In which of the following uterine walls is the powerful muscular layer that contracts during childbirth?
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Hector N. Aguilar, 1 Department of Physiology , University of Alberta , Edmonton, Alberta , Canada Search for other works by this author on: B.F. Mitchell1 Department of Physiology , University of Alberta , Edmonton, Alberta , Canada 2 Department of Obstetrics and Gynaecology , 220 HMRC, University of Alberta , Edmonton, AB , Canada T6G 2S2 Search for other works by this author on: Received: 31 October 2009 Revision received: 29 April 2010
Close Navbar Search Filter Microsite Search Term Search AbstractBACKGROUND Uterine contractile activity plays an important role in many and varied reproductive functions including sperm and embryo transport, implantation, menstruation, gestation and parturition. Abnormal contractility might underlie common and important disorders such as infertility, implantation failure, dysmenorrhea, endometriosis, spontaneous miscarriage or preterm birth. METHODS A systematic review of the US National Library of Medicine was performed linking ‘uterus’ or ‘uterine myocyte’ with ‘calcium ion’ (Ca2+), ‘myosin light chain kinase’ and ‘myosin light chain phosphatase’. This led to many cross-references involving non-uterine myocytes and, where relevant, these data have been incorporated into the following synthesis. RESULTS We have grouped the data according to three main components that determine uterine contractility: the contractile apparatus; electrophysiology of the myocyte including excitation-contraction coupling; and regulation of the sensitivity of the contractile apparatus to Ca2+. We also have included information regarding potential therapeutic methods for regulating uterine contractility. CONCLUSIONS More research is necessary to understand the mechanisms that generate the frequency, amplitude, duration and direction of propagation of uterine contractile activity. On the basis of current knowledge of the molecular control of uterine myocyte function, there are opportunities for systematic testing of the efficacy of a variety of available potential pharmacological agents and for the development of new agents. Taking advantage of these opportunities could result in an overall improvement in reproductive health. IntroductionThe uterus is a hollow organ with a well-differentiated lining layer (endometrium), a thick muscular coat (myometrium) and a serosal outer layer. There has been remarkable progress towards understanding the physiology and clinical pathophysiology of the endometrium and this has resulted in many important interventions to affect conception and contraception as well as menstrual function. In contrast, although there is growing awareness of the potential importance of abnormal function of the uterine muscle layer, there has been relatively little research concerning the role of the myometrium in common disorders of reproduction. Myometrial function may be of vital importance in physiological processes such as sperm and embryo transport and implantation, and in disorders such as dysmenorrhea and endometriosis. At present there is limited understanding of regulation of uterine contractility in the non-pregnant state. Yet, better understanding of this physiology is essential to design and test interventions that can prevent or treat the important clinical problems noted above. To fill in the gaps in our knowledge of uterine physiology in the non-pregnant state, we shall borrow liberally from knowledge gained from experiments using both human and animal models, whether pregnant or not. The goal of this review is to provide an overview of the molecular mechanisms that might regulate uterine contractility, particularly emphasizing recent findings with potential clinical applicability to improvement of reproductive health. MethodsThe initial search strategy involved searching the United States National Library of Medicine (http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed) and matching ‘uterus’ or ‘uterine myocyte’ with ‘calcium signaling’, ‘myosin light chain kinase (MLCK)’, ‘ myosin light chain phosphatase (MLCP)’ or ‘calcium sensitization’. Papers were selected based on the relevance to our objectives as determined by review of the titles and abstracts. After synthesizing a review of this information, key references obtained from these papers were individually reviewed and selected based on their potential relevance to uterine smooth muscle (SM). This information was used to expand the discussion of the regulation of uterine SM. The term ‘uterine contractility’ then was entered and articles were selected based on their clinical relevance to disorders of reproduction in non-pregnant women. Finally, the review was edited and shortened to focus on molecular mechanisms regulating contractility of the non-pregnant uterus with emphasis on information that could be clinically applicable for the improvement of reproductive health. The contractile apparatusAnatomical considerations and uterine contractile activityUterine contractions occur throughout the menstrual cycle in the non-pregnant state and throughout gestation. There are four important parameters that change under various physiological or pathophysiological conditions: frequency, amplitude, duration and direction of propagation. Over the past two decades, considerable information regarding myometrial function in non-pregnant women has been obtained from the use of open-tipped pressure catheter recordings or from three-dimensional ultrasound or magnetic resonance imaging (MRI). Several reviews have described these changes and their potential clinical significance (Brosens et al., 1998; van Gestel et al., 2003; Bulletti et al., 2004; Bulletti and de Ziegler, 2006). Contractile activity in the non-pregnant uterus appears to be fundamentally different than in the pregnant organ. The contractions observed during the menstrual cycle have been termed ‘endometrial waves’ (Ijland et al., 1996). Using a variety of imaging techniques, these contractions appear to involve only the sub-endometrial layer of the myometrium. These observations have led to a new concept of uterine anatomy that encompasses two distinct zones of the myometrium (Fig. 1). Figure 1 Concept of the sub-endometrial layer of myometrium. This thinner, innermost layer of muscle fibers, which are arranged predominantly in a circular configuration around the uterine cavity, is suggested to be of different embryological origin with physiological properties distinct from the more prominent outer layer. The circular sub-endometrial layer may facilitate the changing vectors of ‘endometrial waves’ that might play important roles in common reproductive disorders. The outer layer is likely to be more important in more intense uterine activity including abortion or parturition. In the early follicular phase following menstruation, contractile waves occur once or twice per minute and last 10–15 s with low-amplitude (usually <30 mmHg). As ovulation approaches, the frequency increases to 3–4 per minute. During the luteal phase, the frequency and amplitude decrease perhaps to facilitate implantation. In the absence of implantation of a blastocyst, the contraction frequency remains low but the amplitude increases dramatically (50–200 mmHg) producing labor-like contractions at the time of menstruation. The most fascinating aspect of endometrial waves is the integrated directionality of the SM activity and the changes that occur through the reproductive cycle. These have been classified and are described in greater detail by others (Ijland et al., 1996; van Gestel et al., 2003). In non-primate species, the myometrium consists of two distinct layers—an outer longitudinal layer and an inner circular layer. However, in the human, the myometrial substructure is not so well defined (Huszar and Naftolin, 1984). The outer longitudinal layer is much less distinct and the major thickness of the myometrium is composed of intertwined muscle bundles that frequently surround abundant vascular channels. This histological arrangement may be of vital hemostatic importance following delivery of the hemochorial placenta that is characteristic of primates. Perhaps of particular interest to the physiology of the myometrium in the non-pregnant state, the inner (sub-endometrial) portion of the myometrium has been the focus of compelling research over the past three decades (reviewed in Brosens et al., 1998). In 1983, Hricak used MRI to demonstrate a distinct tissue layer occupying the inner one-third of the myometrium, which appeared as a low intensity signal area that blended into the endometrial stroma (Hricak et al., 1983). During the reproductive years, this ‘junctional’ or ‘sub-endometrial’ layer appears anatomically distinct from the outer, denser myometrium but this distinctiveness is blurred in pre-pubertal and post-menopausal years. Noe et al. (1999) have proposed and provided some evidence to support the view that this junctional layer is also embryologically and functionally distinct from the outer myometrium. They suggest that the inner, junctional myometrium, which is composed of short muscle bundles arranged in a predominantly circular pattern, is derived from the paramesonephric (Mullerian) ducts of the female embryo but the outer, more predominant myometrium originates from non-Mullerian tissue. The junctional myometrium is rich in estrogen and progesterone receptors that are regulated throughout the menstrual cycle (Noe et al., 1999). In contrast, there appears to be no such cyclic changes in sex steroid receptor expression in the thick outer layer of the myometrium, which contains predominantly long muscle fibers arranged longitudinally and is the major contractile tissue during parturition and abortion. More recent and sophisticated MRI studies using diffusion tensor imaging to provide a three-dimensional view confirmed the overall presence of anisotropy, indicating a lack of organization of fibers, but also provided more evidence to support the presence of a distinct inner, sub-endometrial circular layer of fibers throughout the uterine corpus and tubes (Weiss et al., 2006). The proposed junctional zone rationalizes the types of contractile activity observed in the video images from ultrasound or MRI studies. These waves have been described by most investigators as having peristalsis-like character. This is reminiscent of small intestinal peristaltic motility, which is mediated by the actions of distinct inner circular and outer longitudinal muscle layers, although the exact mechanism of coordination for these impulses remains unclear. Interestingly, regarding gastro-intestinal motility, the phenomenon of reverse peristalsis is well described (Andrews and Blackshaw, 2006). Thus, the presence of a functional inner circular layer of muscle fibers could represent a mechanism for this peristaltic and anti-peristaltic activity that is well-documented through the menstrual cycle. The circular arrangement of the muscle fibers may underlie the ability of the contractile activity to travel from fundus to cervix or in the opposite direction, depending on the local hormonal milieu and, undoubtedly, many other factors. Regardless of the presence or absence of physiologically distinct myometrial zones, uterine contractions are dependent on the individual contractile activity of the cellular elements, the uterine myocytes. The remainder of this review will describe the molecular mechanisms that are likely to be involved in this activity. Most myometrial research has focused on changes that occur during pregnancy and in particular, those that might be related to the occurrence of preterm labor (for review see Mitchell and Taggart, 2009). In addition, much of what we know about SM contractility has been derived from studies of vascular SM, from either human or animal sources, or from other SM tissues such as rodent ileum or frog stomach. In this review, we will present findings that are specific to the myometrium as well as information derived from other types of SM. Because of the paucity of information regarding the uterine tissues from non-pregnant women, it is impossible to present a picture of uterine physiology specific to the non-pregnant state. We have attempted to identify studies that have focused specifically on human uterine myocytes, but we have not exhaustively named the species and SM types for findings that are highly likely to be applicable to the human uterine myocyte. SM cells (SMCs) are relatively small and densely packed with myofilaments and associated dense bodies that occupy 80–90% of the cell volume and constitute the contractile machinery (see Fig. 2A and excellent reviews: Gabella, 1984; Morgan and Gangopadhyay, 2001; Gunst and Zhang, 2008). As in all muscle tissue, the predominant proteins expressed in uterine SM are myosin and actin. In skeletal or striated muscle, there is ∼3-fold more myosin than actin. Conversely, SM has more actin than myosin by a factor ranging from 2 to 10 (Gabella, 1984). In uterine SM, there is ∼6-fold more actin than myosin (Word et al., 1993). Figure 2 Smooth muscle (SM) contractile machinery. (A) The smooth muscle cell cytoplasm is densely packed with elements of the contractile machinery (thick and thin filaments), and other structural components (dense bodies, dense bands, intermediate filaments). The network formed by the combination of these elements results in force transduction along the longitudinal axis of the cell and cell shortening. (B) The contractile elements are composed of myosin thick filaments and actin thin filaments anchored to dense bodies. The movement of thin filaments caused by phosphorylation of myosin light chains and subsequent ATP hydrolysis by the myosin II ATPase decreases the distance between anchor points. (C) Myosin II is a hexamer composed of two heavy chains, two essential light chains and two regulatory light chains. Phosphorylation of the two regulatory light chains causes formation of a cross bridge between actin and myosin filaments and also creates a change in the angle of the neck region of myosin II, which causes motion of the actin thin filaments resulting in shortening of the cell. The myofilaments are classified according to their diameter. Thin filaments (6–8 nm diameter) are polymers of globular monomeric actin. Thick filaments (15–18 nm diameter) are made up of myosin. In general, the actin and myosin filaments run in parallel and in the longitudinal dimension of the cell. In contrast, intermediate filaments (10 nm diameter) may be composed of a large number of proteins, although desmin and vimentin are the predominant constituents. Actin thin filamentsMonomeric actin is a soluble globular protein. In cells at rest, ∼80% of the actin is polymerized into actin filaments. There are six isoforms of actin, each expressed from a separate gene. In SMCs, there are two major pools of filamentous actin. The thin filaments that form part of the contractile machinery are predominantly composed of α- and γ-actin (Draeger et al., 1990). These filaments ultimately slide along the myosin thick filaments to shorten the cell during a contraction (Fig. 2B). Another pool of actin (mainly β-actin) constitutes an important structural protein of the cytoskeleton just below the plasma membrane (PM). Although not a part of the classical contractile machinery, this actin polymerizes in the presence of a contractile stimulant and, by strengthening the PM, might play an integral role in development of the mechanical tension generated (Gunst and Zhang, 2008). According to the current concept of uterine SM contractile activity, muscle shortening occurs when the thin filaments exert tension along the longitudinal direction of the cell. This process has three basic requirements: (i) a force is required to move the actin filaments; (ii) the force must be transmitted along the actin thin filaments from the longitudinal poles of the cell towards the cell center; and (iii) the actin filaments must be firmly attached to the cytoskeleton of the myocyte. The myosin motor described in the next section fulfills the first of these functions. The other two functions are filled by specialized structures called dense bodies and dense bands, respectively (Fig. 2A). These electron-dense structures, as viewed by electron microscopy, are found commonly in all SMCs. The dense bodies appear in the cytosol and act to bridge thin filaments together along the contractile plane of the cell. A major protein component is α-actinin. It appears that dense bodies serve as anchors from which the thin filaments can exert force to bring the polar cell membranes towards each other resulting in cell shortening. Interestingly, dense bodies also are associated with β-actin, which is the type found in the cytoskeleton, suggesting that dense bodies may integrate the functions of the contractile machinery and the cytoskeleton during contraction. In comparison, dense bands are associated with the PM. They are composed of a large number of proteins including α-actinin, vinculin and cytoskeletal actin. The actin filaments of the contractile machinery become tethered to the cytoskeleton by virtue of these dense bands, which thus play an important role in transmitting the forces from the contractile units toward the PM to bring about cell shortening. The dense bands form rib-like structures around the circumference of the cell and, towards the pole of the cell, may occupy essentially the entire surface. In the central regions of the cell, the dense bands alternate with bands of caveolae. Dense bands also contain intermediate filaments and bind integrins from the extracellular space. This suggests that they mediate interactions between the contractile machinery and the extracellular matrix. Myosin thick filamentsThe term ‘myosin’ encompasses a large superfamily of genes that share the ability to bind to actin and possess ATPase enzyme activity. The ‘myosin motor’ of human muscle tissue (Fig. 2B and C) is predominantly of the class myosin II (MII, for review, see Eddinger and Meer, 2007). In SM, MII is a hexamer molecule composed of two heavy chains (MHC) and two pairs of myosin light chains (MLC). The MII hexamer consists of three regions. The ‘tail’ domain is made up of the C-terminal ends of the MHCs, which are intertwined in an α-helical rod and form the major constituents of the thick filaments of SMCs. The ‘head’ domain is composed of the globular N-terminal end of the MHCs that protrudes laterally from the filament. The head constitutes the ‘motor domain’ that contains the actin-binding region as well as the ATP hydrolysis site that provides the energy required for force production. The intermediate ‘neck’ domain is the region creating the angle between the head and tail. This hinge-like lever arm is the site of non-covalent binding of the MLCs—one from each pair binds to each MHC. The two MLCs have molecular masses of 20 (MLC20) and 17 (MLC17) kDa. In vascular and uterine SM, MLC20, also known as ‘regulatory light chain’, has a pivotal role in regulating muscle contraction (Gorecka et al., 1976; Arner and Pfitzer, 1999). Its role will be discussed extensively in the following sections. The MLC17 is called the ‘essential light chain’ and its exact function is unclear. However, MLC17 may contribute to the structural stability of the myosin head along with MLC20 and may also play a role in the regulation of contraction through physical interactions with actin that are dependent on the particular MLC17 isoform expressed in a given tissue (Hernandez et al., 2007). The head and neck domains, along with the MLCs, that lean outward from the thick filaments are called cross-bridges to reflect their function as the parts of the myosin macromolecule that interact with the actin filaments during contractile activity. In SM, there is a single gene that codes for the dominant MHC. However, there are splice variants of this gene that result in four distinct SM MHC isoforms (Hamada et al., 1990; Dauvois et al., 1993; Eddinger and Meer, 2007). In addition, SM may contain non-muscle (NM) MHC that can arise from multiple genes (Gaylinn et al., 1989; Eddinger and Meer, 2007). To add further complexity, two variants of MLC17 (MLC17a/b) also exist, as a result of alternate splicing at the MLC17 gene. In contrast, different genes encode the two MLC20 isoforms, one coding for MLC20 that will associate with SM MHC and the other codes for a distinct protein that associates only with NM MHC (Taubman et al., 1987; Gaylinn et al., 1989; Kumar et al., 1989; Eddinger and Meer, 2007). Literally hundreds of permutations of four light and two heavy chains are possible if we allow complete promiscuity amongst all splicing possibilities and combinations of NM and SM MHCs, although it is unlikely that more than a few such combinations are actually used or permitted within a specific SM bed. Despite varying expression ratios of the multiple MHC/MLC20/MLC17 splice variants, a high level of functional specificity can be achieved (Morano 2003; Eddinger and Meer, 2007). Thus, the possibility for fine-tuning of the contractile machinery exists. In this regard, differences in expression of various MII isoforms have been demonstrated to occur in different regions of the same organ (Parisi and Eddinger, 2002). Thus, regional differences in isoform expression could produce slightly different contractility profiles, which may influence the vector of propagation of forces. In the uterus, this could underlie the changes in uterine motility vectors observed during different phases of the menstrual cycle as noted earlier. Clearly, much more research is required to clarify the physiological roles that may be fulfilled for each SM tissue by this heterogeneity of expression and isoform association amongst MII constituents. Once the contraction has occurred, the cross bridge attachments need to be released in order that the muscle can relax. Although less is known about this phenomenon, it appears to be related to dephosphorylation of MLC20. In some situations, the dephosphorylated MLC20 is very slow to allow detachment of the actin from the myosin cross bridge, resulting in a prolonged contraction. This has been referred to as a ‘latch-bridge’ (Hai and Murphy, 1988). This phenomenon may be of great value especially for tonically active SM beds as it would allow them to maintain basal tone through holding in an isometric state without a great energy cost. Phosphorylation of Ser19 on MLC20 causes a conformational change that increases the angle in the neck domain of the MHC, thus mobilizing the cross-bridges and causing the actin thin filament to slide along the myosin thick filament. Upon MII activation, the myosin and actin filaments move by ∼10 nm relative to each other in what is referred to as the power stroke. Through an unknown mechanism, phosphorylation of Ser19 on MLC20 also activates the ATPase activity of the myosin head region to provide the energy to fuel the contraction. Phosphorylation of Thr18 on MLC20 is also possible and may further increase the ATPase activity of MII (Ikebe et al., 1986, 1987, 1988). However, phosphorylation of Ser19 has been the primary interest in studies of regulation of SM contractile activity. This phosphorylation reaction is mediated by the enzyme MLC20 kinase (MLCK), which is predominantly regulated by the intracellular concentration of free calcium ion ([Ca2+]i). These mechanisms are the focus of a subsequent section. Intermediate filamentsIntermediate filaments form the structural network of the cytoskeleton and are largely responsible for the shape and spatio-temporal organization within the cell (Fig. 2A). These filaments may play important roles in signal transduction, contractile activity and other important processes (for review see Taggart and Morgan, 2007; Tang, 2008). Unfortunately, there has been very little research into the intermediate filaments of uterine SMCs. In other tissues, more than 65 separate proteins have been found in intermediate filaments (Hesse et al., 2001). The proportion of vimentin, desmin and the many other constituents of intermediate filaments may vary greatly, both in concentration and distribution, from one cell type to another. Although long considered as part of the cytoskeleton of the cell, there is increasing acceptance that these filaments play a role in force development during contraction of SM tissue. As noted in Fig. 2A, vimentin filaments insert into cytoplasmic dense bodies or dense bands, which also serve as anchors for actin thin filaments. They also insert into PM desmosomes, which are complex intercellular junctions. Thus, when the actin and myosin filaments are activated during a contraction, the intermediate filaments may facilitate the spatial reorganization of the contractile machinery to optimize force development (Wang et al., 2006). In response to uterine contractile activation, vimentin is phosphorylated at Ser56 by p21-activated kinase (Li et al., 2006). This results in some disassembly of vimentin polymers and this may facilitate the spatial reorganization that optimizes force generation. Other proteins of the contractile apparatusIn addition to the constituents of the filamentous structures discussed above, other proteins accessory to the contractile machinery may play important roles in contractile regulation. These proteins are primarily associated with the thin filaments and include tropomyosin, calponin and caldesmon. Tropomyosin is an actin-associated protein that spans seven actin monomers and is laid out end to end over the entire length of the thin filaments. In striated muscle, tropomyosin serves to enhance actin–myosin interactions. However, it has an uncertain role in SM. Calponin may be expressed at levels reaching stoichiometric equivalence with actin, and has been proposed to be a load-bearing protein. Caldesmon may be involved in tethering actin, myosin and tropomyosin, and in so doing may enhance the ability of SM to maintain tension. In addition, caldesmon may be directly involved as a molecular switch for MII ATPase activity dependant on its phosphorylation state. All three of these proteins may have a role in inhibiting MII ATPase activity. For a more thorough discussion regarding these and other important regulatory proteins, the reader is referred to other reviews (Morgan and Gangopadhyay, 2001; Szymanski 2004; Kordowska et al., 2006). Electrophysiology of uterine myocytes (excitation-contraction coupling)Uterine SM has a phasic pattern of contractile activity—maintenance of a resting tone with discrete, intermittent contractions of varying frequency, amplitude and duration. As noted earlier, the state of contractility is regulated predominantly by [Ca2+]i. From a functional, physiological point of view, the regulation of [Ca2+]i can be considered in three phases: maintenance of basal concentrations, which play a role in resting tone of the SM; the marked increase in [Ca2+]i that occurs with contractile agonist stimulation (Fig. 3A); and the restoration of [Ca2+]i to resting state following stimulation (Fig. 3B). In general, these processes are controlled by inter-related ion channel and pump mechanisms. In this section, we will discuss the electrophysiological events underlying this phasic activity. Figure 3 Excitation-contraction coupling in SM. (A) Agonist activation of GPCRs results in opening of receptor-operated (ROC) and voltage-dependent (VDC) PM Ca2+ channels. In parallel, the G-protein Gαq stimulates PLC to cleave PIP2 into DAG and IP3, the latter of which activates a receptor at the level of the SR to induce Ca2+ release from internal stores. These events result in a rise in the internal level of Ca2+ and ultimately activation of MLCK through the intermediary activation of CaM. (B) Ca2+ signals are terminated by extrusion of Ca2+ from the cytosolic compartment or sequestration into internal stores via PM Ca2+ ATPases (PMCA) and SR Ca2+ ATPases (SERCA), respectively. Activation of Ca2+-sensitive K+ channels serves to repolarize the myocyte membrane and induces closure of VDCs, limiting further Ca2+ entry. Internal stores may also be refilled by opening of store-operated channels (SOCE) upon reception of a signal (calcium influx factor, CIF) indicating store-depletion. (C) Ca2+–CaM stimulated phosphorylation of MLC20 is self-limiting through parallel activation of CaMKII by CaM, resulting in inhibitory phosphorylation of MLCK. Dephosphorylation of MLC20 by MLC20 phosphatase (MLCP) results in resetting of the contractile system and relaxation at the level of the tissue. Green, red and black lines depict activation, inhibition and ion movement or ATP consumption, respectively. Maintenance of the resting state (resting membrane potential)The resting membrane potential (Vrest) of uterine myocytes has been recorded between −35 and −80 mV (reviewed in Sanborn, 2000). The ionic currents that maintain this potential and the changes that occur in response to pharmacologic and signaling molecules constitute the complex electrophysiologic network that controls the contractile activity of the uterus. Vrest undergoes rhythmic oscillations, which have been termed ‘slow waves’. These waves reflect the distribution of Ca2+, Na+, K+ and Cl− ions between the intracellular and extracellular spaces and this, in turn, reflects the permeability of the PM to each of those ions (Sanborn, 2000; Khan et al., 2001a, b). Of these relevant ions, the largest electrochemical gradient in the resting state exists for Ca2+, which has 104 greater concentration in the extracellular space as compared with the cytosolic compartment ([Ca2+]i = 0.15 µM compared with 1.5 mM outside the cell). This ensures that the opening of membrane Ca2+ channels stimulated by uterotonins is followed by a rapid and significant rise in [Ca2+]i. Uterine myocyte excitability, as with most other excitable cell types, depends on the movement of Na+, Ca2+ and Cl− ions into the cytosolic compartment from the extracellular space, and of K+ ions in the opposite direction. The former three are concentrated in the extracellular space, whereas the latter is concentrated in the intracellular milieu of SMCs (Sanborn, 1995). The major factors in the establishment of Vrest are the various K+ channels present in the SMCs. A variety of K+ channels with different pharmacologic, kinetic and voltage dependence properties have been identified in human uterine myocytes (Khan et al., 2001a, b). These channels conduct an outward current during periods where the muscle is not active, and thereby maintain Vrest. Further, this outward conductance of K+ repolarizes the membrane post-stimulation, thus decreasing excitability in the absence of a stimulus. Ca2+, voltage and metabolites such as ATP can gate various types of membrane-localized K+ channels, denoted as KCa, KV and KATP, respectively. All of these types of K+ channels have been detected in human myometrium but debate continues as to which channels play predominant roles and how they interact among each other (Anwer et al., 1993; Khan et al., 1997; Khan et al., 2001a, b; Brainard et al., 2005; Aaronson, 2006; Bursztyn et al., 2007; Smith, 2007). Ca2+-sensitive K+ channels may play a key role in regulation of Vrest. These channels, denoted as BKCa (also referred to as maxi-K), are made up of four α and four β subunits, have a large conductance capacity and respond to increased [Ca2+]i as well as changes in PM voltage (Ledoux et al., 2006). The mechanism by which these channels sense the presence of elevated [Ca2+]i is not known. These channels limit cellular excitability by conducting K+ out of the cell when [Ca2+]i rises, thus antagonizing the depolarizing stimulus. Other K+ channels have been studied in myometrium from various species (Inoue et al., 1990; Sanborn 1995; Miyoshi et al., 2004). In particular, several members of the KCNQ family of K+ channels have been observed in non-pregnant murine uterine SM and some appear to have increased expression at the time of progesterone dominance (McCallum et al., 2009). Although their roles are less clearly defined, they could play subtle but important roles in uterine contractility. Certainly, K+ channels are not the sole charge carriers in the myometrium. Many surface proteins have electrogenic properties. For example, currents mediated by ClCa (Ca2+-activated Cl− channels) may play a role in pacemaking, as inhibition of these channels showed alteration in spontaneous and agonist-stimulated contractions in rat myometrium (Jones et al., 2004). Additionally, the expression of Na+ channels and of connexin-43, a main constituent of myometrial gap junctions, has been demonstrated to increase with gestation in human tissues as well as those of rodents (Garfield et al., 1978; Inoue and Sperelakis, 1991; Garfield et al., 1995). Gap junctions serve to interconnect adjacent myocytes both electrically and metabolically (Young, 2007). As may be expected, a mutation in connexin-43 leading to decreased intercellular connectivity reduced the force of myometrial contractions in addition to impairing responsiveness to oxytocin (OT) (Tong et al., 2009). For completeness, it should be noted that Na+ channels which conduct fast depolarizing currents may be involved in enhanced responsiveness to contractile stimuli and in ensuring the rapid and complete electrical propagation of action potentials (APs) in myometrial SM (Sperelakis et al., 1992a, b; Sanborn, 1995). The mRNAs encoding Na+ channel subunits have been found in pregnant rat and human myometrium (George et al., 1992; Boyle and Heslip, 1994), although this type of channel is normally absent from SM. Agonist stimulation (generation of APs)In all excitable tissues, the AP embodied by membrane depolarization is the trigger for many intracellular events. This is also the case in SM where PM depolarization leads to the entry of extracellular Ca2+ which in turn causes [Ca2+]i to rise and contraction to occur (Wray, 1993). Two types of APs have been recorded in myometrial SM from various species—simple APs involving depolarization followed by rapid repolarization, and complex APs, which entail an initial depolarization with a sustained plateau. Different combinations of ionic currents may be at play during these two different patterns of electrical activity (Khan et al., 2001a, b; Bursztyn et al., 2007). Although a single AP is sufficient to induce the propagation of an electrical wave of activity in the myometrium, multiple coordinated depolarizations are necessary for forceful and sustained contractions (Garfield and Maner, 2007). The number of cells involved in the coordinated effort of these clusters encodes the frequency, amplitude and duration of the contraction (Maul et al., 2003). Estrogen treatment has been noted to cause slight depolarization and alter both inward and outward currents in uterine muscle cells from late pregnant rats (Inoue et al., 1999). The concept of a pacemaker in the myometrium has been considered and investigated for many years. Clearly, the uterus is ‘myogenic’ in that it contracts in vivo and in vitro without the need for external stimuli. Decades of research employing a variety of histological techniques have yielded no evidence for the presence of cells with the histological and electrophysiological properties of a functional pacemaker (Gherghiceanu and Popescu, 2005, Hinescu and Hinescu, 2005; Popescu et al., 2005; Radu et al., 2005; Cretoiu et al., 2006; Hinescu et al., 2006, 2007, 2008; Popescu et al., 2006; Mandache et al., 2007; Popescu et al., 2007; Suciu et al., 2007) such as has been described in other tissues including the gut and urethra (Sergeant et al., 2000; Huizinga and Lammers, 2009). The essential issues of the origin of the electrical impulse initiating a myometrial contraction and the regulation of its direction of propagation remain unclear in either the pregnant or non-pregnant uterus. Clearly, much more research is required to understand the regulation, and therefore dysregulation of uterine contractility that causes such a broad and important variety of reproductive disorders noted earlier. Regardless of the origin of the contraction, the individual uterine myocyte contractile activity is mediated by subsequent changes in [Ca2+]i. Thus, regulation of Ca2+ flux across the PM is of ultimate importance to determine the state of contractile activity. Ca2+ is one of the most ubiquitously used second messenger signaling molecules in biological systems. Ca2+ transients regulate a wide range of cellular processes, including fertilization, secretion, proliferation, learning, cytoskeletal rearrangements, gene expression, in addition to SM contraction. In SM, the liberation of Ca2+ from intracellular stores along with the influx of Ca2+ from the extracellular space serve to activate the biochemical pathways which lead to actin–myosin cross-bridging and force development in the presence of ATP. The predominant Ca2+ channels in the uterine myocyte are the L-type Ca2+ channels, which are ubiquitous, large conductance, voltage-operated channels (VOC; Sperelakis et al., 1992a, b). Since Ca2+ is a divalent cation, it contributes to both the chemical and electrical environments of the cell and is itself influenced by electrochemical forces. When the uterine myocyte membrane potential is depolarized to approximately −40 mV, the L-type VOC open to allow a massive influx of Ca2+ (Sanborn, 2000). The resulting rise in [Ca2+]i initiates a chain of events (see below) resulting in a contraction. The myometrium contains other types of Ca2+ channels including isoforms and splice variants of T-type Ca2+ channels (Ohkubo et al., 2005). These channels exhibit faster kinetics than the L-type Ca2+ channel (Sperelakis et al., 1992a, b). Interestingly, myometrial T-type Ca2+ channels have a greater conductance capacity than the L-type Ca2+ channel (Young et al., 1993) and thus were suggested to play a prominent role in AP propagation. In comparison, the L-type Ca2+ channel may be more suited to allow bulk Ca2+ entry over a longer period of time to mediate the signaling effects of Ca2+ as a second messenger. As they become activated at lower (more negative) voltages than L-type Ca2+channels, T-type Ca2+ channels may aid in elevating the PM potential to the threshold necessary for L-type Ca2+ channel activation, which then may lead to firing of myometrial APs. Selective blockage of T-type Ca2+ channels significantly slowed the rate of spontaneous uterine contractions in myometrial strips from late pregnancy (Blanks et al., 2007) supporting a role for these channels in myometrial regulation. In general, uterine agonists interact with a specific G-protein coupled receptor (GPCR) in the myocyte PM resulting in activation of a trimeric G-protein containing a Gαq or Gα11 subunit (Fig. 3; Phaneuf et al., 1993). Activation of this subunit in the uterine myocyte stimulates membrane phospholipase Cβ (PLCβ) (Taylor et al., 1991) to hydrolyze phosphatidylinositol bisphosphate (PIP2) into inositol-trisphosphate (IP3) and diacylglycerol (DAG), which serve as second messengers (Berridge, 1993; Exton, 1996). IP3 interacts with a specific receptor (IP3R) (Furuichi et al., 1989) at the level of the sarcoplasmic reticulum (SR) causing release of Ca2+ from its intracellular storage site (Streb et al., 1983; Gill, 1989) and a subsequent rise in [Ca2+]i. This pathway will be discussed further below. The other product of the reaction, DAG, activates classical and novel protein kinase C (PKC) isoforms. The precise role of the PKC pathway remains unclear although evidence suggests it may play a negative feedback role in uterine myocytes by stimulating internalization and degradation of receptors as well as decreasing transcription of mRNA for new receptor synthesis (Lajat et al., 1998; Ball et al., 2006; Devost et al., 2008). The IP3–IP3R mediated Ca2+ release from the SR is a major contributor to the increase in PM voltage from Vrest to the point where VOC for Ca2+ are opened to trigger an AP. ROCs for Ca2+ or K+ may also contribute to this. Another mechanism, as yet poorly understood, is known as Ca2+-induced Ca2+ release (CICR) whereby the increasing [Ca2+]i sensitizes other Ca2+ channels to open, thus creating a feed-forward loop. This mechanism may involve ryanodine receptors in the SR. The IP3Rs are themselves sensitive to Ca2+ and can mediate CICR (Wray et al., 2003). This activity can give rise to sudden increases in [Ca2+]i that can be observed as spontaneous Ca2+ ‘sparks’ and subsequent Ca2+ ‘waves’ using ion-imaging techniques. Although these phenomena were not detected in uterine SM from pregnant rats (Burdyga et al., 2007), their presence in non-pregnant human myometrium could allow for localized and directional increases in Ca2+ release separated in space from other areas of the cell (McCarron et al., 2004). Such mechanisms could play a role in mediating the variety of patterns of directional myometrial activity seen particularly in the non-gravid uterus. However, despite the fact that the necessary components of CICR are expressed in the non-pregnant and pregnant human uterus (Awad et al., 1997; Taggart and Wray, 1998; Martin et al., 1999; Kupittayanant et al., 2002), the importance of CICR is questionable under physiological conditions (Kupittayanant et al., 2002). A final potential method of regulating Ca2+ release is referred to as store-operated Ca2+ entry (SOCE). Through pathways that are not yet understood, when the intracellular stores of Ca2+ in the SR are emptied, an unknown signal [denoted as ‘calcium influx factor’ (CIF), Fig. 3] is sent to the PM to allow entry of extracellular Ca2+ into the cytosol (Venkatachalam et al., 2002). The channels through which Ca2+ entry occurs in this mechanism are referred to as ‘store-operated channels’ (SOCs). SOCE is likely responsible for the prolonged phase of influx of Ca2+ through the PM, which has been observed following the drug-induced emptying of Ca2+ from the SR. It is likely that this mechanism is more important for longer-term Ca2+ homeostasis rather than for the regulation of the AP activity that occurs on a millisecond time-scale in uterine myocytes. The molecular identity and characterization of the SOC remains unknown. The current carried by some SOCs is termed ICRAC (CRAC: Ca2+ Release-Activated Current). This current is small, reflecting the lower conductance of SOCs, and is non-voltage-dependent but very sensitive to feedback inhibition by Ca2+ (Zweifach and Lewis, 1995). It has been proposed that members of the transient receptor potential (TRP) channel family may be candidates for mediating this current in myometrial SM from pregnant women (Dalrymple et al., 2002). Most known isoforms of the TRP family are expressed in pregnant human myometrium (Yang et al., 2002). SOCE and other aspects of Ca2+ handling in the myometrium have been thoroughly reviewed recently (Noble et al., 2009) and interested readers are referred there for a more complete discussion. As noted above, agonist treatment of uterine myocytes from non-pregnant or pregnant women elevates IP3 concentrations and this causes release of Ca2+ from the SR into the cytoplasm (Luckas et al., 1999). Pharmacological emptying of the SR Ca2+ stores increases tone in human myometrium from late gestation (Kupittayanant et al., 2002). Emptying of the SR Ca2+ store in these same experiments had little if any effect on the cytosolic Ca2+ concentrations achieved or the force generated following OT stimulation. These data suggest that the agonist-stimulated, IP3-mediated release of Ca2+ is a much less important determinant of cyosolic Ca2+ or force generation than the massive influx of Ca2+ from the extracellular space through PM Ca2+ channels. It is suggested that the role of the SR may be primarily that of a sink for Ca2+ clearance from the cytosol after an AP. Thus, the precise role of the SR and the IP3 pathway of agonist signal transduction remain unclear in uterine myocytes obtained from late human pregnancy. Another interesting aspect of myocyte stimulation and subsequent Ca2+ signal generation involves the role of specialized PM microdomains in signal transduction. In general, ‘lipid rafts’ are areas of the lipid bilayer that are rich in cholesterol and therefore move less fluidly in the PM. One type of lipid raft relevant to uterine myocyte biology is termed ‘caveolae’. These structures are associated with and stabilized by a scaffolding protein called ‘caveolin’ that is present at the PM of uterine myocytes (Hagiwara et al., 2002; Ku et al., 2005). Caveolae are enriched in key proteins mediating myocyte excitability, such as BKCa channels (Brainard et al., 2005), that have already been discussed here. The expression of caveolins may be important to regulation of labor in rodent species but may be less significant in humans (Taggart et al., 2000; Riley et al., 2005a, b; Riley et al., 2005a, b). For a more thorough discussion on the topic of caveolae, readers are referred elsewhere (Noble et al., 2006). Myosin light chain kinaseThe events discussed above ultimately yield a marked increase in [Ca2+]i, which is the necessary trigger for activation of calmodulin (CaM), a Ca2+-dependent cytosolic protein which binds four Ca2+ ions (Fig. 3C; Johnson et al., 1996). The 4Ca2+-CaM complex activates the key enzyme MLCK and causes an immediate and marked increase in phosphorylation of MLC20, which activates the contractile machinery (Shojo and Kaneko, 2001). There are three isoforms of MLCK [smooth muscle (smMLCK), skeletal muscle (skMLCK) and cardiac (cMLCK)] (Takashima, 2009). The remainder of this review will deal only with smMLCK. The 4Ca2+–CaM complex assumes a conformation that allows activation of smMLCK and markedly enhances the enzyme activity in phosphorylation of MLC20 (Shojo and Kaneko, 2001). As mentioned previously, smMLCK catalyzes the phosphorylation of the MLC20 on the N-terminus at Ser19 (Kamm and Stull, 2001) to generate phospho-MLC20 (PMLC20). This phosphorylation event is permissive on actin–myosin cross-bridging since it results in both a conformational change in MII from the folded to extended state (Onishi and Wakabayashi, 1982; Craig et al., 1983; Onishi et al., 1983), which may facilitate myofilament formation and further enhance the ATPase activity of MII in vitro (Ikebe et al., 1985). Furthermore, in SM almost the entire pool of MLC20 may be phosphorylated within a few seconds during a maximal stimulus by virtue of the rapid kinetics of smMLCK (Dillon et al., 1981; Hai and Murphy, 1989; Takashima, 2009). The smMLCK isoform is a ubiquitously expressed enzyme encoded by a single gene. There are two isoforms (220 and 130 kDa) arising through use of alternate promoters (Stull et al., 1998). The larger of these two smMLCK isoforms is differentially expressed in embryonic tissues as compared with adult tissues and is also called ‘NM’ or ‘endothelial’ MLCK. This may indicate a different functional role for MLCK activity in the embryo as compared with adult tissues. The 130 kDa smMLCK achieves its highest levels of expression in SM. In both rat and human myometrium, smMLCK inhibition using the inhibitors wortmannin and ML-9 entirely abolished contractions that were induced using OT or depolarization with KCl (Longbottom et al., 2000). These findings indicate that there is no alternative pathway for contraction in uterine SM, and that that MLC20 phosphorylation by smMLCK is both necessary and sufficient for contraction to occur. This is in contrast to the contractile mechanism of skeletal muscle, which depends on Ca2+ availability and requires proteins such as troponin C to undergo a conformational change so as to permit actomyosin complex formation (Gordon et al., 2000). smMLCK contains several phosphorylation target-sites for PKA, PKC and other kinases. PKA-mediated phosphorylation of a site on the CaM-binding region of smMLCK, which impairs the ability of CaM to activate the enzyme, has been shown to decrease uterine contractile activity (Stull et al., 1993). The role of CaM is not limited to the activation of smMLCK. In fact there is evidence that CaM may be involved in regulating membrane channels and Ca2+-ATPases (see next section) that serve to limit the transient rise in [Ca2+]i and therefore aid in resetting the system for the next contraction. Note that the events of Ca2+ influx and contraction are separated temporally. The activation of smMLCK by CaM and movement from the cytosol toward the contractile apparatus may be the rate-limiting steps of contraction (Wray et al., 2003) in terms of the speed of response of the SMC. Restoration of the resting stateCa2+ removal post-contraction is essential to induce relaxation of the SM and to replenish the SR for the next contractile stimulus (Fig. 3B). This is achieved by a variety of mechanisms, including the closure of PM Ca2+ channels and simultaneous extrusion of Ca2+ from the cytosolic compartment into the extracellular space and into intracellular stores via PM Ca2+-ATPase (PMCA) and the SR/ER Ca2+-ATPase (SERCA), respectively. PMCA and SERCA are multi-spanning transmembrane proteins of the P-type Ca2+-ATPases family, which move one Ca2+ ion out of the intracellular compartment and one H+ ion in the opposite direction during each enzymatic cycle, with the aid of ATP hydrolysis (Moller et al., 1996). The unbalanced movement of ionic charge across the membrane helps to maintain Vrest in a hyperpolarized state. In addition, the movement of protons by these enzymes may have implications for pH differences between the intra- and extracellular spaces. One of the main structural differences between PMCA and SERCA is the presence of a large carboxy-terminal tail in PMCA which allows the enzyme to be activated by Ca2+–CaM (Floyd and Wray, 2007). PMCA isoforms 1 and 4 are ubiquitously expressed; whereas there is evidence that isoform 2b may be uniquely expressed in the uterus (Penniston and Enyedi, 1998). Expression of PMCA and SERCA is increased during labor, indicating a possible functional role in parturition for these enzymes (Paul 1998; Taggart and Wray, 1998; Shmigol et al., 1999; Tribe et al., 2000). There are few studies in myometrium from non-pregnant women, but it is possible that regulation of Ca2+ extrusion might alter frequency, amplitude, duration and even direction of uterine contractions. Another important Ca2+-extruding protein is the Na+/Ca2+ exchanger (NCX). This membrane-spanning antiporter harnesses the power of the electrochemical gradient of Na+ established by the Na+/K+ ATPase, for which specific isoforms are expressed in the uterus (Floyd et al., 2003). This Na+ gradient is used to extrude Ca2+ through the PM (Floyd and Wray, 2007). Several experimental approaches have shown that PMCA, SERCA and NCX mechanisms are all important in clearance of the Ca2+ following the peak of the AP regardless of whether the Ca2+ originated from the intracellular stores or from the extracellular space (Taggart and Wray, 1997; Shmigol et al., 1998, 1999). An additional mechanism of Ca2+ clearance from the cytosol involves Ca2+-dependent feedback. This process is voltage and time-dependent and serves to counterbalance excitatory signals (McDonald et al., 1994). Two distinct mechanisms have been demonstrated in uterine SM. First, Ca2+ itself can feed back to inhibit L-type Ca2+ channel function as demonstrated through decreased rates of channel inactivation following the removal of Ca2+ from the experimental medium (Jmari et al., 1986; Sanborn 2000; Wray et al., 2003). Second, Ca2+-dependent feedback may be mediated by CaM. In addition to activating smMLCK, 4Ca2+–CaM can activate a variety of cellular proteins, including CaM-Kinase II (CaMKII) and the protein phosphatase (PP) calcineurin. It has been suggested that CaMKII mediates the facilitatory effects of Ca2+ on the L-type channel (Wu et al., 1999; Dzhura et al., 2000) although the inhibitory effects are mediated through dephosphorylation of an activity-enhancing site on the channel by calcineurin (Schuhmann et al., 1997). Thus, there exists a balance between events facilitating SM contraction and those that serve to dampen the response to Ca2+. In recent years, there has been a significant increase in understanding specific mechanisms that can alter the sensitivity of the myocyte to Ca2+. This will be the focus of the next section. Mechanisms of calcium sensitizationAs mentioned previously, SM contraction is dependent on the state of phosphorylation of MLC20, which is primarily regulated by Ca2+–CaM. However, the concentration of [Ca2+]i does not always parallel the intracellular concentration of phosphorylated MLC20 and/or the degree of contractile activation. In some situations, particularly after stimulation with an endogenous agonist such as OT, a given rise in [Ca2+]i will cause a larger-than-expected force of contraction. This phenomenon is known as ‘Ca2+ sensitization’ (CS) (Somlyo and Somlyo, 1998). The advent of Ca2+-responsive fluorophores enabled the demonstration that the ratio of force output to Ca2+-entry induced by SM agonists was not always constant (Bradley and Morgan, 1987). Further, these agonists were capable of inducing larger amplitude forces compared with depolarizing stimuli such as high K+ solutions (Bradley and Morgan, 1987; Somlyo and Somlyo, 1994). At the biochemical level, PMLC20 concentrations reflect an enzymatic balance between the activities of smMLCK and MLC20 phosphatase (MLCP). Thus, either elevation in smMLCK activity or inhibition of MLCP activity could produce the observed force enhancement. Subsequent experiments demonstrated that inhibition of MLCP is the major mechanism controlling CS (Kitazawa et al., 1989; Noda et al., 1995). Abundant evidence is accumulating to demonstrate that a pathway is activated following stimulation of GPCRs to inhibit MLCP and thus potentiate the PMLC20 generated from the simultaneous activation of MLCK. This pathway involves the small GTPase rhoA and its effector, rhoA-associated kinase (ROK) (Fig. 4). In SMCs, rhoA–ROK activation may be mediated by trimeric G-proteins (Kozasa et al., 1998; Klages et al., 1999). ROK phosphorylation of the subcellular targeting subunit (MYPT1) of MLCP interferes with the ability of the catalytic subunit (PP1c) to act on PMLC20, thereby preventing desphosphorylation. This major pathway of CS is the focus of this section. Figure 4 RhoA and Rho-kinase signaling pathway. Activation of G-protein coupled receptors (GPCRs) leads to recruitment of rhoA to the PM after exchange of GDP for GTP facilitated by a specific rho guanine nucleotide exchange factor (GEF). Rho-associated kinase (ROK) is activated by rhoA in a mechanism proposed to involve trans-autophosphorylation and oligomerization. ROK phosphorylates the myosin targeting subunit (MYPT1) of MLCP at two potential sites (T696, T853) thus promoting dissociation of the holoenzyme and preventing the dephosphorylation of MLC20 by the phosphatase subunit (PP1c) through interrupted targeting. Green, red and black lines depict activation, inhibition and ATP consumption, respectively. RhoA and its associated kinaseRhoA is a small monomeric G-protein and a member of the rho subfamily of the ras superfamily of monomeric GTPases. For activation, rhoA translocates to the PM by virtue of a C-terminal post-translational modification in the form of a prenyl (lipid) moiety (Hori et al., 1991; Fujihara et al., 1997; Lee et al., 2001). The prenyl group confers the ability to interact with the PM and also with guanine nucleotide exchange factors (GEFs). GEFs mediate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on G-proteins such as rhoA, a necessary step resulting in protein activation, which allows interaction with its downstream effectors. Completion of the G-protein signal is achieved by GTPase Activating Proteins (GAPs) that enhance the rate of hydrolysis of the γ-phosphate of the bound GTP. In its inactive state, rhoA is sequestered in the cytosol by rho-guanine nucleotide dissociation inhibitor (rhoGDI). RhoGDI contains a hydrophobic pocket that surrounds the prenyl moiety on the C-terminus of rhoA and prevents its association with the PM and with the activating GEFs (Fukumoto et al., 1990; Bourmeyster et al., 1992; Hancock and Hall, 1993). RhoGDI also diminishes the intrinsic GTPase activity of rhoA (Read et al., 2000) as well as the activating capacity of GAPs (Hancock and Hall, 1993). RhoA can be inhibited by cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-induced phosphorylation at Ser188 mainly by enhancing sequestration by rhoGDI (Lang et al., 1996; Sawada et al., 2001; Ellerbroek et al., 2003). RhoA was implicated as a mediator in the process of CS by experiments demonstrating that this phenomenon was diminished using a specific inhibitor of rhoA (Hirata et al., 1992) and that the molecular mechanism downstream of rhoA involved MLCP inhibition (Kitazawa et al., 1991; Noda et al., 1995). ROK, a serine/threonine kinase (Leung et al., 1996; Matsui et al., 1996), is one of the main signal transduction effectors of rhoA. There are two isoforms (ROK-1 and ROK-2) arising from separate genes and both are expressed in human and rat myometrium (Niiro et al., 1997; Moore et al., 2000; Moran et al., 2002; Somlyo and Somlyo, 2003). ROK is recruited to the PM of responsive cells upon rhoA translocation (Matsui et al., 1996; Sin et al., 1998; Amano et al., 2000; Miyazaki et al., 2006). The activation of ROK appears to involve trans-autophosphorylation and dimerization (Ishizaki et al., 1996; Chen et al., 2002). As mentioned, ROK can inactivate MLCP by phosphorylation of MYPT1 (see the following section) (Noda et al., 1995; Kimura et al., 1996). An ATP-competitive cell-permeable inhibitor of ROK (Y-27632) diminishes spontaneous and agonist-stimulated myometrial contractility in vitro (Fu et al., 1998; Kupittayanant et al., 2001; Tahara et al., 2002). Further, agonist stimulation of uterine myocytes in culture promotes rhoA and ROK recruitment to the PM (Fig. 4) suggesting that these proteins play a role in agonist-induced contractions (Taggart et al., 1999; Lee et al., 2001). However, ongoing experiments in our laboratory indicate that the time course for PMLC20 formation and rhoA activation in agonist-stimulated human uterine SMCs may differ significantly (unpublished data). In addition to its effects on MLCP, ROK can also directly phosphorylate MLC20 on Ser19in vitro leading to enhancement in myosin ATPase activity (Amano et al., 1996; Somlyo and Somlyo, 2003). The physiological relevance of this event has been questioned given that GTP-γ-S induced activation of rhoA did not increase the level of PMLC20 nor contraction to any significant extent in vivo, in the absence of Ca2+ (Somlyo and Somlyo, 2000), suggesting that smMLCK remains a compulsory element in PMLC20 formation. Myosin light chain phosphataseThis key enzyme has been the subject of many recent reviews (Hartshorne et al., 1998; Ceulemans and Bollen, 2004; Hartshorne et al., 2004; Ito et al., 2004; Matsumura and Hartshorne, 2007). The MLCP holoenzyme is a serine/threonine phosphatase that consists of three subunits. The catalytic subunit of 38 kDa is a member of the type 1 protein phosphatase family (PP1c) (Shirazi et al., 1994). As with other members of this phosphatase family, it has broad substrate specificity and therefore the activity of the holoenzyme is determined mainly by the substrate targeting subunit (see below). There are many endogenous peptide inhibitors of the PP1c catalytic subunit but the physiological significance of these with respect to the holoenzyme is unclear (Cohen, 2002). Some evidence suggests that one such endogenous inhibitor may be relevant in myometrial SM. This inhibitor is termed CPI-17 (17-kDa-protein kinase C-potentiated inhibitor of PP1c) and is highly expressed in SM, including human myometrium (Ozaki et al., 2003; Lartey et al., 2007). The inhibitory effect of CPI-17 for the catalytic subunit of MLCP is enhanced by several orders of magnitude through phosphorylation of Thr38 by PKC and also by ROK (Eto et al., 1995), suggesting that phosphorylation of CPI-17 is necessary to ‘activate’ this inhibitor. MYPT1 (also known as the ‘myosin-binding subunit’ Gosser et al., 1997) is highly expressed in SM and has several isoforms resulting from splice variants of a single gene (Okubo et al., 1994; Shimizu et al., 1994; Dirksen et al., 2000; Ogut and Brozovich, 2000, Matsumura and Hartshorne, 2007). This subunit associates with PP1c through a PP-binding motif in its N-terminal region (Terrak et al., 2004). Its major function is to bind PMLC20 and provide access to the catalytic subunit for removal of the phosphate moiety. Phosphorylation of MYPT1 is an important mechanism of regulation of MLCP activity. MYPT1 has two major phosphorylation sites (Thr696, Thr853 in human sequence) that are targets for ROK (Kawano et al., 1999). Phosphorylation of Thr696 causes marked inhibition of PP1c activity, either by interacting with the catalytic site or by causing a conformational change (Hartshorne et al., 1998; Ito et al., 2004). Phosphorylation of Thr853 disrupts the PMLC20 binding motif and thus reduces the ability of MLCP to associate with its target. Phosphorylation of either of these sites has also been shown to disrupt the ability of MYPT1 to target PP1c to a particular subcellular location (Noda et al., 1995; Ichikawa et al., 1996; Kimura et al., 1996; Hartshorne et al., 1998; Wu et al., 2005). It is not clear which of these sites is the predominant mediator of ROK-induced inhibition in uterine myocytes. If they are physiologically relevant, the biochemical determinants mediating CS are likely opposed by mechanisms working to decrease the sensitivity of SM to Ca2+. This concept is termed ‘Ca2+-desensitization’ (CD). Some evidence suggests that CD can be achieved by inhibition of smMLCK through phosphorylation of an inhibitory site leading to a decreased affinity for CaM (Tansey et al., 1994); the candidate kinases in such a mechanism may be dependent on cyclic nucleotides (Murphy and Walker, 1998; Nakamura et al., 2007) and may depend on the expression of a specific isoform of MYPT1 (Payne et al., 2006). Further, these kinases may also target MYPT1 and potentially increase MLCP activity as part of CD and SM relaxation. For example, protein kinase G phosphorylates Ser695 of MYPT1 which causes relaxation, probably by interfering with basal phosphorylation of Thr696 (Nakamura et al., 2007). Further, phosphorylation by PKC of an undetermined residue in the N-terminal portion of MYPT1 reduced its affinity for PP1c and hence the activity of MLCP (Toth et al., 2000). The physiological relevance of CD remains elusive. However, implementation of such a mechanism in tissues such as the human myometrium would aid in ensuring a long period of dormancy during gestation. MYPT1 also can be phosphorylated by a host of other kinases but their physiological roles are uncertain. The variety of phosphorylation targets available on MYPT1 indicates that this protein may be a key convergence point for many signaling pathways involved in contractility modulation, which may be important in both the pregnant and non-pregnant uterus. The third subunit of MLCP is a small peptide of approximately 20 kDa termed sm-M20. Although it may bind MLC20, this does not affect the rate of phosphatase activity (Hartshorne et al., 1998). Its role is unknown. Calcium sensitization in uterine SMCS is becoming increasingly recognized as a potential functional mechanism for the regulation of uterine contractility. Most of the studies have been focused on the uterus during gestation but these same mechanisms may be equally applicable in the non-pregnant state. The mRNAs for rhoA, ROK-1 and ROK-2 are present in the non-gravid uterus and increase during pregnancy (Moore et al., 2000; Tahara et al., 2002; Kim et al., 2003; Riley et al., 2005a, b). Further evidence supporting a physiological role for CS is provided by the presence of antagonism to this system during the quiescent phase of pregnancy. The rnd family of proteins (rnd1-3) consists of monomeric G-proteins with preferential affinity for GTP compared with GDP and with low GTPase activity. By diminishing the availability of GTP, they can interfere with rhoA–ROK interactions resulting in CD (Riento and Ridley, 2003). Protein levels of rnd2 and rnd3 are increased in human myometrium during pregnancy compared with tissues from non-pregnant women (Lartey et al., 2006). Conversely, when assessing mRNA levels using semi-quantitative techniques, pregnant rats had increased concentrations of all three rnd isoforms whereas, in human myometrium, the only increase in mRNA was for rnd1 (Kim et al., 2003). Although this area requires further study, the data are compatible with a role for rnd proteins regulating myometrial activity in pregnancy. Thus, pharmacological modulation of this system in non-pregnant subjects may present a therapeutic opportunity. Various uterotonins have been evaluated for their ability to induce CS. This mechanism appears to be an important component of the potent contractile effects of OT on the human myometrium (Shmygol et al., 2006). Further, OT-stimulated contractions of human myometrium obtained at term elective Caesarean section are inhibited by the ROK inhibitor Y-27632 independently of the change in [Ca2+]i (Woodcock et al., 2004). In addition, ROK inhibition impedes tension development and promotes relaxation without altering the level of [Ca2+]i in spontaneous and agonist-stimulated contractions (Niiro et al., 1997; Vedernikov et al., 2000; Kupittayanant et al., 2001; Moran et al., 2002; Woodcock et al., 2004; Woodcock et al., 2006). Similar observations were made in rat muscle strips stimulated with carbachol (Oh et al., 2003). Finally, rhoA and ROK are translocated to the PM in freshly isolated myometrial cells stimulated with uterine agonists, indicating rhoA–ROK activation (Taggart et al., 1999). Taken together, these data argue for rhoA–ROK as mediating CS in myometrium of different species and, importantly, rhoA–ROK activity as being inducible by physiologically relevant stimuli. On the other hand, there are some data that question the role of ROK in OT-induced contractions in human myometrium obtained at term elective Caesarean section. Y-27632 caused no significant attenuation of force in spontaneous contractions induced by a physiological dose (10 nM) of OT. However, when the muscle strips were pre-exposed to KCl, then treated with a higher dose of OT (100 nM) that is more likely to produce a tetanic contraction, there was a significant suppressive effect of the ROK inhibitor (Kupittayanant et al., 2001). This might suggest that the ROK pathway is more important in promoting force enhancement during tonic rather than phasic contractions. Clearly, more study is necessary to determine the importance of these pathways in basal or stimulated uterine myocyte contractile function. PGF2α has also been shown to induce CS in human myometrium (Woodcock et al., 2006) and in mouse tissues with the accompanying formation of GTP-rhoA (Tahara et al., 2005) again implicating rhoA–ROK as important mediators of CS. Upon PGF2α stimulation of human myometrial strips, peak [Ca2+]i remains unchanged, but peak force of spontaneous contractions is significantly increased. RhoA and ROK are not the only entities studied with regards to their potential involvement in myometrial CS. Activators or inhibitors of PKC might influence myometrial contractility in either the pregnant or non-pregnant states, perhaps through regulation of activity of CPI-17 (Ozaki et al., 2003). Interestingly, CS may not be the only mechanism for enhancing uterine contractility. The myometrium during late pregnancy is capable of producing larger forces per unit of PMLC20 as compared with non-pregnant myometrium (Word et al., 1993). Similar experiments in rats indicate that while pregnant tissues generate higher levels of absolute force, this was not due to a CS effect, but rather a sensitization of the system to PMLC20 itself, although the mechanism of this phenomenon is not yet clear. In summary, mechanisms of CS may be important therapeutic targets for regulation of uterine activity. However, despite the large volume of literature demonstrating the relationships between agonist stimulation and CS, there is little information regarding the physiological role of this mechanism in the uterus. This knowledge gap may be filled with the use of new research tools that are available to analyze the involvement of rhoA–ROK. Recently, mice with genetic deletions for ROK-1 (−/−), ROK-2 (−/−) and a heterozygous ROK-1 (+/−) ROK-2 (+/−) have been produced, although they have not yet been studied with regards to alterations in SM contractility or Ca2+ sensitivity (Thumkeo et al., 2003; Shimizu et al., 2005; Thumkeo et al., 2005). Therapeutic approaches to regulation of uterine contractilityPerhaps the most common gynecological problem associated with uterine motility is primary dysmenorrhea. There is reasonable evidence to support the efficacy of the usual treatments including the contraceptive pill (Wong et al., 2009) or non-steroidal anti-inflammatory drugs (Marjoribanks et al., 2003). The mechanisms of these therapies appear to be directed towards limiting the contractile stimuli to the uterus (through steroid hormonal effects of suppression of inflammatory stimuli) rather than directly interfering with the contractions themselves. Hence, the duration of the effects (and side-effects) of treatment may actually be longer than the duration of the increased uterine contractility resulting in the painful contractions. Furthermore, in view of the intense increases in intrauterine pressure that usually accompany dysmenorrhea, it is likely that it is the outer, predominant zone of the myometrium that is mediating the contractions rather than then inner junctional zone that may be the site of pathophysiology in the other important disorders including abnormal sperm transport, fertilization, implantation, endometriosis and ectopic pregnancy. The recently developed concept that contractile activity in the non-pregnant uterus results from a distinct zone of the inner myometrium emphasizes our lack of understanding of this organ even at the level of its anatomy and histology. Understanding its physiology and pharmacology lags even further behind. Much additional research is necessary before therapeutic agents can be designed or attempted, to increase the clarity of the recommendation on the basis of strong evidence. Even with the greater volume of research information concerning uterine contractility in late gestation, most attempts to prevent or inhibit uterine contractions have had little, if any, beneficial effect and have resulted in common and serious side-effects (Mitchell and Olson, 2004). However, there are some considerations with fair supportive evidence as to where research into therapeutic manipulation of endometrial wave activity in the non-pregnant state could be undertaken immediately. The strongest evidence regarding regulation of the junctional zone of the myometrium concerns the effects of ovarian steroids (Bulletti et al., 1993; Kunz et al., 1998; Fanchin et al., 2000). Perhaps the research strategy with the greatest potential for success would be a systematic assessment of the effects of estradiol and/or progesterone (or their well-characterized antagonists) on endometrial waves at different stages of the reproductive cycle. There is almost a complete void of research data in this potentially fruitful area. Endpoints would include parameters that have both physiological as well as clinical relevance: effects on frequency, amplitude, duration and direction of wave propagations as well as measures of clinical effectiveness. Vaginal suppositories of progesterone will diminish uterine activity at the time of embryo transfer following in vitro fertilization (Fanchin et al., 2001) but the effects on the rate of successful cycles has not been systematically evaluated. An important pharmacological principle is to minimize potential toxicity by maximizing the specificity of the therapeutic agent for the target organ. OT is the most potent and specific contractile agonist and its receptor levels in non-pregnant rat uterine tissue are very high, similar to those at the time of parturition (Arthur et al., 2008). More studies are required to determine the expression of the OT/OT receptor system specifically in the human and particularly in the junctional myometrium through the reproductive cycle. However, vaginally administered OT increases basal uterine tone as well as the frequency and amplitude of cervical–fundal contractions (Kunz et al., 1998; Wildt et al., 1998), but despite obvious potential use to aid sperm transport in normal or donor insemination cycles, it has never been assessed for therapeutic efficacy in this regard. This is an obvious area for increased clinical research. In addition, the possibility that new, orally active anti-oxytocic agents will have a role to play in treating these conditions involving the junctional myometrium or in alleviating dysmenorrhea needs to be appropriately investigated. In a recent case report, the anti-cholinergic hyoscine bromide (another antagonist of the pro-contractile pathway) was used to decrease the frequency of uterine peristalsis and appeared to be of benefit in three women with repeated failures with in vitro fertilization (Kido et al., 2009). At present, there are many well-characterized inhibitors and activators of many of the pathways described in this review. For example, Ca2+ channel blockers can effectively prevent the Ca2+ influx necessary to enable a contraction. Inhibitors of CaM and smMLCK have been thoroughly studied in vitro and have demonstrated the expected reduction in PMLC20 levels. However, there currently are no antagonists of these proteins in use therapeutically. The energy supply could be disrupted using myosin ATPase inhibitors to reduce the rate of active cross bridge cycling. CS mechanisms can be manipulated using well-studied agents that are activators or inhibitors of rhoA, ROK or MLCP. As noted, stimulation of cAMP or cGMP can affect several key factors, particularly smMLCK and MYPT1, which can diminish uterine contractile activity. The concerns with all of these agents relate to their non-specificity and, as has been learned from their use in pregnancy, the resulting potential for significant side-effects. Considering the centrality of many of these protein targets in mediating SM contractility in all muscle beds, it may be difficult to develop pharmacological tools with therapeutic efficacy for uterine disorders without significant advances in drug targeting technologies. Conclusions and future perspectivesThere have been major advances in the understanding of the molecular physiology of SM contractility over the past two decades. The vast majority of research has focused on vascular SM but there appear to be many commonalities with uterine SM. Over a similar time span there has been increasing evidence, using a variety of advanced technologies, describing changing patterns of uterine contractions through different phases of the menstrual cycle. A unique feature of uterine contractility in the non-pregnant state is the apparent change in direction of contractile waves depending on the stage of the reproductive cycle. Indirect evidence suggests this directionality may be important in a wide variety of diseases or dysfunctions of the reproductive system. Recent studies suggest the presence of a unique, sub-endometrial junctional zone of the myometrium that may generate and regulate these ‘endometrial waves’. There is a pressing need to more completely understand human myometrial contractile activity. This is particularly true concerning the important and unique aspects relating to the origin and direction of impulse of contractions. 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More from Oxford Academic Which layer is the powerful muscle layer that contracts to make birthing possible?Myometrium: The highly muscular middle layer. This is what expands during pregnancy and contracts to push your baby out.
Which layer of the uterus is responsible for contractions?Its middle muscular layer is called the myometrium, which is known for its rhythmic contractions which result in 'endometrial waves' in the nonpregnant uterus, Braxton Hicks contractions during pregnancy, and true labor towards the end of the third trimester.
Which layer of the uterine wall is responsible for the muscular contractions involved in childbirth and menstrual cramps?The myometrium is the middle layer of the uterine wall, consisting mainly of uterine smooth muscle cells (also called uterine myocytes) but also of supporting stromal and vascular tissue. Its main function is to induce uterine contractions.
What portion of the uterus is composed of smooth muscle that contracts?The myometrium is the middle and thickest layer of the uterus wall. It is made up mostly of smooth muscle.
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