THG113.31, a specific PGF2alpha receptor antagonist, induces human myometrial relaxation and BKCa channel activation

Background  

PGF2alpha exerts a significant contractile effect on myometrium and is central to human labour. THG113.31, a specific non-competitive PGF2alpha receptor (FP) antagonist, exerts an inhibitory effect on myometrial contractility. The BKCa channel is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate potential BKCa channel involvement in the response of human myometrium to THG113.31.     

Reduced expression of CRH receptor type 1 in upper segment human myometrium during labour

Background  

Corticotropin-releasing hormone (CRH) and CRH-related peptide are shown to modulate uterine contractility through two CRH receptor subtype, CRH-R1 and CRH-R2 during pregnancy. Through different signaling pathways, CRH-R1 maintains myometrial quiescence whereas CRH-R2 promotes smooth muscle contractility. We hypothesized that the expression of CRH receptors in myometrium might be changed during pregnancy and labour.     

Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

Background  

Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour.     

Analysis of Maxi-K alpha subunit splice variants in human myometrium

Background  

Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca^{2 +} homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset.     

The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

Background  

Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium.     

Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells

Background

Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium.

Methods

Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life.

Results

Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs). MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase identified fibroblasts as separate from MICs.

Conclusion

MICs are identified consistently on the boundaries of smooth muscle bundles in both the pregnant and non-pregnant uterus and are distinct from fibroblasts. The uniform distribution of connexin 43 on the cell membrane of MICs, rather than localisation in gap junction plaques, may represent the presence of connexin hemichannels. This antibody specificity may aid future study of this potentially important cell type.     

Ghrelin in the human myometrium

Background  

Ghrelin is a 28-amino acid octanolyated peptide, synthesised primarily in the stomach. It stimulates growth hormone release, food intake and exhibits many other diverse effects. Our group have previously determined that ghrelin inhibited human contractility in vitro. The aim of this study therefore, was to investigate the expression of ghrelin, its receptor, the growth hormone secretagogue receptor type 1 (GHS-R1), ghrelin O-acyltransferase (GOAT) which catalyses ghrelin octanoylation, prohormone convertase 1/3 (PC1/3) responsible for pro-ghrelin processing, in human myometrium, during pregnancy prior to labour, during labour and in the non-pregnant state. Modulation of ghrelin and ghrelin receptor expression in cultured myometrial cells was also investigated.     

Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells

Background  

One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction - more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells - with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.     

Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.     

The focal adhesion protein Hic-5 is highly expressed in the rat myometrium during late pregnancy and labour and co-localizes with FAK

Background  

Myometrial growth and remodeling of the cytoskeleton and focal adhesions during late pregnancy may be critical aspects of myometrial activation and thus labour. Yet our understanding of these aspects is inhibited by the paucity of information concerning the components of focal adhesions in the myometrium. The focal adhesion protein hydrogen peroxide-inducible clone-5 (Hic-5) has recently been found in mononuclear smooth muscle but was not examined in the myometrium during pregnancy. Thus, the goal of this study was to characterize Hic-5 mRNA and protein expression in the rat myometrium during pregnancy and labour.     

Stretch Activates Human Myometrium via ERK,Caldesmon and Focal Adhesion Signaling

An incomplete understanding of the molecular mechanisms responsible for myometrial activation from the quiescent pregnant state to the active contractile state during labor has hindered the development of effective therapies for preterm labor. Myometrial stretch has been implicated clinically in the initiation of labor and the etiology of preterm labor, but the molecular mechanisms involved in the human have not been determined. We investigated the mechanisms by which gestation-dependent stretch contributes to myometrial activation, by using human uterine samples from gynecologic hysterectomies and Cesarean sections. Here we demonstrate that the Ca requirement for activation of the contractile filaments in human myometrium increases with caldesmon protein content during gestation and that an increase in caldesmon phosphorylation can reverse this inhibitory effect during labor. By using phosphotyrosine screening and mass spectrometry of stretched human myometrial samples, we identify 3 stretch-activated focal adhesion proteins, FAK, p130Cas, and alpha actinin. FAK-Y397, which signals integrin engagement, is constitutively phosphorylated in term human myometrium whereas FAK-Y925, which signals downstream ERK activation, is phosphorylated during stretch. We have recently identified smooth muscle Archvillin (SmAV) as an ERK regulator. A newly produced SmAV-specific antibody demonstrates gestation-specific increases in SmAV protein levels and stretch-specific increases in SmAV association with focal adhesion proteins. Thus, whereas increases in caldesmon levels suppress human myometrium contractility during pregnancy, stretch-dependent focal adhesion signaling, facilitated by the ERK activator SmAV, can contribute to myometrial activation. These results suggest that focal adhesion proteins may present new targets for drug discovery programs aimed at regulation of uterine contractility.     

The presence of prostacyclin binding sites in nonpregnant bovine uterine tissue

Myometrium of various animal species makes a considerable amount of prostacyclin (PGI2) which is a potent myometrial and uterine vascular smooth muscle relaxing agent. This action of PGI2 is perhaps mediated by binding to specific receptors, which have never been demonstrated in uterine tissue of any animal species until very recently. The quantitative light microscopic autoradiographic approach used in the present studies demonstrated that while bovine myometrial smooth muscle and uterine vascular smooth muscle contained PGI2 specific binding sites, endometrial and perimetrial cells contained few or no binding sites. The number of binding sites in circular and elongated myometrial smooth muscle and in arteriolar smooth muscle were similar (P greater than 0.05). The PGI2 binding to the uterine cells was greatly reduced (P less than 0.001) following coincubation with excess unlabeled PGI2, but not with its stable metabolite, 6-keto PGF1 alpha, PGE2, PGF2 alpha and leukotriene C4 which bind to nonpregnant bovine uterine tissue, also had no effect of PGI2 binding. In conclusion, nonpregnant bovine uterine tissue contain specific PGI2 binding sites which may mediate its potent relaxing effect on myometrium and uterine vasculature.     

Pennogenin Tetraglycoside Induces Rat Myometrial Contraction and MLC20 Phosphorylation via PLC-IP3 and RhoA/Rho Kinase Signaling Pathways

Background

Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been widely used in China for the treatment of abnormal uterine bleeding. We previously studied the main active constituents of TSSPs and their structure-activity relationships with respect to rat myometrial contractions. Tg (pennogenin tetraglycoside) was identified as one of the active ingredients in TSSPs able to induce rat myometrial contractions. However, the mechanisms underlying the pharmacological actions on uterine activity have not been described clearly.

Methods

Here Tg was screened for effects on contractile activity in isolated uterine strips from estrogen-primed rats and on MLC20 phosphorylation and related signaling pathways in cultured rat myometrial cells as determined by Western blot. Intracellular calcium ([Ca²⁺]_i) was monitored under a confocal microscope using Fluo-4 AM-loaded myometrial cells.

Results

Tg dose-dependently stimulated rat myometrial contractions as well as MLC20 phosphorylation in vitro, which could be completely suppressed by an inhibitor of myosin light chain kinase (MLCK). Use of Ca²⁺ channel blockers and kinase inhibitors demonstrated that Tg-induced myometrial contractions are mediated by activation of the phospholipase C (PLC)-inositol triphosphate (IP3) signaling pathway, resulting in increased MLC20 phosphorylation. Furthermore, Y27632, a specific inhibitor of Rho kinase (ROK), notably suppressed Tg-stimulated myometrial contractions and decreased MLC20 phosphorylation.

Conclusions

These data provide evidence that rat myometrial contractility induced by Tg results from enhanced MLC20 phosphorylation, while both PLC-IP3 and RhoA/ROK signaling pathways mediate the process. These mechanisms may be responsible for the therapeutic effects of TSSPs on abnormal uterine bleeding.     

Up-regulation of p21- and RhoA-activated protein kinases in human pregnant myometrium

The role of small ras homologous GTP-binding proteins in the regulation of smooth muscle contractility has become increasingly apparent but there is still little information about the presence of these proteins in human uterine smooth muscle. Messenger RNAs for p21-activated protein kinase isoforms (PAK1, PAK2, and PAK3) were detectable in both nonpregnant and pregnant human myometrial tissue. However, PAK3 protein was not detectable and the proteins for PAK1 and PAK2 were only detectable in pregnant tissue. Moreover there was a large increase in the constitutively active p34 protein fragment of PAK2 in pregnant tissue. Protein expression of RhoA-activated protein kinases isoforms (ROK1 and ROK2) also increased during pregnancy. Stimulation of RhoA signaling in pregnant myometrial tissue with lysophosphatic acid (LPA) increased the level of myosin light chain (MLC20) phosphorylation. Preincubation of the tissue with C3 toxin inhibited LPA-stimulated MLC20 phosphorylation and lowered the basal phosphorylation level of MLC20. Thus ROKS and PAKS have the potential to regulate uterine contractility and/or load-bearing during human pregnancy.     

Endothelin expression in the uterus

The endothelins (ETs) comprise a family of 21 amino acid peptides, ET-1, ET-2 and ET-3, first demonstrated as products of vascular endothelium. Subsequent work showed that they are also found in non-endothelial cells from a variety of tissues such as breast, parathyroid and adrenal gland. At first, the ETs were recognized for their pressor effects. However, ET administration in vivo initially caused hypotension at low concentrations by triggering the paracrine release of endothelial-derived vasodilators. The ETs exert powerful contractile actions on myometrium and other types of smooth muscle and are mitogenic, or co-mitogenic for fibroblasts, vascular smooth muscle and other cells. Demonstration of extravascular ET in endometrium has revealed a powerful vasoconstrictor which might act on the spiral arterioles to effect a powerful and sustained contraction of vascular smooth muscle. ETs might also contribute to the process of endometrial repair. In addition, the ETs appear to play a fundamental role in the control of uterine function in pregnancy. Effects on myometrial contractility have been implicated in the mechanisms governing the onset of normal and pre-term labour, and the peptides are likely to be key determinants of placental blood flow by binding to vascular smooth muscle receptors in the placenta.     

The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)‐injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT‐PCR/qRT‐PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down‐regulated in myometrium from LPS‐treated‐mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.     

The role of prostaglandins for the coordination of myometrial forces during labour

Systematic studies using a superfusion technique for recording myometrial contractility in vitro have been conducted in our department to explore whether prostaglandins (PG) have a differential action on the different segments of the pregnant uterus and also whether the qualitative and quantitative response undergoes a change during spontaneous labour. Myometrial specimens were excised from the fundal area and from the lower uterine segment at elective caesarean section in the 39th week of pregnancy before commencement of labour and at acute caesarean section during ongoing labour. Before labour PGF2 alpha was without or had a very weak effect on upper segment preparations but was stimulatory on lower segment specimens. PGE2 and PGI2 generally induced a biphasic dose-dependent response (stimulation followed by inhibition). During spontaneous labour PGF2 alpha and PGE2 always stimulated upper segment preparations while the contractile activity of specimens from the lower segment was inhibited by PGE2, PGF2 alpha was generally without effect. PGI2 had the same biphasic action before as during labour. With all reservations for the validity of in vitro experiments, the results favour the hypothesis that initiation of labour in the human involves a qualitative shift in the myometrial reactivity to prostaglandins. These alterations may involve suppression of expulsive forces and perhaps some tightening of the lower uterine segment during pregnancy. Following initiation of labour there is a marked increase in the excitatory action of both PGE2 and PGF2 alpha in the fundal area while the lower uterine segment reacts in a way that favours dilatation.     

Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.