Calcium homeostasis

The phasic nature of uterine contractions is an intrinsic property of uterine smooth muscle and is related to the capacity of myometrial cells to modify [Ca²⁺]_i through reversible activation/inhibition of membrane receptors, ion channels and Ca²⁺ pumps. The main source of Ca²⁺ is the extracellular fluid, but intracellular stores such as the SR have important regulatory functions [71].

Myometrial cells are depolarized by action potentials which provoke the influx of Ca²⁺ through voltage‐operated channels [72,73]. Interestingly, spontaneous action potentials in pregnant human myometrial cells are inhibited in sodium‐deficient or calcium‐free solutions [74–76]. This is due to the essential role of extracellular Ca²⁺ and of membrane Na⁺/Ca²⁺ exchangers in the generation of action potentials and myogenic contractility. Magnesium sulfate is a natural calcium antagonist and a potent L‐type calcium channel inhibitor. Nifedipine is a commonly used pharmacological blocker of external Ca²⁺ entry which also acts on L‐type channels. In the presence of nifedipine both spontaneous and OXT‐induced contractions in myometrial strips from term pregnant women are virtually abolished, confirming the essential role of external Ca²⁺ in the development of phasic uterine contractions in late pregnancy. L‐type calcium channels have been targeted pharmacologically in the management of preterm labour, using magnesium sulfate or nifedipine; these drugs are effective at relaxing the uterus but they lack myometrial selectivity and can cause side effects. The role of T‐type channels in the transmission of action potentials and in the regulation of contraction frequency in myometrial cells has been investigated, but the ubiquitous expression of these channels makes them a poor pharmacological target for the control of uterine activity [77,78].

Intracellular calcium stores

The study of calcium stores in smooth muscle has benefited from the development of fluorescent calcium indicator dyes that allow the measurement of transient rises in free Ca²⁺ in discrete cellular compartments. A number of general observations have been made across many tissues [79] which are relevant to human myometrium.

1. Sporadic increases in [Ca²⁺]_i occur as a consequence of Ca²⁺ release from intracellular stores (e.g. SR) rather than Ca²⁺ entry through the plasma membrane.
   
2. These increases are usually very transient and are followed by Ca²⁺ entry into the cells through voltage‐dependent channels; without Ca²⁺ influx the intracellular stores are rapidly depleted.
   
3. Two types of intracellular stores can be distinguished by the nature of their regulatory mechanisms: those containing the ryanodine receptors (RYRs), activated by ryanodine or caffeine, and the inositol 1,4,5‐trisphosphate receptor (ITPR)‐containing stores. The latter are associated with activation of G protein‐coupled receptors (GPCRs) in the cell membrane and activation of phospholipase C.
   
4. Refilling of intracellular stores may be facilitated by their close proximity to the cell membrane and may be linked to the generation of depolarizing currents.

Several isoforms of RYR and ITPR have been described in human myometrium [42,80]. The RYR acts as a Ca²⁺ channel and studies using human myometrial cells have confirmed the presence of functional RYRs in this tissue [81]. Ryanodine and cyclic ADP‐ribose (cADPR) promote intracellular Ca²⁺ release in myometrial preparations in vitro and potentiate oxytocin‐induced [Ca²⁺]_i increases by facilitating Ca²⁺ entry from the extracellular medium through ‘calcium‐induced calcium release’ [82]. Interestingly, inflammatory mediators such as TNF‐α induce CD38 expression in myometrial cells; this suggests that inflammation may potentiate the parturition cascade by promoting the cADPR system [82].

ITPRs also act as intracellular Ca²⁺ channels and are activated by inositol 1,4,5‐trisphosphate (InsP₃) generated when endogenous myometrial stimulants such as OXT, endothelin or α‐adrenergic agonists bind GPCRs coupled to proteins of the G_q family which activate phospholipase C. InsP₃ binds to myometrial ITPRs and promotes Ca²⁺ release [42,71]. Experiments using myometrial strips from late pregnant women [71] and imaging techniques that allow the visualization of discrete Ca²⁺ fluxes in lipid microdomains [69] suggest that ITPR channel activation is an early event in uterine activation by OXT; however, in the absence of extracellular Ca²⁺ the development of force is short‐lived because intracellular Ca²⁺ stores are soon exhausted; in other words, the development of phasic uterine contractions is absolutely dependent on the availability of extracellular Ca²⁺ to myometrial cells. Under normal physiological circumstances, for example in the absence of inflammation, ITPR channels in the SR appear to be more important for myometrial function than RYR channels [71]. Experiments using myometrial strips from late pregnant women showed that ryanodine had little effect on spontaneous or OXT‐induced contractions [71], suggesting no functional role for RYR in this preparation. Moreover, blocking SR Ca²⁺ uptake with cyclopiazonic acid, an inhibitor of Ca‐ATPase in myometrium, increased contractile force and [Ca²⁺]_i.This suggests that the SR may normally contribute to lowering [Ca²⁺]_i during the relaxation phase of the contractions [71]. The complex role of intracellular Ca²⁺ stores requires further investigation because, in addition to acting as a trigger for agonist‐induced Ca²⁺ mobilization, the SR may act as a ‘sink’ for Ca²⁺ during stimulation and, by enhancing plasma membrane Ca²⁺ efflux, may contribute to homeostatic Ca²⁺ relaxation mechanisms [83].

Electrophysiological mechanisms

A model for the activation of human myometrium based on electrophysiological and receptor mechanisms has been proposed [84], partly based on available experimental evidence [69,85,86]. In this model, activation of uterine contractility during labour is driven by action potentials initiated by slow depolarization of clusters of pacemaker myometrial cells. The action potentials trigger Ca²⁺ entry into the cells through L‐type voltage‐operated channels and the rise in [Ca²⁺]_i provokes contractions, thus linking the electrical signal with force (excitation–contraction coupling). This model envisages that the balance of depolarization and hyperpolarization in the cell membrane is controlled by multiple pumps and channels, including a Na⁺/Ca²⁺ exchanger; a Na⁺/K⁺ pump, and Ca²⁺‐activated K⁺ channels that contribute to relaxation (hyperpolarization). On the other hand, Ca²⁺‐sensitive chloride channels, T‐type channels, and a putative channel that senses whether the level of Ca²⁺ in the SR store is low (store‐operated channel, SOC) can tip the balance of the ‘pacemaker’ towards depolarization [84]. GPCR agonists like OXT can initiate depolarization by generating InsP₃ and emptying the SR store through ITPR channels, thus activating SOC, whereas RYR channels would contribute to contraction directly by increasing [Ca²⁺]_i. Further regulatory refinement of myometrial activation can be provided by Ca²⁺‐ATPases that promote Ca²⁺ extrusion through the plasma membrane (PMCA) or Ca²⁺ uptake into the SR (SERCA).

Some aspects of this model have been reinforced by the abundant experimental evidence in favour of a role for L‐type Ca²⁺ channels as the major route for Ca²⁺ entry into myometrial cells and the unquestionable involvement of the GPCR/phospholipase C/InsP₃ pathway activating ITPR channels in human myometrium [76,87]. However the role of RYR channels in human myometrium is questionable. The presence of Ca²⁺‐activated K⁺ channels in this tissue has been demonstrated but their role in phasic myometrial activity during labour is not clear. An interesting interaction between Ca²⁺‐activated K⁺ channels and α₂‐macroglobulin has been described in myometrial cells whereby in the presence of OXT α₂‐macroglobulin induces oscillations in Ca²⁺‐activated K⁺ channel activity and promotes Ca²⁺ entry via SOC [88]. A number of endogenous Ca²⁺‐ATPase modulators has been described, including phospholamban, a membrane‐associated protein that inhibits SERCA‐mediated Ca²⁺ uptake into the SR. Interestingly, phospholamban can be phosphorylated by cAMP‐dependent kinases leading to its inactivation and the liberation of SERCA activity [83].

Receptor‐regulated myometrial contractility

In addition to the spontaneous uterine activity driven by action potentials, there is strong evidence that receptors and their cell signalling pathways have a physiological influence on the regulation of uterine contractility. The uterus has a rich variety of receptors, many of which are upregulated in pregnancy, and responds to classical hormones and transmitters (e.g. OXT, 5‐hydroxytryptamine) as well as local modulators (e.g. prostaglandins and thromboxanes). The link between the myometrial cell membrane receptor and the initiation of a signalling cascade is, in most cases, a regulatory GTP‐binding protein. G proteins can activate effector enzymes such as phospholipase Cβ (PLCB), adenylyl cyclase (ADCY) or phosphoinositide 3‐kinase. The PLCB pathway is activated by receptors usually coupled to G_q which stimulate myometrial contractility. Endogenous agonists include peptide hormones (OXT, endothelin), prostanoids (PGF_2α, thromboxane A₂), catecholamines, muscarinic agents and inflammatory mediators (bradykinin, serotonin). The response is initiated by the hydrolysis of a receptor‐sensitive pool of phosphatidylinositol 4,5‐bisphosphate (PIP₂) in the cell membrane. The breakdown of PIP₂ generates two molecules with potent signalling effects: InsP₃, which releases calcium form the SR, and 1,2‐diacylglycerol (DAG), which activates protein kinase C (PRKC) and stimulates the phosphorylation of many target proteins. Potential substrates of PRKC include ion channels, calcium pumps, and proteins involved in GPCR function and coupling to phospholipase C, ITPR regulation and elements of the contractile machinery [89]. In addition, DAG has PRKC‐independent effects, for example in human myometrial cells DAG analogues facilitate store‐operated or receptor‐operated Ca²⁺ entry through the cell membrane via L‐type channels and Na⁺/Ca²⁺ exchangers [90].

Some ligands can activate more than one type of myometrial receptor, creating complex responses depending on the relative abundance and affinity of each receptor subtype and the presence of other agonists competing or interacting with related receptors. For each receptor, the effect on myometrial contractility will depend on the integration of these signals and on the physiological state of the organ under different endocrine influences. Changes in myometrial sensitivity to agonists are likely to be important mechanisms for maintaining the balance between uterine quiescence and contractility. The uterus is very sensitive to OXT, which is commonly used for the induction and augmentation of labour; however, many uncertainties remain about its mechanism of action. For instance, analysis of the Ca²⁺–tension relationship in pregnant myometrial strips reveals a strong component of ‘Ca²⁺ sensitization’ during OXT‐induced contractions [91,92], probably involving inhibition of myosin phosphatase. The pathways involve small GTP‐binding proteins of the Rho family and activation of Rho‐dependent kinases [93].

Role of cyclic nucleotides

The importance of cyclic nucleotides (cGMP and cAMP) in smooth muscle function cannot be over‐emphasized. These molecules provide rapid intracellular responses to a variety of signals and have profound effects on regulatory proteins through the activation of specific protein kinases. The role of cyclic GMP as a mediator of nitric oxide (NO) [95] effects in myometrium is a matter of controversy. Initial studies suggested a link between NO production, cGMP generation and uterine relaxation [96]. However, cGMP has a much more potent inhibitory role in tracheal and vascular smooth muscle than in myometrium [97,98]. Intravenous nitroglycerin has been used to relax the uterus during postpartum emergencies [99,100] but the use of NO donors for routine tocolysis is not recommended [101].

Cyclic AMP is produced from ATP in response to activation of GPCRs positively coupled to ADCY, and is known to influence a wide range of physiological events including relaxation of myometrial smooth muscle [102]. The actions of cAMP in myometrial cells, as in other cell types, result from the dynamic interplay of multiple ADCY, phosphodiesterases and protein kinase A (PRKA) isoforms that allow spatial and temporal changes in cAMP levels to be translated into compartmentalized responses within the cell. Most of the effects of cAMP are due to its ability to bind to PRKA. The localization of PRKA activity within cells depends on reversible interactions with ‘A kinase anchor proteins’ (AKAP). Although the evidence that agonist‐mediated increases in cAMP result in uterine relaxation is strong, the mechanism of action of cAMP in myometrium is not well known. PRKA–AKAP complexes have been described in pregnant human myometrium [103–105], but there is little information about the physiological targets that become phosphorylated during cAMP‐induced relaxation. New lines of research suggest that cAMP interacts with progesterone receptors to regulate COX2 expression in human myometrial cells [106].

G protein‐coupled receptors and their second messenger pathways

The uterus has GPCRs for many endogenous agonists and responds with contraction or relaxation depending on the type of G protein and second messenger pathway activated. In general, receptors coupled to G_q are stimulatory through activation of PLCB and receptors coupled to G_s are inhibitory (promote relaxation) via stimulation of ADCY (Table 22.1). Some ligands can activate more than one type of myometrial receptor, creating complex responses depending on the relative abundance and affinity of each receptor subtype and the presence of other agonists competing or interacting with related receptors. G proteins are heterotrimers (αβγ subunits) and the effects of G_α subunits on PLCB, ADCY and other effectors are modulated by the interaction of G_βγ subunits, especially those liberated from G_i/o or G₁₂, with the same or with other effector proteins including ADCY, ARF6, GRK, PLCB, PLCD, phosphatidylinositol kinases and ion channels. For each receptor, the effect on myometrial contractility will depend on the integration of these signals and on the physiological state of the organ under different endocrine influences. Changes in myometrial sensitivity to agonists may be an important mechanism for maintaining the balance between uterine quiescence and contractility. For example, pregnancy induces an increase in the myometrial response to OXT [35] and histamine [95] and a decrease in the response to tachykinins in women [95]. More research needs to be done on the responses of pregnant and non‐pregnant human myometrium to selective GPCR agonists and antagonists to build a complete picture.

Receptor‐mediated uterine relaxation

The β₂‐adrenoceptor (ADRB2) has been a pharmacological target to relax the uterus for many years, using beta‐mimetic drugs. Exposure of human pregnant myometrial strips to the β₂ agonist isoproterenol in vitro results in a significant decrease in contractile force and in the frequency of spontaneous contractions. Similar effects have been obtained with ritodrine, a beta‐mimetic used as a tocolytic agent in preterm labour [107,108]. Table 22.2 lists a number of endogenous ligands that can relax the uterus. Some of these, for example catecholamines and prostaglandins, have complex stimulatory and inhibitory effects due to the presence of multiple receptors in myometrium. Inhibitory receptors such as ADRB2 or the prostaglandin PTGER2 receptors have in common the coupling through G_αs to ADCY. Receptors coupled to G_i/o (e.g. α₂‐adrenoceptors, ADRA2) inhibit ADCY and decrease cAMP production, favouring uterine contractions; however, in many systems prolonged stimulation of G_i/o‐coupled receptors provokes sensitization of ADCY to subsequent stimulation by other G_s‐coupled receptors (heterologous sensitization) [109].

Changes in the sensitivity to endogenous agonists (catecholamines, prostaglandins) operating through ADRB2, PTGER2 and other receptors coupled to cAMP production may be responsible for the transition from uterine quiescence to the onset of labour. This is an attractive hypothesis, although it requires more evidence to support it. Beta‐mimetics (ritodrine, terbutaline, salbutamol) were among the earliest agents used clinically to inhibit uterine contractions, but their efficacy is questionable [110]. ADRB2 receptors are widely expressed in many organs, and consequently the use of beta‐mimetics in pregnancy is associated with potentially serious cardiovascular, neuromuscular and metabolic side effects. There is contrasting new information to be considered before the possible clinical use of PTGER2 agonists to relax the uterus [111].

Desensitization of GPCRs

Sustained stimulation of GPCRs typically causes desensitization (a reduction in response in the face of a constant stimulus), which protects cells from over‐stimulation and involves numerous adaptive mechanisms in characteristic time frames. Within seconds to minutes of stimulation, most GPCRs desensitize as a consequence of receptor phosphorylation by G‐protein receptor kinases (GRKs) or second‐messenger regulated protein kinases which are very abundant in human myometrium [112]. This phosphorylation can inhibit G‐protein activation (i.e. cause receptor desensitization), but the most important effect of GRK‐mediated phosphorylation is to facilitate the binding of arrestin proteins [113]. This not only prevents G‐protein activation but also targets the desensitized receptor for internalization. The consequent reduction in cell surface receptor number can also underlie desensitization in the intermediate time frame of minutes to hours. Over longer periods (hours to days), numerous adaptive processes serve to either reinforce or reverse the earlier desensitization by changing rates of synthesis and degradation of receptors and downstream effectors. Arrestins have many other roles, including acting as scaffolds to facilitate receptor‐stimulated activation of mitogen‐activated protein kinases (MAPKs) in discrete cellular compartments. This mechanism has been confirmed for bradykinin B₂ receptors in human myometrial cells [114].

One of the difficulties with the use of agonists for G_αs‐coupled receptors to promote uterine relaxation is the tendency of GPCRs to desensitize and downregulate. This process has been particularly well studied for the ADRB2 receptor but it has long been known to occur with other utero‐relaxing receptors such as PTGER2 and PTGIR [115]. The loss of tocolytic effect of ritodrine in women in preterm labour has been known for many years [116].