded nanofiber, Group V was ML7 (3x10-5 M) and nifedipine (10-5 M) nanofiber, Group VI was ML7 (3x10-5 M) and nifedipine (3x10-5 M) nanofiber, and Group VII was ML7 (3x10-5 M) and nifedipine (10-4 M) nanofiber. To evaluate the contractile response, the nanofibers loaded with different doses of ML7 and nifedipine were applied onto the tissue strips, and in vitro organ bath experiments were performed. Full-thickness uterine samples were cleared of the serosa and surrounding tissues, and eight strips (3x10 mm) were prepared from each sample. The seven different nanofiber formulations were gently placed and sutured onto the strips, with one strip always kept as the time control. We recorded spontaneous, KCl-induced, and stimulated cumulative oxytocin-induced contractions from all samples in all groups. After completing all experiments, the viability of the strips was checked, and weight measurement was recorded. The administration of drug-loaded polymers resulted in a significant decrease in both the frequency and intensity of spontaneous and induced contractions in all groups (p<0.01). No significant difference was observed between the control group and the non-drug-loaded nanofiber group in post hoc analysis (p=0.704). In terms of amplitude and frequency of contractions, the most significant decrease was observed in group VII at cumulative oxytocin doses compared to the control and non-drug-loaded nanofiber groups (p<0.05). Moreover, group VI also showed a significant decrease in contraction intensity and frequency compared to the control and non-drug-loaded nanofiber groups (p<0.05). While the use of nifedipine and/or ML7-loaded nanofibers decreased both intensity and frequency of contraction, this attenuation was not significant compared to the control and empty polymer groups. However, a more significant inhibition was observed when ML7 was used with nifedipine at doses of 3x10-5 M and 10-4 M. The results indicate that human uterine contractions can be inhibited using calcium channel blocker (nifedipine) and myosin light chain kinase inhibitor (ML7) loaded nanofibers in uterine tissue strips. These results strongly suggested the potential for the development of locally effective and safe controlled drug release systems to prevent premature birth.

Optimal starting dosing regimen of intravenous oxytocin for labor induction based on the population kinetic-pharmacodynamic model of uterine contraction frequency.

Intravenous oxytocin is commonly used for labor induction. However, a consensus on the initial dosing regimen is lac with conflicting research findings and varying guidelines. This study aimed to develop a population kinetic-pharmacodynamic (K-PD) model for oxytocin-induced uterine contractions considering real-world data and relevant influencing factors to establish an optimal starting dosing regimen for intravenous oxytocin. This retrospective study included pregnant women who underwent labor induction with intravenous oxytocin at Peking University Third Hospital in 2020. A population K-PD model was developed to depict the time course of uterine contraction frequency (UCF), and covariate screening identified significant factors affecting the pharmacokinetics and pharmacodynamics of oxytocin. Model-based simulations were used to optimize the current starting regimen based on specific guidelines. Data from 77 pregnant women with 1095 UCF observations were described well by the K-PD model. Parity, cervical dilation, and membrane integrity are significant factors influencing the effectiveness of oxytocin. Based on the model-based simulations, the current regimens showed prolonged onset times and high infusion rates. This study proposed a revised approach, beginning with a rapid infusion followed by a reduced infusion rate, enabling most women to achieve the target UCF within approximately 30 min with the lowest possible infusion rate. The K-PD model of oxytocin effectively described the changes in UCF during labor induction. Furthermore, it revealed that parity, cervical dilation, and membrane integrity are key factors that influence the effectiveness of oxytocin. The optimal starting dosing regimens obtained through model simulations provide valuable clinical references for oxytocin treatment.

Peony Pollen Protects against Primary Dysmenorrhea in Mice by Inhibiting Inflammatory Response and Regulating the COX2/PGE2 Pathway.

Peony pollen contains multiple nutrients and components and has been used as a traditional Chinese medicine with a long history, but the effect of the treatment of primary dysmenorrhea remains to be clarified. The aim of this study is to investigate the therapeutic effect of peony pollen on primary dysmenorrhea mice and the potential mechanism. A uterus contraction model in vitro and primary dysmenorrhea mice were used to evaluate the treatment effect of peony pollen on primary dysmenorrhea. The primary dysmenorrhea mice were treated with 62.5 mg/kg, 125 mg/kg, or 250 mg/kg of peony pollen, and the writhing response, latency period, histopathological changes in the uterus, prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) levels, and infiltration of neutrophils and macrophages were investigated. Protein expression of interleukin 1 β (IL-1β), interleukin 6 (IL-6), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase 1 (mPGEs-1), BCL2-Associated X (Bax), B-cell lymphoma-2 (BCL-2), caspase-3, and cleaved caspase-3 were detected by Western blot, and the oxidative stress related marker malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS) were evaluated. Peony pollen could attenuate spontaneous or oxytocin-induced uterus contractions in vitro. Moreover, peony pollen decreased the writhing times, prolonged the writhing latency, and reduced the pathological damage of uterine tissues. Furthermore, the inflammatory cell infiltration and the protein expression of IL-1β, IL-6, and NLRP3 were decreased. The COX-2/PGE2 pathway was inhibited; oxidative stress and apoptosis in the uterus also improved in the uterus of primary dysmenorrhea mice. Peony pollen exerts a positive effect on primary dysmenorrhea by inhibiting the inflammatory response and modulating oxidative stress and apoptosis by regulating the COX-2/PGE2 pathway.

Inhibitory effect and underlying mechanism of essential oil of Prangos ferulacea Lindl (L.) on spontaneous and induced uterine contractions in non-pregnant rats

Evidence suggests the use of natural compounds as support in the management of uterine contractility disorders. We recently demonstrated that the essential oil of Apiacea Prangos ferulacea (L.) (Prangoil) modulates intestinal smooth muscle contractility. Thus, we aimed to evaluate if Prangoil could also affect the contractility of uterine muscle in non-pregnant rat and to investigate the related action mechanism/s. The effects of the aromatic monoterpenes, β-ocimene and carvacrol, constituents of Prangoil, were also evaluated. Spontaneous contractions and contraction-induced by K+-depolarization and oxytocin in rat uterus were recorded in vitro, using organ bath technique. Prangoil reduced the amplitude of spontaneous contractions as well as responses to KCl and oxytocin. β-ocimene and carvacrol matched oil inhibitory effects. Prangoil effects were not affected by nitrergic and adenylyl cyclase inhibitors or non-specific potassium channel blocker, but they were reduced by nifedipine, L-type calcium channel inhibitor, or 2-aminoethoxydiphenylborate (2-APB), membrane-permeant inositol 1,4,5-triphosphate receptor inhibitor. The response to β-ocimene was reduced by nifedipine and by 2-APB (20 μM), whilst carvacrol inhibitory effect was attenuated only by nifedipine. In conclusion, Prangoil, and its components, β-ocimene and carvacrol, reduced spontaneous and KCl or oxytocin-induced contractions of rat myometrium, mainly modulating extracellular Ca2+ influx through L-Type channels and Ca2+ release from the intracellular store. Further studies could contribute to evaluate the potential use of Prangoil against disorders characterized by abnormal uterine contractions.

Fruit Extract of Oil Palm (&lt;i&gt;Elaeis Guineensis&lt;/i&gt; (Jacq.)) Inhibits Uterine Contractility in &lt;i&gt;Ex-Vivo&lt;/i&gt; Mouse Models

Background: Different parts of Elaeis guineensis (EG) plants are used ethno-medicinally for treating numerous illnesses including abdominal contraction.&#x0D; Objectives: Thus, this study evaluated the inhibitory potential of the fruit-extract of EG on the uterine contractility in ex-vivo mouse models.&#x0D; Material and Methods: The effect of EG extract on spontaneous uterine contractions, oxytocin (OT)-induced uterine contractions, high potassium chloride induced uterine contractions and oxytocin-induced calcium free contraction were evaluated in adult non-pregnant female albino mice.&#x0D; Results: EG in a concentration dependent mode diminished spontaneous uterine contractions, while amplitude and frequency of contractions were inhibited significantly, but the frequency of contraction was at the highest concentration of EG. EG also produced a significant inhibition of the frequency of OT-induced contraction in calciumfree media.&#x0D; Conclusion: The aqueous fruit extracts of EG inhibited uterine contraction in the uterus through inhibition of intracellular calcium stores.

Pepper fruit (Dennettia tripetala [annonaceae]) seed inhibits uterine contractions via blockade of intracellular calcium release and extracellular calcium influx in ex vivo studies

Dennettia tripetala (DT) is a tree found in rain forest belt of Nigeria, Cameroon and Ivory Coast. The seed is commonly used in preparing food for mothers after delivery because it is believed to play a role in postpartum uterine contraction. There is however no scientific evidence validating this claim. Hence, the study aimed to investigate the effects of DT seed on non-pregnant and pregnant uterus using mouse model. The dried seeds of DT were macerated in methanol and the effects of DT extract were investigated on uterus tissues isolated from non-pregnant and pregnant mice. DT extract was examined on spontaneous uterine contraction, contractions induced by oxytocin, and high potassium chloride (KCl)-induced depolarization. Its effect was also evaluated on calcium ion (Ca2+) release from internal stores. The DT extract suppressed spontaneous uterine contractility in both pregnant and non-pregnant uterus in a concentration-dependent manner. It significantly inhibited uterine contractions elicited by high KCl and oxytocin-induced contractions in both pregnant and non-pregnant uterus. The extract also inhibited uterine contractility induced by oxytocin in the Ca2+-free medium in pregnant and non-pregnant uterus. The study has provided scientific evidence that DT seed extract has uterine relaxing effect in both pregnant and non-pregnant mouse uterus possibly by blocking intracellular Ca2+ released and influx of extracellular Ca2+. The result has shown that DT has no benefit in reducing the risk of post-partum hemorrhage, however, it may have potential benefits in management of dysmenorrhea and preterm labour in non-gravid and gravid uteri respectively.

β3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for β3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the β2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that β2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by β2 receptors is unable to provide meaningful tocolysis. The failure of β2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The β3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of β2 agonists as tocolytics and suggests a non-canonical signaling role for β3AR in myometrium. The addition of the β3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to β3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction

The aim of these studies was to investigate the involvement of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.

Assessment of the tocolytic nifedipine in preclinical primary models of preterm birth

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy.

Modulation of Melatonin Receptors Regulates Reproductive Physiology: The Impact of Agomelatine on the Estrus Cycle, Gestation, Offspring, and Uterine Contractions in Rats

Agomelatine is a pharmaceutical compound that functions as an agonist for melatonin receptors, with a particular affinity for the MT1 and MT2 receptor subtypes. Its mode of action is integral to the regulation of diverse physiological processes, encompassing the orchestration of circadian rhythms, sleep-wake cycles, and mood modulation. In the present study, we delve into the intricate interplay between agomelatine and the modulation of estrus cycles, gestation periods, offspring numbers, and uterine contractions, shedding light on their collective impact on reproductive physiology. Both in vivo and in vitro experiments were performed. Wistar Albino rats, divided into four groups: two non-pregnant groups (D1 and D2) and two pregnant groups (G1 and G2). The D1 and G1 groups served as control groups, while the D2 and G2 groups received chronic agomelatine administration (10 mg/kg). Uterine contractions were assessed in vitro using myometrial strips. Luzindole, a melatonin receptor antagonist, was employed to investigate the pathway mediating agomelatine's effects on uterine contractions. In in vivo studies, chronic agomelatine administration extended the diestrus phase (p&lt;0.05) in non-pregnant rats, prolonged the gestational period (p&lt;0.01), and increased the fetal count (p&lt;0.01) in pregnant rats. Additionally, agomelatine reduced plasma oxytocin and prostoglandin-E levels (p&lt;0.01) during pregnancy. In vitro experiments showed that agomelatine dose-dependently inhibited spontaneous and oxytocin-induced myometrial contractions. Luzindole (2 µM) reverse the agomelatine-induced inhibition of myometrial contractions. These findings suggest that agomelatine holds the potential to modulate diverse reproductive parameters during the gestational period, influencing estrus cycling, gestational progression, offspring development, and the orchestration of uterine contractions.

Mechanism of Oxytocin-Induced Contraction in Rat Gastric Circular Smooth Muscle

Oxytocin produces an excitatory effect on gastric muscle through the activation of receptors present on stomach smooth muscle cells. However, the intracellular mechanisms that mediate oxytocin excitatory effects are still largely unknown. Therefore, we aimed to investigate the signaling pathways involved in oxytocin-induced contractions in gastric smooth muscle, shedding light on phospholipase C (PLC)-β1 signaling and its downstream molecules, including inositol 1,4,5- trisphosphate (IP3) and myosin light chain kinase (MLCK). The contractions of gastric smooth muscle from male rats were measured in an organ bath set up in response to exogenous oxytocin 10-7 M, in the presence and absence of inhibitors of the indicated signaling molecules. Oxytocin (10-9-10-5 M) induced dose-dependent stomach smooth muscle contraction. Pre-incubation with atosiban, an oxytocin receptor inhibitor, abolished the oxytocin-induced contraction. Moreover, PLC β1 inhibitor (U73122) and IP3 inhibitor Xestospongin C inhibited oxytocin-induced muscle contraction to various degrees. Verapamil, a calcium channel blocker, inhibited oxytocin-induced contraction, and pre-incubation of the strips, with both verapamil and Xestospongin C, further inhibited the excitatory effect of oxytocin. Chelation of intracellular calcium with BAPT-AM (1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) significantly inhibited the effect of oxytocin on muscle contraction. Finally, pre-incubation of the strips with the Ca2+/calmodulin-dependent protein kinase selective inhibitor STO-609 significantly inhibited the contraction induced by oxytocin. These results suggest that oxytocin directly stimulates its cell surface receptor to activate PLC β1, which in turn liberates IP3, which eventually elevates intracellular calcium, the prerequisite for smooth muscle contraction.

Inhibition of uterine contractility by guanine-based purines in non-pregnant rats.

Growing evidence pointed out that guanine-based purines are able to modulate smooth muscle contractile activity of blood vessels and gastrointestinal tract. Since, so far, possible guanine-based purine modulation of uterine musculature is unknown, the aim of the present study was to investigate in vitro, using organ bath technique, guanosine and guanine effects on spontaneous uterine contraction, and uterine contraction induced by K+-depolarization and oxytocin in a non-pregnant rat. Guanosine, but not guanine, reduced the amplitude of spontaneous contraction of the uterine muscle in a dose-dependent manner. The inhibitory response was antagonized by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a membrane nucleoside transporter inhibitor, but persisted in the presence of theophylline, a nonselective adenosine receptor antagonist, or propanolol, β1/β2 adrenoreceptor antagonist or blockers of a nitrergic pathway. In addition, potassium channel blockers did not influence guanosine-induced effects. Guanosine was able to inhibit the external calcium (Ca2+) influx-induced contraction, but it did not affect the contraction induced by high-KCl solution, indicating that guanosine does not interact with L-type voltage-gated calcium channel. Guanosine prevented/reduced uterine contractions induced by oxytocin, even in the absence of external calcium. In conclusion, guanosine is able to reduce both spontaneous and oxytocin-induced contractions of rat myometrium, likely subsequently to its intracellular intake. The blockade of extracellular Ca2+ influx and reduction of Ca2+ release from the intracellular store are the mechanisms involved in the guanosine-induced tocolytic effects.

Phloroglucinol inhibits oxytocin-induced contraction in rat gastric circular muscle and uterine smooth muscle

Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development. In this study, a PD rat model was established. The effects of phloroglucinol on the contraction of rat gastric circular muscle and uterine smooth muscle induced by oxytocin (OT) were investigated. The writhing response, and levels of oestradiol (E2), prostaglandin e2 (PGE2), and prostaglandin f2α (PGF2α) were determined. The protein and mRNA levels of OT receptor (OTR) were detected. OT showed a significant promoting effect on gastric circular muscle and uterine smooth muscle contraction. However, phloroglucinol strongly inhibited the contraction induced by 10−6 mol/L of OT. We also found that phloroglucinol reduced writhing response and attenuated uterine damage. Compared to the blank group, E2 and PGF2α were significantly increased, but PGE2 was significantly decreased in the PD model group. Phloroglucinol was found to reverse the changes of E2, PGF2α and PGE2. Moreover, phloroglucinol reduced the protein and mRNA levels of OTR. In conclusion, phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The mechanism might be related with the regulation of OTR expression. IMPACT STATEMENT What is already known on this subject? Phloroglucinol is commonly used to alleviate dysmenorrhoea and stomach cramps. However, there is little evidence of phloroglucinol in the mechanism of primary dysmenorrhoea (PD) development. What do the results of this study add? Phloroglucinol could attenuate PD and inhibit the contraction of rat gastric circular muscle and uterine smooth muscle induced by OT. The underlying mechanisms of phloroglucinol for PD treatment may be associated with OTR. What are the implications of these findings for clinical practice and/or further research? These findings provide novel ideas for the role of phloroglucinol in PD development.

Preserved oxytocin-induced myometrium contraction and sensitivity to progesterone inhibition following rat uterus thermal insult. Impact on fertility

Women seeking improved fertility often undergo diagnostic hysteroscopy that could cause uterine thermal injury with unclear impact on uterine contraction, embryo implantation and fertility. We tested whether uterine thermal insult adversely affects myometrium function and contraction related receptors, channels, junctional proteins and remodeling enzymes. Female Sprague-Dawley rats were anesthetized, the left uterine horn was infused with 85 ℃ hot saline (thermal Insult) and the right horn was infused with 25℃ warm saline (control) for 3 min. After 7-days recovery, uterine strips were prepared for tissue histology and measurement of contraction, and mRNA and protein levels of oxytocin receptor, progesterone (P4) receptor A (PR-A), membrane K+ channel TREK-1, junctional protein connexin-43 (CX-43) and matrix metalloproteinases MMP-2 and MMP-9. Uterine tissue histology showed cellular swelling and inflammatory cell infiltration immediately following thermal insult, and recovery with no difference from control 7-days later. KCl (96 mM) and oxytocin (10-13-10-7 M) caused significant contraction that was not different in thermal insult vs control uterine strips. Pretreatment with P4 (10-5 M) for 1 h caused marked inhibition of KCl and oxytocin contraction that was insignificantly greater in thermal vs control uterus. RT-PCR showed decreases in oxytocin receptor, PR-A, TREK-1, CX-43, MMP-2 and MMP-9 mRNA in thermal vs control uterus. Western blots showed decreases in oxytocin receptor, no change in TREK-1 and increased PRA, CX-43, MMP-2, and MMP-9 protein levels in thermal vs control uterus. To assess the impact on fertility, female rats were housed with male rats, and on gestational day 19, the litter size, pup weight and crown-rump length, and placenta weight were not different in thermal vs control uterus. Thus, after thermal insult-induced immediate inflammation and reduced heat-sensitive mRNA expression, the uterus undergoes a recovery and adaptation process involving preserved oxytocin-induced contraction, P4 inhibition and TREK-1 channels. The uterus self-healing process appears to require improved PR-A signaling, intercellular communication via CX-43 and tissue remodeling by MMP-2 and MMP-9. The uterine thermal recovery processes could be essential for maintaining fertility and future pregnancy outcome.

Involvement of a neutrophil-mast cell axis in the effects of Piper malacophyllum (C. PESL) C. DC extract and its isolated compounds in a mouse model of dysmenorrhoea.

The effects of Piper malacophyllum (C. Pesl) C. DC extracts and its isolated compounds were analysed in a mouse model of primary dysmenorrhoea (PD). Female Swiss mice (6-8weeks old) on proestrus were intraperitoneally treated with estradiol benzoate for 3days, to induce PD. Twenty-four hours later, animals were treated 24h later with vehicle, plant extract, gibbilimbol B, 4,6-dimethoxy-5-E-phenylbutenolide, mixture of 4,6-dimethoxy-5-E-phenylbutenolide and 4,6-dimethoxy-5-Z-phenylbutenolide, or ibuprofen. One hour later, oxytocin was injected and the numbers of abdominal writhing were counted. Then, mice were euthanized and uteri were collected for morphometrical and histological analyses. The effects of P. malacophyllum in inflammation were investigated in mouse peritoneal neutrophils culture stimulated with LPS or fMLP (chemotaxis and mediator release). Finally, uterus contractile and relaxing responses were assessed. Similar to ibuprofen, P. malacophyllum extract and isolated compounds reduced abdominal writhing in mice with PD. Histology indicated a marked neutrophil and mast cell infiltrate in the uterus of PD animals which was attenuated by the extract. The compounds and the extract reduced neutrophil chemotaxis and inflammatory mediator release by these cells. Reduced TNF levels were also observed in uteri of PD mice treated with P. malacophyllum. The extract did not affect spontaneous uterine contractions nor those induced by carbachol or KCl. However, it caused relaxation of oxytocin-induced uterine contraction, an effect blunted by H1 receptor antagonist. Overall the results indicate that P. malacophyllum may represent interesting natural tools for reliving PD symptoms, reducing the triad of pain, inflammation and spasmodic uterus behaviour.

Isoindolines and phthalides from the rhizomes of Ligusticum chuanxiong and their relaxant effects on the uterine smooth muscle

Three undescribed isoindoline alkaloids, (+)-(R)-3-butyl-3-ethoxyisoindolin-1-one, (+)-(3S,6S,7R)-3-butyl-6,7-dihydroxy-3-methoxy-4,5,6,7-tetrahydroisoindolin-1-one, and (−)-(3R,6S,7R)-3-butyl-6,7-dihydroxy-3-methoxy-4,5,6,7-tetrahydroisoindolin-1-one, along with nine known phthalides were isolated from a water decoction of the rhizomes of Ligusticum chuanxiong using chromatographic methods. Their structures and absolute configurations were determined by extensive spectroscopic analyses and ECD data calculations. The relaxant effects of the isolated compounds on uterine contractions induced by oxytocin were investigated using a rat uterine smooth muscle contraction model. Furthermore, the effects of riligustilide on extracellular Ca2+ influx and intracellular Ca2+ release were assessed using high-KCl solution-induced and oxytocin-induced uterine smooth muscle contraction in a Ca2+-free balanced salt solution. The results showed that all the tested phthalides had inhibitory effects on oxytocin-induced uterine smooth muscle contraction. Riligustilide, a phthalide dimer, was the most active. Further examinations indicated that riligustilide reduced uterine smooth muscle contraction by inhibiting extracellular Ca2+ influx and intracellular Ca2+ release.

An in vitro Study on the Contractility of Epileptic Myometrium and Effects of Antiepileptic Agents on Oxytocin-Induced Contractions of Myometrium Isolated from Absence Epileptic WAG/Rij Rats.

The aim of this study was to determine whether spontaneous and stimulated contractile activity of myometrium in epileptic rats is different from healthy ones, and whether antiepileptic drugs (AEDs) have any direct influence on myometrial contractility. Myometrial strips from nonpregnant and pregnant adult epileptic WAG/Rij and Wistar rats were suspended in organ bath containing physiological salt solution (37°C and pH 7.4, aerated with 95% oxygen-5% CO2), and isometric contractions were recorded. Effects of cumulative concentrations of selected AEDs including phenytoin, levetiracetam, and valproic acid alone and in combination on oxytocin-induced contractions was examined. Contractile parameters assessed included the area under curve, amplitude, and frequency of contractions, evaluated by 10-min periods. Data were analyzed using one-way analysis of variance and Tukey HSD test. Spontaneous myometrial contractility and responses to oxytocin showed species difference. Compared with that of control Wistar rats, spontaneous contractions of myometrium from nonpregnant epileptic WAG/Rij rats were significantly higher while being significantly lower in pregnant preparations. Upon stimulation with oxytocin, WAG/Rij myometrium showed significantly lower contractile response compared with preparations from healthy control Wistars (p &lt; 0.01). Phenytoin and valproate caused concentration-dependent significant attenuation (p &lt; 0.05) of spontaneous and oxytocin-induced contractions of myometrium from WAG/Rij and Wistar rats, both nonpregnant and pregnant. Myometrial smooth muscle from epileptic rats showed different spontaneous and oxytocin-induced contractility, and AEDs showed contractile modulatory actions, phenytoin being the most and levetiracetam the least effective. Although in vitro, our findings may be of clinical implications with regard to obstetric complications in epileptics and use of AEDs during pregnancy, and warrants further investigations.