Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

LTD₄, a component of the slow-reacting substance of anaphylaxis (Lewis et al., 1980; Samuelsson, 1981), is an extremely potent constrictor of smooth muscle from airways (Dahlén et al., 1980; Sirois et al., 1981; Jones et al., 1982) and vascular tissues (Feigen, 1983).In addition, LTD₄ causes mucus secretion in the airways (Marom et al., 1981). By modulating airway responsiveness and having diverse effects on noncontractile tissues and cells, LTD₄ plays other important roles in the pathophysiology of asthma.

It is well recognized that the smooth muscle contraction is ultimately related to free calcium ion availability; however, the mechanisms by which the contractile agonists elevate the concentration of intracellular calcium ion ([Ca⁺⁺]_i) remain incompletely understood, particularly in airway smooth muscle cells. Classically, there are two basic mechanisms by which the agonists increase the [Ca⁺⁺]_i: via InsP₃-mediated Ca⁺⁺ release from intracellular Ca⁺⁺ store (Somlyo et al., 1988; Berridge and Irvine, 1989; Irvine, 1990) and via an influx of calcium from the extracellular fluid through VOC or ROC (voltage-independent) channels (Benham and Tsien, 1987; Murray and Kotlikoff, 1991). The relative importance of these two mechanisms in the overall LTD₄-induced [Ca⁺⁺]_i increase varies with the cell types, tissues and species. It has been shown that in sheep tracheal smooth muscle cells, the absence of external Ca⁺⁺ did not modify the LTD₄-triggered increase in [Ca⁺⁺]_i, which derived therefore from intracellular Ca⁺⁺ mobilization via the action of InsP₃ (Mong et al., 1988a). On the contrary, the LTD₄-induced [Ca⁺⁺]_i increase in HL-60 and guinea pig ileum muscle is almost completely dependent on the presence of extracellular Ca⁺⁺ (Baud et al., 1987; Oliva et al., 1994).

Previous studies demonstrated that in a Ca⁺⁺-free buffer, LTD₄ did not induce contraction of guinea pig trachea (Weichman et al., 1983; Sirois et al., 1986; Cuthbert et al., 1994). This suggests a major role of the Ca⁺⁺ influx in the LTD₄-induced contraction of guinea-pig trachea. The OC entry blocker, nifedipine, only slightly suppressed the tracheal contractions induced by LTD₄, which suggested a potential role of the ROC in the Ca⁺⁺ influx (Weichman et al., 1983; Cerrina et al., 1983). These observations are supported also by the disappointing results obtained with the use of nifedipine in the therapy for asthma (Ferrari et al., 1989).

Recent studies showed that LTD₄ induces phosphoinositide hydrolysis in guinea pig tracheal smooth muscle cells (Howard et al., 1992), which suggested a role for intracellular Ca⁺⁺ release in the LTD₄-induced [Ca⁺⁺]_i increase. However, no demonstration of [Ca⁺⁺]_i elevations have been reported and the mechanism by which LTD₄ induces [Ca⁺⁺]_i increase in the TSMCs remains essentially undefined. Therefore, the present study was designed to elucidate the roles of intra- and extracellular Ca⁺⁺ sources in the increase of [Ca⁺⁺]_i induced by LTD₄ in the guinea pig TSMCs in culture. We also compared the mechanisms involved in LTD₄-induced [Ca⁺⁺]_i increase with that of BK, another potent constrictor of tracheal smooth muscle, because the effect of BK on [Ca⁺⁺]_i levels in airway smooth muscle cells has been characterized extensively.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. Elastase type IV, collagenase type V, ionomycin, nifedipine, BK and mouse monoclonal -smooth muscle actin antibody were purchased from Sigma Chemical Co., St. Louis, MO. Tissue culture reagents (DMEM/F12 with and without inositol; penicillin and streptomycin) and plasticware were obtained from Gibco Laboratories, Grand Island, NY. myo-[³H]inositol (specific activity 82.5 Ci/mmol) was purchased from Amersham Radiochemical, Arlington Heights, IL. The Fura-2/AM was purchased from Calbiochem Behring, La Jolla, CA. LTD₄ and the compound MK-571 were obtained from Merck Frosst, Pointe-Claire, P.Q., Canada.

Tracheal smooth muscle cell culture. Isolation of the primary smooth muscle cells was based on the method of Devore-Carter et al. (1988). The guinea pigs (Dunkin Hartley, 350-400 g) were sacrificed by cervical dislocation, and tracheae were rapidly placed and rinsed twice in ice-cold HBSS (pH = 7.4) supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ml). The HBSS buffer contained (mM): NaCl, 118.07; KCl, 4.7; KH₂PO₄, 1.18; glucose, 11.1; NaHCO₃, 25; CaCl₂, 2.2; MgCl₂, 1.2 (95% O₂ and 5% CO₂). The tracheae were carefully dissected free of fatty and connective tissues and were opened by cutting the cartilage rings opposite to the strip of smooth muscle. The tracheae were incubated with an enzyme solution containing collagenase type V (2 mg/ml) and elastase type IV (1 mg/ml), under gentle agitation, at 37°C for 30 min. The enzymatically digested tracheae were then passed through a nylon mesh. The resting tracheae fragments were washed with HBSS (10 ml). The collected solution (containing released cells was centrifuged at room temperature at 350 × g, for 5 min. The pellet was resuspended in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) (1:1 v/v) with 10% FBS, penicillin (100 IU/ml) and streptomycin (100 mg/ml). The cells were seeded at 1 × 10⁴ cells/ml in 25 cm² culture flasks and incubated at 37°C, 95% O₂ and 5% CO₂. The viability of cells was evaluated at >95% by Trypan blue exclusion. The medium was changed after 24 h and every 2 days. The cells reached the confluency, usually after 7 to 10 days. At confluency, smooth muscle cells were harvested by washing twice with HBSS without calcium and magnesium followed by a brief incubation in a solution of trypsin (0.05%)-ethylenediaminetetraacetic acid (0.53 mM) at 37°C. The detached cells were centrifuged (300 × g for 5 min) and resuspended in DMEM/F12 with 10% FBS. The cells were plated (1 × 10⁵ cells/ml/2 cm²) for indirect immunofluorescence and [Ca⁺⁺]_i measurement on glass coverslips coated with human placental collagen (type VI) (12 mg/cm²) and, for polyphosphoinositide hydrolysis experiments, in 24-well plates. The subcultured cells became confluent after 3 to 4 days. All the experiments were performed on the first passage cells after 5 days of culture.

Immunocytochemical analysis. The presence of smooth muscle-specific -actin was used to determine the identity and the purity of the cultures. The smooth muscle cells were identified by an indirect immunofluorescence method with -smooth muscle actin monoclonal antibody (Gown et al., 1985). The cells were washed (3 × 5 min) with PBS and fixed in 2% formaldehyde solution for 5 min. The PBS buffer contained (mM): NaCl, 137; KCl, 3.5; Na₂HPO₄, 16.5; NaH₂PO₄, 3.5; glucose, 5.5; CaCl₂, 0.9; MgCl₂, 2 (pH = 7.4). The fixed cells were incubated in PBS containing glycine (100 mM) for 45 min at 4°C, rinsed (3 × 5 min) with PBS containing 1% w/v BSA and then exposed to the primary antibody, anti-smooth muscle -actin (mouse monoclonal) (1:00 dilution in PBS with BSA) for 45 min at room temperature. The cells were washed (3 × 5 min) with PBS and exposed to the second antibody, antimouse immunoglobulin G₂ fragment fluorescein isothiocyanate-conjugate (1:50 dilution in PBS), for 30 min at room temperature in the dark. Finally, the cells were washed (1 × 5 min in PBS) and mounted onto a coverslip with glycerol/PBS [9/1 (v/v)]. Background staining controls were provided by the deletion of primary antibody. The staining of the fixed cells was then observed under a fluorescence microscope (Leica; Wetzlar, Germany) and then photographed.

Measurement of intracellular free Ca⁺⁺ levels. The concentration of intracellular free Ca⁺⁺ was measured by loading the confluent cells with the calcium-sensitive fluorescent dye Fura-2 AM. The cells on individual coverslips were washed twice with PBS and placed in 35-mm Petri dishes with 2 ml of DMEM/F12 containing 3 mM Fura-2/AM (acetoxymethyl ester) and incubated for 30 min at 37°C. The cells were then washed twice with PBS to remove the extracellular dye and then incubated in HEPES buffer (in mM: NaCl, 140; KCl, 5; NaHCO₃, 25; glucose, 5.5; HEPES, 20; MgCl₂, 1; CaCl₂, 1) (pH = 7.4) containing 0.1% BSA for 30 min at 25°C to allow intracellular dye hydrolysis. Loaded coverslips were placed diagonally into quartz cuvettes (Canlab) which were then mounted in a thermostatically (37°C) controlled holder of a Hitachi F-2000 spectrofluorometer. Each cuvette contained 2 ml of HEPES buffer (with 0.1% BSA) and agents were added directly to the cells in a maximal volume of 20 ml. Fluorescence of Ca⁺⁺-bound and unbound Fura-2 was measured with alternating excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. The slitwidths were set at 10 nm for the excitation and at 20 nm for emission. Intracellular free Ca⁺⁺ level was calculated from the ratio (R) of 340 nm/380 nm fluorescent values by use of the equation of Grynkiewcz et al. (1985): where K_d is the affinity of Fura-2 for Ca⁺⁺ (224 nM at 37°C) and F_min380/F_max380 is the ratio ( value) of the fluorescent values obtained at 380 nm in the absence and the presence of saturating [Ca⁺⁺]_i. The maximum and minimum values (R_max and R_min) were determined for calibration purposes by addition of ionomycin (10 mM) and of EGTA (20 mM), respectively. Ionomycin released [Ca⁺⁺]_i in the medium, increasing the concentration of free Ca⁺⁺, whereas the EGTA chelated the calcium ions, giving the minimum fluorescence of the system. Corrections for autofluorescence were made by subtracting the fluorescence produced by unloaded coverslips from the fluorescence data. For experiments requiring Ca⁺⁺ free conditions, the HBSS added to the cuvettes was prepared with Ca⁺⁺ free water, CaCl₂ was omitted and 0.1 mM EGTA was added. Nifedipine was used in these experiments, the HEPES buffer was supplemented with 1 × 10⁵ M nifedipine throughout the Fura-2 hydrolysis and [Ca⁺⁺]_i measurement periods.

Phosphoinositide hydrolysis. The cells in 24-well plates were labeled with myo-[³H]inositol (4.0 × 106 cpm/well) in inositol-free DMEM/F12 for 16-18 h, at 37°C, 95% O₂ and 5% CO₂. The [³H]inositol-labeled cells were washed twice to remove the free inositol and incubated in PBS (200 ml/well) containing 0.01% BSA and LiCl (10 mM) for 10 min, at 37°C, before addition of 50 ml LTD₄, BK or, like controls, PBS. When the antagonist and nifedipine or verapamil were used, they were added 10 and 30 min before the addition of agonists. NiCl₂ was dissolved in a phosphate-free PBS and added 30 min before the stimulation with agonists. In Ca⁺⁺ free experiments, CaCl₂ was omitted from PBS and 0.1 mM EGTA was added. After incubation at 37°C, the agonist stimulations were stopped by addition of 500 ml perchloric acid (175%). The samples were placed on ice for 30 min. The contents (cells and supernatant) of each well were transferred to a series of plastic tubes and centrifuged at 1800 × g for 5 min at 4°C. The perchloric acid-soluble supernatants were transferred in another tubes and were vortexed for 1 min after the addition of 10 ml of ethylenediaminetetraacetic acid (100 mM) and 600 ml of a 1:1 (v/v) mixture of tri-n-octyl-amine and 1,1,2-trichloro-trifluoro-ethane. The tubes were then centrifuged at 1800 × g for 1 min and the aqueous (superior) phases were neutralized with 20 ml of 1 M Tris-HCl (pH = 8.5) and then applied to the columns of the anion exchange resin (Dowex AG 1- X8, formate form, 200 to 400 mesh). The free [³H]inositol fraction was eluted with 12 ml of water. The [³H]InsP₁ and [³H]InsP₂ together with [³H]InsP₃ and [³H]InsP₄ were eluted stepwise with 3 × 4 ml of 0.2 and 1.0 M ammonium formate containing 0.1 N formic acid, respectively. When individual fractions were separated, the [³H]InsP₁, [³H]InsP₂, [³H]InsP₃ and [³H]InsP₄ were eluted with 0.2, 0.5, 0.8 and 1 M ammonium formate/0.1 N formic acid, respectively. The radioactivity of the fractions (of 6 ml each) was measured by liquid scintillation counting.

Statistical analysis. The values are mean ± S.E.M.. The statistical significance was determined by analysis of variance. P  .05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Characterization of the tracheal smooth muscle cells. The primary culture of TSMCs contained a mixed population consisting of smooth muscle cells and small epithelial cell colonies. Morphologically, the smooth muscle cells were spindle shaped at confluence and formed ridges and dense aggregates which conferred to the culture a characteristic "hill and valley" appearance (Chamley-Campbell et al., 1979). This pattern was maintained also for the first subcultured cells. When the primary culture cells were subcultured, the epithelial cells remained attached to the flask, while the smooth muscle cells became detached, usually within 20 to 30 sec. Therefore, the epithelial cells were absent from the first passage cell culture. The identity of the guinea-pig TSMCs was confirmed by the cells staining with -smooth muscle actin monoclonal antibody. The staining of the actin filaments was uniform permitting to evaluate the smooth muscle culture purity at approximately 95%. No background ("non-specific") staining was observed.

LTD₄ and BK-induced changes in the [Ca⁺⁺]_i of TSMCs. Figure 1 shows representative tracings of Fura-2 fluorescence changes induced by LTD₄ (100 nM), determined in the presence of extracellular Ca⁺⁺ in TSMCs. The simultaneous recording of Fura-2 fluorescence at 340 and 380 nm allowed us to evaluate accurately the changes of intracellular Ca⁺⁺ concentration (fig.1A). The ratio R of the dye fluorescence intensities (F₃₄₀ and F₃₈₀) permitted to calculate [Ca⁺⁺]_i independent of total cell dye concentration or sensitivity of the instrument and to eliminate possible artifacts (fig. 1B). The resting level of [Ca⁺⁺]_i in unstimulated TSMCs was 101 ± 18 nM (n = 25). A stable baseline, before the addition of LTD₄ suggested that there was no leakage of Fura-2 outside the cells. A 20 sec delay was observed between the addition of LTD₄ to the cells and the onset of [Ca⁺⁺]_i increase. Stimulation with LTD₄ (1 × 10⁷ M) slowly increased [Ca⁺⁺]_i, which peaked at 326 ± 44 nM (n = 6) within 36 s after challenge (fig. 1B). [Ca⁺⁺]_i slowly declined to a level above the baseline (144 ± 7 nM) (n = 25), which remained until the addition of ionomycin. The maximal and minimal fluorescence were obtained by addition of ionomycin (1 × 10⁵ M) and EGTA (2 × 10² M). LTD₄ (1 × 10⁹-1 × 10⁶ M) induced a concentration-dependent increase in [Ca⁺⁺]_i in TSMCs (fig. 2). Concentration-response curves were obtained with the peak values of [Ca⁺⁺]_i increases induced by LTD₄. The EC₅₀ value was 8 × 10⁹ M (n = 6) and the minimal and maximal responses were obtained with 1 × 10⁹ and 1 × 10⁷ M LTD₄, respectively. Accordingly, the dose of 1 × 10⁷ M LTD₄ was used in subsequent experiments. No difference was observed between the kinetics of [Ca⁺⁺]_i increases induced by various LTD₄ concentrations.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Representative tracings of Fura-2 fluorescence F340, F380 (A) and R (ratio) (B) changes in guinea pig TSMCs stimulated with LTD4 (1 × 107 M), ionomycin (IO) (1 × 105 M) and EGTA (2 × 102 M). Response to LTD4 was obtained in the presence of 1 mM extracellular Ca++. Similar tracings were obtained in at least five separate experiments, each performed with three different cell preparations.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Concentration-response relationship for LTD4-induced increases of [Ca++]i in TSMCs in the presence of extracellular Ca++. The results are expressed as mean ± S.E.M. of the peak increases above the basal levels. The data were obtained from four separate experiments each performed with three different cell preparations. ** P < .001.

6

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Representative recordings of the time course of changes in [Ca++]i induced by LTD4 (1 × 107 M) (A) and BK (1 × 106 M) (B) in Ca++-containing medium. The tracings are from an experiment representative of three, each performed with different cell preparations.

7

6

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Effect of the antagonist MK-571 on the changes in [Ca++]i induced by LTD4. MK-571 (1 × 106 M) was added 5 min before (A) and after (B) addition of LTD4 (1 × 107 M). These tracings are from a single experiment representative of three, each performed with three different cell preparations.

LTD₄ stimulates [Ca⁺⁺]_i increase: dependence on extracellular calcium. To establish whether the responses to LTD₄ were caused by Ca⁺⁺ release from intracellular stores or the result of Ca⁺⁺ influx from extracellular space, the cells were stimulated in the absence of extracellular Ca⁺⁺. There was no change in the resting [Ca⁺⁺]_i level when the cells were incubated in a Ca⁺⁺ free medium. Under these conditions, LTD₄ (1 × 10⁷ M) did not elicit any rise in the [Ca⁺⁺]_i above the basal level (fig. 5A). The LTD₄-stimulated [Ca⁺⁺]_i increase was rapidly reestablished after the addition of CaCl₂ (1 mM) to the Ca⁺⁺-free buffer (fig. 5A). To verify that this [Ca⁺⁺]_i increase is not caused solely by the addition of CaCl₂, the addition order was reversed (i.e., Ca⁺⁺ followed by LTD₄). The addition of CaCl₂ (1 mM) in the Ca⁺⁺ free medium did not induce changes in [Ca⁺⁺]_i, and the subsequent stimulation with LTD₄ (1 × 10⁷ M) increased [Ca⁺⁺]_i levels (fig. 5B). In both cases (fig. 5, A and B), the profile of changes in [Ca⁺⁺]_i induced by LTD₄ was similar to the control (cells incubated in Ca⁺⁺-containing medium).

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Effect of external Ca++ on the changes in [Ca++]i induced by LTD4 (1 × 107 M). Cells were incubated in the absence of external Ca++. The cells were stimulated before (A) and after (B) the addition of CaCl2. These tracings are from a single experiment representative of three, each performed with three different cell preparations.

5

6

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Effect of nifedipine on the change in [Ca++]i induced by LTD4 (A) and BK (B). The cells were pretreated (30 min) with nifedipine (1 × 105 M) and stimulated in its presence. These tracings are from a single experiment representative of three, each performed with three different cell preparations.

LTD₄ and BK-mediated inositol phosphate accumulation. To better understand and correlate the effects of LTD₄ on Ca⁺⁺ mobilization, phosphoinositide metabolism was evaluated. The results shown in figure 7, A to C, demonstrate the kinetics of formation of [³H]InsP₃, [³H]InsP₂₊₃₊₄ (the sum of [³H]InsP₂, [³H]InsP₃ and [³H]InsP₄) and the total [³H]InsP_s (sum of [³H]InsP₂₊₃₊₄ with [³H]InsP₁) in TSMCs stimulated with LTD₄ (1 × 10⁷ M), in the presence of LiCl (1 × 10² M). The basal levels of [³H]InsP₃, [³H]InsP₂₊₃₊₄ and [³H]InsP_s in the TSMCs remained constant during various stimulation times. LTD₄ (1 × 10⁷ M) induced a significant increase in [³H]InsP₃ production in the first 30 sec, reaching a maximum of 193 ± 2% over the basal level after a 2-min stimulation (fig. 7A). The data represent the combined InsP₃ isotypes. After reaching maximal level, the [³H]InsP₃ decreased gradually over the period studied but the levels at 5, 10 and 20 min after the addition of LTD₄ were still significantly higher than the controls. After a stimulation of 30 min, the level of [³H]InsP₃ returned toward the unstimulated level and was not significantly different from control level. When the production of the [³H]InsP₂₊₃₊₄ was evaluated, a similar kinetic value was observed (fig. 7B). LTD₄ induced a significant increase of the [³H]InsP₂₊₃₊₄ level over the incubation period (0.5-30 min). The [³H]InsP₂₊₃₊₄ accumulation increased rapidly, reaching a maximum of 170 ± 7% over basal by 2 min. By increasing the stimulation time, the LTD₄-stimulated [³H]InsP₂₊₃₊₄ formation gradually declined, reaching a level of 125 ± 5% over basal (significantly higher than control) after a 30-min stimulation period. The total [³H]InsP_s accumulation was statistically significant after 30 sec of LTD₄ stimulation (119 ± 2% over basal). The maximal level was reached between 2 and 5 min of stimulation, then gradually declined over time, but remained significantly higher than the control for 30 min. Considering that [³H]InsP₂ and [³H]InsP₄ are the metabolic products of [³H]InsP₃ and the sum of all three had the same kinetics as that of [³H]InsP₃ in all other experiments, individual inositol phosphate species were subsequently not separated. The reported value represents the sum of all three fractions. Because maximal LTD₄-stimulated increase in [³H]InsP₃ was obtained by 2 min, a 2-min stimulation period was used in subsequent experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Time course of [3H]InsP3 (A), [3H]InsP2+3+4 (B) and [3H]InsPs (C) accumulation in the presence of LTD4 (1 × 107 M). The differences between the LTD4-stimulated and the control levels (D) are considered as the net accumulation induced by LTD4. The data are the mean ± S.E.M. from three experiments performed in triplicate. *P < .05; **P < .01.

8

7

9

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of LTD4-receptor antagonist on LTD4-induced dose-dependent synthesis of [3H]InsP2+3+4 (A) and [3H]InsPs (B). The cells were stimulated (2 min) with increasing concentrations of LTD4 in the absence () or after the pretreatment (10 min) with the compound MK-571 (1 × 106 M) (). The values are expressed as percentage of basal accumulation (no addition of LTD4 = 100%) and are the mean ± S.E.M. from three separate experiments performed in triplicate. *P < .05; **P < .01.

6

8

8

9

Dependence of phosphoinositide hydrolysis on extracellular calcium. To assess whether the LTD₄-induced inositol phosphate accumulation, like the [Ca⁺⁺]_i increase, is dependent on the presence of external calcium, the cells were stimulated in the absence of extracellular calcium. Preincubation of the myo-[³H]inositol loaded cells in Ca⁺⁺-free buffer, 10 min before agonist challenge, did not decrease the basal level of inositol phosphates. In the absence of external Ca⁺⁺, the LTD₄ (1 × 10⁹-3 × 10⁷ M) failed to stimulate [³H]InsP₂₊₃₊₄ and [³H]InsP_s synthesis (fig. 9, A and B). Only very small increases in [³H]InsP_s accumulation were detected but were not significantly different respect to the basal values.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Effect of the absence of extracellular Ca++ and NiCl2 on LTD4-induced [3H]InsP2+3+4 (A) and [3H]InsPs (B) synthesis. The cells were stimulated (2 min) with increasing concentrations of LTD4 in the presence of extracellular Ca++ after a pretreatment (30 min) with NiCl2 (5 × 103 M) () or in the absence of extracellular Ca++ (). The values are expressed as percentage of basal accumulation (no addition of LTD4 = 100%) and are the mean ± S.E.M. from three separate experiments performed in triplicate. **P < .01.

3

5

5

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Effect of nifedipine and verapamil on LTD4-induced [3H]InsP2+3+4 (A) and [3H]InsPs (B) synthesis. The cells were stimulated (2 min) with increasing concentrations of LTD4 after a pretreatment (30 min) with nifedipine (NIF) (1 × 105 M) () or verapamil (VP) (1 × 105 M) (). The values are expressed as percentage of basal accumulation (no addition of LTD4 = 100%) and are the mean ± S.E.M. from three separate experiments performed in triplicate. *P < .05; **P < .01.

6

7

3

View larger version (29K): [in this window] [in a new window]   Fig. 11.   Effect of NiCl2 and compound MK-571 on LTD4 (1 × 107 M)- and BK (1 × 106 M)-stimulated [3H]InsP2+3+4 (A) and [3H]InsPs (B) synthesis. The cells were stimulated in the presence of NiCl2 (5 × 103 M) () or after a pretreatment (10 min) with the compound MK-571 (1 × 106 M) () The values are expressed as percentage of basal accumulation (no addition of LTD4 = 100%) and are the mean ± S.E.M. from three separate experiments performed in triplicate. * P < 0.05; ** P < .01vs. control; P < 0.01vs. LTD4.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The present study demonstrates that LTD₄-mediated increase in [Ca⁺⁺]_i is totally dependent on Ca⁺⁺ influx from external source. In the presence of extracellular Ca⁺⁺, LTD₄ stimulation of TSMCs resulted in a dose-dependent increase in [Ca⁺⁺]_i. Our study showed that the increase in [Ca⁺⁺]_i after addition of LTD₄ is a receptor-mediated event, as confirmed by the use of the specific LTD₄ receptor antagonist MK-571. Addition of MK-571 before LTD₄ completely blocked the [Ca⁺⁺]_i increase. Also, the plateau phase of the established LTD₄-induced [Ca⁺⁺]_i increase was reversed by the addition of MK-571. Previous studies demonstrated that the guinea pig tracheal smooth muscle contains at least two (of high and low affinity) specific LTD₄ receptor subtypes (Krell et al., 1983). In fact, our study suggested that the observed [Ca⁺⁺]_i changes were caused by to the binding of LTD₄ with specific receptors. Moreover, the maintenance of the sustained phase requires persistent activation of the LTD₄ receptors.

When the cells were stimulated with LTD₄, a delay between the addition of agonist and the onset of rise in [Ca⁺⁺]_i was observed. [Ca⁺⁺]_i increased to the maximal values and decreased toward the basal level with a slow rate. This kinetics of LTD₄-induced [Ca⁺⁺]_i rise was not concentration-dependent being slow even at the highest concentration of LTD₄. Similar time courses were observed in LTD₄-induced [Ca⁺⁺]_i increase in guinea pig ileal longitudinal muscle (Oliva et al., 1994) and human airway smooth muscle cells (Panettieri et al., 1989). In contrast, rapid LTD₄-induced increases of [Ca⁺⁺]_i levels were observed in DMSO-differentiated U937 cells (Winkler et al., 1988; Saussy et al., 1989), human epithelial cells (Sjlander et al., 1990), sheep tracheal smooth muscle cells (Mong et al., 1988b) and rat basophilic leukemic cells (RBL) (Mong et al., 1988a). By comparison, BK induces a typical increase in [Ca⁺⁺]_i consisting of an initial rapid transient phase (that occurs immediately after addition of agonist) followed by a sustained phase. Similar biphasic responses were observed in the human (Panettieri et al., 1989), bovine (Marsh and Hill, 1993) and canine (Yang et al., 1993b) cultured TSMCs. Farmer et al. (1989) reported that, in guinea pig TSMCs, the BK-induced Ca⁺⁺ mobilization is mediated by a putative B3 receptor. It is well demonstrated that the rapid initial increase in [Ca⁺⁺]_i, due to the activation of BK receptors, is mediated through the release of InsP3 and subsequent mobilization of [Ca⁺⁺]_i from internal stores (Yang et al., 1994; Murray and Kotlikoff, 1991). Therefore, the differences between the patterns of [Ca⁺⁺]_i increases induced by LTD₄ and BK suggest that these two agonists may increase [Ca⁺⁺]_i by two different mechanisms. Thus, our results suggest that Ca⁺⁺ influx rather than intracellular release may be the predominant mechanism involved in LTD₄-induced [Ca⁺⁺]_i increase. Similar differences were observed between the patterns of the guinea pig tracheal contractions induced by LTD₄ (Hedqvist et al., 1980; Sirois et al., 1981) and BK (Rhaleb et al., 1991). For all types of smooth muscle strips, the contractions induced, originally by slow reacting substances (Brocklehurst, 1953; 1962) and further by purified leukotrienes, invariably developed after a longer latency and at considerably slower rate than those induced by another constrictor (i.e., histamine, acetycholine, serotonin, bradykinin) (Hanna and Roth, 1978; Sirois et al., 1981; Bhoola et al., 1989). Therefore, the pattern of changes in [Ca⁺⁺]_i reflects the pattern of contractions induced by LTD₄.

This hypothesis was confirmed by the observation that removal of extracellular Ca⁺⁺, when Ca⁺⁺ is presumably mobilized only from internal stores, completely abolished the rises elicited by LTD₄. These observations are supported also by the finding that guinea pig trachea contractions induced by LTD₄ are dependent on the presence of extracellular Ca⁺⁺ (Weichman et al., 1983; Sirois et al., 1986; Cuthbert et al., 1994). Similarly, in the absence of external Ca⁺⁺, LTD₄ did not induce contractions (Findlay et al., 1982) and [Ca⁺⁺]_i increases (Oliva et al., 1994) in the guinea pig ileal smooth muscle. The same external Ca⁺⁺ dependence of LTD₄-stimulated [Ca⁺⁺]_i rise was observed in HL-60 cells (Baud et al., 1987) and in THP-1 cells (human monocytic leukemia cell line) (Chan et al., 1994). On the contrary, EGTA did not abolish the LTD₄-triggered [Ca⁺⁺]_i increase in sheep TSMCs (Mong et al., 1988b); accordingly, in the presence of external Ca⁺⁺, the transient phase of [Ca⁺⁺]_i increase was rapid. In RBL-1 (Mong et al., 1988a) and U-937 cells (Saussy et al., 1989) the sustained but not the transient phase of [Ca⁺⁺]_i rise induced by LTD₄ was totally inhibited by the absence of external Ca⁺⁺. In neutrophils, LTD₄ induced rise in [Ca⁺⁺]_i exclusively by the release from intracellular stores (Bouchelouche et al., 1990). Thus, the relative contributions of Ca⁺⁺ influx versus intracellular Ca⁺⁺ release in LTD₄-stimulated [Ca⁺⁺]_i increase is different in various tissues and species. In the present study, LTD₄-stimulated [Ca⁺⁺]_i increase in guinea pig TSMCs is completely dependent on Ca⁺⁺ influx from extracellular space.

However, it is well demonstrated that in various cell types LTD₄ stimulates the membrane phosphoinositide hydrolysis with the release of InsP₃, which subsequently mobilizes the Ca⁺⁺ from internal stores (Mong et al., 1988a,b; Saussy et al., 1989; Howard et al., 1992). Our data showed that LTD₄ produced rapid and transient increases in inositol phosphate levels. InsPs synthesis was dose-dependent and was inhibited by the LTD₄ antagonist, MK-571. The inhibition of LTD₄-induced InsP_s synthesis by MK-571 indicated that the interaction with LTD₄ was noncompetitive. Our results are in agreement with those reported by Jones et al. (1989) who demonstrated that the interaction of MK-571 with LTD₄ became noncompetitive at concentrations higher than 5.8 × 10⁸ M MK-571. BK was a more potent stimulator of inositol phosphate formation than LTD₄. However, these data suggest that InsP₃-mediated release of Ca⁺⁺ from intracellular stores may play a role in the [Ca⁺⁺]_i increases induced by LTD₄. Our results also showed that LTD₄ failed to stimulate the synthesis of inositol phosphates in a Ca⁺⁺-free medium. The same dependency for external Ca⁺⁺ was reported in various agonist-stimulated inositol phosphate accumulations in canine TSMCs (Yang et al., 1993a), in neutrophils (Cockcroft et al., 1980), mast cells (Cockcroft and Gomperts, 1980) and guinea pig visceral smooth muscle (Best et al., 1985). In contrast to our results, Saussy et al. (1989) reported that the generation of inositol phosphates by LTD₄ in U-937 cells was unaffected by the absence of external Ca⁺⁺. Our results suggest that the activation of PLC by LTD₄ in guinea pig TSMCs is markedly dependent on the availability of extracellular Ca⁺⁺.

Two possibilities might explain the external Ca⁺⁺ dependency of LTD₄-stimulated PLC in TSMCs. First, it was proposed that the absence of external Ca⁺⁺ can induce a decrease in [Ca⁺⁺]_i that may significantly retard the PLC activity. Best (1986) reported that PLC activity is very sensitive to small changes in [Ca⁺⁺]_i induced by addition of EGTA in extracellular medium. However, our results showed that in a Ca⁺⁺-depleted medium, the basal levels of [Ca⁺⁺]_i and of inositol phosphate accumulation in TSMCs were not reduced. Thus, the activity of PLC in TSMCs was found to be similar either in the absence or in the presence of extracellular Ca⁺⁺. Considering that the LTD₄ binding to its receptors in different tissues is enhanced by the presence of divalent cations (Pong and DeHaven, 1983; Hogaboom et al., 1983), we hypothesized that in the absence of external Ca⁺⁺, LTD₄ did not bind to its receptor to stimulate the PLC. To eliminate this possibility, the cells were stimulated in the presence of external Ca⁺⁺ but the Ca⁺⁺ influx was blocked by the presence of NiCl₂. Under these conditions, LTD₄ did not stimulate the inositol phosphate synthesis. Therefore, these results confirm the second possibility: the increase of Ca⁺⁺ influx into the TSMCs in response to LTD₄ is a prerequisite for phosphoinositide hydrolysis.

In contrast, the pretreatment of the TSMCs with NiCl₂ significantly decreased but did not abolish the BK-stimulated inositol phosphate synthesis. Thus, these results support the former observations which indicated that the mechanisms of transduction of LTD₄ and BK are different.

Pretreatment of cells with nifedipine significantly inhibited the InsP_s synthesis but did not significantly affect the LTD₄-induced [Ca⁺⁺]_i rises. It was reported that nifedipine slightly diminished LTD₄-induced contraction of guinea pig trachea (Cerria et al., 1983; Weichman et al., 1983; Jones et al., 1984; Cuthbert et al., 1994). In contrast, nifedipine significantly attenuated the initial peak and completely abolished the sustained phase of BK-triggered [Ca⁺⁺]_i increase. Our data suggest that LTD₄ stimulates Ca⁺⁺ entry in part via VOC and provide indirect evidence for a role of ROC in [Ca⁺⁺]_i changes.

In conclusion, these studies strongly suggest that LTD₄, acting on specific receptors, stimulates inositol phosphate synthesis and increases the [Ca⁺⁺]_i in TSMCs. An important finding is that both phenomena are totally dependent on the extracellular Ca⁺⁺ influx. Furthermore, the influx of extracellular Ca⁺⁺ precedes and is a requisite for PLC stimulation and, secondarily, may interact synergistically with InsP₃ to increase the [Ca⁺⁺]_i.