Correspondence: Professor A. López, Division of Obstetrics and Gynaecology, Level D, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, UK.
The mechanism of labour is not fully understood and further research into this important physiological process is needed. In some species, notably sheep, parturition is due to activation of the fetal hypothalamic–pituitary–adrenal axis. However, in primates, this axis appears to have a supportive, rather than essential role. Successful parturition requires an increase in coordinated uterine contractility together with changes in connective tissue that allow cervical ripening and dilatation. In most mammals, however, these changes are synchronised by a fall in maternal progesterone levels and a rise in oestrogens. This is not the case in women in whom the onset of labour occurs without apparent changes in circulating steroid levels. The basis of uterine contractility is the interaction between actin and myosin in myometrial smooth muscle cells. This is driven by calcium through Ca2+–calmodulin-dependent myosin light chain kinase (MLCK) activity. Moreover, calcium sensitisation occurs via activation of Rho kinase, a calcium-independent pathway that promotes contractility by inhibiting myosin phosphatase and probably by phosphorylating myosin on the same site as MLCK. Uterine activity can be modulated by many G-protein coupled receptors (GPCRs). For example, receptors coupled to Gαq (oxytocin-, prostanoid FP and TP, endothelin-receptors) stimulate contractility by activating the phospholipase C/Ca2+ pathway; receptors coupled to Gαs (β2-adrenoceptors, prostanoid EP2 and IP, some 5-hydroxytryptamine receptors e.g. 5-HT7) relax the uterus by increasing myometrial cyclic AMP levels; and receptors coupled to Gαi (α2-adrenoceptors, muscarinic, 5-HT1) potentiate contractility, probably by inhibiting cAMP production. Because of its relative abundance in pregnant uterine tissue, the oxytocin receptor is an obvious target for tocolytic therapy. Oxytocin antagonists have been introduced into clinical practice for the management of preterm labour and offer the advantage of uterine selectivity and fewer side effects than conventional beta-agonist therapy.
Despite decades of clinical, physiological and biochemical research by many investigators, the mechanism of parturition in women remains elusive. In some mammalian species, the onset of labour is the result of coordinated endocrine changes in the mother and the fetus. Classical experiments in sheep1 demonstrated that parturition is driven by activation of the fetal pituitary adrenal axis, with increased fetal cortisol secretion2 resulting in the induction of placental P450 C17 enzymes (17α hydroxylase and 17–20 lyase activities)3 that favour the conversion of C21 to C18 steroids. As a consequence, maternal progesterone levels fall and oestradiol levels rise. This endocrine imbalance promotes increased intrauterine production of prostaglandins, cervical softening and the onset of myometrial contractions. Recent evidence4 suggests that fetal adrenal cortisol induces prostaglandin synthase type 2 in placental trophoblast and that the resulting increase in prostaglandin E2 participates in the activation of the P450 cascade. A different mechanism applies to species, such as the goat, rabbit or small rodents who depend on an active corpus luteum for progesterone synthesis throughout pregnancy. In these species, the onset of labour is triggered by luteolysis, which is mediated by activation of prostanoid FP receptors in the corpus luteum by prostaglandin F2α released from the endometrium5. Luteolysis provokes a fall in maternal progesterone levels, which is rapidly followed by uterine activation and labour. In rodents, these changes are associated with a sharp increase in uterine oxytocin receptor levels and many investigators believe that oxytocin has a physiological role in the activation of uterine contractions in labour. The endocrine changes are more complex than was initially thought and it is now known that progesterone metabolism in the uterus and cervix is important for successful parturition. In pregnant mice lacking steroid 5α-reductase type 1, parturition starts at the expected time; there is increased oxytocin receptor density and normal uterine contractility; however the cervix remains closed and the animals cannot deliver6. Progesterone metabolism to 5α-reduced compounds is necessary for the induction of the connective tissue changes that allow cervical softening and dilatation. Interestingly, the induction of 5α-reductase-1 is localised only to the pregnant horn, demonstrating that, at least in small rodents, local regulatory mechanisms, some determined by the conceptus itself, are more important than systemic changes7. Nevertheless, the 5α-reduced steroid allopregnanolone has central effects, and is involved in the regulation of oxytocin release by the maternal neurohypophysis during the peripartum period, through its strong impact on GABA receptor signalling8.
The Fetal Pituitary–Adrenal Axis
Animal experiments demonstrate that the conceptus or fetus(es) have a key role in uterine adaptation to pregnancy and the onset of parturition. However, the factors responsible for parturition in primates remain unknown. The luteolytic mechanism does not apply in women, because the corpus luteum is not required for the last two thirds of gestation. Moreover, the fetal pituitary–adrenal axis, which is very active in late gestation, appears to have a supportive rather than direct role in parturition. This concept arises from observations in anencephalic pregnancies where there is little or no residual pituitary/adrenal function. Detailed analysis of the length of gestation in women carrying single anencephalic fetuses who went into spontaneous labour (not complicated by polyhydramnios, which might cause excessive uterine stretch and override the normal regulatory mechanism) showed that the mean length of gestation was similar to that in women carrying normal singleton fetuses, but with a much wider scatter around the mean9. This demonstrates that spontaneous labour can occur in anencephaly, but suggests that normal fetal pituitary adrenal function is required for the fine-tuning of the timing of parturition. In primates, the fetal adrenal has an essential role in the synthesis of steroids by supplying the placenta with androgen precursors, notably dehydroepiandrosterone sulphate, which are converted to oestrogens by placental sulphatase, aromatase and other enzyme activities. However, there is no induction of placental P450C17 around the time of parturition and the levels of progesterone and oestrogens remain stable. In primates, steroid changes may not be a pre-requisite for the onset of labour. Remarkably, normal pregnancy and parturition have been reported in patients with placental sulphatase or aromatase deficiencies with very low levels of oestrogens10.
The lack of steroid changes in the maternal circulation has prompted other hypotheses to explain the transition from relative uterine quiescence during continuing pregnancy to the initiation of uterine contractions at the onset of labour. These include the possibility of electrophysiological changes in myometrial cells. Myometrial smooth muscle contractions are phasic in nature and are driven by action potentials. The electrophysiological basis for myometrial contractions is not well understood, but it is the subject of investigation by several groups. There is precise synchronisation between electrical activity, influx of calcium inside myometrial cells and the development of tension. Voltage-operated calcium channels and calcium-activated potassium channels have been shown to be involved in the regulation of contractility by allowing calcium entry, or by setting the threshold of activation of the cell membrane, respectively. Changes in the functional activity of ion channels are likely to be an important mechanism to increase contractility in labour11.
Role of G Protein Coupled Receptors
During pregnancy, the uterus is exposed to many hormones and agonists. Many of these are growth factors that operate through tyrosine kinase receptors and mitogen activated protein kinase (MAP kinase) pathways, and are responsible for the hypertrophy and hyperplasia of myometrial cells necessary to accommodate the growing fetus, and the accumulation of contractile proteins necessary for the demands of labour. However, the most abundant receptors in the uterus belong to the G protein coupled receptor (GPCR) family. The GPCRs lack tyrosine kinase activity, but interact with heterotrimeric (α, β, γ subunits) proteins characterised by their capacity to hydrolyse GTP and to activate or inhibit a number of effector enzymes or ion channels, which have strong effects on uterine contractility. Some receptors are clearly stimulatory, for example the oxytocin receptor, the endothelin ETA receptor or the prostanoid FP receptor. These receptors are coupled through proteins of the Gq/11 family to phospholipase C β (PLC-β) whose substrate is a hormone sensitive pool of phosphatidylinositol 4,5-bisphosphate (PIP2) in the cell membrane. The PIP2 hydrolysis generates two second messengers: inositol 1,4,5-trisphosphate, which releases calcium through specific receptors in the sarcoplasmic reticulum, and diacylglycerol, which activates protein kinase C and has multiple effects, including activation of the MAP kinase cascade. The GPCRs are located in the cell membrane but can be internalised through pathways involved in recycling, degradation or further intracellular signalling. The oxytocin receptor is an obvious target for tocolytic therapy because of its high density in pregnant myometrium.
Activation of the PLC-β pathway can initiate contractions in resting tissue and can also increase the frequency and intensity of contractions in spontaneously contracting tissue. The increase in intracellular calcium arising from the sarcoplasmic reticulum store is enough to prime the machinery of contraction, however this store is rapidly depleted and contractions cannot be sustained without calcium entry through the plasma membrane. The modulation of calcium fluxes by GPCRs has a direct effect on contractility and involves very complex interactions between the cell membrane and the sarcoplasmic reticulum12. The sensitivity of the uterus to oxytocin, endothelin or other agonists depends not only on the density of each specific receptor but on the coupling of receptors to Gq and the interaction of receptor/G protein complexes with other signalling proteins in the cell.
Role of Myosin Light Chain Kinase
The basis for contractility in the uterus, as in other types of smooth muscle, is the interaction between actin and myosin, which is regulated by the enzyme myosin light chain kinase (MLCK)13. This enzyme is very sensitive to calcium and when calcium levels are low it rests in an autoinhibited state. However, when calcium increases inside the cell, it binds to calmodulin and the calcium–calmodulin complex activates MLCK, which phosphorylates the regulatory light chains of myosin. Phosphorylated myosin interacts with actin forming a functional structural complex capable of converting the chemical energy of ATP into the mechanical energy of contraction. The reaction is reversible because myosin can be rapidly phosphorylated by a phosphatase leading to relaxation. In phasic smooth muscles, there are mechanisms for the rapid lowering of intracellular calcium by extrusion through the plasma membrane and calcium uptake by the sarcoplasmic reticulum. Thus, successive cycles of contraction and relaxation result from oscillating intracellular calcium levels and the balancing activities of MLCK and myosin phosphatase.
Stimulatory agonists can also enhance uterine contractility through a calcium independent pathway, which involves the activation of small GTPases of the Rho family14,15. Their actions are mediated through Rho kinases, which can phosphorylate myosin phosphatase inhibiting its activity and potentiating the effect of MLCK. This is a mechanism of so called ‘calcium sensitisation’. Rho kinase may be able to phosphorylate myosin light chains directly, further stimulating the development of tension.
Parturition: Activation of Stimulatory Pathways or Loss of Uterine Quiescence?
The uterus is a remarkable organ. For most of the 40 weeks of human pregnancy it expands to accommodate the growing fetus and it remains relatively quiescent. And yet at the time of labour it contracts regularly and forcibly to expel the fetus through the birth canal. The transition from a relatively relaxed state to the onset of contractions can be seen as the result of stimulatory inputs or perhaps as a consequence of the loss or ‘switching off’ of mechanisms that promote relaxation. One such mechanism could be the activation of cyclic nucleotide (cAMP, cGMP) pathways. It is known that cyclic nucleotides promote smooth muscle relaxation through a variety of intracellular reactions involving specific protein kinases. The actions of both cGMP and cAMP in pregnant human myometrium are important16,17, although it could be argued that in comparison to vascular or tracheal smooth muscle, the sensitivity of the uterus to cyclic nucleotides is low18. The most commonly used tocolytic agents (β2-adrenoceptor agonists) act by increasing cAMP production. This review focuses on GPCR activated cAMP signalling pathways. The production of cAMP from ATP is catalysed by the enzyme adenylyl cyclase (AC), which is usually present in clusters in the cell membrane in the vicinity of GPCRs coupled to Gαs. Studies have investigated the possibility that increased positive coupling of receptors to AC may be responsible for uterine quiescence during continuing pregnancy. Data show that GTP-dependent AC activity in myometrial membranes is higher in pregnant than in non-pregnant tissue, and the increased activity is lost in tissues obtained after the onset of labour. Moreover, when the membrane preparations are challenged with prostaglandin E2 there is a further increase in AC activity, which is significantly higher in pregnant compared to non-pregnant tissue, and, again, is lost after the onset of labour19. These data suggest that the coupling of receptors (e.g. prostanoid EP2 receptor) to AC via Gαs is enhanced in pregnancy and the effect of agonists which relax the uterus via cAMP is potentiated. The effect of cAMP is terminated by a phosphodiesterase (PDE) and it is of interest that the activity of PDE4, the isozyme expressed predominantly in pregnant myometrium, is relatively inhibited by the high levels of progesterone in pregnancy20. Thus not only is cAMP synthesis enhanced, but its catabolism is inhibited resulting in long sustained increases in cAMP that promote uterine relaxation. The mechanism by which cAMP dependent protein kinase (PKA) provokes relaxation is not well understood, but it is thought that it targets a variety of substrates, including potassium channels in the cell membrane (causing membrane hyperpolarisation); components of the PLC/calcium pathway, inhibiting PLC-β and promoting calcium extrusion; or perhaps MLCK which may undergo phosphorylation by PKA, losing affinity for calcium–calmodulin and thereby inhibiting the contractile reaction.
The increased coupling of myometrial receptors to Gαs and to AC may be the result of increased expression of Gαs in pregnancy19, but it is thought that the limiting factor in cAMP signalling is the activity of AC itself. There is an unsuspected level of complexity in cAMP signalling pathways in the uterus. This is due to the fact that there are at least nine different isoforms of AC all of which are expressed in myometrial tissue21. At present the reason for this molecular redundancy is unknown. This question is important because although all ACs are stimulated by Gαs, their regulatory susceptibilities are very different (Table 1). For instance, calcium has stimulatory effects on ACs I, II and VIII, but it is inhibitory towards ACs V and VI. ACs II, IV and VII are substrates for PKC which has a stimulatory effect. The function of ACs V and VI is particularly complex. These isoforms are sensitive to receptors (e.g. α2-adrenoceptors, 5-HT1 serotonin receptors, most chemokine receptors), which couple to the inhibitory protein Gαi. Stimulation of these receptors in myometrial smooth muscle potentiates contractility by inhibiting cAMP production. Moreover ACs V and VI can be regulated by βγ-subunits liberated from Gi which is relatively abundant in cells; and are inhibited by PKA in a classical negative feedback effect, whereby synthesis of cAMP leads to a decrease in its rate of production.
Table 1. The nine known adenylyl cyclase isoforms may be divided into one of four subfamilies, according to their modes of regulation.
Abbreviations: AC = adenylyl cyclase. PKC = protein kinase C. PKA = protein kinase A. Gαi= alpha subunit of Gi.
1 (ACs I, III, VIII)
Stimulated by calcium and calmodulin
2 (ACs II, IV, VII)
Activated by G-protein βγ-subunits and phosphorylation by PKC, unaffected by calcium
3 (ACs V, VI)
Inhibited by calcium, G-protein βγ-subunits, Gαi and PKA
4 (AC IX)
Insensitive to either calcium or βγ subunits. May be inhibited by the phosphatase calcineurin
It is not known whether specific receptors coupled to AC, for instance the β2-adrenoceptor or the prostanoid EP2 receptor, use one isoform of AC in preference to another (e.g. ACI versus ACIII or ACVI), or whether any one type of receptor couples to any available AC through Gαs. This question is of fundamental importance in the understanding of how different agonists relax the uterus and help maintain pregnancy until the timely onset of labour. Although all isoforms of AC are present in myometrial cells, in pregnancy there is a relative increase in the expression of the cyclases in groups 2 and 3 (Table 1). This may lead to some selectivity in the design of drugs that target the myometrium to promote relaxation. Currently β2-adrenoceptor agonists (e.g. ritodrine, fenoterol) are the most popular tocolytic drugs, but they cause potentially serious cardiovascular and metabolic side effects in the mother due to the widespread distribution of β2-receptors. Unfortunately, it is not known whether the type of AC associated with β2-adrenoceptors in the uterus is different from that found in the heart or in vascular smooth muscle. In theory, it is possible that there is fidelity between different receptors and AC isoforms (Fig. 1). If this is the case it might be possible to manipulate the system with a degree of pharmacological selectivity because we would know how to influence a particular AC isoform. However, if there is promiscuity between receptors and ACs (Fig. 1; right panel), this approach may not be useful because receptors would be able to couple to any available isoform and the possibility of selective regulation would be lost.
Targeting of cAMP Action in Myometrium
There is much debate about how elevations in the ubiquitous second messenger cAMP are transduced into specific and selective cellular effects. One well-supported hypothesis is that there is compartmentation of cAMP signalling, facilitated by scaffolding proteins whose role is to bring together several components of the signalling pathway to ensure the precise subcellular targeting of cAMP actions22. Receptors and ACs are probably clustered in membrane domains and in indentations termed caveolae, which are coated with specialised proteins called caveolins. A strong association between β2-adrenoceptors and ACV/VI in heart smooth muscle cells has been demonstrated23. This proximity may facilitate specificity of interaction between receptors and individual AC isoforms. Many of the effects of cAMP are mediated by PKA, which is composed of two regulatory (R) and two catalytic (C) subunits. The characteristics of the PKA holoenzymes (R2C2) are largely determined by the structure and properties of their R subunits (I/II), which are differentially distributed in mammalian tissues. This results in the ability of PKA to decode cAMP signals that differ in duration and intensity: PKAI can be activated transiently by weak cAMP signals, whereas PKAII responds to high and persistent cAMP stimulation. A key element in effective PKA activation is the localisation of R2C2 in discrete subcellular domains by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs)24. This ensures that catalytic activity is restricted to a small area, reducing non-specific phosphorylation and ensuring that phosphorylated substrates exert their physiological role (e.g. smooth muscle relaxation).
The AKAP family comprises several gene products and splice variants25. Interestingly, AKAP 5 (also known by its equivalent molecular weight number as AKAP 79 in human and AKAP 150 in rat tissues) contains membrane targeting sequences, which bind phosphatidylinositol-4,5-bisphosphate and provide a model for regulated translocation to the cell membrane of the AKAP signalling scaffold. In the rat, AKAP5 has a role in the regulation of uterine contraction, in early pregnancy AKAP5 targets PKA to phospholipase Cβ3, resulting in the phosphorylation of the enzyme and its inhibition, thus decreasing the stimulatory effect of receptors (e.g. oxytocin) coupled to phospholipase C. In late pregnancy PKA levels decrease, AKAP5 binds a protein phosphatase resulting in dephosphorylation of phospholipase C and a strong contractile effect of oxytocin, which is now fully able to increase calcium inside the cells26. The diversity and specialisation of AKAP scaffolding proteins, together with the differential activation of PKA and the distinct regulatory sensitivities of ACs provides many potential combinations by which cells can respond to receptor activated cAMP generation. It is important to investigate these pathways in uterine smooth muscle cells during continuing pregnancy and at the onset of labour.
Thus, in pregnancy there is increased coupling of relaxing receptors (β2 adrenoceptor, EP2 receptors) to Gαs and AC, favouring cAMP formation, which is amplified by the low, progesterone-inhibited phosphodiesterase (PDE4) activity in the myometrium. This would result in long sustained cAMP elevations, which would be transduced by AKAP assemblies, probably containing PKAII, into activation of regulatory targets that favour uterine relaxation. The nature of these targets in human myometrium remains speculative, but it is likely to involve calcium-activated potassium channels on the cell membrane27, MLCK, inositol trisphosphate receptors or other calcium channels in the sarcoplasmic reticulum. At term, changes in the structure or assembly of cAMP associated proteins would lead to alterations in receptor/effector coupling favouring the transition to phasic uterine contractions at the onset of labour. These changes could occur at the level of AC coupling (e.g. changes in AC isoform in the vicinity of membrane receptors; at the level of AKAP targeting; by altering PKAI/PKAII ratios or by a combination of all these).
Role of Tissue Macrophages
The decidua has a large surface area in contact with the myometrium on one side and the chorion/amnion on the other. During labour, decidual cells increase their output of prostaglandin F2α and it is possible that this contributes to uterine activation. We have shown that decidual macrophages are responsible for the increased PGF2α release during spontaneous labour28. Moreover, tissue macrophages have the capacity to release pro-inflammatory cytokines (IL-1, TNF-α) and respond to bacterial products with increased cytokine/prostaglandin release. Under circumstances of intrauterine infection (chorioamnionitis), decidual macrophages may be involved in preterm labour through the premature release of prostaglandins and other inflammatory mediators in the decidua/fetal membrane area, close to myometrial cells (see Fig. 2).
Under ideal conditions, human parturition must be the result of a precisely coordinated endocrine sequence of events, timed for the best potential outcome for the baby and the mother. These events are likely to involve fetal organs which are functionally integrated with the placenta (e.g. adrenal glands), or whose adequate maturation is vital for the survival of the baby (lungs, liver, brain). Unfortunately we still don't know what these events are or how the signals from the fetal organs reach the target tissues in the uterus and the cervix, where the cascade of events has its effects. However, under some pathological circumstances (infection, antepartum haemorrhage, placental abruption etc.) the onset of labour may be triggered by abnormal mechanisms.
Preterm birth has multiple aetiologies, many of which are the result of unavoidable complications of pregnancy. While the prevention of all preterm births is an unattainable goal, it is realistic to expect better management in the subgroup of spontaneous preterm labour. Nevertheless, even this problem will remain insoluble until the factors responsible for the physiological onset of labour at term are known. Only then will it become clear whether preterm labour results from different mechanisms to those which operate at term, or whether it is the result of the premature activation of the same pathways. For the time being, it is important to develop better tocolytic agents to control preterm contractions when this is indicated (Table 2). Ultimately, however, simply relaxing the uterus is too naıuml;ve and is not going to be good enough. It is still a matter of debate whether tocolytics improve perinatal mortality or morbidity. We need to move a step back in the process and concentrate our efforts on treating the causes of preterm labour. That may mean correcting an endocrine imbalance that leads to increased uterine activity, or the adequate treatment of infection, or the inflammatory response. In my opinion, in order to improve neonatal outcome we need to unravel the mechanism of parturition so that we are able to prevent rather than treat preterm labour.
Table 2. Desired characteristics of an ideal tocolytic drug.