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Joan Mott Prize Lecture |
1 Department of Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Abstract
A better understanding of the mechanisms that generate and modulate uterine contractility is needed if progress is to be made in the prevention or treatment of problems in labour. Dysfunctional labour describes the condition when uterine contractility is too poor to dilate the cervix, and it is the leading cause of emergency Caesarean sections. Recently, insight has been gained into a possible causal mechanism for dysfunctional labour. Study of the physiological mechanisms that produce excitation in the uterus, the subsequent Ca2+ signals and biochemical pathway leading to contraction has underpinned this progress. In this review, I give an account of excitation–contraction signalling in the myometrium and explore the implications of recent findings concerning lipid rafts for these processes. I also discuss how changes of pH are fundamentally enmeshed in uterine activity and biochemistry and explore the effect that pH changes will have on human myometrium. Finally, I present the evidence that acidification of the myometrium is correlated with dysfunctional labour and suggest the processes by which it is occurring. It is only by gaining a better understanding of uterine physiology and pathophysiology that progress will be made and research findings translated into clinical benefit for women and their families.
(Received 10 April 2007;
accepted after revision 26 April 2007; first published online 1 April 2007)
Corresponding author S. Wray: Department of Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK. Email: s.wray{at}liverpool.ac.uk
The uterus is a fascinating but overlooked tool in the making of us all – until it goes wrong. Our ancestors endowed the uterus with god-like magical powers, believing it to roam around the female body causing mayhem and hysteria. While the uterus is no longer regarded as an animal which longs to generate children (‘When it remains barren, it is distressed and straying about in the body and cutting off the breath’, Timaeus, Plato), it is still capable of puzzling and amazing us. Consider the changes that it has to undergo to permit the fertilized embryo to implant and penetrate into the uterine blood supply, to then undergo extreme hyperplasia and hypertrophy to accommodate the growing fetus while not responding to stretch with contraction, before finally producing an ever more insistent series of contractions to produce 10 cm of dilatation of cervix and the delivery of the baby and placenta. It then has to produce an exceptionally long contraction to prevent postpartum haemorrhage, by occluding flow into the sheared vessels. Its final accomplishment is to reverse all the physical changes of gestation in just 6 weeks and eventually to re-commence the monthly changes of the uterine cycle. Amazing!
There are, however, many opportunities for processes to go wrong, with potentially devastating consequences. Myometrial contractions initiated too early in pregnancy underlie premature deliveries. Contractions at term that are too powerful will cause fetal hypoxia and distress, while if contractions are too weak or inco-ordinate the labour will be dysfunctional (dystocic), and an emergency Caesarean section required. The unsolved puzzle is why these aberrant patterns of contractile activity occur. To date we have only limited answers to these questions and have therefore achieved little success at preventing premature or dysfunctional labours and are left having to treat the consequences. In this review, I will briefly cover what insights have been made in understanding myometrial contractility and its control and then focus on the effects of acidity, to show how basic science may translate into clinical benefit.
The focus will be on mechanisms underlying spontaneous activity; for more information on additional pathways produced by agonists, recent reviews should be consulted (Lopez Bernal, 2003; Sanborn et al. 2005).
Pathways to uterine contractility
For significant interaction between myosin and actin in the uterus or any other smooth muscle, serine 19 on the regulatory light chains of myosin must be phosphorylated (Wray et al. 2001, 2003). Although other putative candidates have been suggested, under physiological conditions there appears little doubt that myosin light chain kinase (MLCK) is the selective and dedicated enzyme that brings this about (Moore & Bernal, 2001). Calcium ions binding to calmodulin activate MLCK and therefore initiate the phosphorylation and subsequent cross-bridge cycling. There are two sources for the increase in activator Ca2+: entry across the surface membrane through voltage-gated L-type Ca2+ channels and/or release from the sarcoplasmic reticulum (SR). In those phasic smooth muscles, such as the uterus, where action potentials occur, the resulting depolarization and consequent opening of L-type Ca2+ channels make this the major source of Ca2+ for contraction (Matthew et al. 2004). Each phasic contraction is accompanied by a Ca2+ transient in the uterus, and both the transients and contractions are abolished if L-type channels are blocked (Wray et al. 2003). There is some evidence that T-type Ca2+ channels may contribute to Ca2+ entry in human myometrium (Young et al. 1993). If Ca2+ rises but MLCK is inhibited, uterine contractions also fail (Longbottom et al. 2000). Thus the Ca2+–calmodulin–MLCK pathway is vitally important for uterine mechanical activity (see Fig. 1)
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In addition, even under agonist stimulation and IP3 production, hormones such as oxytocin are unable to produce force in the uterus if Ca2+ entry is inhibited, i.e. any SR Ca2+ release is transient and rapidly depleted (Fig. 2 ; Kupittayanant et al. 2002). While the stimulatory actions of oxytocin on myometrium are often attributed to SR Ca2+ release, this is to ignore its associated actions, which are a stimulation of Ca2+ entry and a decrease of Ca2+ efflux (Soloff & Sweet, 1982). Both actions will increase and/or prolong Ca2+ transients. This is illustrated in Fig. 2, which shows in human myometrium that oxytocin can significantly increase Ca2+ transients and contraction even when the SR is inhibited, that this increase in Ca2+ is through L-type Ca2+ channels, and that even with a functional SR, blocking Ca2+ entry abolishes the effects of oxytocin and Ca2+ transients are no longer produced.
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As mentioned above, when the SR is inhibited, not only are Ca2+ transients and contractions not decreased, but it is observed that Ca2+ and force can be increased. An explanation based on SR Ca2+ releases being targeted to Ca2+-activated K+ (BK) channels on the plasma membrane has been proposed, based on studies of vascular and other smooth muscles. Local SR Ca2+ releases, known as sparks, occur when the RyR open (Cheng et al. 1993). Their characteristics have been described for a number of smooth muscles. These local transient Ca2+ releases may summate to produce global Ca2+ waves in blood vessels and increases in tone, but there is more evidence for their involvement in vasodilatation via BK channels (Nelson et al. 1995; Miriel et al. 1999). The local Ca2+ sparks are thought to increase Ca2+ around BK channels sufficiently to activate them. A close apposition between the SR and plasma membrane may facilitate this mechanism (Blaustein et al. 2002). The opening of BK channels is associated with small hyperpolarizations (STOCs), which will lead to a decreased opening of L-type Ca2+ channels and a fall of Ca2+, hence relaxation (Burdyga & Wray, 2005). Thus, if Ca2+ sparks or BK channels were to be inhibited, then Ca2+ transients and force would be predicted to increase, as was seen in the myometrium.
Such a mechanism has been found in vascular smooth muscles (Nelson et al. 1995) and recently a sparks–BK mechanism was demonstrated to control excitability via effects on the refractory period in ureteric muscle (Burdyga & Wray, 2005). The uterus contains BK channels, and their expression and distribution have been shown to be gestationally regulated (Khan et al. 2001). Furthermore, it has been suggested that this could be an important mechanism for maintaining uterine quiescence before term. However, the essential link between SR, RyR and BK channel activation has not been made in the uterus (Burdyga et al. 2007). Despite studying single myocytes and intact uterine strips, we have not been able to record Ca2+ sparks in the uterus, under conditions in which they are apparent in vascular and ureteric smooth muscle (Burdyga et al. 2007). There are also no other reports in the literature of uterine sparks, save one in a transgenic mouse which was overexpressing RyR3 (Mironneau et al. 2002). Supporting evidence for a lack of the sparks–BK mechanism in the uterus includes: (1) caffeine, an RyR agonist, does not increase Ca2+ and in intact tissues produces relaxation; (2) antagonism of RyR either has no or only a small effect on Ca2+, the effect being an increase; and (3) inhibition of BK channels has little effect on force, producing at most a small increase. It may be that splice variations of RyRs in the uterus render it unable to produce Ca2+ sparks (Dabertrand et al. 2006). Alternatively, there may be sparks but they may be too small or short lived for detection. It is clear, however, that a full understanding of the role of the SR in the uterus requires further insight.
Pathways to uterine relaxation and Ca2+ sensitivity
Biochemically, relaxation of the myometrium follows a reversal of the Ca2+–calmodulin–MLCK pathway. Thus, the myosin light chains are dephosphorylated by myosin phosphatase and Ca2+ falls as L-type Ca2+ channels close and Ca2+ efflux mechanisms are stimulated. This causes a dissociation of Ca2+ from calmodulin and inactivation of MLCK (Wray et al. 2001; Wray et al. 2003).
Calcium efflux occurs via a plasma membrane Ca2+-ATPase (PMCA) and a Na+–Ca2+ exchanger. The uterus expresses both transporters (Kosterin et al. 1994). The Na+–Ca2+ exchanger has a low affinity for Ca2+ but is a high-capacity system. The subplasmalemmal space may have a different concentration of ions, including Ca2+, from bulk cytoplasm (Blaustein et al. 2002). This would help to activate the exchanger under physiological conditions of high Ca2+ and the removal of Ca2+ from the cell. The activity of the Na+–Ca2+ exchanger is determined by the transplasmalemmal Na+ gradient, which in turn depends upon the Na+,K+-ATPase. Experimentally, lowering or removing external Na+ can lead to a reversal of the exchanger and Ca2+ entry. Such Ca2+ entry can trigger SR Ca2+ release in cardiac muscle (Ritter et al. 2003). Given the lack of Ca2+ sparks in the uterus, this mechanism is unlikely to occur in myometrium. The PMCA is expected to work to maintain resting Ca2+ levels and is described as a high-affinity system tuned to keeping Ca2+ low (Matthew et al. 2004). Isoforms 1 and 4, and possibly 2, are expressed in the uterus. Studies on intact uterine preparations and single uterine cells have led to the conclusion that after a rise in Ca2+, the Na+–Ca2+ exchanger extrudes 30% and PMCA 70% of the Ca2+ that entered, and there is no efflux when these pathways are both blocked (Shmigol et al. 1998, 1999).
The SR has been found to act as a Ca2+ sink, taking up Ca2+ to vectorially release it to the two transporters and increasing their rates, but acting alone SERCA cannot lower Ca2+ in uterine myocytes (Shmigol et al. 1999).
Relaxation mechanisms are also a target for agonists. As noted already, oxytocin can inhibit Ca2+ efflux from uterine cells and thereby promote force production (Soloff & Sweet, 1982). Many vasodilators work through the NO–cGMP signalling pathway. It seems that one target in this pathway is myosin phosphatase (MLCP), which is stimulated by cGMP and thereby accelerates the removal of phosphate from myosin, thus inhibiting force. This is known as Ca2+ desensitization (Sanborn et al. 2005).
Conversely, force can be promoted by Ca2+-sensitizing agonists, many of which work by inhibiting MLCP. One of the main mechanisms is by phosphorylation of one of the MLCP subunits by rho stimulation of rho-associated kinase. Such mechanisms are more prominent in tonic smooth muscles than they are in phasic ones, possibly owing to the prominent role of excitability in controlling Ca2+ fluxes in phasic muscles, along with the briefer contractions. Thus, in the uterus, using Y-27632 to inhibit any rho, it has been shown that rho-kinase-stimulated phosphorylation of MLCP produces only a small effect on force (Kupittayanant et al. 2001). Recently, it has been suggested that CP1-17, which also inhibits MLCP, may be effective in myometrium, working through a protein kinase pathway (Sakamoto et al. 2003). Oxytocin may also act in part by Ca2+ sensitization (McKillen et al. 1999). Given the phenotopic changes that occur when smooth muscles are cultured, including loss of L-type Ca2+ channels (Sanborn, 2001) and re-organization of the plasma membrane (Matschke et al. 2006), it is important that fresh tissue and cells are used in investigation into cell signalling pathways wherever possible.
Thus, the uterus, from all species examined to date, including humans, is critically dependent on action potentials leading to Ca2+ entry and phosphorylation of myosin to generate phasic activity. These effects are reversed during relaxation, when Ca2+ leaves the cell on the Na+–Ca2+ exchanger and Ca2+-ATPase, Ca2+ entry falls and MLCP dephosphorylates myosin. The contribution of the SR to these pathways is unclear, as is the production of Ca2+ sparks, and Ca2+ sensitization appears to play a minor role.
Lipid rafts and uterine signalling
Some of the elements of Ca2+ signalling in the uterus and other smooth muscles appear to be associated with special microdomains in the plasma membrane, known as lipid rafts. These are cholesterol- and sphingolipid-rich, detergent-insoluble regions of the membrane. The high cholesterol content decreases fluidity in these regions, leading to them being named ‘rafts’, since they float in the more fluid non-raft regions (Brown & London, 1998). Physiological interest in these biochemically identified microdomains has been stimulated by findings that components of several signalling pathways are preferentially included or excluded from rafts, and the dynamics of this process can constitute turning on or off or modifying those pathways (Pawson & Scott, 1997). Interaction with cytoskeletal proteins such as annexins may also be an important mechanism of regulating rafts (Babiychuk et al. 2002; Draeger et al. 2005).
In addition, when rafts have the protein caveolin-1 associated with them, they form 
-shaped invaginations of the surface membrane, known as caveolae (Taggart, 2001). Smooth muscles have long been known to have a particularly high density of caveolae in their plasma membranes. Thus, although raft signalling mechanisms have been proposed to be applicable to many (if not all) cell types, it seems reasonable to suggest that they may be particularly important in smooth muscle. The first indication that this was so, at a functional level, appears to have been the study of Dreja et al. (2002), who showed that the response of rat tail artery to some, but not all, contractile agonists was affected by whether or not lipid rafts were intact. Then in ureteric smooth muscle it was shown that raft disruption impaired phasic but not tonic contractions (Babiychuk et al. 2004).
It is possible to modify rafts or caveolae by manipulating the cholesterol content of the plasma membrane. Thus, molecules such as β-methyl cyclodextrin (MCD), which can effectively sequester cholesterol into hydrophobic pockets, can cause disruption of rafts and caveolae. Such treatment in the uterus can be shown by electron microscopy to dramatically reduce the number of caveolae, and a decrease in membrane cholesterol can also be shown to occur (Smith et al. 2005). Experimentally, cholesterol can be increased or restored by using MCD saturated with cholesterol. Does disruption of rafts/caveolae have any functional consequence for myometrium?
Our recent data on rat and human myometrium provide strong evidence that raft signalling mechanisms are used by the uterus. Lowering of cholesterol by MCD significantly enhanced both spontaneous and agonist-induced Ca2+ transients and contractions. These effects were reversed by application of MCD–cholesterol (Kendrick et al. 2004; Smith et al. 2005). Conversely, if cholesterol was added to myometrial preparations, Ca2+ transients and contractions fell. Similar results were obtained if cholesterol was lowered by the bacterial enzyme cholesterol oxidase, which resulted in increased contractility (Smith et al. 2005).
Thus, if disruption of lipid rafts enhances Ca2+ signals and phasic contractions in the uterus, it would suggest that normally rafts function to decrease or limit Ca2+ signals. Since K+ currents, by repolarizing the membrane, are one of the most important mechanisms to limit Ca2+ entry, it was proposed that they may function optimally when localized by caveolae (Smith et al. 2005). There are now several reports of K+ channel subunit being associated with rafts (Babiychuk et al. 2004; Brainard et al. 2005). For the uterus, a caveolar location of BK channels is supported by data showing that the 
-subunit is associated with the membrane detergent-resistant fraction in sucrose density gradient experiments (Smith et al. 2005). Presumably, when rafts are disrupted this association with the -subunit is lost and function diminished. We have recently obtained data to support this suggestion. In isolated myocytes, antagonism of BK channels by iberiotoxin decreases outward current (Shmygol et al. 2007) Application of MCD also decreases outward current. If iberiotoxin is added (Shmygol & Wray, 2007) and MCD subsequently applied, little or no further reduction in outward current occurs, suggesting that BK channels were the target of MCD. It was also found that the capacitance of the myocytes was decreased by MCD. Use of membrane-impermeant fluorescent dextrans showed that internalization of the probe occurred with MCD treatment of myocytes.
These data therefore indicate that MCD decreases BK current, possibly by internalization of channel subunits, and will therefore increase the excitability of the myometrium. This in turn will increase the Ca2+ signalling which underlies the increased contractility found with cholesterol manipulation. As stated earlier, however, the influence of BK channels on uterine contractility in intact tissue is rather small and so it is likely that other pathways also play a role. One such other possible mechanism in the uterus affected by lipid rafts might be oxytocin signalling. There is evidence that the high-affinity form of the oxytocin receptor is localized in lipid rafts (Klein et al. 1995), for example. The oestrogen receptor may also have a raft localization and be inhibited in this environment. Disruption of this raft-inhibitory mechanism has been implicated in 17β-oestradiol-stimulated mammary tumorigenesis (Zhang et al. 2005). Oestrogen may also downregulate the number of caveolae and caveolin-1 in the uterus (Turi et al. 2001). It is therefore reasonable to suppose, as Turi et al. (2001) do, that caveolae are increased close to term, although there is evidence contrary to this (Ciray et al. 1995), and further data on this would be very useful. Thus, although these effects of cholesterol manipulation should not be overlooked, or those owing to changes in membrane fluidly, there is now substantial evidence that BK channels and lipid rafts affect uterine activity (Noble et al. 2006).
Cholesterol and obesity in women
Cholesterol manipulation in vitro has been shown to have significant effects on uterine contractility; elevated cholesterol decreases contractility. Obesity is positively correlated with an elevation of plasma cholesterol and low density lipoproteins (Gostynski et al. 2004). It is also known that obese pregnant women have significantly more complications of pregnancy than those with a body mass index (BMI) within the normal range (Weiss et al. 2004). Obese women are also at increased risk of Caesarean section (Crane et al. 1997; Zhang et al. 2007). No clear reason is known about why obese women are likely to require operative delivery. The risk remains even when the weight of the baby and socio-economic factors are controlled for. It has been suggested that adipose deposition in the pelvic floor is responsible, but there is no evidence to support this. In a retrospective analysis of a large data base (> 4000 women), the reason for Caesarean section was noted along with BMI (Zhang et al. 2007). This analysis confirmed previous data, i.e. there is an increased risk of non-elective Caesarean section being required in women with increased BMI, but was also able to show that this occurred because of dysfunctional labour, i.e. poor uterine contractility (Zhang et al. 2007). Thus this is evidence that high BMI is correlated with a decrease in the ability of the uterus to contract forcefully and deliver the fetus. It is tempting to speculate, given the in vitro findings relating to cholesterol manipulation, that elevated cholesterol in obese women is disturbing the lipid raft signalling mechanisms, impairing contractions and producing dysfunctional labours (Noble et al. 2006). It would be useful to determine cholesterol and lipid profiles in obese pregnant women and determine whether this could be used to predict which women would need Caesarean sections.
In summary, uterine caveolae/rafts can be considered important loci for signalling pathways in the uterus. There is likely to be a dynamic regulation that includes or excludes signalling components to rafts and thereby influences the effect on contractility. We have evidence that outward current from BK channels is particularly affected by lipid environment and that this can explain why cholesterol reduction is associated with increased contractility. It is also suggested that since elevation of cholesterol impairs contractility this may account for the uterine dysfunction seen in many pregnant obese women, and explain their increased risk of Caesarean section.
pH changes in the myometrium
The first measurements of intracellular pH (pHi) in the uterus were made in rats using 31P NMR spectroscopy (Dawson & Wray, 1985), providing a value of around 7.1. Further experiments using NMR showed that the uterus was able to decrease perturbations of external pH on pHi; thus, a 1 pH unit change in external pH elicited a 0.3 pH unit change in pHi (Wray, 1988a). Measurement of uterine pHi with pH-sensitive fluorescent indicators, e.g. carboxy-SNARF, confirmed these earlier measurements (Taggart & Wray, 1993b), and subsequent measurements on human myometrium were found to be similar to those in rat, pHi 7.1–7.2 (Parratt et al. 1994). The first in vivo myometrial pHi measurements were made on rats, using NMR spectroscopy (Larcombe-McDouall et al. 1998), and gave values of around 7.25.
Measurements of pHi using fluorescent indicators improved temporal resolution and revealed small (0.04 pH unit) pH transients accompanying each phasic contraction (Taggart & Wray, 1993a). The acidifications lagged the contraction by several seconds and increased in size when contractile activity was increased, e.g. by applying an agonist or high-K+ depolarization. In vivo measurements of pH changes during uterine contraction also showed that significant acidifications (> 0.1 pH unit) occurred with each phasic contraction (Larcombe-McDouall et al. 1998). What underlies these pH changes and what are their functional consequences?
Proton production in the myometrium
During normal myometrial activity, there will be a net production of protons, as occurs in all tissues. The net acid load arises from the biochemical processes supporting cellular functions, notably respiration and contraction (Wray, 1988b). The pH regulatory mechanisms present in the myocytes of the uterus will be active, to give a more-or-less steady pH value. Buffering power has been estimated at around 40 mM per pH unit but there are few data on pH-regulating mechanisms for the uterus of any species (Bullock et al. 1998).
Biochemistry and pH
Acid load in the myometrium will increase with activity and if there is any impairment to oxidative metabolism which causes stimulation of anaerobic glycolysis (Wray, 1990). The reserve of phosphocreatine in uterus is smaller than in striated muscle (approx. 3–5 cf. 20–25 mM, respectively), perhaps reflecting the fact that smooth muscles are not required to perform intense bursts of exercise like striated muscles (Dawson & Wray, 1985). The uterus has a store of glycogen that will support glycolysis should glucose delivery be impaired (Wynn, 1977). A quirk of smooth muscle metabolism is that there is a notable on-going production of lactate during normoxia (Shimizu et al. 2000) and this is the case for uterus (Wray, 1990). If oxygen delivery to the myometrium is impaired then lactate production is further increased (Wray, 1990). Both in vivo and in vitro, decreases in ATP and phosphocreatine (PCr) have been shown to occur during hypoxia (Wray, 1990; Harrison et al. 1995) A significant fall in pHi also occurs; for example, in vivo, 10 min of vascular occlusion reduces pHi by around 0.3 pH unit (Harrison et al. 1995). In vitro, cyanide also produces acidification of the uterus (Taggart & Wray, 1995).
As mentioned above, normal phasic myometrial activity produces transient acidifications. The explanation for this increased proton load is considered to be partly a result of the biochemical processes associated with contraction (e.g. ATP hydrolysis, stimulation of glycolysis). This could account for the changes seen in the in vitro experiments, especially since acidification correlates with contractile activity (Taggart & Wray, 1995). The acidification in vivo is larger than in vitro and therefore additional mechanisms must come into play. We have suggested that these are related to changes in blood flow (Larcombe-McDouall et al. 1999).
pH and uterine blood flow
The contractions of the uterus are sufficiently strong that they occlude the blood vessels coursing within the uterine walls. We have simultaneously measured, in vivo, uterine blood flow, contractions, ATP and pHi (Larcombe-McDouall et al. 1999). As shown in Fig. 3A, dips of around 30–40% in blood flow occur as the uterus contracts. As the uterus relaxes, the uterine vessels are no longer squeezed and the flow is restored. During this contraction–relaxation cycle there is a corresponding pH cycle (Fig. 3B). Given that it has been shown that lactate production is significantly increased during hypoxic conditions (Wray, 1990), it would appear that this may account for the additional acidification found in vivo, i.e. it is due to the biochemical changes of contraction plus the biochemical changes of hypoxia.
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To address the question of the significance of these changes in pH, investigators have imposed pH changes on the uterus and measured the effects on Ca2+ signalling and contractility (Wray et al. 1992; Taggart & Wray, 1998b). Acidification can be produced by including a weak acid in the perfusate, such as butyrate or propionate, or withdrawing a weak base, such as NH4Cl, to produce an acid rebound (Wray, 1993). Using either mechanism in rat myometrial strips, acidification was always associated with a decrement in uterine contractility (Wray et al. 1992; Taggart et al. 1996). Changes of external pH (pHo) will be translated into pHi changes, but with significant attenuation, as described earlier. Considering, therefore, the initial effects on force of changing pHo, it would appear that these can act in the opposite direction to pHi alteration. Thus, external acidification can potentiate contractions (Naderali & Wray, 1999) and alkalizations decrease them (Heaton et al. 1992). These effects may be mediated by pH-sensitive two-pore, K+ channels (Reyes et al. 1998).
Thus, if there are prolonged changes of plasma or extracellular pH, these will change pHi, and if pHi becomes acidic, force falls. This has led to the suggestion that pH changes seen in vivo could feedback and limit the development of further force.
Mechanism
The inhibitory mechanisms acting when proton load is elevated will be related to those mechanisms producing force. As described earlier, Ca2+ entry and phosphorylation by MLCK of myosin light chains are likely to be key control points. Thus, since it seems likely that no biochemical process is unaffected by pH change, it is sensible to ask, which processes are most affected? Measurements of Ca2+ changes during myometrial pH change have shown that changes in Ca2+ can explain most, but not all, of the effects of pH or force (Taggart et al. 1996). Thus, in rat myometrial preparation, the decrements in the phasic contractions mirror those occurring in the Ca2+ transients. In a study of vascular smooth muscle Ca2+, pH and tension were simultaneously measured and this also showed that the effects of pH alteration on tension were via Ca2+ changes (Austin et al. 1996). To next address the question of what causes the change in the Ca2+ transient, examination of the L-type Ca2+ current under acidic conditionsis required, since this gives rise to the Ca2+ transient. In voltage-clamped single uterine cells, the effects of pH change on Ca2+ current can be studied in a controlled manner. Such experiments showed that there were significant effects on the Ca2+ current with intracellular acidification, e.g. a pH reduction of around 0.1 pH unit decreased Ca2+ current by around 50% (Shmigol et al. 1995). Alkalizations increased the Ca2+ current, and there was little effect of pH change on outward K+ current.
Consistent with the effects of pH alteration being via effects on ion channels, inhibition of the SR has no effect on the Ca2+ changes (Taggart et al. 1996). Thus, effects on excitability, especially L-type Ca2+ channels, would appear to underlie the functional effects of altering pHi in the myometrium. Additional effects on Ca2+ sensitivity or outward currents may occur under some conditions (Taggart & Wray, 1998b).
pH changes and human myometrium
The above data and findings were all obtained on animal myometrium. However, recent studies on myometrial biopsies from women undergoing hysterectomy or Caesarean section have shown that similar effects occur when pH is changed. In human myometrium, intracellular acidification decreases and alkalization increases contractility (Parratt et al. 1995). Measurements of intracellular Ca2+ have shown that the changes in these phasic contractions with pH alteration can be accounted for by effects on the Ca2+ transient (Pierce et al. 2003). These conclusions also applied to uterine activity stimulated by oxytocin. Hypoxia was also found to decrease force and Ca2+ signals in human myometrium (Monir-Bishty et al. 2003).
During labour in women, decreases in blood flow are associated with contractions of human myometrium. Indeed, in labour these transient hypoxic dips stimulate a response in the fetus, in which heart rate accelerates (Greiss, 1965; Peebles et al. 1992). Abnormal changes in fetal heart rate, indicative of fetal distress, occur if the uterus contracts too powerfully or for too long a period. Such hyperstimulation can occur with oxytocin administration and is life threatening for the fetus. This can be directly related to the cut-off of oxygen delivery to the uterus as the contraction becomes tonic-like; relaxation is as important as contraction for successful labour.
pH and dysfunctional labour
From the foregoing, it can be seen that repeated periods of hypoxia occur in human myometrium, in vivo. From in vitro and in vivo studies, it can also be seen that the hypoxia will cause a fall in pHi and that this would be expected to decrease Ca2+ signals and thence contractions (Pierce et al. 2003; Earley & Wray, 1993; Larcombe-McDouall et al. 1999). In dysfunctional labours, contractions are too weak or infrequent or too unco-ordinated to dilate the cervix and deliver the fetus. There are very few data concerning what may cause such dysfunctional activity, especially since initial activity in labour may be normal. Dysfunctional labour has been estimated to affect up to 4% of all labours and up to 10% of first labours (Beischer & Mackay, 1986). It is the commonest cause of emergency Caesarean sections and is therefore a significant health and economic issue (Thomas et al. 2000), yet surprisingly little is known about the underlying mechanism. The only treatment for dysfunctional labour is oxytocin administration. Unfortunately, this only works in around 50% of cases and there is no way to predict which women will respond and which will not and end up having a Caesarean section (Blanche et al. 1998).
Given our findings that acidification can impair contractility of human myometrium, we recently posed the hypothesis that acidification in the myometrium underlies dysfunctional labour (Quenby et al. 2004). To test this hypothesis, myometrial capillary blood was taken as the first incision into the uterus was made at the time of Caesarean section. The blood sample was analysed for pH, lactate and oxygen saturation. Four groups were compared: (1) non-labouring women having Caesarean section, e.g. elective Caesarean section; (2) normally labouring women having Caesarean section, e.g. undiagnosed breech; (3) normally labouring women who had received oxytocin, e.g. fetal distress; and (4) women labouring dysfunctionally who had received oxytocin. The data were analysed in a blinded manner and groups 2–4 controlled for length of labours, and no systemic hypoxia was present. All women were hydrated and had no ketosis or hypoglycaemia. There were no differences in the myometrial capillary blood values for women in groups 1–3. Women suffering dysfunctional labour, however, had a highly significantly more acidotic blood sample than all other groups (Fig. 3C). In addition, there was also a decreased partial pressure of O2 and increased lactate in the capillary blood samples. Thus, it can be concluded that women labouring dysfunctionally have a more acidic uterine environment than those labouring normally. Furthermore, the acidification appears to be linked to myometrial hypoxia, since lactate is increased and oxygenation is decreased (Quenby et al. 2004). These data provide the first mechanistic insight into the causes of dysfunctional labour, although they do not explain what caused the hypoxia or acidification. Suggestions would include aberrant recovery of the myometrial vessels to repeated, transient occlusions, or larger acidifications, perhaps resulting from reduced pH buffering power of the myometrium. Changes in lactate dehydrogenize isoforms (1–5) occur towards the end of gestation and have been linked to preparation of the uterus for the hypoxic conditions encountered in labour (Wynn, 1977). It may be that such changes do not occur appropriately in some women, resulting in more adverse effects of the hypoxia on their uteri. Clearly, much more work is needed in this area if we are to be able to predict or prevent dysfunctional labours.
Summary
Good progress is being made in gaining a much better understanding of excitation–contraction coupling in the uterus, with recent data on human myometrium being a welcome addition. There are still key, unresolved issues around the role of the SR and Ca2+-activated ion channels in the uterus. Why are there no sparks, for example? The insights gained have enabled mechanisms to be proposed and tested, relating to the effects of acidity on function. This in turn has led to hypotheses being proposed to test whether acidity may contribute to dysfunctional labours. Evidence from women in labour supports the hypothesis and points the way to further investigations. Thus, we now have the first insight into a mechanism for a condition that has plagued childbirth for millennia. Detailed information on lipid rafts and their role in myometrial activity is now being sought, following the demonstration that cholesterol manipulation has profound effects on Ca2+ transients and contraction. It is suggested that this could produce dysfunctional labour, so often seen in obese women, but further insights are required to test this idea. Similarly, the complex changes in expression of many ion channels, which are necessary for successful pregnancy and labour, may also be imperfect in those women suffering either premature or dysfunctional labour, and this is another area that warrants additional investigation.
We have come a long way from the time when the uterus was thought to be a roaming beast within women's bodies, but the journey into understanding myometrium must continue.
Footnotes
The Joan Mott Prize Lecture was given at the UK Physiological Society meeting at UCL on 6th July 2006.
This article is dedicated to the memory of Professor Robert Harkness (1917–2006), supervisor, friend and scholar.
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Acknowledgements
I am deeply grateful to the Physiological Society for all its support throughout my career and for giving me the opportunity to present the Joan Mott Prize Lecture. I would also like to thank all laboratory members, past and present, for all their hard work and the fun I have had. The work reviewed in this article was supported by Action Medical Research, the MRC, Wellbeing of Women and The Wellcome Trust.
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