The role of the sarcoplasmic reticulum as a Ca²⁺ sink in rat uterine smooth muscle cells

Abstract

1. The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1.

2. Trains of depolarizing voltage-clamp pulses increased [Ca²⁺]_i. The rate of decay of [Ca²⁺]_i was slowed by inhibition of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase with cyclopiazonic acid (CPA). However, if the sarcolemmal Na⁺-Ca²⁺ exchanger and Ca²⁺-ATPase were inhibited then recovery of [Ca²⁺]_i was abolished showing that the SR Ca²⁺-ATPase alone cannot produce decay of [Ca²⁺]_i.

3. In another series of experiments, Ca²⁺ release from the SR was induced with carbachol in a Ca²⁺-free solution. Under these conditions responses to repeated applications of carbachol could be obtained. In the presence of CPA, however, only the first application was effective. This suggests that the SR Ca²⁺-ATPase sequesters a significant amount of Ca²⁺ into the SR.

4. CPA slowed the rate of decay of [Ca²⁺]_i following carbachol addition by > 50%. Again, however, after a brief transient fall, decay was abolished when the Na⁺-Ca²⁺ exchanger and sarcolemmal Ca²⁺-ATPase were inhibited.

5. These data show that, although the SR Ca²⁺-ATPase contributes to the decay of [Ca²⁺]_i, it cannot function effectively in the absence of Ca²⁺ removal from the cell. These data are discussed in the context of the superficial buffer barrier model in which Ca²⁺ is taken up into the SR and then released very close to sarcolemmal Ca²⁺ extrusion sites, i.e. the SR acting in series with the surface membrane extrusion mechanisms. We also suggest that the amount of filling of the SR influences the rate of Ca²⁺ removal.

Contraction of smooth muscle requires an increase of [Ca²⁺]_i. This is derived from Ca²⁺ entry from the extracellular fluid, as well as Ca²⁺ release from the sarcoplasmic reticulum (SR). Correspondingly, relaxation is initiated by the removal of intracellular Ca²⁺ either to the extracellular fluid or to the SR. Transport of Ca²⁺ out of the cell is an active process that depends on the activity of the plasma membrane Ca²⁺-ATPase (PMCA) and the Na⁺-Ca²⁺ exchanger. Both have been found in uterus of different species (Kosterin et al. 1994), but their quantitative importance for Ca²⁺ extrusion from the uterine cell has only recently been examined (Shmigol et al. 1998a). The above studies suggested that both mechanisms made a significant contribution to, and together were entirely responsible for, Ca²⁺ extrusion.

An additional more complex mechanism of Ca²⁺ extrusion has, however, been proposed (van Breemen et al. 1986). It was suggested that the SR may act as a ‘superficial buffer barrier’, by taking up a fraction of the Ca²⁺ that enters the cell through the plasmalemma before it reaches the contractile machinery. In order for the SR to buffer calcium on a steady-state basis, Ca²⁺ must be translocated from the SR lumen to the extracellular space. This translocation is thought to be mediated by the release of accumulated Ca²⁺ from the SR into the narrow space between the SR and plasmalemma (referred to as ‘vectorial Ca²⁺ release’), from where the Na⁺-Ca²⁺ exchanger and PMCA complete the extrusion process (Moore et al. 1993). It is therefore implied that sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) functions in series with the sarcolemmal Na⁺-Ca²⁺ exchanger closely opposed to the peripheral SR, thereby contributing significantly to Ca²⁺ extrusion (Moore et al. 1993; Villa et al. 1993). Evidence supporting such a ‘superficial buffer barrier’ hypothesis has been obtained in vascular smooth muscle cells (Chen & van Breemen, 1993; Nazer & van Breemen, 1998; Rembold & Cheng, 1998) and in gastric fundus (Petkov & Boev, 1996). It is, however, unclear whether or not the SR of uterine smooth muscle cells contributes to the extrusion of Ca²⁺ by means of vectorial calcium release (Taggart & Wray, 1997).

Recently, we have shown that the PMCA and Na⁺-Ca²⁺ exchanger play an important role in the decay of depolarization-induced [Ca²⁺]_i transients in uterine smooth muscle cells (Shmigol et al. 1998a,c). When these two mechanisms were inhibited simultaneously, there was no [Ca²⁺]_i decay after a train of depolarizing pulses. This could be taken as evidence that no other mechanisms contribute to the decay of [Ca²⁺]_i. However, since membrane depolarization greatly increases the total calcium content of the cell, the intracellular calcium accumulating systems may saturate, due to their finite capacity. This saturation could account for the lack of [Ca²⁺]_i transient decay under the inhibition of calcium extrusion. Further insight into the mechanisms responsible for [Ca²⁺]_i transient decay can be obtained by investigating the decay of agonist-induced [Ca²⁺]_i transients, since the release of Ca²⁺ from the SR does not increase the total calcium content of the cell.

Our aim in this study was therefore to evaluate the role of the SR in Ca²⁺ extrusion from uterine smooth muscle cells. In particular we wished to answer the following questions: (i) does the SR contribute to the decay of the depolarization- and agonist-induced [Ca²⁺]_i transients? and (ii) how is the SR Ca²⁺ pump mechanism related to the plasmalemmal Ca²⁺ extrusion mechanisms?

METHODS

Experiments were performed on acutely isolated uterine smooth muscle cells. Female Wistar rats at the end of gestation (19-21 days) were killed by cervical dislocation under CO₂ anaesthesia. The methods of cell preparation, [Ca²⁺]_i measurement and voltage clamp were the same as described in a previous paper (Shmigol et al. 1998b). Freshly dissociated myocytes were kept in KB medium (Klockner & Isenberg, 1985) at 4°C until use. Most data were obtained within the first 8 h after dissociation, although the cells retained their normal physiological properties for up to 36 h.

In the experiments on non-dialysed cells, indo-1 was loaded into the cells by incubating them in KB medium containing 5 μm indo-1 AM and 0.25% of the non-ionic detergent Pluronic F-127 for 15 min at room temperature (19-21°C). Subsequently, the cells were incubated in KB medium for at least 1 h to complete de-esterification of indo-1 AM.

Solutions

During the course of the experiment the cells were continuously superfused with pre-warmed extracellular solution containing (mm): NaCl, 140; KCl, 5.4; CaCl₂, 2; MgCl₂, 1.2; glucose, 10; and Hepes, 10; adjusted to pH 7.4 with NaOH. Nominally Ca²⁺-free solution was prepared by omitting Ca²⁺ and increasing the magnesium concentration to 5 mm. Na⁺-free solution was prepared by equimolar substitution of Tris⁺ for Na⁺ in the extracellular solution. The pipette solution contained (mm): CsCl, 130; MgCl₂, 1; indo-1 pentapotassium salt, 0.1; MgATP 2; and Hepes, 10; adjusted to pH 7.2 with CsOH. The Hanks’ solution used for cell isolation was composed as follows (mm): NaCl, 137; KCl, 5.1; KH₂PO₄, 0.44; Na₂HPO₄, 0.26; glucose, 5.5; and Hepes, 10; adjusted to pH 7.2 with NaOH. KB medium contained (mm): KCl, 40; K₂HPO₄, 10; KOH, 105; taurine, 10; glucose, 11; EGTA, 0.04; and Hepes, 5. Methanesulfonic acid was used to adjust the pH of this solution to 7.2. Carboxyeosin (as the diacetate succinimidyl ester, Calbiochem) was dissolved in methanol to yield a 20 mm stock solution. Carboxyeosin-containing solution was prepared by adding 10 μl of the stock solution to 100 ml of nominally Ca²⁺-free saline. To inhibit the sarcolemmal calcium pump, the cells were pre-incubated with a solution containing 2 μm carboxyeosin for 5 min at room temperature and washed by bath perfusion with a normal extracellular solution for 5–10 min. Carbachol was dissolved in the nominally Ca²⁺-free solution to a concentration of 100 μm and applied from a blunt patch pipette positioned close to the cell under investigation. Cyclopiazonic acid (CPA) was dissolved in DMSO to yield a 10 mm stock solution and kept in a freezer for subsequent use. The 10 μm solution of CPA was prepared daily by dissolving 100 μl of stock solution in 100 ml of Ca²⁺-free Krebs solution and applied to the bath. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and oligomycin were dissolved in ethanol and diluted with Krebs solution to a final concentration of 10 μm. All experiments were performed at 35°C. All chemicals were obtained from Sigma, unless stated otherwise.

Data analysis

It is anticipated that only a small amount of calcium within the cell appears as free ions. The cytosol contains many Ca²⁺-binding molecules that act as rapid, reversible buffers. Although only free calcium ions are transported out of the cytosol, other bound calcium ions dissociate to take their place, slowing the apparent rate of calcium removal. Due to this, the actual rate of [Ca²⁺]_i clearance is much higher than the apparent rate of [Ca²⁺]_i transient decay. Since the cytosolic Ca²⁺-binding properties of uterine smooth muscle cells are still unknown, we cannot calculate the actual rate of Ca²⁺ clearance. We therefore restricted our analysis to the comparison of the rate of [Ca²⁺]_i transient decay by calcium removal systems following physiological and pharmacological manipulation. To characterize the [Ca²⁺]_i transient decay we used the following approach. The decaying phase of the [Ca²⁺]_i transient was smoothed by fitting a linear polynomial (sixth to ninth order) to the original record and the numerical derivative (-d[Ca²⁺]_i/dt) was calculated. The rate constant of [Ca²⁺]_i decay was estimated from the slope of a plot of -d[Ca²⁺]_i/dt vs.[Ca²⁺]_i.

Where appropriate, the results are presented as means ±s.e.m. Otherwise, the traces shown represent typical results from at least five similar experiments, obtained from different cells. Statistical differences were tested using ANOVA. Origin version 5.0 (Microcal Software, Inc., Northampton, MA, USA) was used for data analysis.

RESULTS

The rate of [Ca²⁺]_i decay is affected by inhibition of the SR Ca²⁺ pump

Trains of action potentials arising from spontaneous depolarization trigger uterine contractions in vivo (Parkington & Coleman, 1990). Our first goal therefore was to investigate whether the SR Ca²⁺-ATPase contributes to the decay of depolarization-induced [Ca²⁺]_i transients. We mimicked this with repetitive membrane depolarization (ten 100 ms pulses from −80 to 0 mV at 3 Hz frequency) to activate the sarcolemmal L-type calcium channels and thereby elevate [Ca²⁺]_i (Shmigol et al. 1998b). Each voltage pulse in the train activated an inward calcium current, which was accompanied by an increase in [Ca²⁺]_i (Fig. 1A, control). Between voltage pulses, when the membrane was repolarized to −80 mV, the [Ca²⁺]_i partially declined. During the first six to eight stimuli in the train, there was a summation of individual [Ca²⁺]_i transients as the rise in [Ca²⁺]_i exceeded decline. At the end of the train (i.e. after the seventh pulse in Fig. 1A), these two opposite processes became equal and the [Ca²⁺]_i fluctuated around a steady-state level indicating a dynamic equilibrium between Ca²⁺ entry and Ca²⁺ removal. Upon cessation of stimulation [Ca²⁺]_i decayed towards the baseline. The rate constant of [Ca²⁺]_i decay in control conditions was 0.64 ± 0.04 s⁻¹ (n = 14).

Figure 1. The effect of CPA on the depolarization-induced [Ca²⁺]_i transients.

A, [Ca²⁺]_i transients were elicited by repetitive membrane depolarization in control conditions and then in the presence of CPA, as indicated by the bar. The traces show (from top to bottom) [Ca²⁺]_i, membrane potential (V_m) and transmembrane current (I_m). The smooth lines imposed onto the decaying phase represent polynomial fits to the data. B, the rate of the [Ca²⁺]_i transient decay (-d[Ca²⁺]_i/dt) plotted against [Ca²⁺]_i. The lines represent linear regressions fitted to the data. The slope of these lines gives the rate constant of the [Ca²⁺]_i transient decay.

To assess the role of the SR in Ca²⁺ removal we used an inhibitor of the SERCA pump, CPA (Uyama et al. 1992). In the experiments illustrated in Fig. 1, 10 μm CPA was applied to the cell after recovery from the control transient. Within 60 s of CPA application the base level of [Ca²⁺]_i increased from 75 to 170 nM (Fig. 1A). The rate of the rising phase of the [Ca²⁺]_i transient was not significantly altered in the presence of CPA (see Fig. 1A). The amplitude of the peak [Ca²⁺]_i was significantly decreased from 720 ± 108 nM (control) to 380 ± 56 nM (CPA). However, the decay of the [Ca²⁺]_i transient was slowed, as shown from the slopes of -d[Ca²⁺]_i/dt plotted against [Ca²⁺]_i (Fig. 1B). On average, the resting level of [Ca²⁺]_i increased from 110 ± 25 nM in control to 180 ± 12 nM under CPA application (n = 15). The rate constant of the [Ca²⁺]_i decay after stimulation in the presence of CPA was decreased to 0.30 ± 0.04 s⁻¹ (n = 15) which is 47% of control level (see above).

These data clearly indicate the involvement of the SR in the removal of Ca²⁺ from the cytosol of uterine smooth muscle cells. However, our previous work had indicated that the SR alone could not restore [Ca²⁺] following a depolarization stimulus (Shmigol et al. 1998a). Thus if both the plasma membrane Ca²⁺ pump (PMCA) and the Na⁺-Ca²⁺ exchanger are inhibited, resting [Ca²⁺] increases and the decay of [Ca²⁺] is abolished. An example of this is shown in Fig. 2. Carboxyeosin was used to block the PMCA (Bassani et al. 1995; Shmigol et al. 1998a; Choi & Eisner, 1999), and stepping the membrane voltage above the reversal potential of the Na⁺-Ca²⁺ exchanger was used to prevent extrusion of Ca²⁺ on the exchanger.

Figure 2. Effect of membrane potential on the decay of the depolarization-induced [Ca²⁺]_i transients after inhibition of the PMCA with carboxyeosin.

The traces show (from top to bottom) [Ca²⁺]_i, membrane potential and transmembrane current. Note the lack of decay during depolarization to +50 mV.

Thus although the SR contributes to the decay of [Ca²⁺]_i, it requires the presence of sarcolemmal mechanisms when depolarization is the stimulus for the rise in [Ca²⁺]_i. An attractive explanation for this observation is that the SR Ca²⁺-ATPase operates in series with the plasmalemmal Ca²⁺ extrusion mechanisms. However, two points arise. Firstly, will this be the case when SR release, rather than Ca²⁺ entry, stimulates the rise of [Ca²⁺]? As this will not increase the total amount of Ca²⁺ in the cell, the SR may be capable of lowering [Ca²⁺] without sarcolemmal mechanisms acting. Secondly, we cannot exclude the possibility that the increase in [Ca²⁺]_i observed in carboxyeosin-treated cells during depolarization to +50 mV (Fig. 2) is due to a net entry of Ca²⁺ on the Na⁺-Ca²⁺ exchanger (despite dialysis with a Na⁺-free solution), which might overwhelm the ability of the SR to accumulate Ca²⁺. Working in Ca²⁺-free bathing solution can prevent this entry. However, this means that electrical stimulation cannot be used to increase [Ca²⁺]. Thus in the next set of experiments we worked in such solutions and used an agonist, carbachol, to release Ca²⁺ from the SR, and examined the role of the SERCA pump in the decay of agonist-induced [Ca²⁺]_i transients.

Carbachol-induced [Ca²⁺]_i transient decay: role for plasmalemmal Ca²⁺ extrusion

To elevate [Ca²⁺]_i in the nominal absence of extracellular Ca²⁺ in non-dialysed cells we used carbachol, which activates InsP₃-mediated Ca²⁺ release from the SR (Marc et al. 1992). A typical record of the carbachol-induced [Ca²⁺]_i transient is shown in Fig. 3A. Brief (3–4 s) application of 100 μm carbachol to the cell produced a transient increase in [Ca²⁺]_i followed by decay towards baseline. The rate constant of decay in the control condition was 0.48 ± 0.03 s⁻¹ (n = 52). This is significantly slower than the value obtained following depolarization (see Discussion). Inhibition of the Na⁺-Ca²⁺ exchanger by 5 mm Ni²⁺ or sodium removal from the bath moderately decreased the rate of decay of the [Ca²⁺]_i transient (Fig. 3B). Inhibition of PMCA considerably slowed the decay of [Ca²⁺]_i (Fig. 3C). Figure 3D shows the mean value of the rate constants obtained in this series of experiments. The rate constant of decay was decreased to 58% of control (0.28 ± 0.04 s⁻¹, P < 0.01, n = 21) after carboxyeosin treatment, and to 85% (0.41 ± 0.02 s⁻¹, P < 0.05, n = 18) and 81% (0.39 ± 0.03 s⁻¹, P < 0.05, n = 15) of control with abolition of the Na⁺-Ca²⁺ exchanger with Ni²⁺ or by sodium removal, respectively. The difference between the results obtained with Ni²⁺ and Na⁺-free solution was not statistically significant.

Figure 3. Effect of inhibition of plasmalemmal calcium extrusion on the decay of carbachol-induced [Ca²⁺]_i transients.

All traces were obtained in nominally Ca²⁺-free solution. Application of 100 μm carbachol is indicated by the filled bars beneath the traces. A, a typical [Ca²⁺]_i transient evoked by carbachol in nominally Ca²⁺-free solution. B, superimposed [Ca²⁺]_i transients elicited in the same cell by carbachol application in control and Na⁺-free solution. C, [Ca²⁺]_i transients recorded from another cell in control and after carboxyeosin treatment. D, mean values of the rate constant of [Ca²⁺]_i decay obtained in control, in Na⁺-free solution, in the presence of 5 mm Ni²⁺ and after carboxyeosin (CE) treatment.

Carbachol-induced [Ca²⁺]_i transient decay: effect of CPA

On average, the amplitude of the carbachol-induced [Ca²⁺]_i transient, in the absence of external Ca²⁺, was 710 ± 110 nM (n = 11). When carbachol was applied in zero external Ca²⁺ for a second time after a 30 s interval, the amplitude of the [Ca²⁺]_i transient was 410 ± 70 nM (Fig. 4, transients a and b). Following incubation of the cell in normal Krebs solution for 30 min, to refill the SR, application of CPA increased the basal level of [Ca²⁺]_i from 80 ± 7 to 153 ± 18 nM (n = 9). In the presence of CPA, but absence of external Ca²⁺, a second application of carbachol was ineffective (see transients c and d in Fig. 4). These data indicate that, under control conditions, the SR re-accumulates Ca²⁺ from the cytosol, and suggest that it participates in the decay of the agonist-induced [Ca²⁺]_i transient. Indeed, the rate of decay of the carbachol-induced [Ca²⁺]_i transient obtained in the presence of CPA was slowed compared with control. This is illustrated in Fig. 5, which compares the carbachol-induced [Ca²⁺]_i transients obtained in control and in the presence of CPA. On average, the rate constant of decay fell to 39% of control in the presence of CPA (0.19 ± 0.02 s⁻¹ for CPA, n = 11, compared with 0.48 ± 0.03 s⁻¹ for control). It is therefore clear that the rate of decay of the carbachol-induced [Ca²⁺]_i transient was dependent on SERCA pump activity.

Figure 4. Effect of CPA on [Ca²⁺]_i transients elicited by repetitive application of carbachol in nominally Ca²⁺-free solution.

Two consecutive applications of 100 μm carbachol (indicated by filled bars) elicited [Ca²⁺]_i transients (marked as a and b) when the SERCA was intact. When the SERCA pump was inhibited with CPA (indicated by the open bar) only the first application of carbachol was effective (transients c and d). During the 30 min break in the record, the cell was incubated in normal Krebs solution to replenish the SR.

Figure 5. Effect of CPA on the rate of carbachol-induced [Ca²⁺]_i transient decay in nominally Ca²⁺-free solution.

A, superimposed [Ca²⁺]_i transients obtained in the presence of 10 μm CPA and after a 30 min washout of the same cell with normal Krebs solution (Control). Traces were aligned from the start of carbachol application (filled bar, time zero). B, first derivatives of the decay of the transients shown in A plotted against [Ca²⁺]_i. Continuous lines represent linear regressions fitted to the data.

Carbachol-induced [Ca²⁺]_i transient decay: combined effect of carboxyeosin and Na⁺-free solution

The following experiments were designed to investigate whether the SERCA pump could restore [Ca²⁺]_i following application of carbachol when the plasmalemmal Ca²⁺ extrusion systems were inhibited. In these experiments, the PMCA was inhibited with carboxyeosin and Na⁺-free solution was used to prevent Ca²⁺ extrusion on the Na⁺-Ca²⁺ exchanger. Figure 6 shows a typical record obtained from a carboxyeosin-treated cell (transient a). Both [Ca²⁺]_i transients shown in this figure were obtained in nominally Ca²⁺-free solution containing 140 mm Na⁺. During the decay of the second transient (b in Fig. 6) Na⁺-free solution was applied as indicated by the hatched bar. It can be seen that removal of external Na⁺ halted the decay of [Ca²⁺]. In 19 out of 21 cells challenged with this protocol, zero Na⁺ application was accompanied by a violent contraction and subsequent disintegration of the cell. Restoration of the extracellular Na⁺ concentration led to an irreversible increase in [Ca²⁺]_i and cell death. When Na⁺-free solution was applied between successive applications of carbachol, the resting level of [Ca²⁺]_i was unaffected (Fig. 7). When the cells were then exposed to carbachol, still in a Na⁺-free solution, the magnitude of the response to carbachol was the same as under control conditions (compare transients a and b in Fig. 7). However, following an initial decay, [Ca²⁺]_i started to increase again. Restoring the extracellular Na⁺ did not reverse this increase but rather augmented it and finally the cell died. Similar results were obtained in 15 cells when 100 nM oxytocin was used to initiate the [Ca²⁺]_i transient and 5 mm NiCl₂ was used to inhibit the Na⁺-Ca²⁺ exchanger.

Figure 6. Carbachol-induced [Ca²⁺]_i transients, in nominally Ca²⁺-free solution, following pre-treatment with carboxyeosin.

The cell was capable of restoring [Ca²⁺]_i (transient a) when the Na⁺-Ca²⁺ exchanger was functional. Application of a Na⁺-free solution (hatched bar) during the decay of transient b immediately arrested the [Ca²⁺]_i decay. Restoration of extracellular Na⁺ led to a further increase of [Ca²⁺]_i. Applications of carbachol are indicated by filled bars.

Figure 7. Effect of inhibition of the sarcolemmal [Ca²⁺]_i extrusion systems on the decay of carbachol-induced [Ca²⁺]_i transients in nominally Ca²⁺-free solution.

The PMCA was inhibited with carboxyeosin. Transient a was obtained in the presence of extracellular Na⁺. Transient b was elicited in Na⁺-free solution (application of Na⁺-free solution is indicated by the hatched bar, applications of carbachol by the filled bars). Shortly after the initial decline, [Ca²⁺]_i rose again and the cell died.

These data, and those obtained with depolarization, are compatible with the idea that the SR contributes to the removal of Ca²⁺ from the cytosol by means of vectorial Ca²⁺ transport operating in series with plasmalemmal mechanisms. There is, however, the possibility that not only the SR but also mitochondria operate in this way to remove Ca²⁺ from the cytosol. To investigate whether mitochondria contribute to the decay of carbachol-induced [Ca²⁺]_i transients we performed experiments with the mitochondrial uncoupler CCCP.

Carbachol-induced [Ca²⁺]_i transient decay: role of mitochondria

Cells were treated with 10 μm CCCP to dissipate the proton gradient on the inner mitochondrial membrane. In order to prevent rapid hydrolysis of ATP by the mitochondrial ATP synthase working in reverse, 10 μm oligomycin was also present to inhibit the synthase. Under these conditions, there was no significant effect of the mitochondrial uncoupler on the decay of the carbachol-induced [Ca²⁺]_i transients. This is illustrated in Fig. 8 where two [Ca²⁺]_i transients of similar magnitude obtained from a control cell and from a cell treated with a mixture of CCCP and oligomycin are superimposed. On average the mean values of the rate constants were 0.483 ± 0.035 s⁻¹ in control and 0.496 ± 0.039 s⁻¹ in the presence of CCCP (n = 7). These data indicate that mitochondria do not contribute to the decay of the carbachol-induced [Ca²⁺]_i transients in uterine smooth muscle cells.

Figure 8. Decay of the carbachol-induced [Ca²⁺]_i transient is not affected by the mitochondrial uncoupler CCCP.

A, superimposed [Ca²⁺]_i transients obtained in control and in the presence of 10 μm CCCP and 10 μm oligomycin. Traces were aligned from the start of carbachol application (filled bar, time zero). Both traces were obtained in nominally Ca²⁺-free solution. B, first derivatives of the decay of the transients shown in A plotted against [Ca²⁺]_i. Continuous lines represent linear regression fitted to the data.

DISCUSSION

In smooth muscle the SR must not only release Ca²⁺ from its lumen into the cytosol, in response to a variety of stimuli, but must also re-accumulate Ca²⁺ after the stimulation and during rest. In this study we have found in uterine cells that inhibition of the SERCA pump with CPA greatly slows the decay of [Ca²⁺]_i, but that acting alone it cannot lower [Ca²⁺]_i to resting values. This leads us to suggest that the SERCA pump acts in series with surface membrane efflux mechanisms. A comparison of the decay of [Ca²⁺]_i following depolarization trains or agonist-induced response shows that CPA slowed the decay in both cases. Basal [Ca²⁺]_i was also increased. In both cases inhibition of either the Na⁺-Ca²⁺ exchanger or the PMCA also slowed the decay. Again, in both cases, when these two surface membrane mechanisms were inhibited, the cells could not restore [Ca²⁺]_i to resting levels. However, with carbachol stimulation there was a transient lowering of [Ca²⁺]_i under these conditions, which was not seen when depolarization was the stimulus. The data also showed that when summed, the percentage slowing of decay produced by individually inhibiting the PMCA or Na⁺-Ca²⁺ exchanger totalled 100% with depolarization as the stimulus, but was less than 100% with carbachol. We suggest, for the reasons discussed below, that in both cases the SR acts in series with the surface membrane efflux mechanisms, playing a significant role in the decay of [Ca²⁺]_i, and that the differences between stimulation with depolarization and carbachol are due to the state of filling of the SR.

Role for the SERCA pump in the decay of [Ca²⁺]_i

We found a significant increase in resting [Ca²⁺]_i with CPA. This occurred in the presence (depolarization experiments) and absence (carbachol experiments) of external Ca²⁺. These data are consistent with CPA inhibiting the SERCA pump. Also in agreement with this is our finding that only the first application of carbachol was effective when the SERCA was functionally disabled. When depolarization was the stimulus, we found that CPA reduced the amplitude of the [Ca²⁺]_i transient. Although this manoeuvre might be expected to increase [Ca²⁺] in the cytoplasm, it is countered by the loss of Ca²⁺-induced Ca²⁺ release (CICR) when the SERCA is inhibited. We have previously shown that CICR contributes to the elevation of [Ca²⁺]_i induced by repetitive depolarization (Shmigol et al. 1998b) and by action potentials in intact tissue from pregnant rats (Taggart & Wray, 1998). CICR has also been shown to contribute significantly to the depolarization-induced [Ca²⁺]_i rise in bladder cells, and CPA also reduced the magnitude of [Ca²⁺]_i transients evoked by voltage-clamp steps in these cells (Ganitkevich & Isenberg, 1991; Yoshikawa et al. 1996; Ganitkevich, 1998).

The rate of decay of the [Ca²⁺]_i transient was strongly affected by the application of CPA. The rate constant of the decay was decreased by more than 50% by CPA, irrespective of whether depolarization or agonist was the stimulus for [Ca²⁺] elevation, indicating that the SR plays an important role in shaping the decay of [Ca²⁺]. However, when plasmalemmal Ca²⁺ extrusion was abolished, the [Ca²⁺]_i transient did not return to resting levels. Thus the SERCA, acting alone, cannot lower [Ca²⁺]_i at all following repolarization, and does so only transiently and incompletely following carbachol stimulation. We suggest therefore that Ca²⁺ is taken up into the SR by the SERCA pump and subsequently released towards the PMCA and Na⁺-Ca²⁺ exchanger to be extruded into the extracellular space. Our findings are in agreement with data recently reported in rabbit vena cava (Nazer & van Breemen, 1998). When the Ca²⁺ store has been emptied, as will be the case with agonist stimulation, there will be an initial uptake into the SR. This is presumably the reason why there is a transient lowering of [Ca²⁺] seen under these conditions (Fig. 7).

These data are consistent with a vectorial efflux mechanism in the uterine cells, which raises [Ca²⁺] at the sarcolemmal efflux sites by an amount sufficient to double their rates. Vectorial Ca²⁺ release and efflux from the SR have been proposed in terms of acting as a buffer close to the surface membrane (superficial buffer barrier hypothesis), and we will therefore now discuss how our data relate to this hypothesis.

Superficial buffer barrier hypothesis

This hypothesis proposes that the SR, via its ATPase, takes up a fraction of the Ca²⁺ that enters smooth muscle cells (van Breemen & Saida, 1989). This Ca²⁺ is then preferentially released towards the plasmalemma to be extruded into the extracellular space. This hypothesis therefore predicts that the resting level of [Ca²⁺]_i should increase upon inhibition of the SERCA pump, as we found. It also predicts that the inhibition of the SERCA pump should enhance the amplitude of the depolarization-induced [Ca²⁺]_i transient and facilitate its rate of rise. We did not find such facilitation but as explained above this could be due to removal of CICR. The extra Ca²⁺ that enters following stimulation should be extruded from the cell in order to maintain Ca²⁺ homeostasis. In terms of this superficial buffer barrier hypothesis, the Ca²⁺ entering the cytosol from outside is finally extruded back to the extracellular space by means of vectorial Ca²⁺ efflux. Our data are consistent with such a mechanism. Until now, the superficial buffer barrier hypothesis had only been applied to the [Ca²⁺]_i transients elicited by Ca²⁺ entry from the extracellular space. It was therefore unclear whether the vectorial Ca²⁺ efflux would occur when the total Ca²⁺ content of the cell was not increased. Our experiments with carbachol-induced [Ca²⁺]_i transients, which were performed in calcium-free solution, show, however, that it can also be applied under these conditions. Thus, vectorial Ca²⁺ efflux took place, despite the fact that the total Ca²⁺ content of the cell was not increased (Figs 6 and 7). When both the PMCA and Na⁺-Ca²⁺ exchange were abolished, the application of carbachol led to an irreversible increase in [Ca²⁺]_i and disintegration of the cell.

With carbachol stimulation, inhibition of the PMCA slowed the [Ca²⁺] decay by 42% and inhibition of the exchanger slowed it by around 17%. Unlike our previous data using depolarization as the stimulus (Shmigol et al. 1998a), these percentages do not sum to 100%. This suggests again that when the SR is depleted it takes up Ca²⁺ following stimulation. Depolarization does not deplete the SR and thus this action is not seen (Shmigol et al. 1998a).

What exactly triggers vectorial [Ca²⁺]_i release from the SR is unknown. Background InsP₃ production may be sufficient to activate this process (van Breemen & Saida, 1989). It has been shown in permeabilized rat fibroblast A7r5 cells that the sensitivity of InsP₃ receptors is controlled by luminal Ca²⁺ content (Missiaen et al. 1992). Therefore, the probability of vectorial release by background concentrations of InsP₃ would increase with a rise in the SR Ca²⁺ content.

Our data also suggest that the SR Ca²⁺ storage and release sites in uterine myocytes may be spatially separated. This is evident from the experiments on carboxyeosin-treated cells bathed in Na⁺-free solution (see Fig. 7). The calcium ions can be safely stored within the SR of unstimulated cells when the PMCA and Na⁺-Ca²⁺ exchange are inhibited. The cell maintained a relatively low [Ca²⁺]_i under these conditions. However, when released from this site by carbachol application, calcium ions are sequestered into another part of the SR, from which the vectorial release occurs. With inhibition of plasmalemmal Ca²⁺ extrusion this release manifested itself as the sudden increase in [Ca²⁺]_i seen in Fig. 7. Some of this rise in [Ca²⁺] may also be due to leakage of Ca²⁺ across a damaged surface membrane, as our Ca²⁺-free solution contained no Ca²⁺ chelator, and therefore some Ca²⁺ will be present as a contaminant. Experiments with repetitive carbachol application suggest that only a portion of the Ca²⁺ re-sequestered into the SR is vectorially released towards the plasmalemma and extruded. The remainder is returned to the storage site to be released by subsequent agonist application.

Relation to previous work

Work on uterine plasma membrane vesicles had identified both the PMCA and the Na⁺-Ca²⁺ exchanger but not assessed their contribution to decreasing [Ca²⁺]_i (Kosterin et al. 1994). In intact uterine strips from pregnant rats it was calculated that up to 35% of agonist-induced Ca²⁺ release was extruded by the Na⁺-Ca²⁺ exchanger (Taggart & Wray, 1997). When expressed as a percentage of the total surface membrane contribution (100%), the relative contributions of the Na⁺-Ca²⁺ exchanger and the PMCA to the lowering of agonist-induced Ca²⁺ release were 29 and 71%, respectively. The value for the exchanger is thus very similar to that reported in the intact prepartion (Taggart & Wray, 1997). These estimates, however, differ from those obtained following depolarization trains, where 60-70% of the Ca²⁺ extrusion was attributed to the exchanger and the remainder to the PMCA (Shmigol et al. 1998a). The differences are likely to be methodological. As pointed out in our previous study, in the perfused cells necessary for the depolarization experiments, cell dialysis with a Na⁺-free pipette solution will facilitate Ca²⁺ extrusion on the exchanger. This will cause an over-estimation of its contribution. This can also explain why under control conditions the rate of decay was faster following depolarization than following carbachol stimulation, i.e. the exchanger was facilitated.

Contribution of mitochondria

Experiments with the mitochondrial uncoupler CCCP showed no contribution from the mitochondria to the carbachol-induced decay of [Ca²⁺]_i transients (Fig. 8). There was also little or no change in the rise of [Ca²⁺]. Therefore, in these uterine cells the SR appears to be entirely responsible for the observed changes in [Ca²⁺]_i. This is unlike the data reported in gastric smooth muscle cells where the decay of [Ca²⁺] evoked by depolarization was slowed when mitochondrial function was impaired (Drummond & Fay, 1996; McGeown et al. 1996).

Physiological significance

Ca²⁺ extrusion is an important process ensuring adequate relaxation of smooth muscle. Two transporting systems are responsible for the removal of calcium ions from the uterine cytosol, namely the PMCA and the Na⁺-Ca²⁺ exchanger. We found that, in addition to the above two systems, vectorial Ca²⁺ efflux from the SR plays an important role in maintaining Ca²⁺ homeostasis in uterine cells. The physiological significance of this mechanism is that Ca²⁺ can be removed from the contractile machinery faster than if the PMCA and Na⁺-Ca²⁺ exchanger alone were responsible for this removal. In addition, changes in the filling state of the SR due to the vectorial Ca²⁺ efflux may also modulate membrane excitability and subsequent [Ca²⁺]_i transients.