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Contribution of sarcoplasmic reticular calcium to smooth muscle contractile activation: gestational dependence in isolated rat uterus

Michael J. Taggart and Susan Wray

Received 2 February 1998; accepted after revision 17 April 1998.

	ABSTRACT

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1. The contribution of Ca²⁺ released from the sarcoplasmic reticulum (SR) to smooth muscle contractile activation remains poorly understood. By simultaneously monitoring cytosolic [Ca²⁺] ([Ca²⁺]_i) and force in isolated rat uterine smooth muscle, we report the influence of SR Ca²⁺ release on contractility during conditions (a) of altered SR Ca²⁺ homeostasis and (b) where the only source of activating Ca²⁺ was derived from the SR.

2. In myometria of non-pregnant rats, ryanodine (1-50 µM), a modulator of calcium-induced calcium release (CICR), had no effect on the spontaneous [Ca²⁺]_i or force transients. However, depletion of SR Ca²⁺ by inhibiting the SR Ca²⁺-ATPase (with cyclopiazonic acid (CPA), 20 µM) resulted in an enhancement of spontaneous [Ca²⁺]_i and force transients.

3. In myometria of pregnant rats, although ryanodine had no effect in 40 % of tissues studied it produced a small but significant enhancement of the integrated spontaneous [Ca²⁺]_i and force transient in 60 % of cases. The potentiating effects of CPA were enhanced in myometria of pregnant rats compared with non-pregnant rats, often resulting in maintained [Ca²⁺]_i increases and contraction.

4. In zero external Ca²⁺, agonist-induced SR Ca²⁺ release resulted in transient increases in [Ca²⁺]_i and force. The magnitude of these agonist-induced [Ca²⁺]_i and force changes were significantly enhanced in myometria of pregnant rats. No evidence for agonist-induced Ca²⁺-independent force production was observed.

5. These results indicate that CICR plays little role in SR Ca²⁺ release from the myometrium, and that there are gestational-dependent alterations in the ability of SR Ca²⁺ mobilization to contribute to contractile activation. The implications of these findings for the co-ordination of myometrial [Ca²⁺]_i signalling and contractility are discussed.

	INTRODUCTION

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The phasic spontaneously generated contractions of uterine smooth muscle are critically dependent on extracellular Ca²⁺ influx (Taggart et al. 1996). There is controversy, however, as to whether release of sarcoplasmic reticular (SR) Ca²⁺ plays a significant role in controlling uterine contractility following stimulation. Agonist stimulation can result in IP₃ production and mobilization of SR Ca²⁺ (Anwer et al. 1993) and myo-inositol 1,4,5-trisphosphate (IP₃) introduction to permeabilized myometrial fibres results in transient contractions (Kanmura et al. 1988; Izumi et al. 1996). Thus it appears that agonist-induced Ca²⁺ release (IICR) can influence uterine force production. The importance of Ca²⁺-induced Ca²⁺ release (CICR), however, has been questioned, both in the uterus and other smooth muscles. Evidence for CICR during depolarizations of voltage-clamped isolated myocytes under near-physiological conditions was reported for some smooth muscles (Ganitkivech & Isenberg, 1992; McCarron & Kamishima, 1997) but was absent in others (Ganitkivech & Isenberg, 1995; Kamishima & McCarron, 1996). Caffeine failed to elevate [Ca²⁺]_i in cultured uterine cells (Lynn et al. 1993; Arnaudeau et al. 1994a; Holda et al. 1996), and did not increase force of permeabilized or intact fibres (Kanmura et al. 1988; Savineau & Mironneau, 1990). Furthermore others have reported a lack of [Ca²⁺]_i or force response to ryanodine (Laszlo et al. 1992; Arnaudeau et al. 1994a; Zhuge & Hsu, 1995; Holda et al. 1996) in myometria. Ryanodine, however, was found to elevate [Ca²⁺]_i in cultured cells (Lynn et al. 1993), and to promote release of SR Ca²⁺ from pre-loaded permeabilized cells (Awad et al. 1997). Also, mRNAs encoding ryanodine receptor isoforms have been identified in uterine tissues (Principe et al. 1997; Awad et al. 1997).

Difficulties in interpreting the above data partly arise because of the variety of experimental situations employed, the separate measurements of [Ca²⁺]_i and force, and extrapolating findings from phenotypically altered cultured cells (Awad et al. 1997) to intact contractile tissue. Given the importance to labour of discerning [Ca²⁺]_i signalling and contraction in uterine smooth muscle, it is clearly essential that the contributory role of the SR, and in particular any CICR mechanism, is thoroughly understood. The aim of the present study was, therefore, to simultaneously measure [Ca²⁺]_i and force in uterine smooth muscle in order, for the first time, to directly assess the contribution of Ca²⁺ released from the SR to contractile activation during conditions of altered SR Ca²⁺ handling. We wished to address two particularly important questions. Firstly, during manoeuvres that interfere with SR Ca²⁺ homeostasis what happens to [Ca²⁺]_i and force in intact myometrium? Secondly, are there gestational-dependent differences in the ability of SR Ca²⁺ to contribute to myometrial contractile activation?

Part of this work has been presented to The Physiological Society (Taggart & Wray, 1997a).

	METHODS

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Small strips of longitudinal myometria (1·0 mm long, < 0·5 mm wide, 0·1 mm thick) were dissected from either virgin adult Wistar rats (approximately 250 g) or 20- to 21-day pregnant rats (term is 21 days) that had been killed by cervical dislocation following CO₂ anaesthesia.

Details of the tissue preparation and measurement of [Ca²⁺]_i are described by Taggart et al. (1997). Briefly, muscle strips were incubated for 4 h at room temperature (22-23°C) in Krebs solution (composition (mM): 154 NaCl, 5·4 KCl, 1·2 MgSO₄, 12 glucose, 2 CaCl₂, 10 Hepes; pH 7·4 with NaOH) with 20 µM of the membrane-permeant form of the Ca²⁺-sensitive dye indo-1(Molecular Probes). Strips were then mounted in a small (200 µl) chamber on a Nikon Diaphot inverted microscope and viewed with a × 10 Fluor objective. A metal hook was attached to each end of the muscle strips with one hook fixed to a tension transducer (Grass FT 03) and superperfused with oxygenated Krebs solution at a fast flow rate (6 ml min^-1). Bath volume exchange was complete within 3 s. Tissue was excited (75 W xenon arc lamp) with light of wavelength 340 nm and light emitted at wavelengths 400 nm and 500 nm was recorded via photomultipliers and digitally recorded (Cairn, UK). The ratio of 400 nm : 500 nm emissions was used as an indicator of [Ca²⁺]_i, rather than calibrated absolute values of [Ca²⁺]_i, for reasons detailed previously (Taggart et al. 1997). In all experiments changes in the indo-1 ratio were accompanied by shifts in the opposite direction of 400 nm and 500 nm emission signals. As previously reported for rat myometrial tissues (Taggart et al. 1996, 1997) changes in the indo-1 ratio with cell stimulation preceded force at all time points and contractile activity did not change steady-state tissue autofluorescence intensity.

Experiments examining the influence of modulators of SR function on spontaneous contractions were performed at 37°C. Tissues were equilibrated in Krebs solution for at least 1 h until stable spontaneous [Ca²⁺]_i and force transients were obtained. The effects of caffeine and agonists on SR releasable Ca²⁺ were performed in 0 Ca²⁺ solution (Krebs solution in which CaCl₂ had been omitted and 1 mM EGTA added) at 25°C with the tissues having been equilibrated in Krebs solution for at least 1 h. The protocol for examining the effects of caffeine or agonists in 0 Ca²⁺ solution was as follows: from Krebs solution, the tissue was stimulated for 1 min with a high K⁺ depolarizing solution in which the concentration of KCl was raised to 40 mM by isosmotic replacement of NaCl. [Ca²⁺]_i and force were allowed to recover to pre-stimulated levels and the tissue then bathed in 0 Ca²⁺ solution. Two minutes later, myometrial samples were stimulated with caffeine (10 mM), carbachol (100 µM) or oxytocin (100 nM). In some experiments, repeated exposures to agonists in 0 Ca²⁺ solution were performed. When the ensuing changes in [Ca²⁺]_i and force had returned to pre-stimulation levels, the tissue was returned to Ca²⁺-containing Krebs solution.

All chemicals were obtained from the Sigma Chemical Company (UK) except ryanodine, cyclopiazonic acid (CPA) and iberiotoxin (IbTX; all from Calbiochem, UK).

n refers to the number of animals. Where identical experiments were performed on tissues from the same animal, the data were averaged. Values are expressed as means ± S.E.M. P values of < 0·05 are taken as significant using the appropriate Student's (paired or unpaired) t test.

	RESULTS

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Can caffeine increase force or [Ca²⁺]_i in the uterus?

We investigated whether caffeine could release SR Ca²⁺ and thereby influence myometrial [Ca²⁺]_i and force, in 0 Ca²⁺ solution in order to prevent any effects on transarcolemmal Ca²⁺ entry (Guerrero et al. 1994). Following a control stimulation in high-K⁺ solution, the bathing solution was changed to 0 Ca²⁺ solution. Caffeine (10 mM) was unable to significantly increase [Ca²⁺]_i or force of myometria from non-pregnant (n = 4) or pregnant (n = 4) rats (Fig. 1). This lack of caffeine responsiveness was not due to depletion of the SR Ca²⁺ store as, under identical experimental conditions, carbachol or oxytocin stimulation gave transient increases in [Ca²⁺]_i and force (see later, Figs 5-8).

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Figure 1. Effect of caffeine on SR Ca2+ release and force Simultaneous force (top) and Ca2+ (indo-1 ratio, bottom) recordings showing the effects of high K+ and 10 mM caffeine in myometria from non-pregnant (A) and pregnant (B) rats. Caffeine (Caf; 10 mM) was applied after 2 min exposure to 0 Ca2+ solution.

Can modulators of SR function alter myometrial spontaneous force and [Ca²⁺]_i transients?

The effects of ryanodine on myometria of non-pregnant and pregnant rats. Cumulative application of a range of ryanodine concentrations to myometria of non-pregnant rats failed to alter the profile of either the [Ca²⁺]_i transients or accompanying spontaneous force transients (n = 3; Fig. 2). In an additional series of experiments the effect of 20 µM ryanodine, a concentration which induced a long-lasting sub-conductance state of myometrial SR microsomes when reconstituted into voltage clamped planar lipid bilayers (Martin & Ashley, 1995), was studied in detail. Ryanodine was superfused for at least 20 min to allow any time- and use-dependent action of the compound to be effective. The [Ca²⁺]_i and force transients from three contraction-relaxation cycles before and during prolonged ryanodine application were then averaged and compared as shown in Fig. 3A and B. Ryanodine did not significantly affect the mean integrals of [Ca²⁺]_i (106 ± 5·2 % of control; n = 7) or force (108 ± 3·0 %) transients as shown in Fig. 3C. Additionally, ryanodine had no effect on the spontaneous [Ca²⁺]_i transient amplitudes or durations (106 ± 7·4 and 102 ± 2·9 % of control, respectively) nor the force transient amplitudes or durations (100 ± 0·3 and 98 ± 1·9 % of control, respectively).

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Figure 2. Effect of ryanodine on non-pregnant uterus Spontaneous force and [Ca2+]i transients of myometrium of non-pregnant rat, recorded continually in the presence of cumulative and sequential superfusions of ryanodine (1, 10 and 50 µM).

The effect of ryanodine (20 µM, for at least 20 min) was also examined in myometrium from pregnant rats. In 40 % of cases no changes in [Ca²⁺]_i or force transient profiles were seen; in the remaining 60 %, an enhancement of [Ca²⁺]_i and force transients, as illustrated in the example of Fig. 3 (right panels), was observed although the effects of ryanodine on the amplitude or duration of [Ca²⁺]_i and force transients were not significant. However, as shown in Fig. 3B (right panel), a small significant enhancement of the integrals of both the mean [Ca²⁺]_i (134 ± 15 %; n = 11) and force (123 ± 6·8 %) transients was produced. As in the case of myometria from non-pregnant rats, ryanodine did not affect the half-time for decay of [Ca²⁺]_i (7·6 ± 0·8 s for ryanodine compared with 7·6 ± 0·8 s for control) or force (6·2 ± 0·9 s for ryanodine compared with 6·5 ± 1·2 s for control) transients of myometria of pregnant rats.

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Figure 3. Effects of ryanodine and CPA on spontaneous [Ca2+]i and force transients A, effect of 20 µM ryanodine on spontaneous [Ca2+]i and force transients, from non-pregnant (left panels) and pregnant (right panels) rats. Axis break represents 20 min. B, [Ca2+]i and force transients of 3 contraction-relaxation cycles in control conditions (i) and in ryanodine (ii), denoted by dashed boxes in A, were averaged and superimposed relative to control. C, integrals of the mean [Ca2+]i and force transients of B are plotted with respect to control (100 %). D, effect of cyclopiazonic acid (CPA, 20 µM) on spontaneous force and Ca2+ transients. The mean data of the integrated mean [Ca2+]i and force transients of contraction-relaxation cycles in control conditions and in the presence of CPA are shown.

The effects of cyclopiazonic acid on myometria of non-pregnant and pregnant rats. Cyclopiazonic acid (CPA) is a specific inhibitor of the myometrial SR Ca²⁺-ATPase (Kosterin et al. 1996), and was therefore used to investigate further the role of the SR in cellular function. Application of CPA (20 µM) to myometria of non-pregnant rats, either alone or in addition to 20 µM ryanodine (Fig. 3D), resulted in several clear changes: (i) basal [Ca²⁺]_i was increased to 57 ± 12 % (n = 6) of maximal [Ca²⁺]_i during control spontaneous events, without altering basal force; (ii) the amplitudes (115 ± 4·5 %) and durations (130 ± 5·0 %) of spontaneous contractions were enhanced. Superimposing the mean [Ca²⁺]_i and force transients with control responses illustrates that these changes in force were accompanied by a prolongation of the [Ca²⁺]_i transient in the presence of CPA (132 ± 7·7 %); (iii) the integrals of the mean [Ca²⁺]_i and force transients (140 ± 15 and 189 ± 16 %, respectively) were also enhanced by CPA. The half-time for decay of the spontaneous [Ca²⁺]_i (t0·5) or force (t₅₀) transients were unaltered by either CPA (t0·5 for [Ca²⁺]_i decay 5·8 ± 0·8 s in CPA cf. 5·4 ± 1·0 s; t₅₀ force decay 8·3 ± 2·8 s cf. 11 ± 5·9 s) or by ryanodine (t₅₀ for [Ca²⁺]_i decay 6·8 ± 1·2 s in ryanodine cf. 6·2 ± 1·2 s; t₅₀ for force decay 6·3 ± 0·9 s in ryanodine cf. 7·4 ± 1·1 s). Thus, in non-pregnant animals, although spontaneous myometrial contractile events showed no ryanodine sensitivity, interference of SR function with CPA altered the [Ca²⁺]_i and force transient profiles.

CPA application also resulted in profound changes in spontaneous contractility of myometria from pregnant rats. Basal [Ca²⁺]_i was increased in all tissues to 98 ± 12 % of the spontaneous [Ca²⁺]_i transient amplitude (n = 12). This resulted in an increase in basal tone which, in 75 % of cases, resulted in a cessation of spontaneous contractions (for example, Fig. 3D). The magnitude of maintained contractions induced by CPA were 67 ± 13 % of spontaneous contractile amplitude before CPA addition.

The effects of K⁺ channel blockade. Recently, it has been suggested that elementary spontaneous calcium release from the SR of vascular smooth muscle via ryanodine receptors (Ca²⁺ sparks) results in activation of Ca²⁺-activated K⁺ currents (IK(Ca)) and cell hyperpolarization. Thus, CICR in smooth muscle may act as a feedback mechanism to limit contractility (Nelson et al. 1995). Depletion of SR Ca²⁺ was shown to depolarize and contract vascular smooth muscle in a manner similar to agents which inhibited IK(Ca) (Nelson et al. 1995). We, therefore, investigated the influence of I_K(Ca) inhibition on the CPA-induced changes in [Ca²⁺]_i and force in myometria of pregnant rats, as anothers means to test for CICR.

TEA (1-5 mM), a blocker of K_Ca channels, resulted in a prolongation of the spontaneous [Ca²⁺]_i transients and a consequent enhancement of phasic contractile amplitude and duration (Fig. 4A) without altering basal force. TEA did not, however, prevent the further potentiatory actions of CPA, and both [Ca²⁺]_i and force increased to maintained levels under these circumstances (Fig. 4; n = 4). Iberiotoxin (IbTX; 40 nM), a more potent inhibitor of I_K(Ca), also enhanced Ca²⁺ and force transients, but like TEA did not prevent the potentiatory effects on [Ca²⁺]_i and force of CPA application (Fig. 4B;n = 2). TEA did not alter the effects of ryanodine (n = 5, data not shown).

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Figure 4. The effects of IK(Ca) inhibitors on spontaneous [Ca2+]i and force transients of myometria from pregnant rat A, application of 5 mM TEA and then, in the continued presence of TEA, application of CPA (20 µM). B, application of iberiotoxin (IbTX, 40 nM) and then, in the continued presence of IbTX, CPA (20 µM).

What is the influence of agonist-induced SR Ca²⁺ release on [Ca²⁺]_i and force production?

The above data support little role for CICR from the SR in excitation-contraction coupling during spontaneous depolarizations, particularly in uterus from non-pregnant rats. We also wished to assess the influence of agonists on SR Ca²⁺ release and force production in intact tissues. As agonists can also stimulate Ca²⁺ entry, the experiments were performed in Ca²⁺-free solution to ensure that the Ca²⁺-force relationship was examined when Ca²⁺ release from the SR was the only source of activating Ca²⁺. In order to compare these Ca²⁺ and force responses with those under different physiological conditions, and when IP₃-dependent SR Ca²⁺ release was not operative, a high K⁺ depolarization (2 mM external Ca²⁺) was always also performed.

When the only source of activating Ca²⁺ was derived from the SR, agonist stimulation of myometrial tissues induced transient [Ca²⁺]_i and force changes (Fig. 5). Both these responses were inhibited by prior incubation with CPA (Fig. 5A,n = 4) but were unaffected by the voltage-operated Ca²⁺ channel blockers nifedipine (100 µM; Fig. 5B, n = 3) and ryanodine (20 µM; Fig. 5C, n = 4).

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Figure 5. Agonist-induced SR Ca2+ release and force production in myometria of non-pregnant rats [Ca2+]i and force records in response to exposure to high K+ and then carbachol applied 2 min after exposure to 0 Ca2+ solution followed by: A, CPA (axis break denotes 20 min superfusion in the presence of CPA); B, 100 µM nifedipine (axis break denotes 5 min in the presence of nifedipine); C, ryanodine (axis break denotes 20 min superfusion in the presence of ryanodine).

In addition to elevating [Ca²⁺]_i, agonist stimulation modulates smooth muscle force production by increasing the sensitivity of the myofilaments to Ca²⁺ (Somlyo & Somlyo, 1994). We therefore compared the normalized [Ca²⁺]_i and force profiles during agonist stimulation with the normalized responses in the same tissues to high K⁺ depolarization. Figure 6 represents examples of the resultant plots of normalized ratios versus force for myometria of non-pregnant (Aa-d) and pregnant (Ba-d) animals. It is clear that as [Ca²⁺]_i declines to pre-stimulation levels (indicated by the downward arrows in the hysteresis plots) force remains elevated for a longer period following agonist stimulation than following high K⁺ stimulation. The mean data (n = 20 for non-pregnant rats and n = 14 for pregnant rats) in Fig. 6Ad and Bd illustrate that, when normalized [Ca²⁺]_i had declined by 75 or 100 % from peak stimulation levels, normalized force was significantly greater following agonist stimulation (89 ± 3·3 and 55 ± 8·3 %, respectively, for non-pregnant rats; 70 ± 2·9 and 43 ± 5·6 %, respectively, for pregnant rats; paired t test) compared with that following high K⁺ stimulation (53 ± 5·2 and 10 ± 1·7 %, respectively, for non-pregnant rats; 53 ± 6·7 and 21 ± 7·2 %, respectively, for pregnant rats). In myometria of pregnant rats, such significant differences in force maintenance were apparent when [Ca²⁺]_i had declined from peak stimulation levels by only 50 % (normalized force following carbachol stimulation was 93 ± 2·8 cf. 66 ± 5·2 % following high K⁺ stimulation; Fig. 6Ad and Bd). A further indication that gestational-dependent alterations of SR function exist is illustrated by the data of Fig. 6Ae and Be. Carbachol-induced [Ca²⁺]_i transient amplitudes were greater in myometria of pregnant rats (62 ± 7·6 cf. 42 ± 2·5 % in non-pregnant rats in relation to high K⁺-induced [Ca²⁺]_i changes) as were the [Ca²⁺]_i transient durations (1·4 ± 0·1 cf. 1·1 ± 0·01 min in non-pregnant rats). These changes were accompanied by an approximate doubling of the force transient amplitudes (96 ± 10 cf. 46 ± 3·8 % in non-pregnant rats in relation to high K⁺-induced force changes) and durations (4·0 ± 0·3 cf. 2·5 ± 0·2 min in non-pregnant rats).

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Figure 6. Comparison of the agonist-induced SR Ca2+ release and force responsiveness in myometria of non-pregnant (A) and pregnant rats (B) a, sample traces of carbachol-induced [Ca2+]i and force transients. The [Ca2+]i and force responses in a to high K+, or carbachol in 0 Ca2+, stimuli are normalized and plotted against each other in b and c. The direction of the arrows reflects whether [Ca2+]i was increasing or decreasing. d, normalized [Ca2+]i-force plots of the mean data (n = 14-20). * Significantly elevated force in carbachol compared with KCl for a normalized [Ca2+]i change (P < 0·05, paired t test). e, changes in carbachol-induced [Ca2+]i and force amplitudes (as a % of those obtained in high-K+ solution) and durations are compared for myometria of non-pregnant () and pregnant () rats. * P < 0·05.

Store refilling following agonist application. In myometria of non-pregnant rats we found that one brief (30 s) application of carbachol after 1·5-2 min in 0 Ca²⁺ solution was sufficient to mobilize all the releasable Ca²⁺ (n = 6). Subsequent applications of agonist altered neither [Ca²⁺]_i nor force (Fig. 7). Increasing the time spent in 0 Ca²⁺ solution (3·5-4 min) before applying the first carbachol stimulus in 0 Ca²⁺ solution reduced the magnitude of the increases in [Ca²⁺]_i and force, but significant changes were still evident 4-5 min after 0 Ca²⁺ perfusion (Fig. 7). Thus, time-dependent run-down of the SR Ca²⁺ store could not explain the lack of responsiveness to multiple agonist stimuli in 0 Ca²⁺ solution. Increasing the duration of the carbachol stimuli did not alter the [Ca²⁺]_i or force profiles (n = 4). Figure 7B illustrates the experimental manoeuvres performed in the same tissue whereby carbachol was applied in 0 Ca²⁺ solution for 1 or 4 min. Superimposing the responses demonstrates the identical [Ca²⁺]_i and force profiles under each condition. Such responses were not specific for muscarinic receptor stimulation as one brief application of oxytocin was also sufficient to activate all the SR releasable Ca²⁺ and produce force (Fig. 7C). Subsequent oxytocin exposures did not alter [Ca²⁺]_i or force.

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Figure 7. The effects on [Ca2+]i and force of myometria of non-pregnant rats of varying the time and frequency of agonist exposure in 0 Ca2+ A, the left-hand panel illustrates that one brief (30 s) exposure to carbachol transiently increases [Ca2+]i and force whereas subsequent exposures are ineffective. In the right-hand panel, the tissue was allowed to recover (axis break denotes 5 min) and a first carbachol stimulus was given in 0 Ca2+ at the same time point as the third exposure in the left-hand panel. B, enlarged representation of the [Ca2+]i and force responses, in the same tissue, to a 1 or 4 min carbachol stimulation. Inset, the 4 min responses (dashed lines) are superimposed on the 1 min response. C, a first exposure to oxytocin in 0 Ca2+ transiently increases [Ca2+]i and force whereas a second exposure is ineffective.

Similar responses were seen in myometria of pregnant rats where only one brief application of either carbachol (Fig. 8A and B) or oxytocin (Fig. 8C) was effective in releasing Ca²⁺ and activating force, and subsequent agonist applications failed to alter either parameter. Thus, in myometria of both non-pregnant and pregnant rats, there was no evidence for agonist-induced Ca²⁺-insensitive force production.

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Figure 8. The effects on [Ca2+]i and force from myometria of pregnant rats of repeated agonist exposure in 0 Ca2+ solution A, following a first brief (30 s) exposure to carbachol, which transiently increased [Ca2+]i and force, subsequent brief exposures were ineffective. B, following the [Ca2+]i and force transients in response to an initial brief (30 s) carbachol stimulation, a subsequent longer (3 min) carbachol application had no effect. C, initial exposure to oxytocin in 0 Ca2+ transiently increased [Ca2+]i and force but a prolonged subsequent exposure was ineffective.

	DISCUSSION

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Perhaps the most surprising finding of this study is that no role for CICR could be detected in the uterus of non-pregnant animals. Agonist could produce significant increases in force and [Ca²⁺]_i, from the SR, in the absence of external Ca²⁺. Thus we suggest that IICR is the major process releasing Ca²⁺ from the SR in the rat uterus. Disruption of SR Ca²⁺-ATPase activity (by CPA) markedly affected [Ca²⁺]_i. Our data also show differences between responses in tissues taken from pregnant and non-pregnant animals. This strongly suggests that the SR is modulated by gestational status in the uterus, and that this affects its ability to act as a source of Ca²⁺ for contraction.

By simultaneously monitoring [Ca²⁺]_i and force in longitudinal myometria we have circumvented the interpretative problems associated with previous studies, where either [Ca²⁺]_i or force were measured, which, particularly with reference to the involvement of CICR to uterine excitation- contraction coupling, had contributed to the equivocal conclusions of the findings. By using an intact preparation, rather than cultured cells, it is also easier to extrapolate this data to the in vivo functioning of the uterus.

Caffeine, at a concentration that released SR Ca²⁺ in other smooth muscles (Ganitkivech & Isenberg, 1992), did not alter [Ca²⁺]_i or force in myometria of non-pregnant or pregnant rats even under identical conditions whereby agonists mobilized SR Ca²⁺ and activated contraction. This is in agreement with previous studies in which caffeine failed to alter [Ca²⁺]_i in isolated cells (Lynn et al. 1993; Arnaudeau et al. 1994a) or increase force in multicellular preparations (Savineau & Mironneau, 1990) but where agonist stimulation, or IP₃ application, could (Kanmura et al. 1988; Anwer et al. 1993). Ryanodine was also unable to alter either the [Ca²⁺]_i transient profile associated with spontaneous contractile activity or the [Ca²⁺]_i and force transient responses to agonist activation in 0 Ca²⁺ solution, in myometria of non-pregnant rats. A similar lack of ryanodine sensitivity of agonist-induced SR Ca²⁺ release has been reported in cultured rat and human uterine cells (Arnaudeau et al. 1994a; Holda et al. 1996). CPA, a potent inhibitor of the SR Ca²⁺-ATPase pump (Kosterin et al. 1996), resulted in depletion of SR releasable Ca²⁺, as demonstrated by the inhibition of agonist-induced [Ca²⁺]_i and force transients in the absence of transarcolemmal Ca²⁺ influx. In spontaneously active myometria of non-pregnant rats, CPA application resulted in an increase in basal [Ca²⁺]_i, an effect also observed in bladder smooth muscle (Munro & Wendt, 1994), a prolongation of the spontaneous [Ca²⁺]_i transient duration and, consequently, an increase in the spontaneous contractile amplitude and duration. Neither ryanodine nor CPA altered the half-times for decay of [Ca²⁺]_i or force transients. Therefore, as in stomach smooth muscle (McGeown et al. 1996), the SR of uterine smooth muscle appears not to contribute to [Ca²⁺]_i removal and relaxation following membrane depolarization. Thus, the lack of caffeine and ryanodine sensitivity, coupled with the enhancement of [Ca²⁺]_i and force transients when SR Ca²⁺ is depleted by inhibition of the SR Ca²⁺-ATPase, suggests that the [Ca²⁺]_i increase is derived from sarcolemmal Ca²⁺ entry, and the resultant contractions are not amplified by a CICR mechanism in myometria of non-pregnant rats. SR Ca²⁺ appears to be released by a purely IICR-like mechanism.

In myometria of pregnant rats, ryanodine produced a small but significant enhancement of the integrated spontaneous [Ca²⁺]_i and force transients although in 40 % of tissues no ryanodine sensitivity was observed. We are unclear as to why some tissues were unaffected by ryanodine, but would suggest that variation in the closeness to parturition could be a likely explanation. The effect of ryanodine, but never caffeine, may reflect a caffeine-insensitive ryanodine-sensitive mechanism reported in cultured human uterine tissue (Lynn et al. 1993). Although the localization and abundance of myometrial SR Ca²⁺ release channels under different gestational states are not yet established, mRNA encoding two ryanodine receptor isoforms (RyR2 and RyR3) have been reported in rat uterine tissue (Principe et al. 1997; Chen et al. 1997). However, the recent work of Chen et al. (1997) reported that RyR3 isolated from rabbit uterine tissue (expressed in a non-muscle cell line) exhibited strong caffeine sensitivity. Thus in native myometrial cells there may be an as yet unidentified modulator of RyR altering the caffeine sensitivity. It should be noted, however, that in mice lacking RyR3 expression there was no change in Ca²⁺ sensitivity of vascular smooth muscle force production when compared with wild-type mice (Takeshima et al. 1996).

The effects of CPA were markedly different in myometria of pregnant rats compared with non-pregnant rats. Namely, CPA increased basal [Ca²⁺]_i to such an extent that, in the majority of cases, force transients were abolished and a maintained contraction ensued. The SR volume increases with gestation (Somlyo, 1985) and, in myometria of pregnant rats, is located close to the cell periphery (Broderick & Broderick, 1990). It is possible that these structural changes produce a closer functional coupling of plasmalemmal and SR Ca²⁺ movements and thereby contribute to the gestational-dependent alterations in SR Ca²⁺ mobilization. Although the peripheral and central SR is thought to form a continuous tubular network (Nixon et al. 1994), distinct compartmentalization of ryanodine-sensitive from CPA-sensitive stores within the SR has been reported in cultured smooth muscle (Golovina & Blaustein, 1997). Such a geometric arrangement may partly explain the different ryanodine and CPA sensitivities in myometria of pregnant rats.

Recently, it has been suggested for vascular smooth muscle that elementary SR Ca²⁺ events - Ca²⁺ sparks - enhanced by transarcolemmal Ca²⁺ entry and sensitive to SR Ca²⁺ depletion by ryanodine or thapsigargin, activated I_K(Ca) channels resulting in membrane hyperpolarization (Nelson et al. 1995). With SR Ca²⁺ depletion, no such I_K(Ca) activation could occur and tissue depolarization and contraction resulted. In rat uterine smooth muscle, although Ca²⁺ sparks have not yet been reported, evidence for the presence of I_K(Ca) currents exists (Anwer et al. 1993; Pérez & Toro, 1994). Also, the resting membrane potential of myometria of pregnant rats becomes more positive throughout gestation (approximately -55 to -45 mV; Parkington & Coleman, 1990), closer to the potential for half-activation of I_K(Ca) channel currents (Pérez & Toro, 1994). We found that TEA or IbTX enhanced spontaneous contractions of myometria of pregnant rats, in agreement with Anwer et al. (1993), but that this did not prevent further enhancement of [Ca²⁺]_i and force by CPA. Furthermore, CPA alone did not transiently reduce [Ca²⁺]_i or force transients as may be anticipated if spontaneous outward currents were activated upon SR Ca²⁺ depletion (Nelson et al. 1995). Thus I_K(Ca) alteration cannot solely explain the [Ca²⁺]_i and force changes upon SR Ca²⁺ depletion by CPA. In myometria of pregnant rats, the plateau component of the action potential (approximately -30 mV; Bengtsson et al. 1984) is close to the reported equilibrium potential for chloride ions, and is critically sensitive to alteration of the chloride concentration gradient (Parkington & Coleman, 1990). It is possible, therefore, that CPA-induced SR Ca²⁺ release activates depolarizing calcium-activated chloride currents (ICl(Ca)) resulting in promotion of further Ca²⁺ influx. This notion is supported by the finding of a chloride current activated in rat myometria by SR Ca²⁺ release (Arnadeau et al. 1994b). Another possible source of the maintained [Ca²⁺]_i increase in myometria of pregnant rats following CPA application is activation of a capacitative calcium influx similar to that reported in other smooth muscles (Ohta et al. 1994). Indeed, in 73 % of tissues examined, when SR Ca²⁺ depletion was ensured by agonist activation and prolonged incubation in 0 Ca²⁺ solution, return to Ca²⁺-containing Krebs solution resulted in transient elevations of [Ca²⁺]_i and force above resting levels (data not shown). Experiments are in progress to elucidate further the membrane currents activated following myometrial SR Ca²⁺ release, and preliminary data suggest that CPA does not alter the membrane potential, but prolongs the duration of the action potential (H. W. Mankouri, Th. V. Burdyga, M. J. Taggart & S. Wray, unpublished observations).

In experimental conditions where the only source of activating Ca²⁺ was derived from the SR, agonist stimulation produced transient increases in [Ca²⁺]_i and force. Normalized plots of [Ca²⁺]_i versus force indicated that recovery of force following agonist-induced SR Ca²⁺ release lagged that following high K⁺ stimulation. This agonist sensitization of force reflects a real Ca²⁺ sensitization of the contractile filaments, as no alteration of force in the absence of [Ca²⁺]_i increases (see below) was seen. These results are the first to illustrate agonist-induced Ca²⁺ sensitization of smooth muscle force production as a consequence solely of SR Ca²⁺ release in intact tissue. It may result from increased phosphorylation of the regulatory myosin light chain seen with carbachol (Taggart et al. 1997) possibly via inhibition of phosphatase activity (Somlyo & Somlyo, 1994). The observations that IICR was prominent in myometria of both non-pregnant and pregnant rats, together with the lack of caffeine and ryanodine sensitivity in myometria of non-pregnant rats and slight ryanodine sensitivity in myometria of pregnant rats, suggests that IICR is the major mechanism of physiological SR Ca²⁺ release in this tissue. Indeed, the [Ca²⁺]_i and force responsiveness to agonist mobilization of SR Ca²⁺ increased with gestation.

Experiments performed in the absence of transarcolemmal Ca²⁺ influx illustrated that one brief application of agonist was sufficient to mobilize all the releasable Ca²⁺, in agreement with previous studies (Arnaudeau et al. 1994a, b), and force. Subsequent applications, or lengthening the duration of the initial stimulus, did not alter [Ca²⁺]_i or force. Thus in myometria of non-pregnant or pregnant rats, no evidence could be found for agonist-induced Ca²⁺-insensitive force production, in contrast to the work of others (Matsuo et al. 1989).

In summary, we have illustrated the existence of gestational-dependent changes in the ability of SR Ca²⁺ mobilization to contribute to Ca²⁺ activation of myometrial force. These changes are likely to form part of the myriad mechanisms recruited near term to co-ordinate Ca²⁺ signalling and force production, including: increased Ca²⁺ and Na⁺ current density (Inoue & Sperelakis, 1991; Mironneau, 1994) and Na⁺-Ca²⁺ exchange activity (Taggart & Wray, 1997b), each of which may result in altered SR Ca²⁺ load; increased SR volume (Somlyo, 1985); and enhanced Ca²⁺ sensitivity of force production (Izumi et al. 1996). The gestational-dependent alterations in myometrial SR Ca²⁺ store function reported here add to the expanding heterogeneous expression of functional CICR and/or IICR mechanisms in smooth muscle which can also depend upon the mode of agonist stimulation (Arnaudeau et al. 1996), tissue orientation (Kuemmerle et al. 1993) and species (Burdyga et al. 1995).

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Acknowledgements

This work was supported by a Wellcome Trust Research Career Development Fellowship (M. J. T.).

Corresponding author

S. Wray: Physiological Laboratory, University of Liverpool, Liverpool, L69 3BX, UK.

Email: s.wray{at}liverpool.ac.uk

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