Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats

F. C. Tao, S. Shah, A. A. Pradhan, B. Tolloczko, and J. G. Martin

Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada H2X 2P2


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca2+ mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca2+ mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca2+ transients in Fischer than in Lewis ASM cells. ASM cell B2 receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca2+ in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca2+ transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca2+ transients in Lewis cells. Enhanced Ca2+ mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.

bradykinin; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ASTHMA IS A COMPLEX disease that is characterized by reversible airway obstruction, airway remodelling, airway inflammation, and airway hyperresponsiveness (AHR). The role of atopy in the pathogenesis of asthma has been a topic of intense study in humans and in animal models (16, 21). The cellular players implicated by these studies include dendritic cells, T lymphocytes, and eosinophils, which exert their effects through Th2 cytokines (e.g., IL-2, IL-4, IL-5, IL-13), chemokines (e.g., RANTES, eotaxin), IgE, and leukotrienes (21). However, although allergic sensitization can induce AHR (26), it does not have to be a necessary precondition for AHR to exist. Screens of various normal inbred mice (22) and rat (9) strains revealed heterogeneous airway responsiveness among different strains that suggest that AHR can be a genetically determined trait. Subsequently, AHR in innately hyperresponsive A/J mice was mapped by quantitative trait loci studies to chromosomes 2, 6, 7, 15, and 17 (4, 5, 11). These observations highlight the distinction between genetic and environmental etiologies of asthma and AHR.

In the inbred rat screen for airway responsiveness, Fischer 344 rats were identified to be hyperresponsive to methacholine and serotonin (5-HT) compared with Lewis rats (9, 32). Fischer rats contained more smooth muscle in their airways, but this only partially accounted for this strain's hyperresponsive behavior (10). Comparisons of the excised airways from these two rat strains confirmed that Fischer tracheal rings and intraparenchymal airways in lung explants are also hypersensitive and hyperreactive to muscarinic stimulation in vitro (18, 32). To elucidate signal transduction mechanisms that contribute to genetic AHR, we examined differences in intracellular Ca2+ signaling between Fischer and Lewis airway smooth muscle (ASM) because intracellular Ca2+ mobilization is a critical trigger for ASM contraction (19). We reported that in response to 5-HT, greater constriction of Fischer airways ex vivo was associated with enhanced Ca2+ mobilization in Fischer ASM cells and that this, in turn, may have arisen from lower Ins(1,4,5) P3 5-phosphatase activity in the Fischer cells (29, 30).

In the present study, we postulated that enhanced ASM cell Ca2+ mobilization is a general mechanism of genetic AHR to contractile agonists that mediate contraction through G protein-coupled receptors. To test this hypothesis, we compared the responses of explanted airways and cultured ASM cells from Fischer and Lewis rats to another contractile agonist that signal through a G protein-coupled receptor. Bradykinin (BK) was chosen because it contracts smooth muscle with a slower time course than 5-HT in vivo (13), and it recruits more postreceptor signaling pathways than 5-HT in ASM cells. In ASM, 5-HT couples predominantly to phosphatidylinositide-specific phospholipase C (PI-PLC) (31), whereas BK couples to PI-PLC, phosphatidylcholine-specific phospholipase C (PC-PLC), and phospholipase D (PLD) (25, 27, 28). PI-PLC stimulates Ca2+ mobilization by producing inositol (1,4,5)- trisphosphate [Ins(1,4,3)P3], which releases stored intracellular Ca2+ and 1,2-diacylglycerol, which activates PKC. PKC regulates Ca2+ mobilization by modulating Ins(1,4,3)P3 synthesis (23, 35) and Ca2+ transport at the plasmalemma of ASM cells (15, 33). PC-PLC produces choline phosphate, which has no known effects on Ca2+ mobilization, and 1,2-DAG that can activate PKC. PLD produces phosphatidic acid and choline, neither of which regulates Ca2+ mobilization. The aims of the study were to determine 1) whether BK stimulated greater constriction of Fischer than Lewis intraparenchymal airways ex vivo, 2) whether the greater response was associated with higher Ca2+ transients in Fischer than Lewis ASM cells, and 3) the mechanisms responsible for differences in Fischer and Lewis ASM cell Ca2+ mobilization.


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METHODS
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Lung explant culture. Lung explants were obtained as previously described (32). Excised lungs were inflated to 50% of the total lung capacity with a solution of 1% agarose type VII (Sigma Chemical, St. Louis, MO) in buffered culture medium (BCM). The lungs were chilled at 4°C to allow the agarose to gel inside the airways. The lobes were separated and embedded in a solution of 4% agarose in MEM (GIBCO, Burlington, Canada). After another incubation at 4°C to gel the embedding solution, 0.5- to 1-mm-thick explants were sliced from the lobes using a handheld microtome blade. Only those explants containing a cross-sectional slice of bronchus were kept for study. Explants were cultured in BCM overnight in a humidified 5% CO2 incubator.

Measurement of dynamic airway constriction. Explants screened for intact airway epithelium with beating cilia were individually transferred to single wells of a 6-well plate and bathed with 500 µl of HEPES BCM and maintained at 37°C. In experiments examining the contribution of extracellular Ca2+ to airway narrowing, explants were bathed in Ca2+-free Hanks' buffer containing 1 mM EDTA. The explants were viewed under ×4 magnification with an inverted microscope (model IMT-2; Olympus, Tokyo, Japan) connected to a video monitor and a CCD-200-R camera (Videoscope International, Washington, D.C.). Images of the constricting airways were recorded on an optical disk recorder (Panasonic TZ 2026F, Osaka, Japan), digitized with an 8-bit frame grabber (model PIP1024B; Matrox, Montreal, Canada), and analyzed with Galileo software (Inspiraplex, Montreal, Canada).

Peak airway constriction, the rate of narrowing from rest to 50% of peak constriction, and the time lapse to peak constriction were used as indexes of airway narrowing capacity and velocity. Peak constriction occurred when the airway luminal area reached a minimum following BK and was calculated from A = Pi2/4pi , where A is the luminal area and Pi is the internal perimeter of the airways measured directly from the digitized images. The rate of narrowing during the first half of constriction (v50) was computed as the change in Pi over time (v50 = Delta Pi/Delta t). Both A and v50 were normalized to baseline Pi to eliminate variability due to heterogeneous airway size.

ASM cell culture. Cells were cultured as previously described (31). Tracheae were aseptically excised from the rats and washed with HBSS. The tracheal tube was cut longitudinally opposite the trachealis muscle and digested with 0.2% collagenase IV (Sigma Chemical) and 0.05% elastase IV (Sigma Chemical) in HBSS for 35 min at 37°C. Smooth muscle cells were pelleted, resuspended in 1:1 DMEM:Ham's F12 (GIBCO), supplemented with 10% FBS (GIBCO), and then plated into a T25 flask. At confluence, the cells were subcultured at a seeding density of 5,000 cells/ml onto either 25-mm-diameter round glass coverslips for Ca2+ measurements or 60-mm-diameter plastic dishes for inositol polyphosphate (IPs) measurements. The cells were characterized as smooth muscle by positive immunohistochemical staining of smooth muscle-specific alpha -actin (Sigma Chemical).

Measurement of intracellular Ca2+. Intracellular Ca2+ was measured using fura-2 (Molecular Probes, Eugene, OR). After loading the ASM cells with 5 µM fura-2 AM and 0.02% pluronic acid (Molecular Probes) for 30 min, the coverslips were mounted in a Leiden chamber (Medical Systems, Greenville, NY) that was then placed on the stage of an epifluorescence-equipped inverted microscope (Nikon, Tokyo, Japan). The cells were viewed with a ×40 oil immersion objective, and fura-2 was excited at 340 and 380 nm with a PTI Deltascan 1 dual monochromator illuminator (Photon Technology International, Princeton, NJ). Fluorescence emissions were detected at 510 nm either with a PTI D104 microphotometer or imaged with a CCD-200-R videoscope camera (Videoscope International, Herndon, VA) using commercial (PTI) software. Free intracellular [Ca2+] was calculated according to the formula of Grynkiewicz et al. (14). The contribution of extracellular Ca2+ to the intracellular transient was tested by stimulating the cells in Ca2+-free Hanks' buffer containing 1 mM EDTA. To examine the role of PI-PLC, PC-PLC, and PKC in the BK-mediated Ca2+ transients, cells were pretreated with ET-18-OCH3 (Calbiochem, San Diego, CA), D609 (Calbiochem), or GF109203X (Tocris Cookson, Ballwin, MO)/Go6976 (Calbiochem)/Go6983 (Calbiochem), respectively, for 15 min before BK challenge.

SDS-polyacrylamide gel electrophoresis and Western blotting. Electrophoresis reagents were obtained from Bio-Rad (Mississauga, ON). Confluent Fischer and Lewis ASM cells were rinsed with ice-cold PBS, lysed with 1 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2, 1 mM PMSF, 20 µM leupeptin, 15 U aprotinin/ml, 1 mM sodium orthovanadate), and clarified by centrifugation. Protein concentration was determined, and equal amounts (10 µg) were resolved by 12% SDS-PAGE, transferred onto 0.22-µm pore nitrocellulose filters, and probed with monoclonal anti B2 BK receptor antibodies (BD Transduction Laboratories, Missisauga, ON). Proteins were visualized using a secondary antibody conjugated to horseradish peroxidase (Amersham) and enhanced chemiluminescence (ECL; Amersham Canada, Oakville, ON) on a FluorChem 8000 imaging system (Alpha Innotech, San Leandro, CA). Four independent samples of cells from each rat strain were analyzed by densitometry using FluorChem software.

Measurement of total myo-[3H]-IPs. The technique for measuring myo-[3H]-IPs was adapted from an earlier report (20). Confluent ASM cells were growth arrested and radiolabeled for 48 h with inositol-free DMEM containing 1% FBS and 1 µCi/ml myo-[2-3H]-inositol (Amersham Life Sciences). Cells were washed free of unincorporated myo-[3H]-inositol with Krebs-Henseleit buffer and preincubated with 10 mM LiCl at 37°C for 10 min. The cells were challenged with increasing concentrations of BK in the presence of LiCl for 10 min at 37°C, and the reactions were terminated by incubation with ice-cold 3 M trichloroacetic acid for 30 min on ice. The cells were scraped off the dishes and sonicated on ice. The IPs were extracted by treatment with 1:4 vol/vol of 10 mM EDTA, followed by 1:1 vol/vol of fresh 50% vol/vol trichlorotrifluoroethane/tri-n-octylamine (Sigma Chemical). The aqueous and organic layers were separated by centrifugation at 4,000 g for 10 min at 4°C. The upper aqueous layer was removed and neutralized with 1:5 vol/vol of 60 mM NaHCO3 before recovering the IPs with anion exchange chromatography. Samples were loaded onto columns containing 1 ml formate form AG 1-X8 resin (Bio-Rad). Myo-[2-3H]-inositol and myo-[2-3H]-glycerophosphoinositide were removed by washing with a solution of 60 mM ammonium formate (Fisher Scientific, Ottawa, ON) and 5 mM sodium tetraborate. Myo-[3H]-IPs were eluted with a solution containing 1 M ammonium formate and 100 mM formic acid.

Statistical analysis. Interstrain comparisons of airway narrowing capacities and ASM [Ca2+]i were tested using the Kolmogorov-Smirnoff test. Airway narrowing velocity and ASM cell IP accumulation were compared with ANOVA. P < 0.05 was considered significant.


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ABSTRACT
INTRODUCTION
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Airway constriction to BK. To assess whether BK stimulates Fischer airways to constrict more than Lewis airways, the dynamic constriction of intraparenchymal airways with BK exposure was compared between strains. Both Fischer and Lewis airways constricted to BK in a concentration-dependent manner, but Fischer airways were more sensitive to BK and narrowed to a greater degree than Lewis airways at [BK] > 0.01 µM (P < 0.05) (Fig. 1). Airway narrowing in both strains peaked with 0.1 µM BK. Lewis airways narrowed to a median peak constriction of 72.8% baseline area with 16-20% interquartile variability with 0.1 µM BK and to 79.5% baseline area with 13-16% interquartile variability with 1 µM BK. Fischer airways constricted to 42.2% baseline area with 12-22% interquartile variability with 0.1 µM BK and to 42.7% baseline area with 20% interquartile variability with 1 µM BK. These observations confirmed that Fischer airways narrow more than Lewis airways in response to BK ex vivo.


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Fig. 1.   Intraparenchymal airways from Fischer and Lewis rats constrict to bradykinin (BK). Cross-sectional airways in lung explants were exposed to increasing [BK]. Dynamic constriction was imaged with a CCD-200-R camera. Symbols show median reduction in baseline area, whereas dashed lines indicate 25th and 75th quartile variations in responses of all airways tested at each [BK]. Time is shown on a log scale to highlight early events. open circle , Lewis {n = 9, 18, 25, and 17 with increasing [BK]}. , Fischer {n = 13, 21, 27, and 26 with increasing [BK]}.

The narrowing velocity to 50% peak constriction (v50) was faster in Fischer than Lewis with 0.1-µM BK stimulation, but this difference disappeared with 1 µM BK (Table 1). Despite the faster v50 of Fischer airways with 0.1 µM BK, the time to peak airway narrowing was not shorter in Fischer than Lewis airways, presumably because Fischer airways narrowed more. With 1.0 µM BK, the v50 of Lewis airways increased to match the v50 of Fischer airways (Table 1), but it still took the same amount of time for Lewis airways to reach the same degree of peak constriction as with 0.1 µM BK (Table 2 and Fig. 1). Furthermore, the v50 of Fischer airways was unchanged by increasing [BK] from 0.1 to 1.0 µM (Table 1), yet it took significantly longer for Fischer airways to reach the same peak constriction (Table 2 and Fig. 1). These results indicate that v50 does not solely determine the time lapse to peak constriction and that faster v50 was not essential for differences in narrowing capacity.

                              
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Table 1.   Velocity to 50% of peak constriction


                              
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Table 2.   Time lapse to peak constriction

To determine whether the release of Ca2+ from intracellular stores in the ASM cells was sufficient to trigger the interstrain differences in airway narrowing capacity to BK, the explanted intraparenchymal airways were exposed to BK in the absence of extracellular Ca2+. Fischer and Lewis airways continued to constrict in response to 1 µM BK, but they relaxed immediately on reaching their peak responses (Fig. 2). Lewis airways constricted to 62.7% baseline area with 6-22% interquartile variability, whereas Fischer airways narrowed to 29.6% baseline area with 4-25% interquartile variability. Thus, the narrowing capacity within each strain as well as the differences in narrowing capacity between strains were conserved in the absence of extracellular Ca2+. Although extracellular Ca2+ was necessary to sustain the narrowing, these results indicated that peak narrowing capacity depended on the release of Ca2+ from intracellular stores.


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Fig. 2.   Intraparenchymal airways constrict to BK in the absence of extracellular Ca2+. Lung explants were bathed in Ca2+-free HEPES-buffered culture medium with EDTA before and during BK stimulation. Symbols show median responses, and dashed lines mark 25th and 75th quartile variations in responses of all airways to 1 µM BK. Left: Lewis (n = 11). Right: Fischer (n = 12).

In the absence of extracellular Ca2+ as well, v50 increased significantly in Fischer airways (Table 1), and this was paralleled by a significant decrease in the time lapse to peak constriction (Table 2). Lewis airways also reached their peak constriction significantly faster than in the presence of Ca2+ (Table 2), but without an increase in the v50 (Table 1). Due to the increase in Fischer v50, interstrain differences in v50 appeared with 1-µM BK stimulation (Table 1). These data show that extracellular Ca2+ modulates the rate of narrowing of intraparenchymal airways in lung explants and that this modulation differs between Fischer and Lewis airways.

BK-mediated Ca2+ mobilization in ASM. To determine whether BK stimulated greater Ca2+ mobilization in Fischer than Lewis ASM cells, primary ASM cells cultured from each rat strain were exposed to BK and intracellular Ca2+ responses were measured with fura-2. BK stimulated biphasic, concentration-dependent increases in intracellular Ca2+ in both Fischer and Lewis ASM cells (Fig. 3). Peak Fischer responses were significantly higher than Lewis at all [BK] tested (P < 0.001; Fig. 4). Baseline Ca2+ levels were significantly higher in Lewis cells (P < 0.01). These results confirmed that BK-mediated Ca2+ mobilization is higher in Fischer than Lewis ASM cells.


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Fig. 3.   Representative Ca2+ traces in Fischer and Lewis airway smooth muscle (ASM) cells in response to 1 µM BK. Solid lines, Fischer. Dotted lines, Lewis.



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Fig. 4.   BK-evoked intracellular Ca2+ transients are higher in Fischer than Lewis ASM cells. Fura-2-loaded Fischer and Lewis ASM cells were stimulated with various [BK] for 1'. Responses shown are peak Ca2+ levels of a biphasic transient. Ca2+ was measured by spectrofluorimetric imaging. open circle , Lewis {n = 110, 93, and 100 with increasing [BK]}. , Fischer {n = 90, 112, and 88 with increasing [BK]}. Inset: baseline [Ca2+]i. Lewis, open bar; Fischer, filled bar.

To assess the contribution of extracellular Ca2+ to the interstrain differences in the BK-mediated Ca2+ transient, ASM cells were stimulated with BK in the absence of extracellular Ca2+. Ca2+ responses to 1 µM BK decreased in both rat strains (P < 0.001 in both strains; Fig. 5). More than 90% of Lewis cells had Ca2+ increases <20 nM. Peak Ca2+ levels were also significantly reduced in Fischer cells, and fewer cells responded. Baseline Ca2+ levels in Lewis cells also fell below Fischer levels (P < 0.001). Fischer baseline Ca2+ was the same as when extracellular Ca2+ was present. Significant interstrain differences in baseline and BK-stimulated Ca2+ levels were conserved (P < 0.001). Although the magnitude of Ca2+ transients in both rat strains was highly dependent on extracellular Ca2+, the conservation of interstrain differences suggested that the mechanisms causing these interstrain differences were independent of extracellular Ca2+ levels.


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Fig. 5.   BK-evoked intracellular Ca2+ transients are attenuated in the absence of extracellular Ca2+ in Fischer and Lewis ASM cells. Cells were placed in Ca2+-free Hanks' buffer immediately before BK stimulation for 1'. Responses shown are peak Ca2+ levels of a biphasic transient. open circle , Lewis (n = 198). , Fischer (n = 153). Inset: baseline [Ca2+]i. Lewis, open bar; Fischer, filled bar.

B2 receptor expression. B2 receptor expression was compared between Fischer and Lewis ASM cells by Western blot analysis (Fig. 6). Figure 6A shows a representative blot with two samples from each rat strain. No difference in B2 receptor levels was seen between the strains (Fig. 6B), indicating that the interstrain Ca2+ differences were unrelated to B2 receptor expression.


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Fig. 6.   B2 receptor expression is similar in Fischer and Lewis ASM cells. Cells were lysed, and 10 µg of protein were loaded into each sample lane. Blots were probed with monoclonal anti-B2 receptor antibody. A: representative Western blot. B: densitometry of band intensities averaged for n = 4 from each rat strain.

Effects of PI-PLC or PC-PLC inhibition on BK-induced Ca2+ transients. To establish whether BK activated PI-PLC and PC-PLC to mobilize Ca2+ in Fischer and Lewis ASM cells, these enzymes were inhibited with specific pharmacological antagonists during BK stimulation. Inhibition of PI-PLC with 10 µM ET-18-OCH3 attenuated intracellular Ca2+ responses to 1 µM BK in Fischer (P < 0.001) and Lewis (P = 0.05) ASM cells (Fig. 7, left). Inhibition of PC-PLC with 1 µM D609 also attenuated BK mobilization of Ca2+ in Fischer and Lewis cells (P < 0.001; Fig. 7, right). Baseline Ca2+ levels significantly increased in both strains of rat with all drug pretreatments, but only 10 µM D609 caused substantial deviations from the normal values (Table 3). Both PI-PLC and PC-PLC modulated basal and BK-mediated intracellular Ca2+ levels in Fischer and Lewis ASM cells.


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Fig. 7.   Phospholipase C (PLC) inhibition attenuates Ca2+ mobilization by BK in Fischer and Lewis ASM cells. The effects of ET-18-OCH3, a selective phosphatidylinositide-specific PLC inhibitor, or D609, a selective phosphatidylcholine-specific PLC inhibitor, on Ca2+ responses in Fischer and Lewis ASM cells were tested. Cells were exposed to each drug for 15' before and during 1-µM BK stimulation for 1'. Responses shown are peak Ca2+ levels of a biphasic transient. Left: ET-18-OCH, top: Lewis (DMSO, n = 128; ET-18-OCH3, n = 155), bottom: Fischer (DMSO, n = 120; ET-18-OCH3, n = 140). Right: D609, top: Lewis (DMSO, n = 173; D609, n = 197), bottom: Fischer (DMSO, n = 137; D609, n = 180).


                              
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Table 3.   Effect of PLC inhibitor pretreatment on baseline [Ca2+]i

IP accumulation to BK. To test whether possible differences in BK-mediated PI-PLC activity between the rat strains might account for the interstrain differences in Ca2+, IP accumulation was measured in BK-stimulated Fischer and Lewis ASM cells. BK >0.1 µM increased IPs significantly above baseline in each rat strain (P < 0.05; Fig. 8). However, IP production was similar between the two strains of rat, excluding a direct role for PI-PLC in giving rise to the interstrain Ca2+ differences to BK.


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Fig. 8.   BK stimulates similar modest increases in inositol polyphosphates (IPs) in Fischer and Lewis ASM cells. Cells were pretreated with 10 mM LiCl for 10 min before stimulation with BK for 10 min. IPs were extracted and assayed by column chromatography. open circle , Lewis (n = 6). , Fischer (n = 6). *P < 0.05 BK treated vs. baseline within each rat strain.

Effect of PKC inhibition on BK-induced Ca2+ transients. PI-PLC and PC-PLC both produce 1,2-DAG that can then activate PKC. To investigate the involvement of PKC in the interstrain differences in BK-mediated Ca2+ transients, ASM cells were treated with three different PKC antagonists during BK stimulation. GF109203X (10 µM) had opposite effects in Fischer and Lewis ASM cells. It decreased BK-mediated Ca2+ transients in Fischer ASM cells, whereas it increased the transients in Lewis ASM cells (P < 0.001; Fig. 9, a and b). Go6976 and Go6983 (10 µM) both inhibited Ca2+ responses in Fischer (P = 0.0005 for ±Go6976, P = 0.01 for ±Go6983) and had no effect in Lewis ASM cells (Fig. 9, c-f). These data indicate that PKC regulates Ca2+ mobilization differently in Fischer and Lewis ASM cells.


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Fig. 9.   Ca2+ responses decrease with PKC inhibition in Fischer but not Lewis ASM cells. Cells were treated with DMSO vehicle (open circle ) or inhibitor (black-down-triangle ) for 15' before and during 1-µM BK stimulation. a: Lewis control n = 149, GF109203X n = 157; P < 0.001. b: Fischer control n = 142, GF109203X n = 180; P < 0.001. c: Lewis control n = 29, Go6976 n = 20; P > 0.05. d: Fischer control n = 40, Go6976 n = 20; P = 0.0005. e: Lewis control n = 29, Go6983 n = 10; P > 0.05. f: Fischer control n = 40, Go6983 n = 20; P = 0.01.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present data demonstrate that intraparenchymal Fischer airways are more sensitive and narrow more to BK compared with Lewis airways ex vivo. The interstrain difference in narrowing capacity persisted when the airways were constricted in Ca2+-free buffer, suggesting that differences in narrowing capacity were determined by intrinisic properties of the ASM. We hypothesized that BK mobilized intracellular Ca2+ differently in Fischer and Lewis ASM. Our data show that in both rat strains, BK activated PI-PLC and PC-PLC to mobilize Ca2+. BK-evoked intracellular Ca2+ transients were higher in Fischer than Lewis ASM cells. Inhibiting PKC attenuated Ca2+ transients in Fischer cells, whereas it enhanced or had no effect on transients in Lewis, depending on the inhibitor. These results strengthen the association between AHR and enhanced ASM intracellular Ca2+ mobilization in the Fischer and Lewis model of genetic AHR and suggest that differences in PKC regulation contribute to the interstrain differences in intracellular Ca2+ mobilization by BK.

It is possible that the BK-dependent Ca2+ differences were determined at the receptor level, for example, by variations in receptor/isoform expression. In ASM cells, pharmacological and Scatchard analyses showed only a single B2 receptor binding site (2, 12). Western blot analysis did not show any differences in B2 receptor expression between the two rat strains, indicating that the Ca2+ differences between Fischer and Lewis arose downstream from the receptor.

We previously reported that the airways of Fischer rats are hyperresponsive to 5-HT ex vivo compared with Lewis and that the enhanced airway narrowing was paralleled by enhanced Ca2+ mobilization in Fischer ASM cells (29). The enhanced Ca2+ mobilization to 5-HT in Fischer ASM cells was subsequently attributed to higher Ins(1,4,5)P3 transients caused by lower levels and activities of type I and type II Ins(1,4,5)P3 5-phosphatases (4). Although the discrepancies in the Ins(1,4,5)P3 5-phosphatases must have been also present during BK stimulation, the modest production of IPs with BK activation suggested a minor contribution of Ins(1,4,5)P3-associated pathways in regulating Ca2+ mobilization to BK. Because BK stimulated PC-PLC as well as PI-PLC to mobilize Ca2+, additional regulatory mechanisms may have contributed more to Ca2+ mobilization by this agonist.

In lung explants, Fischer airways narrowed to a greater degree and at a faster rate than Lewis airways in response to BK. The lung explant model is advantageous because it enables sampling of intraparenchymal airways where airflow is limited during bronchoconstriction in vivo (17). The airways also retain many elements of their in vivo milieu including lung parenchyma, which is an important mechanical impedance to airway narrowing, and diverse tissue types that physically and biochemically interact to control airway caliber. However, the complexity of lung explants limits the ability to pinpoint the contribution of each tissue type to the response. To the extent that ASM is the principal effector of airway narrowing, it is likely that the ASM in the explants contributed significantly to the observed outcomes.

In the absence of buffer Ca2+, the interstrain differences in narrowing capacity and v50 persisted, and the time to peak constriction markedly accelerated in both rat strains. These observations indicated that interstrain differences in narrowing capacity and v50 were independent of extracellular Ca2+ but that the rate of narrowing in each rat strain was a function of extracellular Ca2+. The first conclusion suggested that something intrinsic to the tissue gave rise to interstrain narrowing differences. The second conclusion suggested that airway narrowing velocity could be modulated extrinsically. Investigation into mechanisms of extracellular Ca2+-dependent airway narrowing velocity was beyond the scope of our interstrain comparisons but could potentially involve a number of different Ca2+-dependent functions of the various tissues, notably epithelium, in the lung explant.

The greater Ca2+ responses to BK in Fischer than Lewis ASM cells reflect disparities in the population of responsive cells between the two rat strains in vitro. The majority of cells in the Lewis population were poorly responsive or unresponsive to BK, whereas the majority of Fischer cells responded to BK with a Ca2+ transient. This discrepancy in the frequency distribution of responsive cells between the two rat strains was also observed in response to 5-HT (29). Although it is unknown whether the same cells in the population are highly responsive to both agonists or whether different cells have different sensitivities to the two agonists, the overall responsiveness of ASM tissue appears to be determined by the frequency distribution of responsive cells within it. This is assuming that the distribution of responsive cells in culture at confluence reflects the distribution at initial seeding and that responsive and unresponsive cells divide at similar rates. Fischer ASM cells proliferate faster than Lewis ASM cells in response to mitogenic stimuli (37). This might result in greater representation of responsive cells in the Fischer population at confluence in vitro than actually exists in the tissue in situ. Alternatively, associated mechanisms might be driving the same Fischer cells to be Ca2+ hyperresponsive and proliferate faster so the in vitro distribution would accurately reflect the in situ distribution.

Our findings suggest that the alterations in ASM intracellular Ca2+ homeostasis might be associated with the pathophysiology of genetic AHR. Altered Ca2+ homeostasis also seems to play a role in the development of allergic AHR as preexposure to the proinflammatory cytokines TNF-alpha or IL-1beta potentiated contractile agonist-mediated Ca2+ mobilization in ASM cells (16). Other investigators proposed that Ca2+ sensitization of the contractile apparatus, and not the absolute levels of cytosolic Ca2+ per se, is associated with allergic AHR (1). These mechanisms may complement each other because the specific spatial and temporal patterns of the Ca2+ transient dictate different physiological outcomes within one and among different cell type(s) (7, 8). Given the complexity of Ca2+ signaling, it is possible that multiple steps contribute to hyperreactivity. Our observations in innately hyperreactive ASM, in conjunction with observations in sensitized ASM, support a role for increased intracellular Ca2+ mobilization as one possible mechanism contributing to AHR. It would be interesting in future studies to compare the Ca2+ sensitivities of Fischer and Lewis ASM.

When the ASM cells were stimulated with BK in the absence of extracellular Ca2+, significantly fewer cells from both strains showed intracellular Ca2+ transients. Of the responding cells, the transients were also lower than in the presence of extracellular Ca2+. Thus, a portion of the intracellular Ca2+ transient of ASM cells depended on extracellular Ca2+. This finding is consistent with reports showing that PI-PLC and PC-PLC activities require extracellular Ca2+ (3, 24, 28, 34, 36). This was inconsistent with our observations of airway narrowing capacity in explants, which was unaffected by extracellular Ca2+ depletion. The discrepant observations from the two experimental systems might have arisen from a limitation of Ca2+-free buffer to sequester extracellular Ca2+ from the smooth muscle in the lung explant due to the latter's size and tissue complexity. It is possible that the buffer did not penetrate sufficiently into the explant to fully deplete Ca2+ surrounding the ASM, so some extracellular Ca2+ was available to activate the PLCs and replenish Ca2+ leaking from intracellular stores. However, the Ca2+-free buffer markedly accelerated v50 in both Fischer and Lewis explanted airways, indicating that this treatment did affect contractile parameters in the preparation. Another possibility is that other mechanisms downstream of intracellular Ca2+ mobilization, such as different Ca2+ sensitivities of the contractile apparatus, might have conserved the differences in airway narrowing capacity in the lung explants despite lower intracellular Ca2+ transients in the ASM. Unfortunately, we have limited data to conclusively resolve the difficult issue of extrapolating from a single cell preparation to multitissue preparations. To the extent that interstrain differences were conserved in explants and ASM cells under conditions of nominal extracellular Ca2+, these differences appear to be linked with intrinsic differences in ASM.

We explored some of the possible mechanisms responsible for the differences in BK-mediated intracellular Ca2+ mobilization in Fischer and Lewis ASM cells. The data suggest that differences in PKC regulation might account for the interstrain differences in intracellular Ca2+ mobilization by BK because inhibiting PKC with GF109203X (inhibits alpha , beta 1, beta 2, gamma , delta , and epsilon  isoforms), Go6976 (inhibits alpha  > beta  > µ), or Go6983 (inhibits alpha , beta , gamma , delta , zeta ) attenuated responses in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 had no effect on Ca2+ responses in Lewis cells. Thus, in Fischer cells, PKC potentiates Ca2+ transients to BK. In Lewis cells, PKC either attenuates or does not regulate Ca2+ transients to BK; whichever occurs appears to depend on the PKC isoform. More extensive studies will be needed to elucidate the role of different PKC isoforms in regulating the differences in intracellular Ca2+ signaling between Fischer and Lewis ASM cells. A possible difference in PKC regulation of Ca2+ is intriguing because PKC has been shown to differentially regulate growth between Fischer and Lewis ASM cells (38). In those studies, PKCbeta 1 expression was higher while PKCdelta was lower in response to FBS stimulation in Fischer than in Lewis ASM cells. Examining whether the interstrain Ca2+ differences are also due to variation in PKC isoform activity and whether the same isoforms are involved in the differences in growth and contractile signaling could provide a possible link between the increased growth and increased contractility seen in Fischer ASM.

We demonstrated that Fischer airways are hyperresponsive to BK ex vivo and that cultured Fischer ASM cells mobilize more Ca2+ in response to BK. Differences in PKC regulation appear to give rise to the differences in BK-mediated intracellular Ca2+ mobilization between Fischer and Lewis ASM cells. An important limitation of our studies is that the mechanical and biochemical readouts were measured in different model systems, so extrapolating between them is inexact. The lung explants contain multiple tissue types that interact to produce the functional outcome so the behavior cannot be entirely attributable to the ASM. Insofar as the in vitro Ca2+ responses and ex vivo airway narrowing in Fischer were consistently elevated relative to Lewis, we propose that a relationship exists between them and that enhanced ASM cell Ca2+ mobilization may be a convergent mechanism underlying nonspecific, genetic AHR.


    ACKNOWLEDGEMENTS

The authors thank Dr. C. G. Wang for preparation of lung explants and L. Milne for formatting the manuscript.


    FOOTNOTES

This work was supported by CIHR Grant MOP-36334.

Address for reprint requests and other correspondence: J. G. Martin, Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2 (E-Mail: James.Martin{at}McGill.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 6, 2002;10.1152/ajplung.00023.2002

Received 18 January 2002; accepted in final form 4 September 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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