Store-operated Ca²⁺ entry and depolarization explain the anomalous behaviour of myometrial SR: effects of SERCA inhibition on electrical activity, Ca²⁺ and force.

In the myometrium SR Ca(2+) depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca(2+) sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca(2+)-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca(2+) transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca(2+)]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca(2+) spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca(2+) and depolarization continued a point was reached when electrical and Ca(2+) spikes and phasic contractions ceased, and a maintained, tonic force and Ca(2+) was produced. Lanthanum, a non-selective blocker of store-operated Ca(2+) entry, but not the L-type Ca(2+) channel blocker nifedipine (1-10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca(2+) depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca(2+) release is coupled to marked Ca(2+) entry, via store operated Ca(2+) channels, leading to depolarization and enhanced electrical and mechanical activity.

Introduction

Uterine contractility is a direct consequence of the underlying electrical activity in myometrial cells. Therefore, the regulation of electrical activity in myometrial cells plays a crucial role in determining the onset, the duration and the strength of uterine contractions during labour. Thus, understanding the mechanisms that modulate membrane potential is of major importance [1], [2], [3], [4]. Recently Gravina et al. [5] in a study of mitochondria in mouse myometrium showed that inhibiting SERCA was associated with depolarization. We have previously shown that sarcoplasmic reticulum (SR) Ca2+ load has a profound effect on intracellular Ca2+ signals [6], [7]. In isolated rat uterine myocytes an increased SR Ca2+ load, produced by maintained depolarization, inhibited spontaneous Ca2+ signals. A decreased SR luminal Ca2+ load, by inhibiting SERCA, activated intracellular Ca2+ spikes. In intact tissue the same results were found; there was a potentiation of Ca2+ transients and contractions when SERCA was inhibited by cyclopiazonic acid (CPA) [8]. In pregnant rat uterus CPA stopped the phasic contractions and transformed activity to tonic-like, associated with a large increase in the baseline Ca2+ [8]. Although Gravina et al. showed a depolarization with CPA in mouse [5], which might be expected to increase force and Ca2+, the absence of a Ca2+ sparks-STOCs (spontaneous transient outward current) in myometrium [9], make it difficult to appreciate what the underlying mechanism may be. Our previous data suggested the presence of store operated Ca2+ entry (SOCE) under some conditions [10]. The presence of Stim-Orai proteins in myometrium [11] and Trp homologues [11], [12], adds to the suggestion that SOCE may operate in the myometrium. Previous workers had suggested this (e.g. [12], [13], [14]) but to date, no direct demonstration supporting SOCE, has been made in myometrium.

Further understanding of how the Ca2+ load in the myometrial SR influences Ca2+ signalling and contractility, and the mechanisms linking SR Ca2+ depletion to SOCE is needed to help us explain their relationship to electro-mechanical coupling in myometrium. We hypothesized that Ca2+ release from the SR, induced by CPA or agonists, could activate SOCE leading to depolarization of the uterine smooth muscle cells, which would be a stimulant at low level and inhibitory at high levels, on action potentials. The main aims of this study were therefore to investigate the effects of SERCA pump inhibition and agonists (carbachol) on electrical activity, Ca2+ signalling and force in the myometrium.

Methods

Wistar pregnant rats (∼22 days of gestation) were humanely killed by CO2 anaesthesia followed by cervical dislocation, in accordance with UK Home Office legislation. The uterus was removed and longitudinal myometrial strips dissected, 3–5 mm × 1 mm [15]. For measurement of Ca2+, the uterine strips were incubated in the membrane-permeant form of Fluo-4-AM, or Indo-1AM (15 μM, Invitrogen) dissolved in dimethyl sulphoxide premixed with Pluronic F127 (final concentration of 0.01%). Loading was performed at room temperature for 3 h with the cuvettes wrapped in black tape and rotated at 30 rpm. Tissue samples were then removed from the loading medium and placed in physiological saline solution for at least 30 min to allow cleavage of Fluo-4AM to Fluo-4 and Indo-1AM to Indo-1 by intracellular esterases. For Ca2+ measurement using Indo-1, the tissues were excited at 340 nm and the Indo-1 fluorescence emitted at 400 and 500 nm was recorded at a sampling rate of 1 KHz, using Axoscope software (version 10.0) [16]. The ratio of these signals (F400/F500) provides a measure of [Ca2+]i and provides stable recording of Ca2+ signals for hours which are less effected by movement artefacts, leakage and photobleaching than single wavelength indicators. When Fluo-4 was used for Ca2+ measurement the tissue samples were excited at 488 nm and Fluo-4 fluorescence emitted at 510 nM recorded. Ca2+ signal was expressed as pseudo-ratio F/F0 where F0 is the background fluorescence and F is the changes of fluorescence in the region of interest where cytoplasmic Ca2+ was increased [17].

For simultaneous recording of Ca2+ signalling and force the myometrial strips were loaded with either Indo-1 or Fluo-4, clipped at both ends using aluminium foil clips, and attached to a force transducer (WPI) at one end and a stainless steel hook, fixed to the bottom of the experimental chamber at the other end. The force transducer was attached to a 3-D manipulator (Narashige, Japan), on the confocal system, allowing movement of the strip in the X–Y–Z directions, to position it in the focal plane of the objective and apply optimal stretch. In both systems a resting tension of 40% of active maximal force induced by high-K+ depolarization, was applied to the strips. Superfusion of the uterine strips in the chamber was performed by applying either positive pressure (Confocal system, Perkin Elmer) or gravity feed (Photometric system, Cairn) multiple syringe systems connected to common manifold. Solution was removed by suction at the other end of the chamber. All experiments were performed at 33–34 °C, as this was optimal temperature for signalling and being near physiologic. For confocal imaging fast Nipkow disc-based confocal imaging attached to a high sensitivity (iXon Andor) CCD camera, was used to enable acquisition of images at 60–200 fps and thereby accurate measurement of temporal and spatial characteristics of Ca2+ signalling in the muscle cell bundles. To measure temporal and spatial characteristics of Ca2+ signalling in individual myocyes, a 60× water objective (NA 1.2) was used, while low-power dry objectives (4×, NA 0.13; 20×, NA 0.7) were used to measure temporal and spatial characteristics of Ca2+ signalling in whole uterine muscle bundles. Data acquisition was performed using Andor iQ software.

For simultaneous force, Ca2+ signalling and electrical activity measurements, a modified tissue bath was used, as detailed elsewhere [18], [19]. Briefly the bath became a sucrose-gap chamber with a cover slip at its base, to enable the optical measurements to be made.

Strips were left until spontaneous contractions became regular and stable. If the tissue failed to spontaneously contract after 60 min, but was responsive to high-K+, it was classified as quiescent (about 30% of strips). In all protocols contraction induced by 40 mM high-K+ applied for 40 s was used to normalise both force and Ca2+ data, taken as 100%.

The standard physiological solution was a HEPES-buffered modified Krebs solution containing (in mM): NaCl, 120.4; KCl, 5.9; CaCl2, 2; MgSO4, 1.2; HEPES, 11.6; and glucose, 11; pH 7.4 adjusted with NaOH. Solutions with increased [K+] were obtained by replacing Na+ by equimolar K+. The Ca2+-free solutions contained 2 mM EGTA. In solutions where La3+ or Gd3+ was used, MgSO4 was replaced by MgCl2.

Values are given as means ± SEM where n is the number of animals. Differences were considered significant for P < 0.05 using the appropriate Student's t test or ANOVA.

Results

Effects of cyclopiazonic acid on electrical activity, Ca2+ signalling and force

The effects of CPA on electrical activity, Ca2+ signalling and force recorded simultaneously from Fluo-4 loaded uterine strips using confocal imaging combined with double sucrose gap method, were studied (n = 4).

Fig. 1 shows that under control conditions the smooth muscle of rat myometrium is spontaneously active and generated bursts of electrical activity, consisting of slow depolarisations, superimposed with spikes. Each action potential was coupled to and correlated with, the generation of Ca2+ spikes. In agreement with our previous studies [20], the Ca2+ spikes could be seen to propagate and underlined the development of phasic contractions (Fig. 4A).

Fig. 1

Effects of CPA (20 μM) on electrical activity (bottom trace), Ca2+ transient (middle trace) and force (top trace) of Fluo-4 loaded uterine strips. (i), (ii), (iii) distinct phases of CPA-induced changes of electrical activity, Ca2+ signalling and force respectively.

Fig. 4

Effects of CCh on electrical activity, Ca2+ transients, and force in Fluo-4 loaded uterine smooth muscle strips. (A) Images showing uterine bundles at rest (i), at a peak of asynchronized Ca2+ release phase (ii), and during the synchronized Ca2+ spike activity (iii). (B) Typical records showing CCh-induced changes in electrical activity (bottom trace), Ca2+ transients (middle trace), and force (top trace) at rest (i), at a peak of asynchronized Ca2+ release phase (ii), and during the synchronized Ca2+ spike activity (iii), respectively.

Inhibition of SERCA by CPA (20 μM) had three distinct effects on electrical activity, Ca2+ signalling and force (Fig. 1). During the first stage (Fig. 1i) CPA produced a gradual rise of baseline [Ca2+] accompanied by membrane depolarization, and an increase in the duration (128.4 ± 4.5%) of the burst of the action potentials. This lead to a prolongation of the burst of Ca2+ spikes (132.7 ± 7.2%) associated with a markedly increased duration (138.5 ± 8.2%) and small increase in the amplitude of contractions (114.5 ± 4.4%), which were still phasic at this time (Fig. 1i). The number of spontaneous phasic contractions observed in the presence of CPA varied from 1 to 4, after which contraction became titanic (Fig. 1ii). During the second stage which occurred after ∼5 min in CPA, baseline Ca2+ was further increased, the membrane further depolarized, and the action potentials associated with Ca2+ spikes and muscle twitching, were generated continuously (Fig. 1ii). In stage three (Fig. 1iii), as the membrane depolarization continued, this decreased the amplitude of, and then abolished action potentials, resulting in a decrease in the amplitude and then abolition of Ca2+ spikes, and a partial relaxation of tension (Fig. 1iii). As the action potentials, Ca2+ spikes and muscle twitching ceased, a steady state tonic contraction remained, associated with a maintained, significant rise of baseline Ca2+ (68.3 ± 14.2%) of spontaneous Ca2+ response and membrane depolarisation which reached maximal level at this time.

In summary, reducing luminal Ca2+ by inhibition of SERCA leads to a significant depolarization, increased intracellular Ca2+ signalling and produced a tonic contraction in myometrium.

Effects of nifedipine

The large elevation of baseline Ca2+ and force induced by CPA could be caused by Ca2+ influx via store-operated and/or voltage operated Ca2+ channels. In order to assess the contribution of Ca2+ influx via voltage gated Ca2+ channels, the effect of the selective blocker nifedipine, was investigated. These experiments (and those shown in Fig. 3) were conducted with Indo-1 loaded myometrial strips to enable longer protocols to be accomplished than is usually accomplishable with Fluo-4 and confocal recording. In Fig. 2, Fig. 3 the tissue was initially stimulated with a high-K+ solution, to determine maximum activation and ensure the SR was Ca2+ loaded.

Fig. 3

Effects of Ca2+ antagonists on changes in intracellular Ca2+ and force induced by readmission of external Ca2+ following 2 min treatment by CPA of Indo-1 loaded uterine smooth muscle bundles. (A) and (B) Typical records showing effects of nifedipine and La3+ (10 μM), respectively. (C) Mean data showing % of inhibition by nifedipine (light grey bar) and La3+ (grey bar) of force and Ca2+ transients induced by readmission of external Ca2+, expressed as percentage of high-K+ responses taken for 100%.

Fig. 2

Effects of nifedipine (10 μM) on CPA-induced Ca2+ transients and force in uterine smooth muscle cells. (A) Typical record showing selective inhibition of Ca2+ spikes (bottom trace) and force (top trace) by nifedipine. Note that inhibition of L-type voltage gated Ca2+ channels by nifedipine unmasks steady state rise of voltage independent baseline Ca2+ and force. (B) Mean data showing % of inhibition by nifedipine (grey bar) relative to control (black bar) of CPA-induced force and Ca2+ transients.

Fig. 2A shows the effects of CPA culminating in the continuous discharge of the Ca2+ spikes, sustained elevation of base line Ca2+ and tonic contraction, and is very similar to the data obtained confocally with Fluo-4 (Fig. 1). Application of nifedipine (10 μM) resulted in an immediate termination of Ca2+ spikes and a more than 50% relaxation of tension (mean data in Fig. 2B, n = 5, data are statistically different from preceding control values). It can however also be seen that there remains a significant elevation of tonic force and Ca2+ in the continued presence of nifedipine, suggesting significant contribution of voltage independent Ca2+ entry to CPA induced Ca2+ signal, in the pregnant rat myometrium.

The nifedipine insensitive rise of Ca2+ could be due to Ca2+ entry via SOCE stimulated by a release of Ca2+ from the SR, or some other mechanism, e.g. mitochondrial Ca2+ release. We therefore next investigated if SR Ca2+ release in the absence of external Ca2+, could reproduce the data seen in the presence of Ca2+ entry.

Effects of 0—Ca2+

CPA was applied for 120 s in Ca2+-free solution. As Fig. 3 shows (typical record of changes of intracellular Ca2+ and force in Indo-1 loaded strips, n = 7), when applied in Ca2+-free solution, CPA induced a small rise of intracellular Ca2+, caused presumably by an increased Ca2+ leak from the SR. Readmission of external Ca2+ produced a large elevation of the baseline Ca2+ and force, which gradually declined. Superimposed on these baselines were phasic contractions, associated with bursts of Ca2+ spikes (Fig. 3A). These data are consistent with Ca2+ depletion initiating SOCE. Unlike nifedipine, a non-selective blocker of SOCE, La3+ (10 μM) blocked both the sustained rise of intracellular Ca2+ and phasic contractions (n = 4, Fig. 3B). Fig. 3C shows the mean data, comparing nifedipine and La3+, under the same protocol.

Effects of agonists

Our CPA data suggests that Ca2+ release and Ca2+ entry coupling is involved in causing depolarization when the SR Ca2+ store is depleted. Agonists such as oxytocin and carbachol, have also been reported to release Ca2+ from the SR, causing depletion of luminal Ca2+ [7], [21], [22]. Agonists also produce uterine smooth muscle membrane depolarization [2]. Fig. 4 shows simultaneously recorded changes in response to carbachol (10 μM), of membrane potential, Ca2+ (Fluo-4) and force. As described for CPA, carbachol produced a slow depolarization, which upon reaching threshold, triggered repetitive firing of action potentials (Fig. 4B). These electrical changes were associated with a rise in basal calcium and repetitive Ca2+ spikes, and a substantial increase in phasic force, which plateaued and then declined as the membrane started to repolarise and action potentials and Ca2+ spikes ceased. As also indicated in Fig. 4, the confocal record detects an initial rise in calcium, presumably from the SR, which was not synchronised (Fig. 4Aii) and did not spread throughout the myometrial cells and therefore was not associated with a rise of force (Fig. 4B,i).

In order to exclude the possible involvement of receptor operated channels in the tissue responses to carbachol, we made use of carbachol's relatively fast (compared to oxytocin), on and off rates of binding to its muscarinic receptor, and applied if for just 5 s, to quiescent preparations. For these experiments we also increased the flow rate to 25 ml/min, to allow rapid (1.5 s) exchange of the tissue bath, and achievement of a relatively well synchronised Ca2+ release from the SR.

Using this protocol, as shown in Fig. 5A, there is a rapid release of Ca2+, with no significant change of membrane potential. This was followed by a sustained depolarization, maintained elevation of Ca2+ and large contraction. Fig. 5B shows these events on an expanded time scale. It can be seen that, as with CPA, action potentials then cease towards the peak of the depolarization, along with Ca2+ spikes. As the membrane started to repolarize action potentials reappeared, presumably as depolarization induced inactivation of L-type Ca2+ channels is removed, and these were associated with rises of Ca2+ and small increases of force (Fig. 5A). After about 3–4 min there was complete repolarisation of the cell membrane and termination of the action potentials and Ca2+ transients and a relaxation of force.

Fig. 5

Changes in electrical activity, Ca2+ transients and force induced by brief (5 s) application of CCh in Fluo-4 loaded uterine smooth muscle bundles. (A) and (B) Changes in electrical activity (bottom trace), Ca2+ transients (middle trace) and force (top trace) induced by CCh on normal and expanded time scale, respectively; (C) Typical records of CCh-induced Ca2+ transients in control, and in the presence of nifedipine and La3+.

Thus at a time when the carbachol receptor is not activated, but the store has released Ca2+ in response to agonist, Ca2+ entry and depolarization are stimulated, resulting in contraction. Application of nifedipine under these conditions, i.e. brief application of carbachol, had no effect on the Ca2+ release from the SR (Fig. 5C) but had a small effect on the sustained component of Ca2+ entry. Data obtained with La3+ showed that it significantly inhibited this sustained component of the nifedipine resistant Ca2+ transient induced by carbachol (Fig. 5C).

Discussion

The control of uterine contractility is paramount for successful timing of parturition and delivery of the baby. The importance of electrical activity to both the development and coordination of uterine contractions is appreciated, but the interaction between the SR and electrical activity is not understood. This in turn had limited understanding of the role of the SR and its luminal Ca2+ content in Ca2+ signalling in the myometrium. Consistent with previous studies we found that depletion of SR luminal Ca2+ increases contractile activity in rat myometrium. We show that this is due to low luminal Ca2+ stimulating plasmalemmal Ca2+ entry predominantly via store operated entry, and crucially, that this causes depolarization. We demonstrated this by depleting SR Ca2+ in two ways; inhibition of SERCA with CPA to promote Ca2+ leak from the SR, and agonist application to produce IP3 and Ca2+ release through IP3R channels. In addition we show how the changes in membrane potential and action potential discharge frequency can explain the complex pattern of changes in both the Ca2+ signal and force, as the SR depletes of Ca2+. These findings increase our understanding of how agonists act to modulate electrical activity and force, and by revealing differences from other smooth muscles, especially vascular, reveal potential targets for new and specific therapies to control uterine activity.

Our confocal recordings of Ca2+ in intact myometrial strips confirmed our previous finding of the lack of Ca2+ sparks in myometrium under any conditions i.e. with SR loaded with Ca2+, with agonist present and during large and sustained depolarization and Ca2+ entry [9]. In addition this technique was able to show that SR Ca2+ release is sufficient to produce small local rises in Ca2+, which are undetected by global measurements of Ca2+ obtained with photometric systems. These local rises were not coordinated and did not produce detectable changes in uterine force. This demonstrates the need for depolarization to synchronize Ca2+ entry, which is spread via gap junctions to produce coordinated cross bridge cycling and contraction [23], [24].

The importance of the SR in Ca2+ signalling and contraction has been elucidated in several smooth muscles, with details of its contribution to agonist stimulation and excitability being understood [25]. For example we have shown that increases in luminal SR Ca2+ following a stimulus, leads to an increase in Ca2+ sparks and activates large Ca2+-activated K+ channels (BK) in ureteric myocytes [26]. This causes membrane hyperpolarization and the relative refractory period in the ureter [18]. In several vascular preparations it has been shown that SR Ca2+ releases activate BK channels, promote hyperpolarization and reduce tone [27], [28], [29]. These links between the SR and excitability in smooth muscle are considered to be functionally important and affected by disease such as hypertension and dyslipedaemia [25], [30]. In the myometrium the contribution of the SR to both function and excitability has been more elusive to uncover [24]. As a Ca2+ store in the myometrium, possessing both ryanodine (RyR) and IP3 receptors [16], agonist stimulated SR Ca2+ releases and augmentation of force, were assumed to be the SR role, along with being a sink for Ca2+ during relaxation [31]. Direct measurements of luminal Ca2+ content in isolated uterine myocytes demonstrated that agonists produced Ca2+ releases, sufficient to deplete most of the Ca2+ store and activate SERCA [22]. Investigations on rat and then human myometrium however failed to demonstrate anything other than, at best, a tiny role for Ca2+-induced Ca2+ release (CICR) [6]. Thus the model developed in cardiac muscle, whereby a small amount of L-type Ca2+ entry triggers a substantial rise of cytoplasmic Ca2+ via RyR-mediated SR Ca2+ release, and produces contraction, could not be supported in the myometrium. Subsequent findings that the RyR expressed in the myometrium are non-functional splice variants of RyR3 [32], and that there were no Ca2+ sparks in myometrial myocytes [9], provided an explanation for the lack of effective CICR in myometrium, as demonstrated in intact tissue. The question therefore remained, what mechanism underlies the increase in force and Ca2+ signalling observed when CPA is applied to the myometrium? Although BK channels are expressed in myometrium [33], as well as small conductance Ca2+-activated K+ channels (SK) [34], the lack of Ca2+ sparks in myometrium removes one of their major routes of activation, although some activation via Ca2+ entry remains possible [9], [35]. However pharmacological and functional studies from our group [34] and others [36], show only modest effects on force and membrane potential, when these channels are inhibited in the myometrium. Thus as noted in recent reviews [10], [37] it was not possible to give a rigorous explanation for the effects of SR Ca2+ release on uterine force.

As Ca2+ sensitization i.e. alteration of the relationship between force and [Ca2+] appears not to be marked in myometrium under physiological conditions (e.g. [24]), changes in excitability i.e. membrane potential appeared the most likely cause of increased Ca2+ and force with SR Ca2+ depletion. Making measurements of electrical activity in intact myometrium is technically challenging, due to the highly contractile nature of the tissue, and hence there are not extensive studies in the literature [1], [2], [3], [38], [39]. Without the insight provided by making these measurements and simultaneously determining the related force or Ca2+ changes, direct interpretation of data fundamental to myometrial physiology, such as role of the SR Ca2+ store, cannot be made. We used the sucrose gap method of determining membrane potential changes [18], [19]. This method has the disadvantage that absolute measurements of potential are not made but has the considerable advantage that impalement of intracellular electrodes do not have to maintained over periods of intense contractile activity. As seen in the figures, good fidelity recordings were achieved, that enabled burst of action potentials over a range of sizes and membrane potential changes. Our findings of a depolarization with CPA is consistent with the report of Gravina et al. [5], who using microelectrodes reported a mean depolarization with 10 μM CPA of 22 mV, which was reduced to just 3 mV when nifedipine was used to block voltage-gated Ca2+ channels.

Our data show and explain the complex interactions between SR Ca2+ release and depletion and electrical activity in the myometrium with agonist binding to G protein-coupled receptors, and support the following interpretation. Upon agonist binding and IP3 production there is Ca2+ release from the SR. This lowers luminal SR Ca2+ and stimulates Ca2+ entry, and depolarization. Our data with nifedipine shows that although it abolishes action potentials, it does not affect membrane potential and only a small part (about 25-35%) of the sustained Ca2+ entry via L-type Ca2+ channels. Our data therefore suggests that the majority of the entry is through either store or receptor operated Ca2+ channels. Both sorts of channels are present in myometrium [24] and recent data has confirmed expression of the necessary components for SOCE, STIM and Orai proteins, in myometrium [11]. Our data with brief agonist application (5s), sufficient to release Ca2+ from the SR but not instigate further signalling pathways, such as receptor operated channels, show that it is store operated Ca2+ entry that is activated and underlying the rise of Ca2+ stimulated when the SR is depleted. This entry produces depolarization, and explains the return of the membrane potential to resting levels, when this entry is blocked. The depolarization resulting from SOCE will cause a sustained rise of Ca2+ and once the depolarization reaches their threshold, will trigger opening of L-type Ca2+ channels and action potentials. We suggest that it is this interplay between on the one hand SR Ca2+ depletion and plasma membrane Ca2+ entry and depolarization, and on the other, store refilling, signal termination and repolarization, that leads to the activation of action potential bursts size and oscillations of intracellular Ca2+ and force. The properties of the L-type Ca2+ channels in myometrium will also shape the changes in Ca2+ and force, as they are initially opened by the depolarization produced by SOCE, but then begin to inactivate [6]. Inactivation of these channels in myometrial cells is both voltage and Ca2+ dependent [40].

In summary, this paper shows that the SR contributes to agonist stimulation of force in the myometrium by IP3 gated Ca2+ release lowering luminal Ca2+ and stimulating Orai-stim mediated SOCE. This Ca2+ entry can produce a sustained depolarization, rise of Ca2+ and force, upon which evoked opening and then closing of L-type Ca2+ channels, produce bursts of action potentials, Ca2+ spikes and phasic contractions.