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OtherPROSTAGLANDINS, LEUKOTRIENES AND OTHER EICOSANOIDS

Mechanism of Action of Leukotriene D4 on Guinea Pig Tracheal Smooth Muscle Cells: Roles of Ca++ Influx and Intracellular Ca++ Release

Daniela Dumitriu, Stéphane Prié, Sylvie G. Bernier, Gaétan Guillemette and Pierre Sirois
Journal of Pharmacology and Experimental Therapeutics March 1997, 280 (3) 1357-1365;

Abstract

The effects of leukotriene D4 (LTD4) on the concentration of intracellular cytosolic free calcium ([Ca++]i) and on phosphoinositide hydrolysis were studied in cultured guinea pig tracheal smooth muscle cells. In Fura-2-loaded cells, LTD4(10−9–10−6 M) induced concentration-dependent changes in [Ca++]iconsisting of a slow, transient increase followed by a sustained phase. Preincubation of cells with LTD4 receptor antagonist MK-571 (10−6 M) blocked the increase in [Ca++]i. Similarly, LTD4-induced inositol phosphate ([3H]InsPs) synthesis was transient, concentration-dependent and inhibited by the LTD4 antagonist. In the absence of extracellular Ca++, LTD4 failed to induce [Ca++]i increases and [3H]InsPs formation. Accordingly, NiCl2 completely inhibited the LTD4-stimulated [3H]InsPs synthesis. Nifedipine (10−5 M) had a slight inhibitory effect on [Ca++]i increase but significantly reduced (40–50%) the [3H]InsPs accumulation. These findings indicate that LTD4-stimulated inositol phosphate synthesis and [Ca++]i increases in tracheal smooth muscle cells are receptor-mediated events and are dependent on the availability of extracellular Ca++. It is suggested that Ca++ influx plays a major role in the LTD4signal transduction mechanism.

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: viaInsP3-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 (Ferrariet 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

Materials.

Elastase type IV, collagenase type V, ionomycin, nifedipine, BK and mouse monoclonal α-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-Carteret 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 α-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 α-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 α-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):[Ca2+]i=Kd·(R−Rmin)(Rmax−R)·Fmin 380Fmax 380 where K d is the affinity of Fura-2 for Ca++ (224 nM at 37°C) and Fmin380/Fmax380 is the ratio (β value) of the fluorescent values obtained at 380 nm in the absence and the presence of saturating [Ca++]i. The maximum and minimum values (Rmax and Rmin) 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 × 10−5 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

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 α-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 × 10−7 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 × 10−5 M) and EGTA (2 × 10−2 M). LTD4 (1 × 10−9–1 × 10−6 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 × 10−9 M (n = 6) and the minimal and maximal responses were obtained with 1 × 10−9 and 1 × 10−7 M LTD4, respectively. Accordingly, the dose of 1 × 10−7 M LTD4 was used in subsequent experiments. No difference was observed between the kinetics of [Ca++]i increases induced by various LTD4 concentrations.

Figure 1
Figure 1

Representative tracings of Fura-2 fluorescence F340, F380 (A) and R (ratio) (B) changes in guinea pig TSMCs stimulated with LTD4 (1 × 10−7 M), ionomycin (IO) (1 × 10−5 M) and EGTA (2 × 10−2 M). Response to LTD4was 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.

Figure 2
Figure 2

Concentration-response relationship for LTD4-induced increases of [Ca++]iin 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.

To compare the effect of LTD4 on [Ca++]i with that of another potent smooth muscle constrictor, the response to BK (1 × 10−6 M) was examined. In contrast with LTD4, BK induced a typical biphasic [Ca++]i response without detectable latency. A rapid transient [Ca++]i increase to a peak of 457 ± 50 nM (n = 6) was reached within 15 s and was followed by a sustained elevation (152 ± 9 nM) (n = 6) above the basal level (fig.3).

Figure 3
Figure 3

Representative recordings of the time course of changes in [Ca++]i induced by LTD4 (1 × 10−7 M) (A) and BK (1 × 10−6 M) (B) in Ca++-containing medium. The tracings are from an experiment representative of three, each performed with different cell preparations.

Responses to maximal (1 × 10−7 M) LTD4stimulation were prevented by 1 × 10−6 M MK-571 (specific LTD4 receptor antagonist) (fig.4A), whereas the addition of MK-571 during the plateau phase returned [Ca++]i levels to basal (fig.4B).

Figure 4
Figure 4

Effect of the antagonist MK-571 on the changes in [Ca++]i induced by LTD4. MK-571 (1 × 10−6 M) was added 5 min before (A) and after (B) addition of LTD4 (1 × 10−7 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 × 10−7 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 × 10−7 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).

Figure 5
Figure 5

Effect of external Ca++ on the changes in [Ca++]i induced by LTD4(1 × 10−7 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.

To investigate the source of external Ca++ involved in [Ca++]i response to LTD4, the TSMCs were pretreated (30 min) with nifedipine (1 × 10−5 M), a selective voltage-dependent Ca++-channel blocker. The amplitude and the duration of LTD4-induced increase in [Ca++]iwere not significantly reduced by the pretreatment with nifedipine (fig. 6A). In BK (1 × 10−6M)-stimulated [Ca++]i increase, nifedipine significantly reduced the initial transient and the sustained increases by 33 ± 8% (297 ± 33 nM) (n = 3) (P < 0.05 as compared with control) and 100%, respectively (fig. 6B).

Figure 6
Figure 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 × 10−5 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 figure7, 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]InsPs (sum of [3H]InsP2+3+4 with [3H]InsP1) in TSMCs stimulated with LTD4 (1 × 10−7 M), in the presence of LiCl (1 × 10−2 M). The basal levels of [3H]InsP3, [3H]InsP2+3+4 and [3H]InsPs in the TSMCs remained constant during various stimulation times. LTD4 (1 × 10−7 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+4accumulation increased rapidly, reaching a maximum of 170 ± 7% over basal by 2 min. By increasing the stimulation time, the LTD4-stimulated [3H]InsP2+3+4formation gradually declined, reaching a level of 125 ± 5% over basal (significantly higher than control) after a 30-min stimulation period. The total [3H]InsPs 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.

Figure 7
Figure 7

Time course of [3H]InsP3(A), [3H]InsP2+3+4 (B) and [3H]InsPs (C) accumulation in the presence of LTD4 (1 × 10−7 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.

LTD4-stimulated inositol phosphate formation was dose dependent (fig. 8) and the 50% maximal effective concentration (EC50) for the synthesis of [3H]InsP2+3+4 and [3H]InsPs was 1 × 10−8 M. The maximum increases in [3H]InsP2+3+4 (fig.8A) and [3H]InsPs (fig. 8B) were achieved at 1 × 10−7 M LTD4 and were 195 ± 2% and 156 ± 8% over the basal (unstimulated, 100%) level, respectively. The LTD4 was effective in stimulation of PIP2 hydrolysis at concentrations over than 1 × 10−9 M.

Figure 8
Figure 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 × 10−6 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.

To confirm that LTD4-induced inositol phosphate accumulation is mediated via the LTD4 receptors in TSMCs, the LTD4 receptor antagonist MK-571 was used to study agonist-induced PI hydrolysis. MK-571 did not induce [3H]InsPs formation at the concentration of 1 × 10−6 M. Pretreatment of the cells with MK-571 significantly attenuated the LTD4-stimulated [3H]InsP2+3+4 and [3H]InsPs formation (fig.8). Maximal synthesis of [3H]InsP2+3+4 and [3H]InsPs obtained in the presence of the antagonist was significantly lower (a 40% inhibition) than that obtained in its absence, and the EC50 values were 3 × 10−8 M and 1 × 10−8 M, respectively. The minimal inositol phosphates synthesis in the pretreated cells was observed with 3 × 10−9 M LTD4.

Dependence of phosphoinositide hydrolysis on extracellular calcium.

To assess whether the LTD4-induced inositol phosphate accumulation, like the [Ca++]iincrease, 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 × 10−9–3 × 10−7 M) failed to stimulate [3H]InsP2+3+4 and [3H]InsPs synthesis (fig. 9, A and B). Only very small increases in [3H]InsPsaccumulation were detected but were not significantly different respect to the basal values.

Figure 9
Figure 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 LTD4in the presence of extracellular Ca++ after a pretreatment (30 min) with NiCl2 (5 × 10−3 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.

Based on these observations, further studies of the effects of NiCl2 (VOC and ROC blocker) and nifedipine and verapamil (VOC blockers) on the inositol phosphate synthesis stimulated by LTD4 were made. Pretreatment of the cells with NiCl2, nifedipine or verapamil did not change the basal production of inositol phosphates. NiCl2 (5 × 10−3 M) blocked (100% inhibition) LTD4-stimulated synthesis of [3H]InsP2+3+4 and [3H]InsPs (fig. 9, A and B). Pretreatment of the cells with nifedipine (1 × 10−5 M) resulted in a significant inhibition of LTD4-stimulated [3H]InsP2+3+4 and [3H]InsPs synthesis by 40 and 50%, respectively (at intermediary and maximal doses) (fig.10, A and B). The results obtained with the cells pretreated with verapamil (1 × 10−5 M) were similar to those obtained with nifedipine, except that verapamil was less potent (fig. 10, A and B). However, the EC50 andE max were not modified by the presence of nifedipine or verapamil.

Figure 10
Figure 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 LTD4after a pretreatment (30 min) with nifedipine (NIF) (1 × 10−5 M) (•) or verapamil (VP) (1 × 10−5 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.

BK (1 × 10−6 M)-induced [3H]InsP2+3+4 and [3H]InsPs formation increased by 868 ± 36 and 301 ± 2% over the basal level, respectively (fig.11, A and B). The effect of BK on [3H]InsP2+3+4 and [3H]InsPs was found to be four and two times greater than that of LTD4 (1 × 10−7 M), respectively. BK-stimulated synthesis of inositol phosphates was significantly (P < .01) higher than that induced by the maximal dose of LTD4. Moreover, the LTD4 receptor antagonist MK-571 did not significantly affect the effect of BK on inositol phosphate production (fig. 11). Pretreatment of the cells with NiCl2 (5 × 10−3 M) significantly reduced the BK-stimulated [3H]InsP2+3+4 and [3H]InsPs synthesis by 40% (P < .01) (fig.11A) and 25% (P < .05) (fig. 11B), respectively.

Figure 11
Figure 11

Effect of NiCl2 and compound MK-571 on LTD4 (1 × 10−7 M)- and BK (1 × 10−6 M)-stimulated [3H]InsP2+3+4(A) and [3H]InsPs (B) synthesis. The cells were stimulated in the presence of NiCl2 (5 × 10−3 M) (▨) or after a pretreatment (10 min) with the compound MK-571 (1 × 10−6 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

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 LTD4is 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; Saussyet 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 LTD4and 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 (Hedqvistet al., 1980; Sirois et al., 1981) and BK (Rhalebet 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++]ireflects 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 (Monget al., 1988b); accordingly, in the presence of external Ca++, the transient phase of [Ca++]i increase was rapid. In RBL-1 (Monget 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 versusintracellular 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 InsPssynthesis 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 × 10−8 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, Saussyet 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 NiCl2significantly 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 InsPs 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 (Cerriaet al., 1983; Weichman et al., 1983; Joneset 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 partvia 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.

Footnotes

  • Send reprint requests to: Pierre Sirois, Ph.D., Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.

  • Abbreviations:
    LTD4
    leukotriene D4
    BK
    bradykinin
    [Ca2+]i
    cytosolic calcium ion concentration
    TSMCs
    tracheal smooth muscle cells
    VOC
    voltage-operated calcium channels
    ROC
    receptor-operated calcium channels
    [3H]InsPs
    sum of inositol mono-, bi-, tri- and tetraphosphates
    MK-571
    (3-(((3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl) ((3-(dimethyl amino-3-oxo propyl)thio)methyl)thio) propanoic acid
    HEPES
    N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
    DMEM-F12
    Dulbecco’s modified Eagle’s medium and nutrient mixture F-12
    FBS
    fetal bovine serum
    PBS
    phosphate-buffered saline
    Fura-2AM
    Fura-2 pentaacetoxymethyl ester
    EGTA
    ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid
    BSA
    bovine serum albumin
    HBSS
    Hanks’ balanced salt solution
    F
    fluorescence
    R
    ratio of the fluorescence at 340 and 380 nm
    PLC
    phospholipase C
    • Received June 21, 1996.
    • Accepted November 25, 1996.

References

  1. ↵
    1. Baud L.,
    2. Goetzl E. J.,
    3. Koo C. H.
    (1987) Stimulation by leukotriene D4 of increase in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells. J. Clin. Invest. 80:983–991.
  2. ↵
    1. Benham C. D.,
    2. Tsien R. W.
    (1987) A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328:275–278.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Berridge M. J.,
    2. Irvine R. F.
    (1989) Inositol phosphates and cell signalling. Nature 341:197–205.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Best L.
    (1986) A role for calcium in the breakdown of inositol phospholipids in intact and digitonin-permeabilized pancreatic islets. Biochem. J. 238:773–779.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Best L.,
    2. Brooks K. J.,
    3. Bolton T. B.
    (1985) Relationship between stimulated inositol lipid hydrolysis and contractility in guinea-pig visceral longitudinal smooth muscle. Biochem. Pharmacol. 34:2297–2301.
    OpenUrlCrossRefPubMed
  6. Bhoola, K. D., Bewley, J., Crothers, D. M., Cingi, M. I. and Figueroa, C. D.: Kinin receptors on epithelial cells and smooth muscle of the trachea. Adv. Exp. Med. Biol. 247A: 421–427, 189..
  7. ↵
    1. Bouchelouche P. N.,
    2. Ahnfelt-Ronne I.,
    3. Thomsen M. K.
    (1990) LTD4 increases cytosolic free calcium and inositol phosphates in human neutrophils: inhibition by the novel LTD4 receptor antagonist, SR2640, and possible relation to modulation of chemotaxis. Agents Actions 29:299–307.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Brocklehurst W. E.
    (1953) Occurrence of an unidentified substance during anaphylactic shock in cavy lung. J. Physiol. (Lond.) 120:16.
    OpenUrl
  9. ↵
    1. Brocklehurst W. E.
    (1962) Slow reacting substance and related compounds. Prog. Allergy 6:539–558.
  10. ↵
    1. Cerrina J.,
    2. Advenier C.,
    3. Regnier A.,
    4. Floch A.,
    5. Duroux P.
    (1983) Effects of diltiazem and other Ca2+ antagonists on guinea-pig trachea muscle. Eur. J. Pharmacol. 94:241–249.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Chamley-Campbell J. H.,
    2. Campbell G. R.,
    3. Ross R.
    (1979) The smooth muscle cell in culture. Physiol. Rev. 59:1–61.
    OpenUrlFREE Full Text
  12. ↵
    1. Chan C. C.,
    2. Ecclestone P.,
    3. Nicholson D. W.,
    4. Metters K. M.,
    5. Pon D. J.,
    6. Rodger I. W.
    (1994) Leukotriene D4-induced increases in cytosolic calcium in THP-1 cells: dependence on extracellular calcium and inhibition with selective leukotriene D4 receptor antagonists. J. Pharmacol. Exp. Ther. 269:891–896.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Cockcroft S.,
    2. Gomperts B. D.
    (1980) The ATP4- receptor of rat mast cells. Biochem. J. 188:789–798.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Cockcroft S.,
    2. Bennett J. P.,
    3. Gomperts B. D.
    (1980) f-MetLeuPhe-induced phosphatidylinositol turnover in rabbit neutrophils is dependent on extracellular calcium. FEBS Lett. 110:115–118.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Cuthbert N. J.,
    2. Gardiner P. J.,
    3. Nash K.,
    4. Poll C. T.
    (1994) Roles of Ca2+ influx and intracellular Ca2+ release in agonist-induced contractions in guinea pig trachea. Am. J. Physiol. 266:L620–L627.
    OpenUrlAbstract/FREE Full Text
    1. Dahlén S. E.,
    2. Hedqvist P.,
    3. Hammarström S.,
    4. Samuelsson
    (1980) Leukotrienes are potent constrictors of human bronchi. Nature (Lond.) 288:484–486.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Devore-Carter D.,
    2. Morway P. F.,
    3. Weiss E. B.
    (1988) Isolation and characterization of guinea pig tracheal smooth muscle cells that retain differentiated function in long term subculture. Cell Tissue Res. 251:325–331.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Farmer S. G.,
    2. Burch R. M.,
    3. Meeker S. A.,
    4. Wilkins D. E.
    (1989) Evidence for a pulmonary B3 bradykinin receptor. Mol. Pharmacol. 36:1–8.
    OpenUrlAbstract
  19. ↵
    1. Feigen L.
    (1983) Differential effects of leukotrienes C4, D4 and E4 in the canine renal and mesenteric vascular beds. J. Pharmacol. Exp. Ther. 225:682–688.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Ferrari M.,
    2. Olivier M.,
    3. DeGasperi M.,
    4. Lechi A.
    (1989) Differential effects of nifedipine and diltiazem on methacholine-induced bronchospasm in allergic asthma. Ann. Allergy. 63:196–200.
    OpenUrlPubMed
  21. ↵
    1. Findlay S. R.,
    2. Leary T. M.,
    3. Parker C. W.,
    4. Bloomquist E. J.,
    5. Scheid C. R.
    (1982) Contractile events induced by LTD4 and histamine in smooth muscle a function of Ca2+ utilization. Am. J. Physiol. 243:C133–C139.
    OpenUrlFREE Full Text
  22. ↵
    1. Gown A. M.,
    2. Vogel A. N.,
    3. Gordon D.,
    4. Lu P. L.
    (1985) A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. J. Cell. Biol. 100:807–813.
    OpenUrlAbstract/FREE Full Text
    1. Grynkiewicz G. M.,
    2. Poenie M.,
    3. Tsien R. Y.
    (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440–3450.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Hanna C. J.,
    2. Roth S. H.
    (1978) The guinea pig tracheobronchial spiral strip: Responses to selected agonists. Can. J. Physiol. Pharmacol. 56:823–827.
    OpenUrlPubMed
  25. ↵
    1. Hedqvist P.,
    2. Dahlén S. E.,
    3. Gustafsson J.,
    4. Hammarstrom S.,
    5. Samuelsson B.
    (1980) Biological profile of leukotrienes C4 and D4. Acta Physiol. Scand. 110:331–333.
    OpenUrlPubMed
  26. ↵
    1. Hogaboom G. K.,
    2. Mong S.,
    3. W. U. H-L.,
    4. Crooke S. T.
    (1983) Peptidoleukotrienes: Distinct receptors for leukotriene C4 and D4 in the guinea-pig lung. Biochem. Biophys. Res. Commun. 116:1136–1143.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Howard S.,
    2. Chan-Yeung M.,
    3. Martin L.,
    4. Phaneuf S.,
    5. Salari H.
    (1992) Polyphosphoinositide hydrolysis and protein kinase C activation in guinea pig tracheal smooth muscle cells in culture by leukotriene D4 involve a pertussis toxin sensitive G-protein. Eur. J. Pharmacol. (Mol. Pharmacol. Sect.) 227:123–129.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Irvine R. F.
    (1990) “Quantal” Ca2+ release and the control of Ca2+ entry by inositol phosphates: A possible mechanism. FEBS Lett. 263:5–9.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Jones T. R.,
    2. Davis C.,
    3. Daniel E. E.
    (1982) Pharmacological study of the contractile activity of leukotriene C4 and D4 on isolated human airway smooth muscle. Can. J. Physiol. Pharmacol. 60:638–643.
    OpenUrlPubMed
  30. ↵
    1. Jones T. R.,
    2. Denis D.,
    3. Comptois R.
    (1984) Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea. Prostaglandins 7:939–959.
  31. ↵
    1. Jones T. R.,
    2. Zamboni R.,
    3. Belley M.,
    4. Champion E.,
    5. Charette L.,
    6. Ford-Hutchinson A. W.,
    7. Frenette R.,
    8. Gauthier J.-Y.,
    9. Leger S.,
    10. Masson P.,
    11. McFarland C. S.,
    12. Piechuta H.,
    13. Rokach J.,
    14. Williams H.,
    15. Young R. N.,
    16. DeHaven R. N.,
    17. Pong S. S.
    (1989) Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist. Can. J. Physiol. Pharmacol. 67:17–28.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Krell R. D.,
    2. Shung-Tsai B.,
    3. Berdoulay A.,
    4. Barone M.,
    5. Giles R. E.
    (1983) heterogeneity of leukotriene receptors in guinea-pig trachea. Prostaglandins 25:171–178.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Lewis R. A.,
    2. Drazen J. M.,
    3. Austen K. F.,
    4. Clark D. A.,
    5. Corey E. J.
    (1980) Identification of the C(6)-S-conjugate of leukotriene A with cysteine as a naturally occurring slow reacting substance of anaphylaxis (SRS-A). Importance of the 11-cis-geometry for biological activity. Bochem. Biophys. Res. Commun. 96:271–277.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Marom Z.,
    2. Shelhamer J. H.,
    3. Bach M. K.,
    4. Morton D. R.,
    5. Kaliner M.
    (1981) Slow reacting substances, leukotrienes C4 and D4, increase the release of mucus from human airways in vitro. Am. Rev. Respir. Dis. 126:449–454.
    OpenUrl
  35. ↵
    1. Marsh K. A.,
    2. Hill S. J.
    (1993) Characteristics of the bradykinin-induced changes in intracellular calcium ion concentration of single cultured bovine tracheal smooth muscle cells. Br. J. Pharmacol. 110:29–35.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Mong S.,
    2. Wu H-L.,
    3. Wong A.,
    4. Sarau H. M.,
    5. Crooke S. T.
    (1988a) Leukotriene D4 receptor-mediated phosphoinositol hydrolysis and calcium mobilization in rat basophilic leukemic cells. J. Pharmacol. Exp. Ther. 247:803–813.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Mong S.,
    2. Miller J.,
    3. Wu H-L.,
    4. Crooke S. T.
    (1988b) Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells. J. Pharmacol. Exp. Ther. 244:508–515.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Murray R. K.,
    2. Kotlikoff M. I.
    (1991) Receptor-activated calcium influx in human airway smooth muscle cells. J. Physiol. 435:123–144.
    OpenUrlPubMed
  39. ↵
    1. Oliva D.,
    2. Accomazzo M. R.,
    3. Giovanazzi S.,
    4. Nicosia S.
    (1994) Correlation between leukotriene D4-induced contraction and cytosolic calcium elevation: a quantitative and simultaneous elevation in smooth muscle. J. Pharmacol. Exp. Ther. 268:159–166.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Panettieri R. R.,
    2. Murray R. K.,
    3. DePalo L. R.,
    4. Yadvish P. A.,
    5. Kotlikoff M. I.
    (1989) A human airway smooth muscle cell line that retains physiological responsiveness Am. J. Physiol. 256:C329–C335.
    OpenUrl
  41. ↵
    1. Pong S.-S.,
    2. DeHaven R. N.
    (1983) Characterization of a leukotriene D4 receptor in guinea pig lung. Proc. Natl. Acad. Sci. U.S.A. 80:7415–7419.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Rhaleb N.,
    2. Rouissi N.,
    3. Drapeau G.,
    4. Jukic D.,
    5. Regoli D.
    (1991) Characterization of bradykinin receptors in peripheral organs. Can. J. Physiol. Pharm. 69:938–943.
    OpenUrlPubMed
    1. Samuelssön B.
    (1981) Leukotrienes: A novel group of compounds including SRS-A. Prog. Lipid Res. 20:23–30.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Saussy D. L.,
    2. Sarau H. M.,
    3. Foley J. J.,
    4. Mong S.,
    5. Crooke S. T.
    (1989) Mechanisms of leukotriene E4 partial agonist agonist activity at leukotriene D4 receptors in differentiated U-937 cells. J. Biol. Chem. 264:19845–19855.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Sirois P.,
    2. Lauziere M.,
    3. Braquet P.
    (1986) Further studies on the mechanism of action of leukotrienes and histamine on guinea-pig lung parenchyma. Role of calcium, phospholipase and methyl-transferase. Prostaglandins 31:1117–1133.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Sirois P.,
    2. Roy S.,
    3. Tétrault J. P.,
    4. Borgeat P.,
    5. Picard S.,
    6. Corey E. J.
    (1981) Pharmacological activity of leukotrienes A4, B4, C4 and D4 on elected guinea-pig, rat, rabbit and human smooth muscles. Prostaglandins Med. 7:327–340.
    OpenUrlCrossRefPubMed
    1. Söjlander A.,
    2. Grönroos E.,
    3. Hammarström S.,
    4. Andersson T.
    (1990) Leukotriene D4 and E4 induce transmembrane signaling in human epithelial cells. J. Biol. Chem. 265:20976–20981.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Somlyo A. P.,
    2. Walker J. W.,
    3. Goldman Y. E.,
    4. Trentham D. R.,
    5. Kobayashi S.,
    6. Kitazawa T.,
    7. Somlyo A. V.
    (1988) Inositol triphosphate, calcium and muscle contraction. Philos. Trans. R. Soc. B Biol. Sci. 320:399–404.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Weichman B. M.,
    2. Muccitelli R. M.,
    3. Tucker S. S.,
    4. Wasserman M. A.
    (1983) Effect of calcium antagonists on leukotriene D4-induced contractions of the guinea-pig trachea and lung parenchyma. J. Pharmacol. Exp. Ther. 225:310–315.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Winkler J. D.,
    2. Sarau H. M.,
    3. Foley J. J.,
    4. Crook S. T.
    (1988) Leukotriene D4-induced homologous desensitization in basal and differentiated U-937 cells: Characterization with the partial agonist leukotriene E4 and assessment receptor reserve. J. Pharmacol. Exp. Ther. 247:54–62.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Yang C. M.,
    2. Chou S-P.,
    3. Wang Y-Y.,
    4. Hsieh J-T.,
    5. Ong R.
    (1993a) Muscarinic regulation of cytosolic free calcium in canine tracheal smooth muscle cells: Ca+2 requirement for phospholipase C activation. Br. J. Pharmacol. 110:1239–1247.
    OpenUrlPubMed
  52. ↵
    1. Yang C. M.,
    2. Yo Y. L.,
    3. Wang Y. Y.
    (1993b) Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors. Br. J. Pharmacol. 110:983–988.
    OpenUrlPubMed
  53. ↵
    1. Yang C. M.,
    2. Hsia H. C.,
    3. Hsieh J.-T.,
    4. Ong R.,
    5. Luo S. F.
    (1994) Bradykinin-stimulated calcium mobilization in cultured canine tracheal smooth muscle cells. Cell Calcium 16:59–70.
    OpenUrlCrossRefPubMed
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OtherPROSTAGLANDINS, LEUKOTRIENES AND OTHER EICOSANOIDS

Mechanism of Action of Leukotriene D4 on Guinea Pig Tracheal Smooth Muscle Cells: Roles of Ca++ Influx and Intracellular Ca++ Release

Daniela Dumitriu, Stéphane Prié, Sylvie G. Bernier, Gaétan Guillemette and Pierre Sirois
Journal of Pharmacology and Experimental Therapeutics March 1, 1997, 280 (3) 1357-1365;
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