A Modified Hai–Murphy Model of Uterine Smooth Muscle Contraction

We extend and analyze the Wang and Politi modified Hai–Murphy model of smooth muscle cell contractions to capture uterine muscle cell response to variations in intracellular calcium concentrations. This model is used to estimate values of unknown parameters in uterine smooth muscle cell cross-bridging. Uterine motility is responsible for carrying out important processes throughout all phases of the nonpregnant female reproductive cycle, including sperm transport, menstruation, and embryo implantation. The modified Hai–Murphy partial differential equation model accounts for the displacement of myosin cross-bridge heads relative to their binding sites. This model was originally developed for the study of airway contractions; we now extended it for use in modeling nonisometric uterine contractions. Our extended model incorporates cross-bridge position and contractile velocity into the original model, resulting in more accurate modeling of the initial stages of contraction and modeling nonisometric contractions. Numerical simulations show that the contraction rate in our extended model is faster than the original Hai–Murphy model. These simulations provide quantitative estimates for the increased level of responsiveness of our extended model to intracellular calcium concentrations. The extended model and new parameter estimates for the cross-bridging can be coupled with uterine flow models to advance our understanding of embryonic motility and intrauterine flow.     

Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid

In the lung, the contraction of smooth muscle, or bronchospasm, is generally caused by an immunologic insult resulting in mast cell degranulation and the release of histamine, slow reacting substances, and other mediators of inflammation (1). Although the immediate response is bronchospasm, continued activation of this sequence of events results in a chronic inflammatory disease. In the uterus, numerous conditions can result in smooth muscle contraction. One major pathophysiological syndrome associated with increased uterine tone and severe rhythmic contraction is primary dysmenorrhea (2). In this disease state, prostaglandins have been shown to play a major role in these contractions (3,4), and inhibitors of cyclooxygenase have proven beneficial in clinical practice (5). Both dysmenorrhea and cervical ripening have been likened to inflammatory reactions due to varying degrees of vasodilation, invasion by inflammatory cells, proliferation of fibroblasts and smooth muscle contraction (6,7). Metabolism of arachidonic acid (AA) via cyclooxygenase to prostaglandins and thromboxanes and via lipoxygenase to hydroxyeicosatetraenoic acids (HETEs) and leukotrienes is an integral part of both the acute and chronic inflammatory reaction in the lung or uterus. The material reviewed here examines the effect of endogenous leukotrienes on both the lung and uterus and suggests that other smooth muscles and pathophysiological states may be more involved with the lipoxygenase pathway of AA metabolism than previously believed.     

Uterine contractions depend on KIT-positive interstitial cells in the mouse: genetic and pharmacological evidence

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit W-v/Kit W-v compared with Kit+/+ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit W-v/Kit W-v and Kit+/+ mice. Ex vivo treatment of Kit+/+ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.     

Effects of adlay hull extracts on uterine contraction and Ca2+ mobilization in the rat

Dysmenorrhea is directly related to elevated PGF(2alpha) levels. It is treated with nonsteroid antiinflammatory drugs (NSAIDs) in Western medicine. Since NSAIDs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative medicine. Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for treating dysmenorrhea. However, the relationship between smooth muscle contraction and adlay extracts remains veiled. Therefore, we investigated this relationship in the rat uterus by measuring uterine contraction activity and recording the intrauterine pressure. We studied the in vivo and in vitro effects of the methanolic extracts of adlay hull (AHM) on uterine smooth muscle contraction. The extracts were fractionated using four different solvents: water, 1-butanol, ethyl acetate, and n-hexane; the four respective fractions were AHM-Wa, AHM-Bu, AHM-EA, and AHM-Hex. AHM-EA and its subfractions (175 microg/ml) inhibited uterine contractions induced by PGF(2alpha), the Ca(2+) channel activator Bay K 8644, and high K(+) in a concentration-dependent manner in vitro. AHM-EA also inhibited PGF(2alpha)-induced uterine contractions in vivo; furthermore, 375 microg/ml of AHM-EA inhibited the Ca(2+)-dependent uterine contractions. Thus 375 microg/ml of AHM-EA consistently suppressed the increases in intracellular Ca(2+) concentrations induced by PGF(2alpha) and high K(+). We also demonstrated that naringenin and quercetin are the major pure chemical components of AHM-EA that inhibit PGF(2alpha)-induced uterine contractions. Thus AHM-EA probably inhibited uterine contraction by blocking external Ca(2+) influx, leading to a decrease in intracellular Ca(2+) concentration. Thus adlay hull may be considered as a feasible alternative therapeutic agent for dysmenorrhea.     

Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.     

Paeonol alleviates primary dysmenorrhea in mice via activating CB2R in the uterus

Background and purposePrimary dysmenorrhea is the most common gynaecologic problem in menstruating women and is characterized by spasmodic uterine contraction and pain symptoms associated with inflammatory disturbances. Paeonol is an active phytochemical component that has shown anti-inflammatory and analgesic effects in several animal models. The aim of this study was to explore whether paeonol is effective against dysmenorrhea and to investigate the potential mechanism of cannabinoid receptor signalling.Experimental approachDysmenorrhea was established by injecting oestradiol benzoate into female mice. The effects of paeonol on writhing time and latency, uterine pathology and inflammatory mediators were explored. Isolated uterine smooth muscle was used to evaluate the direct effect of paeonol on uterine contraction.Key resultsThe oral administration of paeonol reduced dysmenorrhea pain and PGE2 and TNF-α expression in the uterine tissues of mice, and paeonol was found to be distributed in lesions of the uterus. Paeonol almost completely inhibited oxytocin-, high potassium- and Ca²⁺-induced contractions in isolated uteri. Antagonists of CB2R (AM630) and the MAPK pathway (U0126), but not of CB1R (AM251), reversed the inhibitory effect of paeonol on uterine contraction. Paeonol significantly blocked L-type Ca²⁺ channels and calcium influx in uterine smooth muscle cells via CB2R. Molecular docking results showed that paeonol fits well with the binding site of CB2R.Conclusions and implicationsPaeonol partially acts through CB2R to restrain calcium influx and uterine contraction to alleviate dysmenorrhea in mice. These results suggest that paeonol has therapeutic potential for the treatment of dysmenorrhea.     

Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.     

Contraction of cultured human uterine smooth muscle cells after stimulation with endothelin-1

To our knowledge, the problem of how to maintain isolated smooth cells in a "contractile" phenotypic state without deviation after subculturing has yet to be resolved. The present study characterized the in vitro contractile response of human uterine smooth muscle cell to endothelin-1, which induces contractions in isolated uterine strips. Contractile effects were qualitatively investigated using silicone rubber substrata. Endothelin-1 was able to distort and reduce the wrinkles in the silicone surface. Contractions were also quantified by measuring the resulting change in the collagen lattice area. Endothelin-1 significantly increased the contractile response in a dose-dependent manner by selectively activating endothelin A receptors. When myometrial cells were cultured within collagen lattices, a microfilament-disrupting agent, cytochalasin B, abolished contractions, and no change was observed in smooth muscle alpha-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.     

Effect of nitrendipine on isolated uterus and other smooth muscles of the rat

Increasing concentrations of nitrendipine were found to inhibit various types of muscular activation (electrical stimulation, acetylcholine, oxytocin, potassium chloride), as well as the spontaneous rhythmic activity of the isolated rat uterus. The degree of the inhibitory effect of nitrendipine depends on the type of activation. Nitrendipine showed an exceptionally high efficacy in inhibiting contractions induced by electrical stimulation and of spontaneous rhythmic activity. For inhibition of these contractions even osmolar concentrations of nitrendipine were sufficient. The relaxant effect of nitrendipine depended on the concentration of extracellular calcium and the time of incubation of nitrendipine in the bathing medium. Nitrendipine showed high selectivity for the uterine smooth muscle because in a very high concentrations is exerted an insignificant relaxation of the other isolated smooth muscles (oesophagus, urinary bladder). Our experiments indicate that nitrendipine might have a role in therapy of premature delivery and abortion because of its great selectivity for the uterine smooth muscle. Addition of calcium into the medium restores completely all types of muscular activation after the inhibitory action of nitrendipine except its depressive action on the phasic component of oxytocin-induced contractions. These findings that individual types of activation, after inhibitory action of nitrendipine, are reestablished in various degrees by the addition of calcium into the medium, are also an additional confirmation about the existence of various types of calcium channels.     

Dietary Selenium Influences Calcium Release and Activation of MLCK in Uterine Smooth Muscle of Rats

We sought to elucidate the effects of different concentrations of dietary selenium on calcium ion release, MLCK levels, and muscle contraction in the uterine smooth muscle of rats. The selenium (Se) content of blood and of uterine smooth muscle tissues was detected by fluorescence spectrophotometry. Ca²⁺ content was measured by atomic absorption spectroscopy. Calmodulin (CaM) and MLCK RNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Dietary Se intake increased the Se levels in the blood and in uterine smooth muscle tissues and increased the Ca²⁺ concentration in uterine smooth muscle tissues. The addition of Se also promoted CaM expression and enhanced MLCK activation in uterine smooth muscle tissues. In conclusion, Ca²⁺, CaM, and MLCK were regulated by Se in uterine smooth muscle; Se plays a major role in regulating smooth muscle contraction in the uterus.     

天花粉对小鼠子宫肥大细胞超微结构的影响

本研究用雌性中国１号小鼠１２只,分实验组和对照组,实验组分别腹腔注射天花粉后处死,取子宫组织作超薄切片,电镜观察。在天花粉作用下,常见子宫肥大细胞脱颗粒,呈功能活化状态,并与子宫平滑肌细胞紧密相邻。提示肥大细胞可能通过脱颗粒释放组胺促进子宫平滑肌的收缩。同时,也见肥大细胞与子宫结缔组织中的成纤维细胞,淋巴细胞等密切接触,提示成纤维细胞和淋巴细胞与天花粉作用下子宫肥大细胞的数量增多有关。     

Immunocytochemical localization of galanin in the rat male and female genital tracts and motor effects in vitro

Galanin, a recently discovered neuropeptide, was studied in the rat male and female reproductive tracts by immunocytochemistry and in vitro pharmacology. Nerve fibers containing galanin immunoreactivity were most abundant in the female paracervical tissue, where they surrounded non-immunoreactive ganglion cells. Galanin nerves were also found in the uterus and Fallopian tubes, as well as in the vas deferens. When tested in vitro galanin contracted the smooth muscle of both the uterine horn and cervix. Galanin also slightly potentiated the response to electrical field stimulation in preparations from the uterine cervix and vas deferens, but it had no effect on the seminal vesicle. Galanin-(1–10), an N-terminal residue of galanin, also contracted the uterine horn, though higher concentrations were required. The neurally induced contractions were not influenced by galanin-(1–10) in any of the smooth muscle preparations tested. The muscle receptors mediating the direct contractile effects in the uterine horn seem to require the N-terminus of galanin, while the neuromodulatory effects on the electrically induced contractile activity seem to need the C-terminal part or the whole galanin molecule. Galanin may thus function as a neuromediator in the rat male and female genital organs.     

Complex effects of imatinib on spontaneous and oxytocin-induced contractions in human non-pregnant myometrium

Human myometrium includes two important cell populations involved in its contractility: smooth muscle fibers and interstitial cells. The pacemaking mechanism is not yet identified, but it is possible that myometrial smooth muscle cells contract in response to a signal generated by c-kit positive interstitial cells. The aim of this study was to investigate the effects of imatinib as a c-kit receptor antagonist on the spontaneous or oxytocin (OT) induced contractions of human non-pregnant myometrium in vitro. Myometrial strips were obtained from non-pregnant women (reproductive age) undergoing hysterectomy for benign indications. The strips were suspended in organ baths for recording of isometric tension. Imatinib effects were assessed on spontaneous contraction and after preexposure to OT.Direct exposure of myometrial strips to imatinib inhibits both amplitude and frequency of contractions (80-320 μM) in a dose dependent manner. Amplitude reverted back to 90% of the baseline amplitude by consequent addition of imatinib (until 480 μM). Total inhibition of myometrial contraction was obtained after addition of OT 60 nM. If myometrium was pre-exposed to OT (320 nM), imatinib 80-160 μm increased amplitude, while decreasing frequency. These data provide evidence that telocytes may be involved as modulators of the spontaneous contractions of the non-pregnant human uterus, via a tyrosine-kinase independent signaling pathway.     

Myometrial characteristics of the syrian hamster uterine smooth muscle cell line,SHM

Summary We describe several characteristics of a novel smooth muscle cell line, SHM (Syrian hamster myometrium) derived from a primary uterine leiomyosarcoma which was induced by chronic estrogen plus androgen treatment of a female Syrian (golden) hamster. To determine the usefulness of the SHM cell line as a model for understanding myometrial function and its regulation, we have examined the morphologic and immunocytochemical properties of these cells, and the ability of uterotonic agonists to activate transmembrane signaling via phosphoinositide hydrolysis. The SHM cells exhibited a spindle-shape, smooth musclelike morphology when subconfluent, and a more compact, stellate shape at confluence. Like primary myocytes, SHM cells expressed the intermediate filament desmin and the contractile protein alpha smooth muscle actin, but not the epithelial antigen cytokeratin. Norepinephrine and bradykinin, which stimulate contraction and inositol polyphosphate production in the uterus, also stimulated inositol polyphosphate production in SHM cells. The maximal phosphoinositide signaling responses were lower in SHM cells compared with primary hamster uterine myocytes. We conclude that the SHM cell line exhibits primary uterine myocyte characteristics, and may therefore be a useful system for examining the mechanisms through which myometrial functions are regulated.     

Tools,techniques, and future opportunities for characterizing the mechanobiology of uterine myometrium

The myometrium is the smooth muscle layer of the uterus that generates the contractions that drive processes such as menstruation and childbirth. Aberrant contractions of the myometrium can result in preterm birth, insufficient progression of labor, or other difficulties that can lead to maternal or fetal complications or even death. To investigate the underlying mechanisms of these conditions, the most common model systems have conventionally been animal models and human tissue strips, which have limitations mostly related to relevance and scalability, respectively. Myometrial smooth muscle cells have also been isolated from patient biopsies and cultured in vitro as a more controlled experimental system. However, in vitro approaches have focused primarily on measuring the effects of biochemical stimuli and neglected biomechanical stimuli, despite the extensive evidence indicating that remodeling of tissue rigidity or excessive strain is associated with uterine disorders. In this review, we first describe the existing approaches for modeling human myometrium with animal models and human tissue strips and compare their advantages and disadvantages. Next, we introduce existing in vitro techniques and assays for assessing contractility and summarize their applications in elucidating the role of biochemical or biomechanical stimuli on human myometrium. Finally, we conclude by proposing the translation of “organ on chip” approaches to myometrial smooth muscle cells as new paradigms for establishing their fundamental mechanobiology and to serve as next-generation platforms for drug development.     

Collagenase production by smooth muscle: correlation of immunoreactive with functional enzyme in the myometrium

A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti-collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti-smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra- or extra-cellularly.     

Neuropeptides in the female genital tract: effect on vascular and non-vascular smooth muscle

The presence of vasoactive intestinal polypeptide (VIP), substance P (SP), somatostatin, enkephalin, and avian pancreatic polypeptide (APP) in nerves in the female genital tract raises the question of their physiological significance as neurotransmitter substances. We have examined the effect of these peptides on non-vascular uterine smooth muscle in vivo as well as in vitro, and the effect on blood flow in the genital tract of rabbit and cat. SP caused a dose-dependent increase in mechanical and myoelectrical activity, an action which could be antagonized by VIP. Substance P, leu-enkephalin and VIP induced a concentration related increase in blood flow of the uterus, where VIP seems to be the most potent vasodilator. Neither the effects on vascular nor on non-vascular smooth muscle were inhibited by adrenergic nor cholinergic blocking agents. APP was able to inhibit the VIP-induced vasodilation in rabbits. These findings suggest that several peptides are involved in the local nervous control of both uterine contractions and haemodynamic events.     

The Role of Cellular Coupling in the Spontaneous Generation of Electrical Activity in Uterine Tissue

The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.