Role of sarcoplasmic reticulum Ca²⁺ content in Ca²⁺ entry of bovine airway smooth muscle cells

Abstract

Depletion of intracellular Ca²⁺ stores induces the opening of an unknown Ca²⁺ entry pathway to the cell. We measured the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) at different sarcoplasmic reticulum (SR) Ca²⁺ content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca²⁺ in the extracellular medium generated a time-dependent decrement in [Ca²⁺]i which was proportional to the reduction in the SR-Ca²⁺ content. This SR-Ca²⁺ level was indirectly determined by measuring the amount of Ca²⁺ released by caffeine. Ca²⁺ restoration at different times after Ca²⁺-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca²⁺]i. This increase in [Ca²⁺]i was considered as Ca²⁺ entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca²⁺ content was higher than 50% (2 and 4 min in Ca²⁺-free medium), and significantly (p<0.01) accelerated (>1.0 nM/s) when SR-Ca²⁺ content was lower than 50% (6 and 10 min in Ca²⁺-free medium). Thapsigargin significantly induced a higher rate of this Ca²⁺ entry (p<0.01). Variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by a Mn²⁺ quench approach. Ca²⁺ restoration to the medium 4 min after Ca²⁺ removal did not modify the Mn²⁺ influx. However, when Ca²⁺ was added after 10 min in Ca²⁺-free medium, an increment of Mn²⁺ influx was observed, corroborating an increase in Ca²⁺ entry. The fast Ca²⁺ influx was Ni²⁺ sensitive but was not affected by other known capacitative Ca²⁺ entry blockers such as La³⁺, Mg²⁺, SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2⁺ channels with methoxyverapamil or by the sustained K⁺-induced depolarisation. The slow Ca²⁺ influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx, which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry; and B) The fast Ca²⁺ influx, observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive.

Introduction

In airway smooth muscle (ASM) intracellular free calcium concentration ([Ca²⁺]i) has an important role mediating excitation-contraction coupling. In this tissue two ways have been described to increase the [Ca²⁺]i. One involves the extracellular Ca²⁺ entry through receptor-operated and voltage-sensitive (both L and T type) Ca²⁺ channels (Kajita and Yamaguchi 1993; Cuthbert et al. 1994; Montaño et al. 1996; Janssen 1997). The other mechanism requires intracellular Ca²⁺ release mainly from sarcoplasmic reticulum (SR) via inositol 1, 4, 5-trisphosphate- and ryanodine-sensitive channels (Baron et al. 1984; Madison et al. 1998; Bazán-Perkins et al. 1998, 2000).

Endoplasmic/sarcoplasmic reticulum Ca²⁺ depletion is associated to Ca²⁺ influx. This process, known as capacitative Ca²⁺ entry, was formerly observed in non-excitable cells by Putney (1986). In human ASM cells, Amrani and co-workers (1995) observed an increase of [Ca²⁺]i after SR-Ca²⁺ depletion suggesting a capacitative Ca²⁺ entry mechanism.

A direct relationship between the SR-Ca²⁺ content and the temporal course of Ca²⁺ entry has been demonstrated in non-excitable cells (Jacob 1990; Hofer et al. 1998; Sedova et al. 2000) and in excitable cells, such as pancreatic β-cells (Dyachok and Gylfe 2001). However, it is unknown if a similar relationship occurs in ASM.

Currently, there is no consensus regarding the efficiency and selectivity of capacitative channel blockers, but cations like La³⁺ and Mg²⁺ have been reported to block this Ca²⁺ entry in excitable cells (Yoshimura et al. 1997; Yang 1998). Ni²⁺, an inorganic Ca²⁺ channel blocker, has also been used to inhibit the capacitative Ca²⁺ entry in non-excitable (Hoth and Penner 1993; Kerschbaum and Cahalan 1998) and excitable cells (Lee et al. 2002; McDaniel et al. 2001).

Recently, some substances as 2-aminoethyldiphenyl borate (2-APB) and the 1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) have been used as capacitative Ca²⁺ entry blocker in vascular myocytes and mast cells, respectively (Franzius et al. 1994; Peppiatt et al. 2003; Wakabayashi et al. 2003). The aim of the present work was to study, in bovine ASM cells, the effect of different SR-Ca²⁺ content on Ca²⁺ entry of depolarised and non-depolarised cells, and to investigate the effect of 2-APB, SKF 96365, La³⁺, Mg²⁺ and Ni²⁺ in this phenomenon.

Materials and methods

Preparation of tracheal smooth muscle

Bovine tracheas were obtained from a local slaughterhouse and transported to the laboratory in cold, oxygenated, physiological salt solution (PSS) with the following composition (mM): 118 NaCl, 25 NaHCO₃, 4.6 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2 CaCl₂ and 11 glucose. Animals were sacrificed according the Mexican laws. Tracheal smooth muscle was dissected free of adhered fat and connective tissue using a stereoscopic microscope Nikon SWZ-10 (Tokyo, Japan), at room temperature in oxygenated PSS.

Tracheal myocytes isolation

Airway smooth muscle cells were obtained from bovine trachea as follows. Approximately 200 mg smooth muscle were minced, placed in 5 ml Hanks solution (Gibco BRL, Rockville, MD) containing 2 mg cysteine and 0.05 U ml^-1 papaine, and incubated for 10 min at 37°C. The tissue was washed with Leibovitz's solution (Gibco) to remove the enzyme excess, and then placed in PSS containing 0.144 mg ml^-1 of a highly purified collagenases and neutral proteases mixture (Liberase Blendzyme 2, Roche, Indianapolis, IN) during ~12 min until dispersed cells were observed. This procedure allowed us to obtain cells with consistent levels of resting [Ca²⁺]i (Montaño et al. 2003). Then, cells were loaded with 0.5 µM fura 2/AM in low Ca²⁺ (0.1 mM) at room temperature (22–25°C). After 1 h, cells were allowed to settle into a heated perfusion chamber with a glass cover in the bottom. This chamber was mounted on a Nikon inverted microscope (Diaphot 200, Tokyo, Japan) and the cells adhered to the glass were continuously perfused at a rate of 2–2.5 ml/min with PSS (37°C, equilibrated with 95% O₂/ 5% CO₂, pH 7.4) and containing 2 mM Ca²⁺.

[Ca²⁺]i measurement

Cells loaded with fura 2 were exposed to alternating pulses of 340 and 380 nm excitation light, and emission light was collected at 510 nm using a PTI microphotometer (Photon Technology International, Princenton, NJ). Background fluorescence was automatically subtracted and determined by removing the cell from the field before starting the experiments. The fluorescence acquisition rate was 0.5 s. [Ca²⁺]i was calculated according to the formula of Grynkiewicz and co-workers (1985). The K _d of fura 2 was assumed to be 386 nM (Kajita and Yamaguchi 1993). The mean 340/380 fluorescence ratios Rmax and Rmin were obtained by exposing the cells to 10 mM Ca²⁺ in presence of 10 µM ionomycin and in Ca²⁺-free PSS with 1.11 mM EGTA, respectively. Rmax was 11.2 and Rmin 0.37. The fluorescence ratio at 380 nm light excitation in Ca²⁺-free PSS and Ca²⁺ saturated cells (ß) was 5.2.

Experimental design

Bovine ASM cells stimulated with 10 mM caffeine or 10 µM carbachol (Cch) in PSS, were afterwards perfused with nominal Ca²⁺ free medium for about 10 min. During this period, caffeine was added at 2, 4, 6 or 10 min. In other set of experiments, following a similar protocol, Ca²⁺ was restored at the same intervals, 2, 4, 6 and 10 min. Cells used in these intervals are referred in the text as groups 2, 4, 6 and 10. In some cells of 10-min group, Ca²⁺-free medium was supplemented with 1 µM thapsigargin. Other cells from groups 4 and 10 were previously depolarised by substitution of 118 mM NaCl with 122 mM KCl or incubated with 30 µM of methoxyverapamil hydrochloride (D-600), an L-type Ca²⁺ channel blocker. For each interval we used different cells.

Additionally, in groups 4 and 10, we explored the effect of 100 µM 2-APB, 20 µM SKF 96365, 4 mM Mg²⁺, 0.2 mM La³⁺ or 1 mM Ni²⁺, a well known capacitative Ca²⁺ entry blockers (Hoth and Penner 1993; Franzius et al. 1994; Yoshimura et al. 1997; Yang 1998; Bootman et al. 2002; Kawanabe et al. 2002; Peppiatt et al. 2003; Wakabayashi et al. 2003), during Ca²⁺ entry after SR-Ca²⁺ depletion. In La³⁺ and Mn²⁺ experiments the PSS had the following composition (mM): 138 NaCl, 5 HEPES, 4.6 KCl, 1.2 MgCl₂, 1.2 KH₂PO₄, 2 CaCl₂ and 11 glucose, the pH 7.4 was adjusted with NaOH (10 M).

Finally, using groups 4 and 10, variations in Ca²⁺ influx after SR-Ca²⁺ depletion were estimated more directly by Mn²⁺ (50 µM) quench approach. In this experiments 360 nm excitation light was used.

Drugs

Caffeine, fura 2/AM, EGTA, ionomycin, HEPES, methoxyverapamil hydrochloride, thapsigargin, lanthanum chloride heptahydrate, nickel chloride hexahydrate, magnesium chloride anhydrous and manganese chloride tetrahydrate were obtained from Sigma (St. Louis, MO). SKF 96365 was obtained from Biomol (Plymouth Meeting, PA). 2-APB was obtained from Calbiochem (La Jolla, CA). Liberase Blendzyme 2 was obtained from Roche (Indianapolis, IN). Ionomycin and fura 2/AM were dissolved in DMSO (final concentration 0.025%). In control experiments, use of DMSO alone had no effect.

Statistics

One-way analysis of variance and Tukey's test for multiple comparisons were used. In some experiments we used Student's t-test for paired data. Two-tailed p values <0.05 were considered statistically significant. Ca²⁺ entry rate was calculated for each experiment using straight linear regression and calculating the slope of each recording during Ca²⁺ restoration and expressed as nM/s. All values in the text and illustrations correspond to mean ± SEM.

Results

Effect of external Ca²⁺ removal on the [Ca²⁺]i

The resting [Ca²⁺]i in bovine ASM cells was 148±6 nM (n=90). Caffeine (10 mM) addition during 2 min resulted in a transient Ca²⁺ peak (630±10 nM, n=31; Fig. 1a). Cch (10 µM) incubation induced a similar transient response (472±96 nM, n=4). Removal of external Ca²⁺ lead to a continuous reduction in the [Ca²⁺]i until a steady state was reached after 10 min (22±6 nM, n=5; Fig. 1b). This decrement in [Ca²⁺]i was associated with a significant and proportional reduction of the caffeine Ca²⁺ transient responses observed at 2, 4 and 6 min in Ca²⁺ free PSS when compared with their respective control group in normal PSS (n=6; Fig. 1c). At 10 min, the response to caffeine was totally absent whilst Cch response was only 4% of its response in normal PSS (n=6 and 4 respectively, Fig. 1c, d). These results corroborated that the SR-Ca²⁺ stores were almost completely depleted after 10 min in Ca²⁺ free medium.

Fig. 1a–d.

External Ca²⁺ removal decreased the intracellular free-Ca²⁺ concentration ([Ca²⁺]i) and the SR-Ca²⁺ content in isolated tracheal myocytes. (a) Original trace of caffeine-induced Ca²⁺ transient peak. (b) Typical recording from a myocyte incubated in Ca²⁺-free PSS. Recovery of basal [Ca²⁺]i, suggesting Ca²⁺ entry, was initiated after external Ca²⁺ (2 mM) was restored. (c) Ca²⁺ responses induced by caffeine (10 mM) after different incubation periods in Ca²⁺-free medium. (d) Ca²⁺ response induced by carbachol (10 µM) after 10 min in Ca²⁺-free medium. For details see text. *p<0.01

Restoration of extracellular Ca²⁺ (2 mM) produced, in all groups, a return to the initial [Ca²⁺]i basal levels (Fig. 2). This increase in [Ca²⁺]i was considered as a Ca²⁺ entry to the cell. The rate of Ca²⁺ entry in groups 6 and 10 min was significantly faster (p<0.01, fast Ca²⁺ entry) than those rates calculated at other times (slow Ca²⁺ entry). An additional group of 10 min that was incubated with 1 µM thapsigargin showed a faster Ca²⁺ entry rate (p<0.01) when compared with its control group (10 min, Fig. 2). The long lasting depolarisation of the ASM cells by substitution of NaCl by high potassium solution to induce inactivation of L and T-type Ca²⁺ channels and inversion of the Na⁺/Ca²⁺ exchanger, did not appear to change Ca²⁺ entry rate, since this phenomenon was still faster after 10 min (1.48±0.21 nM/s, p<0.01, n=4) compared with the 4-min group (0.19±0.06 nM/s, n=6). Indeed, we did not observe any statistical difference in Ca²⁺ entry in these groups when compared with their respective non-depolarised cells.

Fig. 2.

Temporal course of [Ca²⁺]i increment after Ca²⁺ restoration of four different cell groups that have been incubated in Ca²⁺-free medium during different periods. These values suggest the existence of two different mechanisms for Ca²⁺ entry. An additional group of 10 min was incubated with 1 µM thapsigargin (Thap, an SR-Ca²⁺ ATPase blocker). †,†† p<0.05, 0.01, respectively, between group 10, 6 vs. 2, 4 groups. *p<0.01 between 10, 6 and Thap groups. § p<0.01 between Thap group vs. all groups

When Mn²⁺ (50 µM) was present in the PSS, Ca²⁺ restoration to the cells after 4-min incubation in Ca²⁺ free medium did not modify the fluorescence at 360 nm (n=5; Fig. 3a), i.e., we did not observe an additional Mn²⁺ influx. However, when Ca²⁺ was added after 10 min incubation in Ca²⁺-free medium, we noticed an increment of Mn²⁺ influx (n=5, Fig. 3b), corroborating an increase in Ca²⁺ entry.

Fig. 3a, b.

Original recordings of Mn²⁺ quenching (50 µM) using 360 nm excitation light in cells perfused with Ca²⁺-free medium during 4 (a) and 10 min (b). Arrows indicate when 2 mM Ca²⁺ was added in the perfusion medium. Ca²⁺ restoration to the medium modifies Mn²⁺ influx only 10 min after Ca²⁺ removal, suggesting the existence of a membrane Ca²⁺ pathway during the fast Ca²⁺ recovery

Effect of La³⁺ and Mg²⁺ in Ca²⁺ entry

In non-stimulated myocytes, incubation with La³⁺ (0.2 mM) or Mg²⁺ (4 mM) during 10 min did not modify the [Ca²⁺]i basal levels. Some cells were incubated in Ca²⁺ free PSS during either 4 or 10 min (as described above) and the Ca²⁺ entry rate was determined by restoring this ion in the external medium in the presence of these two cations. Figure 4 shows the results of the 10-min group (n=9, 5, respectively) and it can be observed that Ca²⁺ entry rates were not modified when cells were incubated with the capacitative Ca²⁺ blockers. Similar lack of effect of La³⁺ and Mg²⁺ was observed in the 4-min group, which had an average value of 0.37±0.14 and 0.38±0.13 nM/s, n=6, 4, respectively, when compared with its corresponding control group: 0.30±0.10 and 0.38±0.09 nM/s, respectively.

Fig. 4a–c.

La³⁺ (0.2 mM, n=9) and Mg²⁺ (4 mM, n=4) did not affect the fast Ca²⁺ entry observed in bovine airway smooth muscle cells (group of 10 min). (a) Original recording showing the lack of effect of La³⁺ on the Ca²⁺ entry. (b, c) Average data of the temporal course of Ca²⁺ entry during Ca²⁺ restoration in two different experimental conditions

Effect of Ni²⁺ in Ca²⁺ entry

Ni²⁺ (1 mM) addition to PSS induced a slow [Ca²⁺]i decrement in non-stimulated myocytes, and after 5 min perfusion, a new steady state of [Ca²⁺]i was observed (79±11 nM, n=6; Fig. 5a–c). Ni²⁺ removal restored Ca²⁺ basal levels even when cells were submitted to Ca²⁺-free medium (Fig. 5a, b). Under the new steady state induced by Ni²⁺, two groups of cells were incubated in Ca²⁺ free PSS during either 4 or 10 min and the Ca²⁺ entry rate was determined by restoring this ion in the external medium (n=5 each group; Fig. 5c, d). In the first group a rate of 0.30±0.11 nM/s was obtained, which was not different than the rate values of the control group of 4 min (without Ni²⁺, see Fig. 2). The calculated rate of the second group was slower (0.16±0.03 nM/s, n= 6) than that of the control group of 10 min (Fig. 2).

Fig. 5a–d.

Ni²⁺ (1 mM) decreased the basal [Ca²⁺]i and slowed down the fast Ca²⁺ entry triggered by Ca²⁺ restoration in bovine airway smooth muscle cells. Ca²⁺ addition restored [Ca²⁺]i basal levels in cells that were previously incubated with Ni²⁺ in normal (a) and Ca²⁺-free PSS (b). (c) Original recording showing the effect of Ni²⁺ in Ca²⁺ entry after Ca²⁺ restoration. (d) Time course of Ca²⁺ entry during Ni²⁺ incubation in cells with slow (4 min) and fast (10 min) Ca²⁺ influx

It has been proposed that Ni²⁺ induces fura 2 quenching (Merritt et al. 1989). In Fig. 5a we can observe that the signal from 340 and 380 nm during Ni²⁺ perfusion was not modified i.e., Ni²⁺ did not enter to the cell.

Effect of D-600, 2-APB and SKF-96365 in Ca²⁺ entry

Incubation of the myocytes with D-600 (30 µM, n=4), 2-APB (100 µM, n=5) or SKF 96365 (20 µM, n=5) during 10 min did not modify the [Ca²⁺]i basal levels. In the 10-min group, the fast Ca²⁺ entry was not modify by D-600, 2-APB or SKF 96365 (Figs. 6 and 7a). Similar results were observed in the 4-min group where the slow Ca²⁺ entry (0.38±0.09 nM/s, n=4) was not modified by D-600 (0.30±0.12 nM/s), or 2-APB (0.72±0.21 vs. control 0.39±0.09 nM/s, n=5). Contrary to this last effect, SKF 96365 significantly reduced the Ca²⁺ entry rates and the [Ca²⁺]i basal level was not restored, i.e., the slow Ca²⁺ entry was blocked in the 4-min group (Fig. 7b, n=6).

Fig. 6a, b.

Effect of D-600 (30 µM) and 2-APB (100 µM, a capacitative Ca²⁺ entry blocker) in the fast Ca²⁺ entry observed in bovine airway smooth muscle cells (group of 10 min). Both drugs, D-600 (a) and 2-APB (b) did not modify the Ca²⁺ entry rate when compared with their respective control groups

Fig. 7a, b.

Effect of SKF 96365 (20 µM) in the fast and slow Ca²⁺ entry observed in bovine airway smooth muscle cells. In (a) we can observe that SKF 96365 did not modify the fast Ca²⁺ entry, but significantly inhibited the slow Ca²⁺ entry (b)

Discussion

External Ca²⁺ removal in bovine ASM cells induced a slow and continuous [Ca²⁺]i decrement until a new baseline was achieved. This slow [Ca²⁺]i fall was associated with a similar reduction in the SR-Ca²⁺ content. When external Ca²⁺ was restored, an increase in [Ca²⁺]i was observed, which would imply a Ca²⁺ entry to the cell. This Ca²⁺ entry had a faster rate (>1 nM/s) when the SR-Ca²⁺ content was less than 50% and was sensitive to Ni²⁺. However, when the SR-Ca²⁺ content was above 50%, Ca²⁺ entry rate was slower (~0.3 nM/s) and sensitive to SKF 96365. Both influxes, the slow and the fast, were not blocked by 2-APB, La³⁺, Mg²⁺ or voltage Ca²⁺ channels inhibition.

In non excitable cells such as endothelial (Jacob 1990, Sedova et al. 2000), RBL/1 cells, BHK/21 fibroblasts (Hofer et al. 1998) and excitable cells as mice pancreatic β cells (Dyachok and Gylfe 2001), it has been observed that capacitative Ca²⁺ entry is specifically controlled by the Ca²⁺ content of intracellular stores, i.e., the rate of Ca²⁺ entry varies inversely with the degree of fullness of these stores. Opposite to what happens in these cells, in ASM cells the rate of Ca²⁺ entry does not appear to vary with the degree of fullness of the SR-Ca²⁺, since we found two patterns of Ca²⁺ entry, the slow and the fast influx. This would imply that ASM uses different mechanisms to regulate this Ca²⁺ entry than those used in previous mentioned cells. In this regard, it has been reported in RBL cells (non excitable cells) that opening of Ca²⁺-release-activated Ca²⁺ (CRAC) channels occurs only when the endoplasmic reticulum Ca²⁺ content reaches a thershold level (Parekh et al. 1997). It is possible that in ASM cells capacitative Ca²⁺ influx signaling is also activated when SR-Ca²⁺ stores reach a threshold level, and this one seems to be when the SR-Ca²⁺ content was less than 50%.

Mg²⁺ and La³⁺, two capacitative Ca²⁺ entry blockers in vascular myocytes (Yoshimura et al. 1997) and mast cells (Hoth and Penner 1993), did not modify the Ca²⁺ entry rate in ASM cells with slow and fast Ca²⁺ entry. These results would imply that in ASM the channels involved in this Ca²⁺ entry are not sensitive to these blockers.

The fast Ca²⁺ entry appears to be sensitive to Ni²⁺. In our experiments, we observed that Ni²⁺ produced a gradual [Ca²⁺]i decrement until a new steady state of [Ca²⁺]i was reached. This effect might be mediated by its known action as Ca²⁺ antagonist that involves the blockade of voltage-sensitive and receptor-operated Ca²⁺ channels, and the Na⁺/Ca²⁺ exchange (Cuthbert et al. 1994; Hoya and Venosa 1995). The effect of Ni²⁺ on Ca²⁺ entry after SR depletion is, however, unlikely mediated by such an action, hypothesis that is supported by the following: 1) we found that the slow and fast Ca²⁺ entry, was not affected by prolonged depolarisation or incubation with D-600, suggesting that voltage-sensitive L- and T-type Ca²⁺ channels and the Na⁺/Ca²⁺ exchanger are not important in this phenomenon; 2) receptor-operated Ca²⁺ channels require the action of an agonist to be opened and this is not the case.

Recently, in vascular smooth muscle from aorta and pulmonary artery, it was found that Ni²⁺ abolished the capacitative Ca²⁺ entry (Lee et al. 2002; McDaniel et al. 2001), suggesting that this cation is an appropriate antagonist for excitable cells. In our study, we observed that Ni²⁺ only abolished the fast Ca²⁺ entry. Thus, these observations support the hypothesis that Ni²⁺ is blocking the putative capacitative Ca²⁺ entry pathway, which is only present when the SR-Ca²⁺ content is lower than 50%. On the other hand, since Ni²⁺ did not block the slow Ca²⁺ entry, we could suggest that this is a non capacitative phenomenon, probably the so called passive influx. The results with 2-APB, recently described as a capacitative Ca²⁺ inhibitor (Prakriya and Lewis 2001), in the slow Ca²⁺ entry support our hypothesis that this Ca²⁺ influx is a non capacitative phenomenon since the drug did not block it. In this context, SKF 96365, a compound with several effects such as store-operated and receptor-operated Ca²⁺ channels blocker (Merritt et al. 1990; Prakriya and Lewis 2002), significantly reduced this slow Ca²⁺ entry. This effect of SKF 96365 on the passive Ca²⁺ entry was also previously reported in human leukemic HL-60 cells (Leung et al. 1996). In this last work SKF 96365 almost completely blocked (basal) Mn²⁺ entry. These results suggested the participation of Ca²⁺ channels in the slow Ca²⁺ entry, however further experiments are required to determine the type of Ca²⁺ channels involved.

In order to corroborate that during the fast Ca²⁺ entry an additional Ca²⁺ pathway was activated, we used the Mn²⁺ quench approach to confirm more directly the Ca²⁺ influx. We found an increase in Mn²⁺ influx when Ca²⁺ was restored in cells with SR-Ca²⁺ content lower than 50%, suggesting a change in Ca²⁺ entry that probably was due to a capacitative Ca²⁺ phenomenon. In cells with SR-Ca²⁺ content higher than 50%, Mn²⁺ quench did not change when Ca²⁺ was added to PSS. Thus, Ca²⁺ influx that contributes to restore the intracellular Ca²⁺ levels in these cells seems not to be related to the activation of an additional Ca²⁺ pathway, confirming that this slow Ca²⁺ entry is passive.

Our observations also indicate that in the fast Ca²⁺ entry, the rate of [Ca²⁺]i increase after reintroduction of extracellular Ca²⁺ could be influenced by sequestration of Ca²⁺ by the SR, because we found that the blockade of SR-Ca²⁺ pump with thapsigargin significantly accelerates the rate of [Ca²⁺]i increase. The SR might be playing a similar role in experimental cells with slow Ca²⁺ influx. Unfortunately, experiments with thapsigargin in these later groups would have been difficult to interpret because this maneuver also depletes the SR.

In conclusion, we observed that in bovine ASM cells Ca²⁺ influx after SR-Ca²⁺ depletion has two rates: A) The slow Ca²⁺ influx which occurred in cells with more than 50% of their SR-Ca²⁺ content, is sensitive to SKF 96365 and appears to be a non-capacitative Ca²⁺ entry (passive entry). B) The fast Ca²⁺ influx observed in cells with less than 50% of their SR-Ca²⁺ content, is probably a capacitative Ca²⁺ entry and was only Ni²⁺-sensitive. Both mechanisms were not blocked by La³⁺, Mg²⁺, 2-APB, and voltage-Ca²⁺ channels were also not involved.