INTRODUCTION

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Because the principal trigger for contraction of smooth muscle is an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), mechanisms generating increases in [Ca²⁺]_i have been subject to intensive investigation. Depending on the stimulus, a rise in [Ca²⁺]_i may reflect Ca²⁺ entry from the outside, release from an internal store, or both. Ca²⁺ entry across the plasma membrane occurs through Ca²⁺ channels. In smooth muscle, three types of Ca²⁺ channel exist: a low-voltage, rapidly inactivating, small conductance channel (T-type), and a dihydropryidine-sensitive, high-threshold, large conductance channel (L-type; Benham et al., 1987; Vivaudou et al., 1988). A third type of Ca²⁺ channel directly operated by agonists and insensitive to voltage has also been proposed (Benham and Tsien, 1987). In addition to Ca²⁺ fluxes across the plasmalemma, Ca²⁺ may also be released from an internal store by agonist-generated IP₃ (Horowitz et al., 1996) or by a Ca²⁺-induced Ca²⁺ release mechanism (Kamishima and McCarron, 1997).

After stimulation [Ca²⁺]_i returns to the resting levels, permitting relaxation. However, although their importance is well appreciated, the exact efflux or uptake routes of Ca²⁺ removal in smooth muscle remain unclear. Four transport systems are thought to be involved in Ca²⁺ removal. A Ca²⁺ pump and a Na⁺-Ca²⁺ exchanger in the plasma membrane are believed to extrude Ca²⁺, whereas a Ca²⁺ pump in sarcoplasmic reticulum (SR) and a Ca²⁺ uniporter in mitochondria sequester Ca²⁺. In cardiac myocytes, Ca²⁺ removal occurs largely by Ca²⁺ pumps in the SR and, to a lesser extent, by a Na⁺-Ca²⁺ exchanger in the sarcolemma (e.g., Wier, 1990); the contributions of Ca²⁺ pumps in the cell membrane and the Ca²⁺ uniporter in the mitochondria are relatively small (Bassani et al., 1992). In smooth muscle, however, the Ca²⁺ removal pathways are less well defined. This prompted the present study, in which Ca²⁺ removal mechanisms have been examined in resistance artery smooth muscle. Evidence is provided that Ca²⁺ pumps in the SR and cell membrane, but not the Na⁺-Ca²⁺ exchanger, are important in Ca²⁺ removal. Part of the study has already been published as an abstract (McCarron and Kamishima, 1997).

	MATERIALS AND METHODS

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Drugs and statistics

Fura-2 pentapotassium salt was purchased from Molecular Probes (Eugene, OR), and Bay K 8644, ryanodine, and thapsigargin were from Calbiochem-Novabiochem (Nottingham, England). Bay K 8644 was dissolved in ethanol to make a 1 mM stock solution. The final concentration of ethanol in bathing solution was 0.05%. Ryanodine and thapsigargin were dissolved in dimethyl sulfoxide (DMSO) to produce stock solutions of 30 mM and 500 µM, respectively. The final concentration of DMSO in experimental solutions was 0.1% in both cases. Dithioerythritol, collagenase Type F, and hyaluronidase Type I-S were purchased from Sigma Chemical (Dorset, England). Papain was obtained from Worthington Biochemical Corporation (Freehold, NJ). When appropriate, the data were expressed as means ± SEM of n cells, and significant difference was detected using Student's unpaired or paired t-test (p < 0.05).

Cell dissociation

Male Sprague-Dawley rats (225-350 g) were killed by pentobarbitone sodium overdose (150 mg kg¹, I.P.). The brain was removed and placed in a solution containing (mM) 137 NaCl, 5 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 11 glucose (pH adjusted to 7.4 with NaOH). Superior cerebral arteries (diameter  150 µm) were dissected, and single smooth muscle cells were dissociated as previously described (Quayle et al., 1994). Briefly, a low-Ca²⁺ solution was used for cell dissociation (mM): 80 Na glutamate, 54 NaCl, 5 KCl, 1 MgCl₂, 0.1 CaCl₂, 10 HEPES, 10 glucose, and 0.2 EDTA. pH of the low-Ca²⁺ solution was adjusted to 7.3 at room temperature with NaOH to provide pH 7.4 at 35°C. The arteries were first treated with (mg ml¹) 1.7 papain and 0.7 dithioerythritol for 30 min at 35°C, then further digested with (mg ml¹) 1.7 type F collagenase and 1 type I-S hyaluronidase for 20 min. The arteries were then rinsed with enzyme-free low-Ca²⁺ solution, and single cells were dispersed by triturating the arteries with a fire-polished Pasteur pipette. The cell suspension was stored in a refrigerator and used the same day.

Voltage-clamp technique

Command pulses were applied in tight-seal whole-cell recording mode (Hamill et al., 1981). Unless otherwise stated, the extracellular solution consisted of (mM) 80 Na glutamate, 40 NaCl, 20 tetraethylammonium (TEA) chloride, 1.1 MgCl₂, 3 CaCl₂, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with NaOH). In most cases, the composition of the pipette solution was (in mM) 145 CsCl, 3 MgCl₂, 3 Na₂ATP, 10 HEPES, and 0.04 Fura-2 pentapotassium salt (pH adjusted to 7.2 with CsOH). Where the role of Na⁺-Ca²⁺ exchanger was studied, Na⁺ was eliminated from both the bathing and the pipette solutions. Hence, the composition of the bathing solution was (mM) 120 choline Cl, 20 TEA Cl, 1.1 MgCl₂, 3 CaCl₂, 10 HEPES, and 10 glucose (pH adjusted to 7.4 with CsOH). The composition of the pipette solution was (mM) 145 CsCl, 3 MgATP, 10 HEPES, and 0.04 Fura-2 (pH adjusted to 7.2 with CsOH). Ryanodine was added to the pipette solution, and thapsigargin was applied to both pipette and bathing solutions. Whole-cell currents were amplified with an Axopatch 1D (Axon Instruments, Foster City, CA), filtered at 500 Hz, and sampled at 1.5 kHz with pCLAMP software (version 6.0.1; Axon Instruments). In the standard protocol, a 1.6-s voltage pulse to 0 mV was applied from a holding potential of 70 mV to trigger Ca²⁺ influx (I_Ca) through voltage-dependent Ca²⁺ channels. All experiments were performed at room temperature (18°C-22°C).

Ca²⁺ microfluorimetry

High temporal [Ca²⁺]_i measurement was carried out using a PTI deltascan (Photon Technology International, London) as described previously (Kamishima and McCarron, 1996). Cells were dialyzed with a pipette solution containing 40 µM membrane-impermeable Fura-2 pentapotassium salt. Cells were illuminated with alternating UV light (340/380 nm; 9-nm bandpass) at 100 Hz, and emission signals were obtained at 510 nm (60-nm bandpass, complete ratio obtained at 50 Hz). The K_d for Fura-2 was determined as 280 nM from an in vitro calibration (Kamishima and McCarron, 1996), as were R_min and R_max; the latter were decreased by 15% to make up for the viscosity of intracellular milieu (Poenie, 1990). Background fluorescence was measured when the tight seal was formed, but before achieving whole-cell configuration and subtracted from the fluorescent counts during the experiments.

Calculation of Ca²⁺ removal rate

Unless otherwise stated, a depolarizing pulse to 0 mV was given from a holding potential of 70 mV. Upon repolarization to 70 mV, the increased [Ca²⁺]_i returned to the basal level (Fig. 1). The declining phase of the Ca²⁺ transient was fitted with high-order polynomial equations, and the Ca²⁺ removal rate was calculated as the negative derivative of the polynomial by averaging the slope of two adjacent data points (Kamishima and McCarron, 1996). The calculated Ca²⁺ removal rate was expressed either as a function of time, where time = 0 is the instant of repolarization, or of [Ca²⁺]_i.

View larger version (17K): [in this window] [in a new window]   FIGURE 1   Depolarization-evoked increases in [Ca2+]i and rates of Ca2+ removal. A depolarizing pulse to 0 mV from a holding potential of 70 mV (middle trace) triggered an increase in [Ca2+]i (upper trace). The elevated [Ca2+]i returned to the resting level after the termination of the pulse. The declining phase of the Ca2+ transient was fitted to a high-order polynomial, and the rate of Ca2+ removal was determined from the negative derivative of the fit. The Ca2+ removal rate was expressed either as a function of measured [Ca2+]i (lower left-hand panel) or time, where time = 0 is the first data point after repolarization (lower right-hand panel). The Ca2+ removal profile consisted of three phases. The initial fast phase of Ca2+ decline (first phase) was followed by a plateau (second phase). Just before [Ca2+]i returned to the basal level, the Ca2+ removal rate increased. This third phase appears as an upward hump in the lower panels.

	RESULTS

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Ca²⁺ removal in control cells

Fig. 1 illustrates a typical Ca²⁺ transient (upper trace) triggered by a depolarizing pulse to 0 mV from 70 mV (middle trace) under control conditions. [Ca²⁺]_i returned to baseline on repolarization to 70 mV. The decay of the Ca²⁺ transient displayed a characteristic three-phase pattern (McGeown et al., 1996). After repolarization, Ca²⁺ removal was initially fast (first phase), then slowed (second phase), but before [Ca²⁺]_i was restored to the basal level, the removal rate again accelerated (third phase). When plotted as a function of [Ca²⁺]_i (Fig. 1, lower left panel) or time (Fig. 1, lower right panel), the third phase is apparent as an upward hump.

Thapsigargin and ryanodine inhibit third-phase Ca²⁺ removal

The role of internal stores in the decline of [Ca²⁺]_i was determined by using the store uptake inhibitor thapsigargin (Thastrup et al., 1990). However, at concentrations over 200 nM, thapsigargin also inhibits the plasma membrane Ca²⁺ current (Rossier et al., 1993; Shmigol et al., 1995) and blocks Ca²⁺-induced Ca²⁺ release (Kamishima and McCarron, 1997). Therefore, in the presence of thapsigargin, the depolarization-evoked Ca²⁺ transient would be reduced substantially. To overcome this problem, 0.5 µM Bay K 8644 was included in the bathing solution to enhance the opening of voltage-dependent Ca²⁺ channels, thus allowing comparison of Ca²⁺ transients of similar magnitudes. (Bay K 8644 0.5 µM in the absence of thapsigargin did not alter the profile of Ca²⁺ removal (n = 3).) Fig. 2 depicts one such experiment. Depolarization to 0 mV (middle trace) increased [Ca²⁺]_i (upper trace). [Ca²⁺]_i sharply declined immediately after repolarization. However, [Ca²⁺]_i declined without the acceleration of Ca²⁺ removal before returning to the baseline (i.e., phase three was absent). Indeed, the calculated removal rate, shown in the lower panels (Fig. 2), supports this suggestion. To compare the third phase, the Ca²⁺ removal rate was measured at the peak of the third phase. Both thapsigargin (500 nM) and ryanodine (30 µM; a plant alkaloid that should "short circuit" internal stores by locking the release channels in a subconductance state; Smith et al., 1988) significantly (p < 0.05) slowed the peak rate of decline in the third phase. Thus the control rate was 28.6 ± 3.4 nM s¹ (n = 14), whereas the rate in the presence of thapsigargin was 6.4 ± 3.1 nM s¹ (n = 5). The averaged peak third-phase removal rate seen when ryanodine was included in the patch pipette filling solution was also significantly (p < 0.05) reduced from control rates (7.7 ± 2.7 nM s¹; n = 14). These results indicate that Ca²⁺ sequestration by the SR contributes to the third phase of removal.

View larger version (19K): [in this window] [in a new window]   FIGURE 2   Ca2+ removal after inhibition of Ca2+ store uptake by thapsigargin (500 nM). The third phase (hump; see Fig. 1) is no longer present (lower panels). Whereas phase 1 of removal is still apparent, the third phase is not. The experiment was carried out in the presence of 0.5 µM Bay K 8644 to produce a Ca2+ transient whose magnitude matches that of control cells.

Ryanodine and thapsigargin do not affect first-phase Ca²⁺ removal

To examine the contribution of internal stores to the first phase of Ca²⁺ removal, the time required for the transient to fall by 25% was measured. This measure was preferred to the peak rate of decline in phase 1, because, in some cells, I_Ca was not completely inactivated by the end of the pulse. This resulted in a brief suppression of first-phase Ca²⁺ removal rate because of a "Ca²⁺ tail" produced by a tail current. Although the contamination of the Ca²⁺ tail was brief, because I_Ca rapidly deactivates at 70 mV, it compromised the accurate detection of the peak first-phase Ca²⁺ removal rate occurring at the instant of repolarization. Therefore, the first phase of removal was summarized by using the time needed for the transient to decrease by 25% (t_0.25; Fig. 3, inset). For example, if [Ca²⁺]_i increased from a resting value of 100 nM to 700 nM, then t_0.25 is the time required to reach a [Ca²⁺]_i of 550 nM. This measure reflected phase one of removal, but was sufficiently far from the time of repolarization to avoid complication of the analysis of [Ca²⁺]_i decline by the Ca²⁺ tail. Measurements from thapsigargin-treated and ryanodine-treated cells were combined, because each treatment yielded a similar inhibition of the peak third phase removal rate. Fig. 3 (upper panel) depicts the average t_0.25 for controls and thapsigargin or ryanodine-treated cells. The average t_0.25 for control cells was 3.4 ± 0.6 s (n = 14) and was not significantly different from the thapsigargin- or ryanodine-treated cells (3.0 ± 0.5 s, n = 17). Thus Ca²⁺ uptake by the SR does not contribute to the first phase of removal.

View larger version (32K): [in this window] [in a new window]   FIGURE 3   Comparison of t0.25 and t0.8-0.9 in the absence and presence of thapsigargin or ryanodine. The inhibition of Ca2+ uptake by sarcoplasmic reticulum did not affect t0.25, but significantly prolonged t0.8-0.9. The inset illustrates t0.25 and t0.8-0.9.

To determine whether measurement of time (t_0.25) was sufficiently sensitive to detect alterations in removal rates, the effect of store disruption on removal times during phase 3 was also examined. In this case, the time lapsing between 80% and 90% of the transient decay (t_0.8-0.9) was used (Fig. 3. inset, and in example above, time required for [Ca²⁺]_i to fall from 220 nM to 160 nM). The rate of decline was significantly slowed during phase 3, as determined by using t_0.8-0.9 as a measurement of Ca²⁺ removal. Thus the control value was 1.8 ± 0.2 s, whereas the time after thapsigargin or ryanodine treatment was 6.0 ± 1.6 s (Fig. 3, lower panel). Because t_0.8-0.9 appears to detect the inhibition of the third phase by store disruption, t_0.25 may reasonably reflect first-phase Ca²⁺ removal.

Summary of Ca²⁺ uptake by the SR

The contribution of Ca²⁺ uptake by the SR was examined as a function of [Ca²⁺]_i (Fig. 4). The average Ca²⁺ removal rate for controls (n = 14) was significantly faster than that for thapsigargin- or ryanodine-treated cells (n = 17) up to 350 nM [Ca²⁺]_i, supporting the proposal that the SR Ca²⁺ pump is a high-affinity Ca²⁺ clearance mechanism (Kargacin and Kargacin, 1995).

View larger version (27K): [in this window] [in a new window]   FIGURE 4   Comparison of the rate of Ca2+ removal as a function of [Ca2+]i, in the absence or presence of thapsigargin or ryanodine. The third phase removal rate was significantly (p < 0.05) faster in control cells than in those treated with store Ca2+ uptake inhibitors, as shown by *.

Na⁺ dependence of [Ca²⁺]_i decline

The low-affinity Na⁺-Ca²⁺ exchanger is an important Ca²⁺ removal mechanism in some (McCarron et al., 1994; McGeown et al., 1996) but not in other (Ganitkevich and Isenberg, 1991; Fleischmann et al., 1996) smooth muscle cells. Ca²⁺ removal through the Na⁺-Ca²⁺ exchanger requires extracellular Na⁺, so to examine the exchanger's contribution to [Ca²⁺]_i decline, particularly during phase 1, extracellular Na⁺ was replaced with choline⁺ (Fig. 5). Depolarization to 0 mV (middle trace) evoked a Ca²⁺ transient (upper trace). The overall profile of the Ca²⁺ transient was virtually indistinguishable from that of the control cells (Fig. 1), suggesting that the Na⁺-Ca²⁺ exchanger does not significantly contribute to the Ca²⁺ removal process. When the Ca²⁺ removal rate was expressed as a function of [Ca²⁺]_i or time, all three phases were still clearly evident (Fig. 5, lower panels). Indeed, the average peak third phase Ca²⁺ removal rate in the presence of choline⁺ was 25 ± 5 nM s¹ (n = 8), and was not significantly different from the control rates (29 ± 3 nM s¹). Similarly, neither t_0.25 (2.0 ± 0.6 s, n = 8) nor t_0.8-0.9 (1.5 ± 0.2 s) was significantly different in the presence or absence of Na⁺ (Fig. 6). Thus neither the first phase nor the third phase was affected by Na⁺ replacement.

View larger version (18K): [in this window] [in a new window]   FIGURE 5   Depolarization-evoked Ca2+ increases and the rate of [Ca2+]i after substitution of Na+ with choline+. Na+ removal did not alter the Ca2+ removal rate (lower panels), and all three phases are present.

View larger version (22K): [in this window] [in a new window]   FIGURE 6   Comparison of t0.25 and t0.8-0.9 in the Na+-containing (control) and Na+-free choline+-bathed cells. The inhibition of the Na+-Ca2+ exchanger did not affect either t0.25 or t0.8-0.9. The inset illustrates t0.25 and t0.8-0.9.

Summary of Ca²⁺ removal in Na⁺-free solution

Fig. 7 summarizes the rate of Ca²⁺ removal, in the presence and absence of Na⁺, plotted as a function of [Ca²⁺]_i. The average Ca²⁺ removal rate of choline⁺-bathed solution (n = 8) was not significantly different from that of the control cells (n = 14), supporting the suspicion that Ca²⁺ clearance through the Na⁺-Ca²⁺ exchanger is not important in superior artery smooth muscle cells over the [Ca²⁺]_i range examined.

View larger version (25K): [in this window] [in a new window]   FIGURE 7   Comparison of the Ca2+ removal rates expressed as a function of [Ca2+]i under control conditions and in the presence of choline chloride, a substitute for Na+. No detectable difference in removal rate occurred.

Summary of Ca²⁺ removal by Ca²⁺ pumps in SR and cell membrane

In rat superior cerebral artery smooth muscle cells, inhibitors of store Ca²⁺ uptake, attenuated the third phase. Therefore, the difference obtained by subtracting the Ca²⁺ removal rate of thapsigargin- or ryanodine-treated cells from the control value should represent Ca²⁺ uptake rate by the SR (Fig. 8). It appears that the store's contribution to Ca²⁺ clearance occurs at low [Ca²⁺]_i. There was no detectable contribution of the Na⁺-Ca²⁺ exchanger. By elimination, therefore, the Ca²⁺ removal rates observed in the presence of thapsigargin or ryanodine seem consistent with extrusion by Ca²⁺ pumps in the cell membrane (Fig. 8). It is conceivable, however, that some Ca²⁺ removal processes are time dependent as well as [Ca²⁺]_i dependent. Hence it is possible that the store Ca²⁺ uptake requires slow up-regulatory mechanisms and is not observed immediately after the repolarization, when [Ca²⁺]_i is high.

View larger version (24K): [in this window] [in a new window]   FIGURE 8   A summary of Ca2+ removal rate by Ca2+ pumps. The removal by the sarcoplasmic reticulum Ca2+ pump was determined by subtracting removal rates of thapsigargin- or ryanodine-treated cells from those of controls (open circles). The filled circles represent the removal rate of thapsigargin- or ryanodine-treated cells. Because no detectable Ca2+ clearance through the Na+-Ca2+ exchanger was detected, Ca2+ removal in these cells presumably occurs through Ca2+ pumps in the cell membrane.

	DISCUSSION

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The results provide evidence that both the SR Ca²⁺ pump and the plasma membrane Ca²⁺ pump are important removal mechanisms in resistance artery smooth muscle. The data also indicate that the Na⁺-Ca²⁺ exchanger contributes little to the restoration of resting [Ca²⁺]_i levels after depolarization-evoked increases in [Ca²⁺]_i.

Ca²⁺ clearance in cardiac myocytes, neurons, and adrenal chromaffin cells has been the subject of detailed investigation (e.g., Balke et al., 1994; Friel and Tsien 1994; Herrington et al., 1996; Babcock et al., 1997). In smooth muscle cells, however, less is known about the Ca²⁺ removal mechanisms. The Ca²⁺ pumps of the internal Ca²⁺ stores have been proposed to have primary responsibility for removing increased [Ca²⁺]_i after the termination of excitatory stimuli (Kargacin and Kargacin, 1995). However, others have proposed that the Na⁺-Ca²⁺ exchanger is physiologically important in smooth muscle cells (e.g., Blaustein et al., 1986; McCarron et al., 1994) or that the sarcolemma Ca²⁺ pump is the main route in clearing Ca²⁺ (e.g., Raemaekers and Wuytack, 1996). Such varying conclusions may be ascribed to differences in tissue, species (Eggermont et al., 1988), or experimental/analytical approach.

Because Ca²⁺-induced Ca²⁺ release contributed substantially to the depolarization-induced increase in [Ca²⁺]_i in the resistance artery used in this study (Kamishima and McCarron, 1997), it seemed likely that Ca²⁺ pumps in the stores play an important role in Ca²⁺ removal. Ca²⁺ uptake by the stores was detected as a delayed up-regulation of Ca²⁺ removal (the third phase). The [Ca²⁺]_i range over which the SR removes Ca²⁺ is consistent with the notion of a high-affinity Ca²⁺ removal mechanism.

The role of Na⁺-Ca²⁺ exchange in regulating Ca²⁺ in smooth muscle has been tenaciously debated. Clear evidence from several studies has demonstrated that a Na⁺-Ca²⁺ exchanger can substantially alter [Ca²⁺]_i in smooth muscle, but only when in reverse mode (Ca²⁺ entry mode; e.g., Aaronson and Benham, 1989). Fewer studies have demonstrated that a Na⁺-Ca²⁺ exchanger can regulate [Ca²⁺]_i when operating in forward mode (Ca²⁺ extrusion mode). In the gastric smooth muscle cells of the toad, forward mode Na⁺-Ca²⁺ exchanger activity did remove [Ca²⁺]_i at higher concentrations (>300 nM; McCarron et al., 1994; McGeown et al., 1996). However, in equine tracheal myocytes and guinea pig bladder cells, the Na⁺-Ca²⁺ exchanger did not play a significant part in Ca²⁺ clearance from the cytosol (Fleischmann et al., 1996; Ganitkevich and Isenberg, 1991).

In the present study the Na⁺-Ca²⁺ exchanger made no detectable contribution to the removal of Ca²⁺. Thus Na⁺ replacement with choline⁺ did not affect the rate of Ca²⁺ removal over the [Ca²⁺]_i range tested (Fig. 7). Furthermore, an inward current, which should accompany the rapid decay of Ca²⁺ through Na⁺-Ca²⁺ exchanger activity, was not detected (not shown). Finally, Ca²⁺ removal was voltage independent. For example, holding the membrane potential at 120 mV after the voltage pulse to 0 mV should attenuate the rate of Ca²⁺ removal (McCarron et al., 1994). On repolarization to 70 mV from +120 mV, an accelerated rate of Ca²⁺ removal would be expected. However, in three cells in which this high-voltage protocol was tested, the inhibition of Ca²⁺ removal during high voltage was not observed. Indeed, the Ca²⁺ removal rate just before the voltage was changed from +120 mV to 70 mV was 18.7 ± 1.6 nM s¹, not significantly different from that just after the voltage change (18.6 ± 1.1 nM s¹, n = 3).

Investigations of Ca²⁺ removal processes have frequently relied on measurements such as muscle relaxation times, [Ca²⁺]_i decay times, or rate constants of decline. Each of these measurements will almost certainly vary as the [Ca²⁺]_i range over which the measurement is made changes. Experiments designed to inhibit a removal system, which also independently alter resting [Ca²⁺]_i, could alter rate constants or time measurements regardless of changes in Ca²⁺ removal. Unless they are well controlled, such measurements will distort the contributions of the underlying removal mechanisms. Complicating matters further, when rate constants or time is used to measure decline, is the probability that more than one mechanism is operating in parallel to clear Ca²⁺ from the cytosol. Thus inhibition of one removal pathway will increase the substrate (Ca²⁺) available to the other systems with a possible increase in their removal rates. Again, this could frustrate the determination of the underlying removal mechanisms. The method of analysis of removal is, therefore, of considerable importance, and velocity plotted against [Ca²⁺]_i, in all probability, is the most reliable.

Together, the results presented highlight the importance of the plasma membrane Ca²⁺ pump and SR Ca²⁺ pump in regulating [Ca²⁺]_i in smooth muscle and support the idea that Na⁺-Ca²⁺ exchange may contribute little to Ca²⁺ removal in mammalian smooth muscle.