Mechanisms that regulate [Ca²⁺]_i following depolarization in rat systemic arterial smooth muscle cells

Abstract

1. We have used the patch-clamp technique in combination with fluorimetric recording to study the mechanisms that regulate intracellular Ca²⁺, [Ca²⁺]_i, following depolarization in cells isolated from the rat femoral artery.

2. Depolarization to 0 mV from a holding potential of −70 mV increased [Ca²⁺]_i. Little Ca²⁺ release from sarcoplasmic reticulum, SR, was detected during depolarization since application of 30 μM ryanodine, a Ca²⁺-release inhibitor, had no significant effect on total Ca²⁺ buffering power.

3. Upon repolarization to −70 mV, 7 out of 13 cells showed three phases of Ca²⁺ removal; an initial rapid first phase, a slow second phase, and a faster third phase. Six cells, in which Ca²⁺ recovered quickly, lacked the third phase. The third phase was also absent in cells treated with a SR Ca²⁺-pump inhibitor, cyclopiazonic acid.

4. The peak first-phase Ca²⁺ removal rate observed upon repolarization to −70 mV was significantly reduced in cells treated with a mitochondrial Ca²⁺ uptake inhibitor, carbonyl cyanide m-chlorophenylhydrazone. However, an ATP-synthase inhibitor, oligomycin B, had no significant effect.

5. The Ca²⁺ removal rate was little affected by clamping the cell at +120 mV rather than −70 mV, suggesting that Ca²⁺ removal processes are largely voltage independent. Also, little inward current was associated with Ca²⁺ clearance, indicating that Ca²⁺ removal does not involve an electrogenic process.

6. Our results suggest that Ca²⁺-induced Ca²⁺ release contributes little to the elevation of Ca²⁺ in these cells. The SR Ca²⁺ pump may contribute to Ca²⁺ removal over a low [Ca²⁺]_i range in cells where [Ca²⁺]_i remains high for long enough, while mitochondrial Ca²⁺ uptake may be important when [Ca²⁺]_i is high.

The contractile status of arterial smooth muscle cells is primarily determined by the level of intracellular free Ca²⁺ ([Ca²⁺]_i). An increase in [Ca²⁺]_i may be achieved by the opening of Ca²⁺ permeable channels in the cell membrane or by release of Ca²⁺ from internal storage sites. Numerous electrophysiological studies have been carried out to address the former process while development of Ca²⁺ sensitive probes such as fura-2 has greatly aided understanding of the latter mechanism.

The mechanisms by which Ca²⁺ is cleared from the cytoplasm are much less well understood. Upon the termination of a stimulus, the raised [Ca²⁺]_i must return to its resting level. This is an important process not only because it allows the cell to respond to the next excitatory signal, but also because a prolonged elevation in [Ca²⁺]_i is generally harmful to the cell. Also, inhibition of Ca²⁺ removal can be as important as Ca²⁺ influx and release in elevating [Ca²⁺]_i since [Ca²⁺]_i is determined by the balance between Ca²⁺ entering and leaving the cytosol. Despite this, little is known about Ca²⁺ removal mechanisms in smooth muscle cells.

Recently, the combination of the patch-clamp technique and microfluorimetry has proven to be useful in examining Ca²⁺ homeostasis. Typically, a depolarizing voltage pulse is applied to open voltage-dependent Ca²⁺ channels, and the resultant increase in [Ca²⁺]_i measured simultaneously using fura-2 applied from the patch pipette. Upon repolarization, Ca²⁺ influx through Ca²⁺ channels (I_Ca) will be terminated rapidly due to deactivation of the channels, allowing the Ca²⁺ removal mechanisms to be studied in isolation. From studies carried out so far, the importance of each Ca²⁺ removal pathway appears to vary between different types of smooth muscle. For instance, Ca²⁺ extrusion through Na⁺-Ca²⁺ exchange is important in toad stomach smooth muscle cells (McCarron et al. 1994; McGeown et al. 1996) but not in guinea-pig bladder cells (Ganitkevich & Isenberg, 1991), equine airway smooth muscle cells (Fleischmann et al. 1996), or rat cerebral arterial smooth muscle cells (Kamishima & McCarron, 1998). On the other hand, Ca²⁺ uptake by sarcoplasmic reticulum, SR, is a significant part of Ca²⁺ clearance in rat cerebral artery (Kamishima & McCarron, 1998) but not in toad stomach smooth muscle cells (McGeown et al. 1996). Also, recent studies suggested that Ca²⁺ uptake by mitochondria plays an important part in Ca²⁺ removal (Drummond & Fay, 1996; McGeown et al. 1996; Drummond & Tuft, 1999; McCarron & Muir, 1999) though this Ca²⁺ clearance mechanism has been regarded as not physiologically important by many investigators.

In the study of Ca²⁺ homeostasis, the differences between preparations are not limited to Ca²⁺ removal mechanisms. In guinea-pig urinary bladder (Ganitkevich & Isenberg, 1992) and rat cerebral arterial smooth muscle cells (Kamishima & McCarron, 1997), the amount of Ca²⁺ elevated by sarcolemmal calcium current, I_Ca, is amplified by further Ca²⁺ release from SR (Ca²⁺-induced Ca²⁺ release). In other preparations, however, Ca²⁺-induced Ca²⁺ release is of little significance, for example in rat portal vein, equine trachea and toad and guinea-pig stomach (Fleischmann et al. 1994; Guerrero et al. 1994; Kamishima & McCarron, 1996; Kim et al. 1997). In guinea-pig coronary artery, Ca²⁺-induced Ca²⁺ release is only detectable if I_Ca is enhanced (Ganitkevich & Isenberg, 1995). Thus, unlike Ca²⁺ handling in cardiac myocytes where the mechanisms appear similar in most preparations (Wier, 1990), each type of smooth muscle may have distinctive characteristics in Ca²⁺ handling. Despite this, little comprehensive characterization of Ca²⁺ homeostasis in smooth muscles is available.

In this paper we characterize Ca²⁺ homeostasis in smooth muscle cells of a systemic artery, the rat femoral artery. Our results suggest that Ca²⁺-induced Ca²⁺ release plays little part in the elevation of [Ca²⁺]_i following depolarization in these cells. Ca²⁺ removal through the SR Ca²⁺ pump appears to become important over the low [Ca²⁺]_i range in cells where [Ca²⁺]_i remains high for long enough to activate time-dependent processes. Furthermore, mitochondrial Ca²⁺ uptake seems important when Ca²⁺ load is high. On the other hand, it appears unlikely that a substantial part of elevated [Ca²⁺]_i is cleared through Na⁺-Ca²⁺ exchange. Part of this study has been presented as a communication (Kamishima et al. 1999).

METHODS

Cell dissociation

Male Wistar rats (180–300 g) were rendered unconscious by exposure to a rising concentration of CO₂, and killed by exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedures) Act, 1986. The femoral artery was dissected in a physiological solution containing (mM): 137 NaCl, 0.44 NaH₂PO₄, 0.42 Na₂HPO₄, 4.17 NaHCO₃, 5.6 KCl, 1 MgCl₂, 2 CaCl₂, 10 Hepes, and 10 glucose (pH adjusted to 7.4 with NaOH). Single smooth muscle cells were dissociated using a low-Ca²⁺ salt solution containing (mM): 80 sodium glutamate, 55 NaCl, 6 KCl, 2 MgCl₂, 0.2 CaCl₂, 10 Hepes, 10 glucose, and 0.2 EDTA (pH was adjusted to 7.3 with NaOH at room temperature so that it is 7.4 at 35°C). The femoral artery was digested as previously reported (Quayle et al. 1994). First, the femoral artery was incubated for 30 min at 35°C in the low-Ca²⁺ salt solution containing (mg ml⁻¹): 1.7 papain, 1 elastase (Type II-A), and 0.7 dithioerythritol. Then, the femoral artery was further digested for 20 min at 35°C in the low-Ca²⁺ solution containing (mg ml⁻¹): 1.7 collagenase (Type F) and 1 hyaluronidase (Type I-S). The artery was rinsed with enzyme-free low-Ca²⁺ solution, and single smooth muscle cells were dispersed by triturating the artery with a fire-polished Pasteur pipette. The cell suspension was stored in a refrigerator and used the same day.

Electrophysiology

Membrane currents were recorded using the conventional whole-cell patch clamp technique (Hamill et al. 1981). This configuration permitted the application of the membrane-impermeant fura-2 potassium salt into the cell via the pipette solution. In most experiments, the composition of the extracellular solution was (mM): 80 sodium glutamate, 40 NaCl, 20 tetraethylammonium (TEA) chloride, 1.1 MgCl₂, 3 CaCl₂, 10 Hepes, and 30 glucose (pH adjusted to 7.4 with NaOH, at room temperature, 18–25°C). Unless otherwise stated, the intracellular solution contained (mM): 145 CsCl, 3 MgCl₂, 3 Na₂ATP, 10 Hepes, and 0.05 fura-2 pentapotassium salt (pH adjusted to 7.2 with CsOH, at room temperature). When the concentration of Hepes was raised to 30 mM, no osmotic compensation was made. Whole-cell currents were amplified using an Axopatch 200A (Axon Instruments, Foster City, CA, USA), filtered at 500 Hz, and sampled at 1.5 kHz using custom-made software. All test drugs were added to the extracellular solution except ryanodine which was added to the intracellular solution. In most experiments, the membrane potential was held at −70 mV, and a depolarizing pulse to 0 mV was applied for 1.8 s to evoke a Ca²⁺ current, through voltage-dependent Ca²⁺ channels. All experiments were carried out at room temperature.

Microfluorimetry

High temporal resolution of [Ca²⁺]_i measurements was achieved as previously described (Kamishima & McCarron, 1996), except that we used a PTI deltaRAM (Photon Technology International Inc., London) in the present study. A single cell was loaded with 50 μM fura-2 potassium salt and alternately illuminated with UV light of 340 and 380 nm at 100 Hz (8 nm bandpass). The emission signals were detected at 510 nm (40 nm bandpass), and complete ratio measurements were obtained every 20 ms. For the determination of the K_d of fura-2, 10 mM EGTA and varying amounts of CaCl₂ were added to the intracellular solution to produce solutions containing a range of free Ca²⁺ (Kamishima & McCarron, 1996). From the in vitro calibration, the K_d for Ca²⁺ binding to fura-2 was calculated as 112 nM. The fluorescence ratios in the absence of Ca²⁺, R_min, and in the presence of saturating Ca²⁺, R_max, were also determined in vitro.R_min was obtained with the intracellular solutions containing 10 mM EGTA and no CaCl₂ while R_max was measured with 10 mM EGTA and saturating CaCl₂. These values were decreased by 15 % to adjust for viscosity (Poenie, 1990). Background fluorescence was measured after seal formation but before achievement of the whole-cell configuration, and subtracted from the signals obtained during measurements.

When the effect of ryanodine was tested, the cell was dialysed with ryanodine-containing internal solution for at least 5 min. Other drugs were applied to the bath solution, and typically 4 min waiting period was given before the commencement of experiments. The results are shown as means ±s.e.m. of n cells. When appropriate, Student's unpaired t test was used to examine significant differences (P < 0.05).

Drugs

Fura-2 pentapotassium salt was purchased from Molecular Probes Inc. (Eugene, OR, USA). Ryanodine and cyclopiazonic acid (CPA) were obtained from Calbiochem-Novabiochem Ltd. Both were dissolved in dimethyl sulphoxide (DMSO) to make 30 mM and 10 mM solutions, respectively. The final concentrations of DMSO in the experimental solutions were both 0.1 %. Nimodipine and BayK8644 were purchased from Research Biochemicals International (Natick, MA, USA) and dissolved in DMSO and ethanol to make 10 mM and 0.5 mM stock solution. The final concentrations of DMSO and ethanol in the bathing solutions were both 0.1 %. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and oligomycin B were obtained from Sigma. Both were dissolved in DMSO to make 1 mM and 3 mM solutions. The final concentrations of DMSO in the experimental solutions were both 0.1 %. Papain was obtained from Worthington Biochemical Corporation (Lakewood, NJ, USA). Dithioerythritol, elastase (Type II-A), collagenase (Type F) and hyaluronidase (Type I-S) were purchased from Sigma.

RESULTS

Depolarization evoked Ca²⁺ influx through voltage-dependent Ca²⁺ channels and increased [Ca²⁺]_i

Ca²⁺ influx through voltage-dependent, dihydropyridine-sensitive Ca²⁺ channels provides a useful means to examine Ca²⁺ homeostasis since it provides a rapid, well-controlled increase in [Ca²⁺]_i. Therefore we made simultaneous measurements of membrane currents and Ca²⁺ transients using smooth muscle cells isolated from the rat femoral artery. Figure 1 shows one such experiment. The membrane potential was held at −70 mV, and a depolarizing pulse to 0 mV was applied for 1.8 s. This triggered an inward current (Fig. 1, lower traces) and an increase in [Ca²⁺]_i (Fig. 1, upper trace). Upon repolarization, [Ca²⁺]_i returned to the basal level. The time course of the inward current suggests that it consisted largely of Ca²⁺ influx through voltage-dependent Ca²⁺ channels. This notion was supported by the observations that both inward current and the increase in [Ca²⁺]_i were blocked completely by Ca²⁺ channel antagonists 0.5 mM Cd²⁺ (n = 3, data not shown) or 10 μM nimodipine (n = 2, data not shown).

Figure 1. Inward current and increase in [Ca²⁺]_i in response to a depolarizing pulse.

A single rat femoral arterial smooth muscle cell was voltage clamped, and a 1.8 s depolarizing pulse to 0 mV was applied from a holding potential of −70 mV. This evoked an inward current (lower trace, and shown on an expanded timescale below) and an increase in [Ca²⁺]_i (upper trace). Upon repolarization, elevated [Ca²⁺]_i returned to the baseline.

When I_Ca and [Ca²⁺]_i are determined simultaneously with good time resolution, the relationship between Ca²⁺ entering the cell and Ca²⁺ appearing in the cell can be analysed (see Ganitkevich & Isenberg, 1995; Kamishima & McCarron, 1996; Kim, et al. 1997). Thus, the expected total Ca²⁺ increase due to I_Ca was calculated as:

(1)

where [integral]-I_Cadt is total charge entry, F is Faraday's constant, and η is the cell volume, estimated as 2.6 pl. It was assumed that at the end of the 1.8 s pulse to 0 mV, most of the Ca²⁺ channels were inactivated since usually little tail current and concomitant [Ca²⁺]_i increase were observed at the instant of repolarization. Thus, I_Ca was time integrated as the area under the curve taking the level at the end of the pulse as the baseline (see Kamishima & McCarron, 1996). The dotted line in Fig. 2a shows the time course of the expected increase in [Ca²⁺]_i. Figure 2a also shows the measured increase in [Ca²⁺]_i obtained by subtracting the resting [Ca²⁺]_i from the measured [Ca²⁺]_i (continuous line). Although the time course of these lines were similar, the magnitude of the expected increase in [Ca²⁺]_i far exceeded that of the measured increase in [Ca²⁺]_i. Thus, the expected increase in [Ca²⁺]_i is shown in micromolar (right hand ordinate) while the measured increase in [Ca²⁺]_i is shown in nanomolar (left hand ordinate). This indicates that the Ca²⁺ flux through voltage-dependent Ca²⁺ channels was more than enough to account for the observed increase in [Ca²⁺]_i as described in other preparations (Neher & Augustine, 1992; Ganitkevich & Isenberg, 1995; Kamishima & McCarron, 1996; Kamishima & McCarron, 1997; Kim et al. 1997).

Figure 2. I_Ca does not trigger Ca²⁺ release from the sarcoplasmic reticulum during depolarization.

A, the continuous line shows the measured increase in [Ca²⁺]_i (difference between the measured [Ca²⁺]_i and the resting [Ca²⁺]_i, left hand ordinate), plotted against time for the first 1 s of the depolarizing pulse. The dotted line shows the expected increase in [Ca²⁺]_i calculated from I_Ca using eqn (1) (right hand ordinate). The time courses of the two lines are similar, but note that the expected increase in [Ca²⁺]_i is shown in micromolar while the measured increase in [Ca²⁺]_i is shown in nanomolar. B, relationship between the expected and measured increase in [Ca²⁺]_i in control cells (n = 5). The plot was made from the data obtained during the first 200 ms of the depolarization. During this period, the relationship was linear (regression coefficient = 0.96 ± 0.01, n = 5), and therefore the total Ca²⁺ buffering power was calculated as the reciprocal of the slope of the fit for each cell. C, relationship between the expected and measured increases in [Ca²⁺]_i in cells treated with ryanodine (30 μM, regression coefficient = 0.96 ± 0.01, n = 5). The values for the total buffering power were not significantly different between control and ryanodine-treated cells, suggesting the Ca²⁺-induced Ca²⁺ release contributes little to the elevation of [Ca²⁺]_i.

I_Ca does not induce further Ca²⁺ release from SR

The observation that the amount of Ca²⁺ entering the cell through Ca²⁺ channels exceeded Ca²⁺ appearing in the cytosol does not preclude the possibility that I_Ca triggers further Ca²⁺ release from SR (Ca²⁺-induced Ca²⁺ release). While it is well known that Ca²⁺-induced Ca²⁺ release plays a crucial part in cardiac excitation-contraction coupling (e.g. Fabiato, 1983), its importance in smooth muscle cells seems to vary among preparations. We therefore examined the contribution of Ca²⁺-induced Ca²⁺ release to the elevation of [Ca²⁺]_i that occurs during depolarization in rat femoral artery smooth muscle cells.

First, the relationship between the expected and measured increases in [Ca²⁺]_i was examined in control cells. The analysis was carried out using measurements obtained during the first 200 ms of the depolarization since it is unlikely that the rate of Ca²⁺ removal changes substantially over this short period (Guerrero et al. 1994). Thus, the measured increase in [Ca²⁺]_i was plotted as a function of expected increase in [Ca²⁺]_i as summarized in Fig. 2B. The relationship was well fitted with a linear regression. From such a relationship, it is possible to calculate how many Ca²⁺ ions must enter the cell in order to provide an increase of one free Ca²⁺ ion (the Ca²⁺ buffering power, Guerrero et al. 1994). Thus, Ca²⁺ buffering power was obtained as the reciprocal of the slope of each linear fit. The Ca²⁺ buffering power calculated using this method consists of both the intrinsic buffering power of the cell and the fura-2 buffering power. In control cells, the total Ca²⁺ buffering power was 257 ± 49 (n = 5) indicating that one in 257 Ca²⁺ ions entering the cell appears as free Ca²⁺, and the remaining Ca²⁺ binds to intrinsic Ca²⁺ buffer and fura-2. During the first 200 ms of the depolarizing pulse, the measured [Ca²⁺]_i increased by about 80 nM while the expected increase in [Ca²⁺]_i was about 28 μM. Of this expected increase in [Ca²⁺]_i, about 14 μM is calculated to bind to fura-2 under our experimental conditions. Therefore, the buffering power of fura-2 is about 50 % of the total Ca²⁺ buffering power. Thus, the intrinsic Ca²⁺ buffering power of rat femoral artery smooth muscle cell is about 130, not dissimilar to the values reported using guinea-pig coronary artery (150, Ganitkevich & Isenberg, 1995) and rat portal vein cells (114, Kamishima & McCarron, 1996), but somewhat higher than the values reported using equine airway (∼15-50, Fleischmann et al. 1996) and guinea-pig urinary bladder cells (46, Ganitkevich, 1996).

Next, the total Ca²⁺ buffering power was measured in cells treated with an inhibitor of Ca²⁺-induced Ca²⁺ release, ryanodine. Ryanodine (30 μM) was included in the pipette solution, and I_Ca and the corresponding Ca²⁺ transient was evoked by depolarization to 0 mV as for the control cells above. The relationship between the expected and measured increases in [Ca²⁺]_i was also linear as shown in Fig. 2C, and the total buffering power calculated as the reciprocal of the slope was 236 ± 52 (n = 5), not significantly different from that of the control cells. If there was significant Ca²⁺-induced Ca²⁺ release, the measured buffering power of the ryanodine-treated cells would have been significantly larger than that of the control cells. Thus, our results indicate that I_Ca triggers little Ca²⁺ release from the SR in rat femoral arterial smooth muscle cells during depolarization.

Effects of inhibition of the SR Ca²⁺ pump on the profile of Ca²⁺ removal

[Ca²⁺]_i is determined by the balance between Ca²⁺ entering and leaving the cytosol. While the former has been a focus of intensive research and thus is relatively well understood, little is known about the latter. Hence, the Ca²⁺ removal mechanisms were examined in the next set of experiments. First, the profile of the removal mechanism was studied under control conditions. The declining phase of the Ca²⁺ transient was fitted with a polynomial function (Fig. 3a, upper trace, continuous line), and the removal rate obtained as the negative time derivative of the fit. The result was plotted against either time (Fig. 3a, bottom left), where time = 0 is the instant of repolarization, or measured [Ca²⁺]_i (Fig. 3a, bottom right). In this cell, the overall Ca²⁺ removal profile resembles those reported in rat cerebral artery (Kamishima & McCarron, 1998) and toad stomach smooth muscle cells (McGeown et al. 1996). The Ca²⁺ removal rate was fast immediately after repolarization (phase 1) and then became somewhat slower (phase 2). Before [Ca²⁺]_i returned to the resting level, the rate of Ca²⁺ removal increased (phase 3), and this appears as an upward hump in the Ca²⁺ removal profile (Fig. 3a, bottom traces). Thus, Ca²⁺ clearance in this cell appears to consist of three phases, possibly suggesting that the process involves more than one mechanism.

Figure 3. A delayed increase in Ca²⁺ removal rate was observed in a control but not in a CPA-treated cell.

A, the declining phase of the Ca²⁺ transient was fitted with a 9th order polynomial (upper trace, continuous line), and the Ca²⁺ removal rate was calculated as its negative derivative. The result is expressed either as a function of time (bottom left), where time = 0 is the instant of repolarization, or of measured [Ca²⁺]_i (bottom right). In this cell, the removal profile consisted of three phases. The removal rate was fast immediately after the repolarization (phase 1) and then somewhat slowed down (phase 2). Before the [Ca²⁺]_i returned to the resting level, the removal rate increased, and this appears as an upward hump (phase 3). B, the Ca²⁺ removal profile in a cell treated with CPA. Note the absence of the upward hump (phase 3) in the bottom traces.

Since it has been reported recently that Ca²⁺ uptake by SR is important for Ca²⁺ clearance in rat cerebral arterial smooth muscle cells (Kamishima & McCarron, 1998), the effect of an inhibitor of SR CaATPase, cyclopiazonic acid (CPA), was examined. Application of 10 μM CPA significantly increased resting [Ca²⁺]_i to 68 ± 15 nM (n = 11) from its value in control cells (35 ± 3 nM, n = 19). When the removal rate was calculated from a polynomial fit (Fig. 3B, upper trace, continuous line), no obvious upward hump was observed (Fig. 3B, bottom traces). Thus, inhibition of the CaATPase in SR appears to slow down the rate of the third phase of Ca²⁺ removal, with little effect on the first phase.

Figure 4 summarizes Ca²⁺ removal rates obtained from 13 control cells and 10 CPA-treated cells. Ca²⁺ removal rates are expressed as a function of measured [Ca²⁺]_i in 25 nM increments. It appears that CPA reduced the Ca²⁺ removal rate over the low [Ca²⁺]_i range, up to 200 nM, with little effect at higher [Ca²⁺]_i. This observation is consistent with the finding in rat cerebral artery that, when the SR Ca²⁺ uptake was inhibited by thapsigargin (500 nM) or ryanodine (30 μM), the Ca²⁺ removal rate was reduced in the [Ca²⁺]_i approximate range of 150–275 nM with little change at higher concentrations (Kamishima & McCarron, 1998). However, while a significant difference was detected between control cells and store-disrupted cells from the cerebral artery, our present results from rat femoral artery do not reach statistical significance.

Figure 4. Ca²⁺ removal rates in control and CPA-treated cells.

The Ca²⁺ removal rate is plotted as a function of measured [Ca²⁺]_i at 25 nM intervals. •, mean ±s.e.m. results from 13 cells under control conditions. □, results from 10 cells treated with CPA.

SR Ca²⁺ uptake is a time-dependent process

The lack of a significant difference in Ca²⁺ removal rate between control cells and cells treated with CPA is not easy to reconcile with the finding that CPA treatment significantly elevated resting [Ca²⁺]_i. From Fig. 4, it appears that the characteristic feature of the third phase of Ca²⁺ removal, the upward hump (Fig. 3a), is less prominent when measurements from several cells were averaged (filled circles in Fig. 4). Indeed, it seems this is because there are two types of Ca²⁺ removal profile among control cells.

The third phase, a delayed increase in the Ca²⁺ removal rate at lower [Ca²⁺]_i, was observed in 7 out of 13 control cells while the remaining 6 cells did not show a clear upward hump. In rat cerebral artery smooth muscle cells where a significant contribution of SR Ca²⁺ uptake was detected, it was suggested that Ca²⁺ uptake by SR is facilitated in a time-dependent manner by elevated [Ca²⁺]_i (Kamishima & McCarron, 1998). This notion appears to be useful in explaining the two types of Ca²⁺ removal profile seen in our control cells. In cells which developed the third phase, its peak occurred at 20.4 ± 3.4 s (n = 7) after repolarization (see Fig. 3a). On the other hand, 5 out of 6 cells which did not develop the third phase returned to the resting [Ca²⁺]_i in less than 20 s. Figure 5 shows the Ca²⁺ removal profile from one such cell.

Figure 5. The third phase does not develop in a control cell where increased [Ca²⁺]_i returned to the resting level quickly.

Removal profile of a control cell which lacked the third phase. The elevated [Ca²⁺]_i returned to the resting level within 20 s. The peak third phase in the control cell shown in Fig. 3A, however, occurred around 25 s.

If the hypothesis that the third phase develops when [Ca²⁺]_i remains high for long enough is appropriate to explain that there are two kinds of Ca²⁺ removal in the control cells, then a control cell without the third phase should develop an upward hump when a higher Ca²⁺ load is received. This possibility was tested by subjecting control cells without the third phase to a Ca²⁺ channel opener, BayK8644. Figure 6 shows one such experiment. The Ca²⁺ removal profile was observed before and after application of 0.5 μM BayK8644. Initially, this control cell did not develop a clear upward hump (Fig. 6a, bottom traces), and elevated [Ca²⁺]_i returned to the resting level in less than 20 s (Fig. 6a, bottom left trace). After treatment with BayK8644, however, the same cell received an increased Ca²⁺ load (Fig. 6B, upper trace), and the third phase is seen as an upward hump (Fig. 6B, bottom traces). The appearance of the third phase after application of BayK8644 was observed with another cell which initially did not develop the third phase. Thus, it appears that the absence of the third phase in some of the control cells is not due to the lack of this Ca²⁺ removal pathway in these cells, but rather that raised [Ca²⁺]_i returned to the baseline before the development of the third phase. As shown in Fig. 3B, however, the third phase does not appear to develop in a cell treated with CPA, even when it took more than 40 s to return to the resting [Ca²⁺]_i, twice as long as the time to the peak of the third phase in control cells without CPA. Thus, in rat femoral artery elevated [Ca²⁺]_i may be cleared by the SR Ca²⁺ pump provided that [Ca²⁺]_i remains high for long enough for this pump to be activated.

Figure 6. An enhanced Ca²⁺ load induces the third phase in a control cell which initially did not develop an upward hump.

A, the declining phase of the Ca²⁺ transient was fitted with a 9th order polynomial (upper trace, continuous line), and the Ca²⁺ removal rate was calculated as its negative derivative. The result is expressed either as a function of time where time = 0 is the instant of repolarization (bottom left) or of measured [Ca²⁺]_i (bottom right). In this cell, elevated [Ca²⁺]_i returned to the resting level in less than 20 s, and no obvious third phase was observed. B, Ca²⁺ removal profile of the same cell as in A after Ca²⁺ load was enhanced with the application of Ca²⁺ channel opener, BayK8644 (0.5 μM). It took about 30 s for the raised [Ca²⁺]_i to return to the baseline (bottom left), and the third phase is now apparent (bottom traces).

High voltage has little effect on Ca²⁺ removal

Some Ca²⁺ removal mechanisms such as the Na⁺-Ca²⁺ exchanger are voltage dependent while others such as the Ca²⁺ pumps in the plasma membrane are not. Therefore, the voltage dependence of Ca²⁺ removal was examined. As shown in Fig. 7, when the membrane potential was held at +120 mV rather than −70 mV following a depolarization to 0 mV, no apparent inhibition of Ca²⁺ removal was observed. Indeed, when the membrane potential was switched to −70 mV from +120 mV, no facilitation in Ca²⁺ removal was noted. If a voltage-dependent Ca²⁺ removal process had been suppressed at +120 mV, the sudden appearance of another Ca²⁺ removal mechanism upon the voltage jump to −70 mV would have appeared as a kink in the Ca²⁺ removal profile. Similar results were obtained in two other cells. Thus, it appears that the Ca²⁺ clearance in rat femoral arterial smooth muscle cells largely utilizes voltage-independent mechanisms.

Figure 7. High voltage does not affect the Ca²⁺ removal profile.

Following a depolarization to 0 mV, the membrane potential was clamped at +120 mV, then repolarized to −70 mV (middle trace). When the removal rate was determined from the polynomial fit (upper trace, continuous line), high voltage did not inhibit Ca²⁺ clearance (bottom trace).

Inhibition of mitochondrial Ca²⁺ uptake reduces the peak first phase Ca²⁺ removal rate

Mitochondria can act as a Ca²⁺ uptake compartment due to the large electrochemical gradient in favour of Ca²⁺ accumulation. However, the notion that mitochondria sequester Ca²⁺ under physiological conditions has been largely dismissed by many investigators. Nonetheless, recent re-appraisal of mitochondrial Ca²⁺ uptake in various preparations including smooth muscle cells has indicated that mitochondria play an integral part in Ca²⁺ regulation (Drummond & Fay, 1996; McGeown et al. 1996; Drummond & Tuft, 1999; McCarron & Muir, 1999). Thus, the role of mitochondria in the Ca²⁺ clearance mechanism was examined.

To inhibit mitochondrial Ca²⁺ uptake, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used. CCCP is an ionophore which collapses mitochondrial potential by making the membrane permeable to protons. Thus, when the driving force on Ca²⁺ is eliminated by CCCP, mitochondria will no longer sequester Ca²⁺. When 1 μM CCCP was applied, the resting [Ca²⁺]_i was significantly increased to 88 ± 7 nM (n = 13). In the presence of 1 μM CCCP, the Ca²⁺ removal rate was examined. One example is shown in Fig. 8A. It appears that the initial rapid Ca²⁺ removal, observed in the control cells (e.g. Figs 3A and 5), is substantially reduced when mitochondrial Ca²⁺ uptake is inhibited. Thus, the peak first-phase Ca²⁺ removal rate was measured at the instant of repolarization, and the values in the presence and absence of CCCP were compared. The peak first-phase Ca²⁺ removal rate of CCCP-treated cells was 14.2 ± 2.1 nM s⁻¹ (n = 14), significantly different from the values obtained with control cells (52.3 ± 11.7 nM s⁻¹, n = 13).

Figure 8. Inhibition of mitochondrial Ca²⁺ uptake reduces the peak first phase Ca²⁺ removal rate.

A, Ca²⁺ transient evoked in the presence of a mitochondrial Ca²⁺ uptake inhibitor, CCCP. The declining phase of the Ca²⁺ transient was fitted with a 9th order polynomial (upper trace, continuous line), and the Ca²⁺ removal rate was calculated as its negative derivative. The result is expressed either as a function of time (bottom left), where time = 0 is the instant of repolarization, or of measured [Ca²⁺]_i (bottom right). In this cell, the initial rapid decay in [Ca²⁺]_i, seen as the first phase in the control cell (e.g. Fig. 3), appears to be slowed. B, application of oligomycin B, an inhibitor of mitochondrial ATP synthase, did not suppress Ca²⁺ removal rate during the initial rapid phase.

To test the possibility that CCCP exerts its effect through acidification of the intracellular milieu as reported in guinea-pig urinary bladder (Ganitkevich, 1999), the concentration of Hepes in the pipette solution was increased. Raising the Hepes concentration to 30 mM to reduce possible acidification had no effect on the profile of Ca²⁺ removal in CCCP-treated cells (n = 3, data not shown). Thus, it is unlikely that the observed CCCP effect was due to a reduction in intracellular pH in rat femoral arterial smooth muscle cells.

When the proton gradient in the mitochondrial membrane is removed by CCCP, ATP is no longer produced by mitochondria. Since 3 mM Na₂ATP is included in the patch pipette, the inhibition of ATP production by mitochondria also seems unlikely to contribute to reduction in the peak first-phase Ca²⁺ removal rate. Nonetheless, under some conditions CaATPase may utilize ATP which is either compartmentalized or bound to the site of action (Steenbergen & Fay, 1996). Hence, the effect of oligomycin B on the Ca²⁺ removal profile was examined. Oligomycin blocks mitochondrial ATP synthase without affecting the membrane potential. Application of 3 μM oligomycin B did not have a significant effect on resting [Ca²⁺]_i (36 ± 8 nM, n = 11). When the Ca²⁺ removal profile was examined in the presence of oligomycin (Fig. 8B), the peak first-phase Ca²⁺ removal rate did not appear to be reduced. The average peak first-phase Ca²⁺ removal rate determined from 10 oligomycin-treated cells was 32.8 ± 5.0 nM s⁻¹, not significantly different from the control values. Thus, it seems likely that the reduction in the peak first-phase Ca²⁺ removal rate observed in CCCP-treated cells is due to the inhibition of mitochondrial Ca²⁺ uptake.

DISCUSSION

Though the role of SR in Ca²⁺ homeostasis has been a focus of intense interest, little is known about its precise function in smooth muscle. Thus, one of the aims of the current study was to examine the contribution of SR to the depolarization-induced elevation of [Ca²⁺]_i via Ca²⁺-induced Ca²⁺ release.

Since the application of caffeine (25 mM) produced a large Ca²⁺ transient whose peak [Ca²⁺]_i often exceeded 1 μM (n = 4, data not shown), rat femoral artery smooth muscle cells clearly contain caffeine-sensitive SR. However, the total Ca²⁺ buffering power of ryanodine-treated cells was not significantly different from that of control cells. Thus, Ca²⁺-induced Ca²⁺ release appears to contribute little to the elevation of [Ca²⁺]_i during a depolarization in this preparation. In a preparation where a significant contribution of Ca²⁺-induced Ca²⁺ release was identified (Kamishima & McCarron, 1997), I_Ca was more effective in releasing Ca²⁺ at smaller depolarizations. This observation was attributed to the larger size of the unitary current at less positive voltages. To detect the amplitude of currents through single Ca²⁺ channels, the ryanodine receptors must be located in close proximity to voltage-dependent Ca²⁺ channels. Indeed, Carrington et al. (1995) have shown that dihydropyridine and ryanodine receptors are co-localized in guinea-pig bladder smooth muscle cells, preparations where Ca²⁺-induced Ca²⁺ release was previously identified (Ganitkevich & Isenberg, 1992). On the other hand, such a spatial arrangement of ryanodine receptors and Ca²⁺ channels may be absent in tissues such as the femoral artery where little Ca²⁺-induced Ca²⁺ release is detected.

The role of the SR in Ca²⁺ removal is somewhat more elusive. In 7 out of 13 control cells, the temporal profile of Ca²⁺ decline followed the pattern reported in toad stomach and rat cerebral arterial smooth muscle cells (McGeown et al. 1996; Kamishima & McCarron, 1998). [Ca²⁺]_i declined rapidly following repolarization (phase 1), and then the removal rate was somewhat slowed down (phase 2). Before the [Ca²⁺]_i returned to the resting level, the Ca²⁺ removal rate increased, appearing as an upward hump in the removal profile (phase 3). On the other hand, the remaining 6 cells did not show a well-defined third phase, while cells treated with the SR Ca²⁺ pump inhibitor, CPA, showed little third phase. It appears that the lack of the third phase in some of the control cells is not due to the absence of this Ca²⁺ removal pathway in these cells but rather that Ca²⁺ had declined too rapidly for the development of the third phase. In fact, control cells which initially had no apparent third phase developed an upward hump after their Ca²⁺ load was enhanced by the application of BayK8644 (Fig. 6). In other preparations where Ca²⁺ removal mechanisms were examined in detail, the third phase appears to be a time-dependent process. In the case of rat cerebral artery, the third phase is due to Ca²⁺ uptake by the SR, and this process seems time dependently regulated (Kamishima & McCarron, 1998). In the case of toad stomach smooth muscle cells, the third phase is due to delayed increase in mitochondrial Ca²⁺ uptake (McGeown et al. 1996). These authors later reported that the activation of mitochondrial Ca²⁺ uptake is controlled by Ca²⁺-calmodulin and calmodulin-dependent protein kinase II (McGeown et al. 1998). Thus, it is probably reasonable to suggest that the third phase, Ca²⁺ uptake by the SR, was observed in rat femoral arterial cells only when [Ca²⁺]_i was elevated for long enough to activate Ca²⁺ pumps in the SR. Consistent with this hypothesis, the average peak third phase occurred at about 20 s while in 5 out of 6 cells which did not develop the third phase, [Ca²⁺]_i returned to the resting level in less than 20 s. Thus, Ca²⁺ uptake by SR in femoral artery may contribute to Ca²⁺ clearance if [Ca²⁺]_i is elevated for long enough. On the other hand, rat cerebral artery smooth muscle cells appear to take up Ca²⁺ into SR more readily (Kamishima & McCarron, 1998), and this may be because a significant amount of Ca²⁺ is released from the SR during depolarization in this preparation (Kamishima & McCarron, 1997). Accordingly, it is possible that SR Ca²⁺ uptake may become more important in femoral artery cells if [Ca²⁺]_i is elevated by Ca²⁺ release from the SR, for example by inositol 1,4,5-trisphosphate-induced Ca²⁺ release in response to receptor activation.

When SR does remove Ca²⁺, it appears to do so at the lower range of [Ca²⁺]_i. On the other hand, inhibition of mitochondrial Ca²⁺ uptake by CCCP significantly reduced the peak first-phase Ca²⁺ removal rate immediately following repolarization. It seems unlikely that the effect of CCCP is due to the acidification of the cells since increasing pH buffer capacity did not prevent reduction in the peak first-phase Ca²⁺ removal rate caused by CCCP. Also, the observed effect is unlikely to be due to the inhibition of ATP production by mitochondria, since oligomycin B did not have a significant effect on the peak first-phase Ca²⁺ removal rate. Reduction of the Ca²⁺ removal rate by CCCP was also reported in guinea-pig colonic smooth muscle cells (McCarron & Muir, 1999). In these intestinal smooth muscle cells, application of CCCP slowed the Ca²⁺ removal rate when [Ca²⁺]_i was higher than 300 nM, but not when [Ca²⁺]_i was lower (McCarron & Muir, 1999). Similarly, mitochondrial Ca²⁺ uptake seems to become more important in cultured aortic myocytes when [Ca²⁺]_i is higher (Monteith & Blaustein, 1999). On the other hand, inhibition of mitochondrial Ca²⁺ uptake had no effect on the initial rapid decay in [Ca²⁺]_i, but reduced the third phase in toad stomach smooth muscle cells (McGeown, et al. 1996). Thus, the [Ca²⁺]_i range over which mitochondrial Ca²⁺ removal is more prominent may vary among smooth muscle preparations.

The voltage dependence of Ca²⁺ removal mechanisms was also examined. Clamping the membrane potential to +120 mV following depolarization to 0 mV had no apparent effect on the Ca²⁺ removal rate. Thus, voltage-dependent processes appear to contribute little to Ca²⁺ removal in this preparation. Furthermore, little inward current was detected during the declining phase of the Ca²⁺ transient (data not shown). Combined, these observations suggest that it is unlikely that Na⁺-Ca²⁺ exchange plays a major role in Ca²⁺ removal in this preparation. Lack of significant Ca²⁺ removal through the Na⁺-Ca²⁺ exchanger has been reported in other types of mammalian smooth muscle preparations (Ganitkevich & Isenberg, 1991; Fleischmann et al. 1996; Kamishima & McCarron, 1998). However, Na⁺-Ca²⁺ exchange may be important in some preparations (e.g. Aaronson & Benham, 1989; Leblanc & Leung, 1995).

In summary, Ca²⁺ uptake by both SR and mitochondria may play an important part in Ca²⁺ removal. Yet since elevated [Ca²⁺]_i is cleared from cells treated with inhibitors of Ca²⁺ sequestration, other mechanisms must also contribute to Ca²⁺ removal. Although not tested directly in the present study, most certainly Ca²⁺ pumps in the cell membrane are important in Ca²⁺ clearance (Carafoli, 1991; O'Donnell & Owen, 1994; Kamishima & McCarron, 1998). Since these three mechanisms can potentially work in parallel, multiple Ca²⁺ removal pathways ensure that [Ca²⁺]_i is lowered effectively following depolarization.