INTRODUCTION

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Cysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators formed through the 5-lipoxygenase pathway of arachidonic acid (1), and may contribute to the pathogenesis of asthma (2). In particular, cysteinyl-LTs are very potent constrictors of human bronchi (HB) not only in vitro (5), but also in vivo, in both normal and asthmatic individuals (6).

Among the cysteinyl-LTs, LTD₄ has been studied most extensively, and is known to act through specific G-protein-coupled receptors (7, 8), inducing phosphoinositol hydrolysis (9, 10) and an increase in the cytosolic Ca²⁺ concentration (intracellular [Ca²⁺]; [Ca²⁺]_i) (11). The latter effect in particular occurs in tracheal smooth-muscle cells (14).

Differences exist between cell types with respect both to the role of Ca²⁺ and to the mechanisms of the LTD₄-induced increase in [Ca²⁺]_i (15). For instance, LTD₄ can induce either Ca²⁺ influx through the plasma membrane without any Ca²⁺ release from intracellular stores (11, 12), or Ca²⁺ release without influx (16), and, in many cell types can induce both Ca²⁺ influx and release (9, 13, 14).

The role of Ca²⁺ in muscle contraction has been investigated for many years: Ca²⁺-dependent phosphorylation of myosin light chain is a major pathway for the regulation of smooth-muscle contractile force (17). However, the force/ Ca²⁺ ratio is variable (18), and the mechanism of action of agonist-induced smooth-muscle contraction (pharmacomechanical coupling) may consist of an alteration of the sensitivity of the contractile apparatus to Ca²⁺ (19). In some instances, agonist-triggered contraction can occur with little or no change in [Ca²⁺]_i (17, 20). In airway smooth muscle (ASM), including that of the human bronchus, the Ca²⁺-sensitivity of the contractile apparatus is very high and displays high positive cooperativity (21); quite often, the tonic activation of ASM by a variety of agonists depends on Ca²⁺ influx from the extracellular space (22, 23). However, despite the existence of voltage-operated calcium channels (VOC) in human airways (24), Ca²⁺-entry blockers are relatively ineffective in inhibiting bronchoconstriction in asthma, indicating that airway narrowing in asthma is mediated mainly by other means than VOCs. Accordingly, it has recently been suggested (25) that LTD₄ contracts human bronchi through a receptor-operated Ca²⁺ channel (ROC).

The aim of the present study was to clarify whether variations in [Ca²⁺]_i play a major role in LTD₄-induced contraction of HB, or whether other signal-transduction mechanisms are involved. For this purpose, we compared the in vitro contraction of isolated HB with increases in [Ca²⁺]_i and protein kinase C (PKC) activation in isolated smooth-muscle cells obtained from the same tissue.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All experiments were performed in the presence of 10 mM cysteine to prevent metabolism of LTD₄ (26).

Cell Isolation Procedure

Smooth-muscle cells from HB were isolated as previously described (27). Briefly, macroscopically normal fragments of lung were obtained at thoracotomy. Third-order bronchi were removed, under sterile conditions, the connective tissue and the epithelium were removed, and the smooth muscle was cut into pieces weighing approximately 10 mg each. The explants were grown at 37° C in a humidified atmosphere of 5% CO₂ in Medium 199, with the addition of 20% (vol/vol) fetal calf serum (FCS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were grown in monolayers in minimum essential medium (MEM) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and were used between passages 3 and 8. The cells stained positively with an antibody to smooth-muscle -actin.

Measurements of [Ca²⁺]_i

Human bronchial cells were seeded onto coverslips and used when they reached 100% confluence. The cells were incubated for 45 min at 30° C in the dark, with 5 µM Fluo3 acetoxymethyl ester (Fluo3/AM) (28) in MEM plus 0.03% Pluronic F-127, 2.5 mM probenecid, and 10 mM 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethanesulfonic acid (HEPES). After loading, cells were washed twice with saline solution (NaCl 145 mM, KCl 5 mM, MgCl₂ 1 mM, CaCl₂ 1.8 mM, HEPES 10 mM, glucose 10 mM; pH 7.4) plus 2.5 mM probenecid, and were kept at room temperature for 15 min to complete hydrolysis of the Fluo3/AM. The coverslips were then transferred to a spectrofluorometer (Perkin Elmer LS5; Perkin Elmer Italia, Monza, Italy) cuvette, and the fluorescence of the cell preparations was monitored at 30° C (506-nm excitation, 530-nm emission). In order to evaluate [Ca²⁺]_i from fluorescence readings, F_max (maximal fluorescence of the system) was obtained by adding 2.7 µM ionomycin and 100 µM digitonin and F_min was obtained by measuring the autofluorescence of cells that were not exposed to Fluo3/AM (29). [Ca²⁺]_i was determined according to the method of Tsien and colleagues (30), with K_d = 646 nM, which was calculated by assuming a linear dependence on temperature (29). Increases in [Ca²⁺]_i were expressed as the ratio of the stimulated over the basal level (s/b).

Preparation of Isolated HB

Macroscopically normal HB (2- to 3-mm diameter) were obtained from patients undergoing thoracic surgery for pulmonary carcinoma, and were prepared as previously described (31). Briefly, the bronchi were dissected free of parenchyma and blood vessels, and were cut helically into strips 2 to 3 mm wide and about 10 mm long. The strips were suspended in organ baths containing Tyrode's solution (composition in mM: NaCl, 140; KCl, 3; CaCl₂, 1; MgCl₂, 0.05; NaH₂PO₄, 0.5; glucose, 8.4; NaHCO₃, 12; pH 7.4) at 37° C, which was bubbled with 95% O₂- 5% CO₂. Contractions were measured with a Basile 7004 isometric force transducer (Ugo Basile, Comerio, Italy) and recorded on a Basile Gemini 7070 polygraph. Bronchial strips were set at an initial tension of 1 g and repeatedly washed over a 60-min equilibration period. Contractions are expressed as the percent of the contraction induced by 300 µM acetylcholine (ACh). When necessary, the Tyrode's solution was replaced after ACh administration, with a solution lacking Ca²⁺ and containing 1 mM ethylene glycol-bis-(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1 µM thapsigargin to eliminate intra- and extracellular calcium. At the end of experiments with calcium- depleted strips, contractility was verified by addition of 30 mM BaCl₂.

Cell Contraction

Cells were grown in monolayers in Petri dishes as described earlier, and were used at submaximal (95%) confluence. Cell contraction was recorded by measuring the increase in intercellular spaces by means of a TV camera connected to an inverted light microscope (Axiovert; ×20 lens; Zeiss, Jena, Germany) coupled to an image analyzer (a Macintosh computer [Apple Computer, Inc., Cupertino, CA] running NIH Image v.1.62, developed by Dr. Wayne Rasband, of the National Institutes of Health, Bethesda, MD) through a Data Translation Inc. (Marlboro, MA) QuickCapture frame grabber card. Images were collected for 15 min (one frame every 30 s). After 5 min of recording of the control state, either histamine or LTD₄ was added. When necessary, intra- and extracellular Ca²⁺ was removed as described earlier.

Image intensity is reported on a scale of 255 Gray Units with 0 for white and 255 for black. The background intensity level (empty Petri dishes) was determined to be in the range of 0 to 50 of Gray Units, and all Gray-level readings of 50 or lower were converted to a red color to represent intercellular spaces and to compute the percent increase in intensity over the basal state.

Laser Scanning Confocal Fluorescence Microscopy

Cells were grown in monolayers on chamber slides and loaded with Fluo3/AM as described earlier. The chamber slide was placed on a thermostatically controlled copper plate (at 30° C) on the stage of a confocal microscope (MRC-600; Bio-Rad, Microsciences Division, Hemel Hempstead, UK) coupled to an upright epifluorescence microscope (Optiphot-2; Nikon, Tokyo, Japan). The indicated concentrations of histamine or LTD₄ were added to the cells with a microsyringe. Intracellular Fluo3 fluorescence in single living cells was measured by confocal microscopy. Argon ion laser excitation was used at 488 nm, and the power of the laser was kept at low values (< 0.1 mW) to preserve cell viability and to avoid possible saturation of the fluorescence signal. Fluorescence emission was detected in the photon-counting mode with a photomultiplier (S20 Thorn-EMI-9890; Bio-Rad Microsciences Division, Hemel, Hempstead, UK) through a long pass filter at wavelengths above 515 nm. The indicator fluorescence was followed in a field of view of about 400 × 300 µ², in which about 10 cells were inspected at the same time through a ×20 air objective. The scan speed of the laser excitation was set to 0.25 s for a frame of 384 × 256 pixels. Under these conditions, a single scan of the field of view enabled us to obtain an excellent fluorescence image at a confocal aperture of about 2 mm and a numerical aperture of 0.4. A temporal resolution of 2 s was used. The time course of the indicator fluorescence was followed by using the time series option of the MRC-600 COMOS software.

Image fluorescence intensities were recorded on a scale of 256 Gray Units (from 0 for black to 255 for peak white). False colors were assigned to four ranges of Gray-Unit values, corresponding to four ranges of Ca²⁺ concentrations, as shown in Figure 7. The time dependences of fluorescence intensities were studied in selected areas inside single cells (about 40 to 80 µ² wide), in which the mean Ca²⁺ concentrations were evaluated.

View larger version (40K): [in this window] [in a new window]   Figure 7.   Western blot analysis done with antibodies to PKC- and PKC- on membrane proteins from HASM cells incubated with vehicle, LTD4, or histamine. (A) Effect of LTD4 (0.3 µM) or histamine (100 µM) on translocation of PKC- to the membrane. (B) Effect of LTD4 (0.3 µM) or histamine (100 µM) on translocation of PKC- . (C) Time-course of PKC- translocation with 0.3 µM LTD4 (duplicate lanes are shown in this case). (D) Effect of increasing concentrations of LTD4 on translocation of PKC- . The last two lanes represent the effect of 10 µM SKF 104353 on translocation of PKC- in cells stimulated with 0.3 µM LTD4 or in resting cells, respectively. Vehicle was MEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM cysteine. In (D) vehicle contained also 0.7% DMSO.

Evaluation of PKC Translocation

Smooth-muscle cells were incubated for 18 h in culture medium without FCS, and were then treated with the agents being investigated at 37° C for 5 min, unless otherwise indicated. The incubation was stopped at 4° C and the cells were washed twice in cold phosphate-buffered saline (PBS). Cells were then lysed at 4° C for 30 min in 20 mM Tris-HCl, pH 7.4, containing 2 mM EGTA, 20 mM ethylenediamine tetraacetic acid (EDTA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and a set of protease inhibitors (Complete; Boehringer Mannheim, Mannheim, Germany). The lysed suspension was centrifuged at 100,000 × g for 60 min, with the supernatant comprising the cytosolic fraction. The pellet was resuspended in lysis buffer plus 0.1% Triton X-110 at 4° C for 45 min, and centrifugation was then repeated, with the resulting supernatants representing the membrane fraction. The proteins in the cytosolic and membrane fractions were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (running gel: acrylamide 11%), and were electroblotted onto nitrocellulose paper. PKC isozymes were evaluated by Western blot analysis, utilizing polyclonal antibodies raised against PKC- (at a dilution of 1:1,500) and PKC- (at a dilution of 1:1,000) for 1 h at 25° C. After washing with Tris-buffered saline (TBS) containing 0.1% Tween-20, the sheets were incubated (1 h, 25° C) with peroxidase-conjugated antirabbit IgG (1:3,000) and washed as described previously. Antigen- antibody complexes were detected by enhanced chemiluminescence. Results were analyzed with a computer-assisted image analysis program (NIH Image v.1.62), and were expressed as percents of PKC translocation versus basal conditions.

Statistical Analysis

Statistical analysis of the concentration-response curves for [Ca²⁺]_i versus histamine or LTD₄ was done with the ALLFIT computer program (32), which calculates lower and upper plateaus, slope, and EC₅₀ values, and allows the comparison of two or more curves. Selection of the best fitting model and evaluation of the statistical significance of parameter differences was based on the F test for the extra-sum-of-squares principle (33). All curves shown in the illustrations were computer generated.

Statistical comparison of multiple groups was done through one-way analysis of variance followed by Bonferroni's post hoc test. A statistical level of significance of p < 0.05 was accepted. Data are expressed as mean ± SE. Each experiment was performed at least three times and, when possible, in triplicate.

Materials

TBS, PBS, Hanks' balanced salt solution (HBSS), Medium 199, Eagle's MEM, trypsin-EDTA, EGTA, penicillin (10,000 U/ml), streptomycin (10 mg/ml), L-glutamine (200 mM), H7, dimethylsulfoxide, ACh, histamine, probenecid, atropine, pyrilamine, L-cysteine, thapsigargin, phorbol-dibutyrate ester (PDBu), anti-smooth-muscle -actin antibody, peroxidase-conjugated antirabbit IgG, PMSF, and DTT were from Sigma Chemical Co., St. Louis, MO. Antibodies to the PKC- and PKC- isoforms, were from Boheringer Mannheim. LTD₄ was from Cayman Chemical Co., Ann Arbor, MI. All salts for Tyrode's solution were from Merck, Darmstadt, Germany. FCS was from PBI International, Milan, Italy. Disposable culture flasks, Petri dishes, and filters were from Corning Glassworks, Corning, NY. Fluo3/AM and pluronic F-127 were purchased from Molecular Probes, Eugene, OR. MK 571 was a generous gift from Dr. A. W. Ford-Hutchinson of Merck Frosst, Point Dorval, PQ, Canada, and SKF 104353 was kindly provided by Dr. A. von Sprecher of the Research Department, Pharmaceutical Division, Novartis Ltd., Basel, Switzerland.

	RESULTS

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Contraction of Human Airway Smooth-Muscle Cells and of Isolated Strips of HB

Contraction of human airway smooth-muscle (HASM) cells in monolayers was recorded by measuring the increase in intercellular spaces by means of an image analyzer coupled to a light microscope. LTD₄ was able to contract HASM cells to the same extent in the presence of Ca²⁺ (30 ± 8% [mean ± SE] increase in intercellular spaces, n = 6; Figures 1A and 1B) as in its absence (which was effected by pretreatment with 1 mM EGTA and 300 nM thapsigargin, to eliminate extra- and intracellular Ca²⁺, respectively) (28 ± 3%, n = 3, data not shown). The time-course of the increase in intercellular space in the same field is shown in Figure 1C. On the contrary, the effect of histamine, albeit generally greater than that of LTD₄ (66 ± 9%, n = 6; Figures 1D and 1E), was greatly decreased by the absence of Ca²⁺ (26 ± 9%, n = 3, data not shown). Figure 1F shows the time course of the effect of histamine. The concentration of thapsigargin used and the preincubation time were sufficient to deplete the sensitive cytoplasmic stores of Ca²⁺ as assessed by [Ca²⁺]_i measurement (data not shown).

View larger version (78K): [in this window] [in a new window]   Figure 1.   Light microscopic images of HASM cells made with a false-color map. Cell images are given in a scale of 256 Gray-Unit levels (from 0 for white to 255 for black); of red color (0 to 50 Gray-Unit levels) represents intercellular spaces. Cells were used at submaximal (95%) confluence. (A) Control cells. (B) Same field as in A after stimulation with 0.3 µM LTD4 (8 min). (C ) Time-course of the percent increase in intercellular spaces induced by LTD4 (data from A and B); frames were recorded at 30-s intervals. (D) Control cells. (E ) Same field as in D after stimulation with 100 µM histamine (5 min). (F ) Time-course of the percent increase in intercellular spaces induced by histamine (data from D and E ); frames were recorded at 30-s intervals. The experiments were performed in the presence of 1 mM extracellular Ca2+.

Both LTD₄ (Figure 2A) and histamine (Figure 2B) caused marked concentration-dependent bronchoconstriction with comparable efficacy in isolated strips of HB. The calculated EC₅₀ values for LTD₄ and histamine are summarized in Table 1.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Concentration-response curves of agonist-induced contraction (A and B) of isolated human bronchi, and [Ca2+]i increase (C and D) in HASM cells. (A and C) LTD4. (B and D) Histamine. Insets in C and D are representative tracings of the individual effect of agonist concentrations in inducing [Ca2+]i increases. Curves in A and B were constructed with strips from seven different lung specimens. Curves in C and D were obtained from four to seven experiments on cells obtained from three to five different lung specimens, respectively.

[Ca²⁺]_i Increase in a Population of HASM Cells and in Single HASM Cells

In a population of HASM cells, LTD₄ (Figure 2C) was able to elicit only a very modest, if any, transient increase in [Ca²⁺]_i (maximum [Ca²⁺]_i increase: s/b = 1.8 ± 0.35). On the contrary, the response to histamine (Figure 2D) was concentration-dependent (EC₅₀ = 30 µM), with a marked increase in [Ca²⁺]_i (maximum increase: s/b = 8.9 ± 0.89). Representative traces showing the [Ca²⁺]_i increases evoked by 1 µM LTD₄ and 30 µM histamine, respectively, are shown as insets in Figures 2C and 2D. The response was completely abolished by 5 min of preincubation with specific antagonists (i.e., 1 µM MK 571 and 30 µM mepyramine) (data not shown).

[Ca²⁺]_i transients in single HASM cells loaded with Fluo3 were studied with confocal fluorescence microscopy. Figure 3 shows the false-color images of cells under basal conditions (Figure 3A) and at 40 s (Figure 3B), corresponding to the peak [Ca²⁺]_i value, after stimulation with 100 µM histamine. Figures 3C and 3D (48 s) show the results obtained with 1 µM LTD₄. Figures 4A and 4B show the mean [Ca²⁺]_i increase in response to histamine and LTD₄, respectively, as a function of time, measured in the cells indicated in Figure 3, as well as the effect elicited by the vehicle alone (0.1% EtOH; Figure 3C). It is clear that the effect of LTD₄ was not much greater than that of the appropriate control, whereas histamine elicited a much greater increase in [Ca²⁺]_i. Furthermore, the specific antagonist SKF 104353 (10 µM) decreased the LTD₄ response by only a slight degree (data not shown).

View larger version (47K): [in this window] [in a new window]   Figure 3.   False-color confocal fluorescence microscopic images of HASM cells loaded with the fluorescent Ca2+ probe Fluo3. (A) Resting cells. (B) Appearance at 40 s after the addition of 100 µM histamine. (C ) Resting cells. (D) Appearance at 48 s after the addition of 1 µM LTD4. Fluorescence intensities were recorded on a scale of 256 Gray-Unit levels and divided into four ranges, corresponding to four ranges of [Ca2+]i concentrations, to which pseudocolors were assigned. Images shown are representative of seven or eight other experiments (for LTD4 and histamine, respectively) on cells obtained from three to five different lung specimens.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Mean [Ca2+]i as a function of time with (A) 100 µM histamine and (B) 1 µM LTD4. Measurements were made in the cells indicated in Figure 3. (C ) Effect of vehicle (0.1% EtOH) for LTD4 alone.

Role of PKC in Contraction

Figures 5A through 5D show the contraction tracings for isolated strips of HB prepared from adjacent segments challenged with LTD₄ (0.3 µM). Compound H7 (50 µM), an inhibitor of PKC, did not affect LTD₄-induced contraction in the presence of Ca²⁺ (Figure 5B), whereas in the absence of Ca²⁺ it abolished the residual contraction usually present (compare Figures 5C and 5D). Moreover, in the absence of Ca²⁺, repeated in vitro administration of the PKC activator PDBu (1 µM) completely inhibited LTD₄-induced contraction of bronchial strips (Figure 5E).

View larger version (36K): [in this window] [in a new window]   Figure 5.   Representative tracings of contraction of isolated strips of HB in response to 0.3 µM LTD4. (A and B) Tracings in the presence of Ca2+ in the absence (A) and in the presence (B) of 50 µM H7. (C through E ) Tracings in the absence of Ca2+ in the absence (C ) and in the presence (D) of 50 µM H7. (E ) Tracing made after repeated administration of 1 µM PDBu. The tracings are representative of two or three other tracings.

When the strips were stimulated with 0.3 µM LTD₄, the removal of extra- and intracellular Ca²⁺ markedly decreased, but did not abolish, the contraction (Figures 6 and 5C). On the contrary, the effect of PDBu, which induced a slowly developing contraction (Figure 5E), was not affected by removal of Ca²⁺ (Figure 6). In the case of histamine, no contraction was detectable in the absence of Ca²⁺ (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 6.   Contraction of isolated strips of HB in the presence or absence of intracellular and extracellular Ca2+. Bars represent the mean ± SE. Closed bars: LTD4 at 0.3 µM; hatched bars: PDBu at 1 µM. *p < 0.05 versus addition of Ca2+ (one-way analysis of variance, n = 4).

Translocation of Different Isoforms of PKC

Figure 7 shows a representative Western blot analysis performed with polyclonal antibodies to PKC-, a calcium-dependent isoform of PKC, and to PKC- , a calcium-independent isoform of the enzyme, on membrane proteins of HASM cells incubated with either LTD₄ or histamine. LTD₄ at 0.3 µM induced marked activation of the calcium-independent PKC- (170 ± 15%; Figure 7B) and, to a lesser extent, of the calcium-dependent PKC- isoform (116 ± 17%; Figure 7A), whereas incubation with 100 µM histamine produced marked translocation only of the calcium-dependent PKC- isoform (514 ± 181%; Figure 7A). The effect of LTD₄ on PKC- was time-dependent, being maximal after 5 min of incubation and progressively decreasing at 15 and 30 min (Figure 7C), thus suggesting that prolonged incubation induces downregulation of this isoform of the kinase. Figure 7D shows a Western blot analysis of membrane proteins of cells incubated with increasing concentrations of LTD₄. LTD₄ (5 nM and 0.3 µM) increased the translocation of PKC- by 143% ± 21 and 105 ± 16, respectively. In addition, 10 µM of SKF 104353 was able to counteract the effect of the highest concentration of LTD₄.

As expected, removal of Ca²⁺ abolished LTD₄-induced translocation of PKC-, but it did not affect the translocation of PKC- (Figure 8).

View larger version (41K): [in this window] [in a new window]   Figure 8.   Western blot analysis done with antibodies to PKC- and PKC- on membrane proteins from HASM cells incubated with 0.3 µM LTD4 in the presence or absence of extracellular Ca2+. Effect of LTD4 on translocation of PKC- (A) and PKC- (B) in the presence (first two lanes) and absence (last two lanes) of Ca2+. Control samples contained MEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM cysteine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

LTD₄ is a potent constrictor of smooth muscle, including bronchial smooth muscle. In most tissues and cells, this effect has been shown to be receptor-mediated and coupled to an increase in [Ca²⁺]_i (7, 15). Smooth-muscle contraction has been directly correlated with either an increase in [Ca²⁺]_i (in the guinea pig ileal longitudinal muscle [12]) or with an increased hydrolysis of phosphatydyl inositol in lung [8]). On the contrary, we found that a discrepancy exists between contraction and increased [Ca²⁺]_i in bronchial tissue. In fact, LTD₄ was able to contract strips of human bronchus in vitro to an extent comparable with that elicited by histamine, albeit with much higher potency, but was much less efficient in inducing an increase in [Ca²⁺]_i in isolated smooth-muscle cells obtained from the same organ.

One possible explanation for this discrepancy would be the loss during cell isolation and culture of the LTD₄ receptor that was responsible for contraction. However, this hypothesis can be ruled out, because the cultured cells actually responded to LTD₄ with measurable contractions that were inhibited by MK-571, a specific LTD₄ receptor antagonist. Another possible explanation would be a heterogeneity in the [Ca²⁺]_i response in the cell population examined, with the cells either not being synchronized in their Ca²⁺ response or with few cells expressing the receptor and therefore responding with an increase in [Ca²⁺]_i. Laser scanning confocal fluorescence microscopy allowed us to measure [Ca²⁺]_i variations in single HASM cells, and demonstrated that the percentage of cells responding to LTD₄ with increases in [Ca²⁺]_i was not lower than that responding to histamine. Furthermore, the time-course of Ca²⁺ variation in response to LTD₄ did not vary significantly from cell to cell. The data obtained at the single-cell level therefore confirmed those obtained in the overall cell population, showing a marked difference in the peak [Ca²⁺]_i level with histamine as opposed to LTD₄, and excluding a heterogeneous cell response as the cause of the discrepant effect of LTD₄.

Thus, our data demonstrate a dissociation between LTD₄-induced contraction and increased [Ca²⁺]_i elevation and suggest that Ca²⁺-independent mechanisms contribute to LTD₄-triggered bronchoconstriction in human airways. Indeed, measurement of single-cell contraction by light microscopy shows that the removal of extra- and intracellular Ca²⁺ elicits only a minor reduction in cell contraction induced by LTD₄ from that observed in the presence of Ca²⁺. On the contrary, histamine-induced contraction was substantially reduced (by more than 50%) in the absence of Ca²⁺. This indicates that in isolated HASM cells, the histamine response depends largely on the presence of Ca²⁺, whereas the LTD₄ response is largely Ca²⁺-independent.

In seeking a Ca²⁺-independent mechanism for LTD₄-mediated bronchoconstriction, we postulated an involvement of PKC in LTD₄-mediated signal transduction. Indeed, it is known that: (1) LTD₄ can activate PKC (7, 34); (2) LTD₄-triggered contraction in HB is rather slow, but is sustained in time; (3) PKC is involved in the sustained phase of smooth-muscle contraction in general (35) and in HASM cells in particular (36, 37); and (4) HASM cells express different isoforms of PKC (38), some of which (, ,  , and ) are Ca²⁺ independent.

Thus, using polyclonal antibodies against specific PKC isoforms (i.e., PKC-) as representatives of the classical Ca²⁺- dependent isoforms of PKC, and against PKC- as a representative of the Ca²⁺-independent isoforms, we investigated their activation. LTD₄ was able to translocate PKC- and, to a lesser extent, PKC-, whereas histamine was able to translocate only PKC-. The activation of PKC- by LTD₄ was concentration- and time-dependent. The decrease in PKC- translocation at 15 and 30 min, with respect to 5 min, might suggest a desensitization of the enzyme. This result is in agreement with the tachyphylaxis displayed by LTD₄ as a contractile agent (39). Furthermore, inhibition of LTD₄-induced translocation of PKC by SKF 104353, a cysteinyl-LT₁ antagonist, showed that the activation was receptor-mediated.

To further link PKC activation and contraction of HB, we evaluated the effect of PDBu, a PKC activator, on strips of HB. PDBu was able to contract HB to the same extent in the presence as in the absence of extra- and intracellular Ca²⁺. These data are apparently at variance with the previously reported (37, 40) finding that removal of extracellular Ca²⁺ abolished PDBu-induced HB contraction; however, the investigators who reported this generated cumulative concentration-response curves for PDBu, in which desensitization of PKC yields a response lower than the one obtained by single-dose administration, as also confirmed by Yang and Black (40). In our hands, the slowly developing and sustained contraction of HB seemed to be Ca²⁺-independent.

Interestingly, in the absence of Ca²⁺, LTD₄ was able to contract HB to the same extent as was PDBu, thus suggesting the involvement of a Ca²⁺-independent PKC isoform in LTD₄-mediated signal transduction. This is confirmed by the complete inhibition, in the absence of Ca²⁺, of the LTD₄ response caused by PDBu-induced desensitization of PKC. In addition, the PKC inhibitor H7 abolished the slow and sustained contraction triggered by LTD₄ in the absence of Ca²⁺. The relatively minor effect of H7 on the response to LTD₄ in the presence of Ca²⁺ suggests the involvement of mechanisms other than PKC activation (possibly activation of myosin light-chain kinase [41]) in the rapid phase of LTD₄ contraction. Thus, the LTD₄-induced response in HB consists of both a Ca²⁺-dependent phase (responsible for the onset of contraction) and a slower, Ca²⁺-independent phase (responsible for the maintenance of contractile tone). On the contrary, histamine-induced contraction of HB is completely Ca²⁺-dependent.

In conclusion, our data, taken collectively, indicate that in human airways, LTD₄-induced contraction is at least in part independent of increases in Ca²⁺, and this might represent an extreme form of the increase in Ca²⁺ sensitivity induced by agonists (19). Such Ca²⁺-independent contraction involves the activation of one of the novel isoforms of PKC (i.e., PKC- ), and indeed, PKC activation has been proposed as a mechanism of increased force development at constant [Ca²⁺]_i (42). The nature of the transduction pathway involved will be the subject of future research. However, one can speculate that the LTD₄ receptor is coupled to phospholipase D activation, yielding diacylglycerol formation (and thus PKC activation) without concomitant formation of Ca²⁺. Additionally, our data support the existence of a link between LTD₄-induced activation of a Ca²⁺-independent PKC and bronchial smooth-muscle contraction. This pathway, which is specific for LTD₄ (being abolished by two different receptor antagonists and not shared by histamine), represents a novel transduction mechanism for cysteinyl-LTs.