Karen Noble

Depletion of membrane cholesterol eliminates the Ca2+‐activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes

Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl‐β‐cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca2+ signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19–21 rats showed that outward K+ current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol‐saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K+ channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca2+‐sensitive K+ channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca2+ level ([Ca2+]i transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca2+ signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol‐rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca2+ signalling and uterine contractility.

Lipid rafts, the sarcoplasmic reticulum and uterine calcium signalling: an integrated approach

The pathways involved in Ca2+ signalling in the uterus remain incompletely understood, impairing our ability to prevent preterm and difficult labours. In this review we focus on two elements in the pathway of Ca2+ signalling that have recently emerged as playing important roles: membrane lipid rafts and the sarcoplasmic reticulum. We examine the evidence for lipid rafts in the uterus and discuss their functional role. We suggest that the increases in cytosolic [Ca2+] and contractility that occur with raft disruption are due, at least in part, to effects on large conductance Ca2+‐activated K+ (BK) channels that are localized to rafts. The role of the SR in contributing to subsarcolemmal cytosolic microdomains in uterus is evaluated, along with its interactions with ion channels on the plasma membrane. Thus, signalling microdomains play an important, but incompletely understood, role in the uterus, and integrating them into other Ca2+ signalling pathways is a challenge for further research. We suggest that the role of the SR changes in pregnancy, from promoting quiescence via BK channels or SR Ca2+ uptake, to promoting Ca2+ entry and contractility at term, and relate data on lipid rafts to clinical outcome in obese pregnant women.

A review of recent insights into the role of the sarcoplasmic reticulum and Ca entry in uterine smooth muscle

The uterine sacroplasmic reticulum (SR) takes up and stores calcium [Ca], using an ATPase (SERCA) and the Ca-buffering proteins, calsequestrin and calreticulin. This stored Ca can be released via IP(3)-gated Ca channels. Decreases in luminal Ca concentration [Ca] have been directly measured following agonist stimulation. During spontaneous contractions however, there appears to be no involvement of the SR, as Ca entry and efflux across the plasma membrane account for these phasic contractions. After over-viewing current knowledge concerning SR structure and function, we highlight three areas of research which suggest new ways of looking at the role of the SR in the uterus, although they may be controversial or speculative at the moment. Firstly, we review the evidence for the function, if any, of Ca-induced SR Ca release channels, the ryanodine receptor (RyR) and the lack of Ca sparks (the elemental release events from RyRs), in the uterus. Secondly, we ask does regulation of SERCA by the accessory protein, phospholamban, occur in the uterus and what is the effect of knocking out phospholamban on uterine activity? Thirdly, we address the question of when and how store-operated Ca entry occurs in the myometrium. By analogy with other, usually less excitable tissues, is there a mechanism that links store Ca depletion to plasma membrane Ca entry in smooth muscle cells within intact uterus and is it physiologically relevant and regulated? Are the recently described proteins ORAI and STIM-1 involved in uterine store-operated Ca entry? We end the review by integrating these new insights with previous data to present a new working model of the SR in the uterus.

Sex Hormones and Excitation–Contraction Coupling in the Uterus: The Effects of Oestrous and Hormones

In this review, we examine how far the increased understanding that we have of the events in excitation contraction can explain the effects of the oestrous cycle and sex hormones on uterine function. Observational studies of electrical and mechanical activity in the rat myometrium have shown a relative quiescence during pro‐oestrous, with little propagation of any electrical events. Thus, uterine activity can be said to approximately inversely reflect plasma 17β‐oestradiol concentrations. We show that Ca2+ signalling and mechanical activity are greatest in metoestrous and dioestrous compared to pro‐oestrous and oestrous. These data are discussed in terms of hormonal effects on Ca2+ and K+ channels. Finally, the influence of sex hormones on lipid rafts and caveolae are considered and discussed in relation to recent findings on their role in uterine signalling and contractility, and cholesterol levels and obesity.

In situ calcium signaling: no calcium sparks detected in rat myometrium.

Abstract:  Controlled uterine smooth muscle activity is essential for our reproductive health. While we understand reasonably well the steps that produce contraction following a rise in intracellular [Ca], the mechanism controlling excitability and thus the rise of Ca, is less well understood. Here we examine the role of the internal Ca sore, the sarcoplasmic reticulum (SR), and its relation to surface membrane ion channels. We show that despite having a well‐developed SR, the rat uterus does not produce the elemental and local Ca signals, known as Ca sparks. This in turn has consequences for excitability, as the negative feedback loop between these Ca signals and Ca‐activated K (BK) channels on the surface membrane is lost. This may be important for producing the powerful long‐lasting contractions of the uterus required during labor.

Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium

Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels.

The role of the sarcoplasmic reticulum in neonatal uterine smooth muscle: enhanced role compared to adult rat

Little is known about contractile activity, response to agonists or excitation‐contraction coupling in neonatal smooth muscle. We have therefore investigated 10‐day rat uterus to better understand these processes, and compared it to adult uterus to elucidate how control of contractility develops. Spontaneous contractions are present in the 10‐day neonatal uterus, although they are not as large or as regular as those present in adult tissues. External Ca2+ entry via L‐type Ca2+ channels is the sole source of Ca2+ and is essential for the spontaneous activity. The neonatal uterus was responsive to carbachol or prostaglandin F2α application; it showed a marked stimulation and a clear dissociation between the force and Ca2+ changes. Such sensitization was not apparent in adult rat myometrium. The sarcoplasmic reticulum (SR) had more releasable Ca2+ and contributed more to the response to agonists in neonatal compared to adult tissues. Thus, Ca2+ entry as opposed to SR Ca2+ release contributed much less to the uterine response to agonists in the neonatal, compared to adult tissues. Inhibition of the SR by cyclopiazonic acid also caused a more vigorous increase in Ca2+ and contractile activity, particularly frequency, in the neonatal compared to the adult uterus. Taken together these data suggest that: (1) spontaneous activity is already present by day 10, (2) receptor‐coupling and excitation‐contraction signalling pathways are functional, (3) the SR and Ca2+ sensitization mechanisms play a more prominent role in the neonate, and (4) there is a shift to a greater reliance on Ca2+ entry and excitability with development of the myometrium.

What do we know about what happens to myometrial function as women age

Much has been written about the effects of aging on reproductive function, especially female fertility. Much less is known about how aging may affect the contractility of the smooth muscle within the uterus, the myometrium. The myometrium is active through a woman’s entire life, not just during pregnancy. Here we will discuss briefly the contractile functions of the uterus and the changes it undergoes throughout the stages of a woman’s life from menstruation and the menopause, before evaluating the evidence for any changes in myometrial contractility and responses as women age, with a particular focus on women of advanced maternal age. We present original contractility analysis for the widest data set for human myometrium so far examined, and determine inherent spontaneous activity as well as responses to depolarisation and stimulation with oxytocin. Our data show that in the non-pregnant state there is a significant decrease in contractility for both spontaneous and depolarised-induced contractions, with age. We suggest that muscle atrophy and down regulation of Ca channels may account for this. Interestingly in pregnant myometrium we found a wide range of contractile ability between women and little evidence for decreased spontaneous activity between the ages of 25–40. Oxytocin responses appear to be more affected by aging, a finding that is consistent with previously reported clinical findings, and may partly be the result of membrane lipids such as cholesterol, increasing as women age. The marked differences between the age-related decline of force beyond age 30 in non-pregnant uterus, and the lack of difference in the pregnant state over this period, shows that the uterus retains its ability to respond to gestational hormones. The growth of the pregnant uterus and increase in content of myofibrillar proteins, may abolish any previous age-related force deficit. This finding is consistent with what is apparent for postmenopausal women in their 50s and 60s; that with the appropriate hormonal stimulation the uterus can allow an embryo to implant, and then without further intervention, carry the foetus to term. It is tempting therefore to speculate that unlike other well documented declines in female reproductive functions with age, the myometrium remains able to function into a woman’s 7th decade.

Progress in understanding electro‐mechanical signalling in the myometrium

In this review, we give a state‐of‐the‐art account of uterine contractility, focussing on excitation–contraction (electro‐mechanical) coupling (ECC). This will show how electrophysiological data and intracellular calcium measurements can be related to more modern techniques such as confocal microscopy and molecular biology, to advance our understanding of mechanical output and its modulation in the smooth muscle of the uterus, the myometrium. This new knowledge and understanding, for example concerning the role of the sarcoplasmic reticulum (SR), or stretch‐activated K channels, when linked to biochemical and molecular pathways, provides a clearer and better informed basis for the development of new drugs and targets. These are urgently needed to combat dysfunctions in excitation–contraction coupling that are clinically challenging, such as preterm labour, slow to progress labours and post‐partum haemorrhage. It remains the case that scientific progress still needs to be made in areas such as pacemaking and understanding interactions between the uterine environment and ion channel activity.

Myometrial physiology – time to translate?

What is the topic of this review? To provide an overview of the current understanding of myometrial contractility and give examples of where this has or is close to being applied clinically. What advances does it highlight? Examples of where myometrial science has been translated into clinical practice/patient benefit are discussed, as well as including suggestions of how it can be advanced.