Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...

University of Liverpool

Prolongation of pregnancy i.e. going more than 10 days over the estimated due date, complicates up to 10% of all pregnancies and is associated with increased risk to both mother and fetus. Despite the obvious need for contractions of the uterus to end pregnancy, there have been no studies directly examining the role of uterine smooth muscle, myometrium, in the aetiology of prolonged pregnancy. This study tested the hypothesis that the intrinsic contractile characteristics of myometrium taken from women with prolonged pregnancy (>41 weeks and 3 days) was reduced compared to those delivering at term (39-41 weeks). We recruited women undergoing Caesarean Section (CS) delivery either pre-labour (n = 27) or in labour (n = 66) at term or postdates. The contractile ability of the postdates myometrium, whether spontaneous or elicited by oxytocin or high-K solution, was significantly reduced compared to term myometrium. These differences remained when adjusted for parity and other maternal characteristics. The findings remained significant when expressed per cross sectional area. Histological examination revealed no differences between the two groups. The contractile differences were however related to intracellular Ca transients suggesting an effect of [Ca] on reduced force production in the postdates group. In summary, myometrium from prolonged pregnancies contracts poorly in vitro even when stimulated with oxytocin and in active labour. Responses to high K(+) and measurements of Ca suggest that alterations in excitation contraction coupling, rather than any histological changes of the myometrium, may underlie the differences between term and postdates myometrium. We show that postdates pregnancy is associated with poor myometrial activity and suggest that this may contribute to increased myometrial quiescence and hence, prolonged gestation.

Poor spontaneous and oxytocin-stimulated contractility in human myometrium from postdates pregnancies.

Much has been written about the effects of aging on reproductive function, especially female fertility. Much less is known about how aging may affect the contractility of the smooth muscle within the uterus, the myometrium. The myometrium is active through a woman's entire life, not just during pregnancy. Here we will discuss briefly the contractile functions of the uterus and the changes it undergoes throughout the stages of a woman's life from menstruation and the menopause, before evaluating the evidence for any changes in myometrial contractility and responses as women age, with a particular focus on women of advanced maternal age. We present original contractility analysis for the widest data set for human myometrium so far examined, and determine inherent spontaneous activity as well as responses to depolarisation and stimulation with oxytocin. Our data show that in the non-pregnant state there is a significant decrease in contractility for both spontaneous and depolarised-induced contractions, with age. We suggest that muscle atrophy and down regulation of Ca channels may account for this. Interestingly in pregnant myometrium we found a wide range of contractile ability between women and little evidence for decreased spontaneous activity between the ages of 25-40. Oxytocin responses appear to be more affected by aging, a finding that is consistent with previously reported clinical findings, and may partly be the result of membrane lipids such as cholesterol, increasing as women age. The marked differences between the age-related decline of force beyond age 30 in non-pregnant uterus, and the lack of difference in the pregnant state over this period, shows that the uterus retains its ability to respond to gestational hormones. The growth of the pregnant uterus and increase in content of myofibrillar proteins, may abolish any previous age-related force deficit. This finding is consistent with what is apparent for postmenopausal women in their 50s and 60s; that with the appropriate hormonal stimulation the uterus can allow an embryo to implant, and then without further intervention, carry the foetus to term. It is tempting therefore to speculate that unlike other well documented declines in female reproductive functions with age, the myometrium remains able to function into a woman's 7th decade.

What do we know about what happens to myometrial function as women age?

Over half of twin pregnancies in US and UK deliver prematurely but the reasons for this are unclear. The contractility of myometrium from twin pregnancies has not been directly investigated. The objective of this research was to determine if there are differences in the contractile activity and response to oxytocin, between myometrium from singleton and twin pregnancies, across a range of gestational ages. Furthermore, we wished to determine if contractile activity correlates with increasing level of stretch, using neonatal birth weights as a marker of uterine stretch.

A comparison of the contractile properties of myometrium from singleton and twin pregnancies.

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.

In this short review, we discuss how recent insights into myometrial physiology may be taken forward and translated into much-needed novel therapies for problems associated with labour. We consider excitation-contraction coupling in the myometrium and how this relates to our understanding of the changes that occur to produce myometrial contractions and successful labour. We then discuss how this information has already been used in the development of drugs to either stimulate or relax the myometrium, to address the needs of women with either slow (dystocic) labours or threatened preterm labours, respectively. We next present the data showing how basic physiological findings pertaining to hypoxia and lactate production have been taken and translated into a tool for predicting and thus better managing difficult labours. We then highlight examples of where physiological research has started to provide mechanistic insight into clinical problems associated with labour and parturition (obesity, diabetes, advanced maternal age, postdate and twin pregnancies) and suggest how these findings could be translated into new therapies for difficult labours.

Myometrial physiology--time to translate?

For successful birth, contractions need to become progressively stronger. The underlying mechanisms are unknown, however. We have found that a novel mechanism, hypoxia-induced force increase (HIFI), is switched on selectively, at term, and is essential to strengthening contractions. HIFI is initiated as contractions cyclically reduce blood flow and produce repeated hypoxic stresses, with associated metabolic and transcriptomic changes. The increases in contractility are a long-lasting, oxytocin-independent, intrinsic mechanism present only in the full-term pregnant uterus. HIFI is inhibited by adenosine receptor antagonism and blockade of cyclooxygenase-2 signaling, and partially reproduced by brief episodes of acidic (but not alkalotic) pH. HIFI explains how labor can progress despite paradoxical metabolic challenge, and provides a new mechanistic target for the 1 in 10 women suffering dysfunctional labor because of poor contractions.

Hypoxia-induced force increase (HIFI) is a novel mechanism underlying the strengthening of labor contractions, produced by hypoxic stresses.

We compared the relaxant effect of 2 known tocolytics; indomethacin and atosiban and progesterone, on pregnant human myometrial spontaneous and oxytocin-induced contractions from singleton and twin pregnancies. All agents exerted a concentration-dependent relaxant effect on myometrial contractions. There was no significant difference in the concentration-response curves between singletons and twins for progesterone or indomethacin on spontaneous contractions or atosiban on oxytocin-induced contraction. Under oxytocin however, the concentration-response curves for indomethacin and progesterone were significantly shifted to the right for both amplitude of contraction (P < .01) and activity integral (P < .01). When compared to singleton myometrium however, the concentration-response curves were significantly shifted to the right in the twin myometrium group (P < .05 progesterone and P < .001 indomethacin). We conclude that a greater concentration of progesterone and indomethacin is required to inhibit oxytocin-induced myometrial contractions in twins compared to singletons in vitro. The differences noted in the tissue pharmacologies may have implications for the successful prevention or inhibition of preterm labor in twin pregnancy.

Differing In Vitro Potencies of Tocolytics and Progesterone in Myometrium From Singleton and Twin Pregnancies.

People living in the tropical rain forest of South-Western Nigeria use Rinorea dentata (P. Beauv.) Kuntze (Violaceae) in ethno-veterinary medicine to facilitate parturition. There are no evidence-based pharmacological investigations for the uterotonic activity of this plant.

Ethnobotanical survey of Rinorea dentata (Violaceae) used in South-Western Nigerian ethnomedicine and detection of cyclotides.

Preterm birth at <37 weeks of gestation is the most common and costly complication of pregnancy and remains the leading cause of neonatal morbidity, death, and reduced achievement in surviving infants. Magnesium sulfate is 1 class of tocolytics for threatened preterm labor; however, its clinical efficacy has been questioned. Twin pregnancies are at increased risk of preterm delivery compared with singleton gestations, which suggests that there is twin-specific risk to preterm delivery in twins. The prevention strategies that are applied to singleton pregnancies, however, have not been shown to be effective in twin pregnancies.

The combination tocolytic effect of magnesium sulfate and an oxytocin receptor antagonist in myometrium from singleton and twin pregnancies.

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide.

Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduction, cardiovascular function, social behavior, memory, and learning through four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across rodents and humans for translational studies. We identified a selective and more stable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and vasopressin. [Se-Se]-oxytocin-OH displayed similar potency to oxytocin but improved selectivity for OTR, an effect that was retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OH produced a more regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicating a potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In conclusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systems and is a promising new lead for therapeutic development. Our medicinal chemistry approach may also be applicable to other peptidergic signaling systems with similar selectivity issues.

Sarah Arrowsmith

Harris-Wellbeing Preterm Birth Research Centre,
Department of Cellular and Molecular Physiology,
Institute of Translational Medicine,
Harris-Wellbeing Preterm Birth Research Centre, Department of Cellular and Molecular Physiology, Institute of Translational Medicine

University of Liverpool

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development