Functional effects of protease-activated receptor-2 stimulation on human airway smooth muscle

Linda S. Chambers1, Judith L. Black1, Philip Poronnik2, and Peter R. A. Johnson1

1 Department of Pharmacology and 2 Department of Physiology and Kolling Institute, University of Sydney, New South Wales 2006, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The protease-activated receptor (PAR)-2 is present on the smooth muscle and epithelium of human airways and can be activated by mast cell tryptase, trypsin, or the PAR-2 activating peptide (AP). Trypsin and the PAR-2 AP induced contractions in human isolated airways, and these contractions were potentiated in the presence of the cyclooxygenase (COX) inhibitor indomethacin. Trypsin also increased the contractions to histamine in airways from sensitized (allergic) patients but not from nonsensitized (nonallergic) patients. Tryptase purified from human lung, skin and lung recombinant beta -tryptases, trypsin, and the PAR-2 AP all increased DNA synthesis in human airway smooth muscle (HASM) cells. Activation of PAR-2 by tryptase, trypsin, and the PAR-2 AP did not induce PGE2 release from HASM cells. Trypsin and the PAR-2 AP increased the levels of intracellular calcium in HASM cells, with desensitization evident after treatment with either agonist. In conclusion, activation of PAR-2 can induce contractions of human airways, potentiate contractions to histamine, and induce proliferation and therefore may contribute to airway diseases such as asthma.

tryptase; proliferation; contraction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROTEASE-ACTIVATED RECEPTORS (PARs) are a family of G protein-coupled, seven-transmembrane-domain receptors that are activated by proteolysis (reviewed in Refs. 16, 17). These receptors are cleaved within the extracellular amino-terminal domain, forming a new amino terminus that functions as a tethered ligand that binds to and activates the receptor. Four PARs have been identified to date. PAR-1 and PAR-3 are activated by thrombin (23, 41), PAR-2 requires cleavage by trypsin (31) or mast cell tryptase (30), and PAR-4 is activated by thrombin and trypsin (43). Synthetic peptides corresponding to the tethered ligand sequences of PAR-1, PAR-2, and PAR-4 can also be used to activate their respective receptors.

PAR-2 has been identified on the bronchial epithelium and smooth muscle cells in human airways (14). Studies in animal models have produced conflicting results as to the functional outcome of PAR-2 activation in the airways. Cocks et al. (11) reported that activation of PAR-2 with trypsin or the mouse PAR-2 activating peptide (AP; SLIGRL-NH2) induces relaxation of mouse precontracted bronchial rings. These relaxations were cyclooxygenase (COX) and epithelium dependent, and hence it was hypothesized that activation of PAR-2 on the airway epithelium induces release of epithelial PGE2. In a separate study, Lan et al. (27) found similar results in mouse airways, but the relaxations were not altered by removal of the epithelium. In guinea pigs, activation of PAR-2 induced bronchoconstriction in vivo and either contraction or relaxation in vitro (33), whereas in sensitized sheep, activation of PAR-2 appears to induce bronchoconstriction (1).

The role of PAR-2 in human airways has yet to be determined; however, one of the activators of PAR-2, mast cell tryptase, may be an important mediator in asthma. Elevated levels of tryptase have been detected in the sputum (28) and bronchoalveolar lavage fluid (42) from asthmatic patients. Johnson et al. (24) have previously demonstrated that tryptase increases contractions to histamine in sensitized (allergic) but not in nonsensitized (nonallergic) human isolated airways. Tryptase is also a potent stimulator of proliferation in dog tracheal smooth muscle cells in culture (9). Whether the effects of tryptase in human airways in vitro are mediated via PAR-2 has yet to be investigated.

In this study, we examined the effects of PAR-2 activation with tryptase (tryptase purified from human lung and skin and lung recombinant beta -tryptases), trypsin, and PAR-2 AP on two key properties of human airways, namely changes in the tone of bronchial segments and proliferation of human airway smooth muscle (HASM) and the role of COX in these responses.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sensitization test. Human lung tissue was obtained from patients undergoing lung transplantation, lobectomy, or pneumonectomy. Approval for all experiments using human lungs was provided by the Human Ethics Committee of the University of Sydney (New South Wales, Australia) and the Central Sydney Area Health Service. Sensitization status was determined for every patient from whom lung tissue was obtained. Experiments were usually performed ~4-18 h after excision of the lung tissue from the patient. Bronchial rings measuring 1-4 mm in internal diameter and 5 mm in length were dissected free from the surrounding parenchyma and were suspended in 5-ml organ baths containing Krebs-Henseleit solution (118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2 · 2H2O, 1.2 mM MgSO4 · 7H2O, 1.2 mM KH2PO4, 25.5 mM NaHCO3, and 11.1 mM D-glucose), maintained at 37°C, and aerated with 5% CO2 in O2 (4, 24). Each tissue was equilibrated against a load of 0.5-2 g as determined by tissue size (5). Tissues were washed a minimum of five times at 15-min intervals until a stable baseline tone was established. Changes in tension were measured isometrically with Grass FT03 force transducers and recorded with an analog-to-digital recording device (MacLab).

A volume of 10 µl of a standard skin prick test solution of Dermatophagoides pteronyssinus [30,000 allergen units (AU)/ml], Timothy Phleum pratense (1:20 wt/vol), Alternaria tenuis (1:10 wt/vol), and cat pelt (10,000 bioequivalent AU/ml) allergens was added to the bronchial rings. These particular allergens were chosen because >97% of the atopic population of Australia exhibits a positive skin prick test to their application (32). Tissues from patients with cystic fibrosis were also tested with Aspergillus mix (1:10 wt/vol; A. fumigatus, A. nidulans, A. niger, and A. terreus) because many of these patients are allergic to Aspergillus. Patients were classified as sensitized if their tissues contracted in response to one or more of these five allergens. If the tissues from a patient did not contract after the addition of these allergens but did contract subsequently in response to a maximal concentration of acetylcholine (ACh; 1 mM), the patient was classified as nonsensitized.

Contractile responses to PAR-2 activation. Bronchial rings from 23 patients [age 49 ± 18 (SD) yr] were set up in organ baths as for the sensitization test. Once the tissues had been washed and a stable baseline was achieved, an initial response to a maximal concentration (1 mM) of ACh was elicited to ensure that the tissue was responsive and to allow the response to trypsin, the PAR-2 AP, or histamine to be expressed as a percentage of this ACh response. After the contraction had plateaued, the tissues were washed repeatedly a minimum of four times until baseline tone was reestablished. Increasing concentrations of trypsin (10-9 to 10-3 g/ml) were then added. A bolus concentration of the PAR-2 AP (100 µM) was also tested in separate experiments. To examine the effect of inhibiting COX, tissues were incubated with indomethacin (2.5 µM) for 20 min before the addition of trypsin or the PAR-2 AP.

To investigate the effect of trypsin on the responses to histamine, the tissues were incubated with 1, 10, or 100 µg/ml of trypsin for 15 min. Increasing concentrations of histamine were then added (1 nM to 3 mM) to these tissues, and the responses were compared with untreated control tissues. Treatments tested in these studies were carried out on duplicate tissues.

Airway smooth muscle cell isolation. Airway smooth muscle cell cultures were established from 30 patients [age 48 ± 18 (SD) yr] from airways separate from those used for sensitization testing as previously described (22). Smooth muscle bundles were dissected free from the surrounding tissue and placed as explants in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20 U/ml of penicillin, 20 µg/ml of streptomycin, 2.5 µg/ml of amphotericin B, and 10% fetal bovine serum (FBS) in a humidified incubator (5% CO2 in air) at 37°C.

The smooth muscle cells were grown to confluence and then passaged at 7- to 10-day intervals with Mg2+/Ca2+-free Hanks' balanced salt solution and 0.05% trypsin in 1 mM EDTA disodium salt (25). The smooth muscle cells were identified by examination of their morphology under a light microscope and by positive staining with a specific smooth muscle alpha -actin immunofluorescent antibody (25) and a calponin antibody (18).

Assay for tryptase activity. The activities of lung-purified and recombinant tryptase were determined with the synthetic peptide substrate N-p-tosyl-Gly-Pro-Lys p-nitroanilide (24). Two microliters of tryptase were added to 1 ml of the substrate solution [0.05 M Tris · HCl (pH 7.6), 0.12 M NaCl, 20 µg/ml of heparin, and 0.1 mM N-p-tosyl-Gly-Pro-Lys p-nitroanilide] at 37°C. The rate of change of absorbance at 405 nm was recorded with a spectrophotometer. From this rate of change, the activity of tryptase was determined as previously described (24).

Proliferative responses to PAR-2 activation. The cells were seeded in 96-well plates at a density of 104 cells/cm2 in DMEM supplemented with 10% FBS. After growth to confluence for 7-8 days in 10% FBS, the medium was changed to 1% FBS for 24 h to equilibrate the cells (22). The various conditions to be tested were then added to quadruplicate wells: 10-11 to 10-8 M tryptase, 0.05-50 U/ml of trypsin, and 10-8 to 10-3 M PAR-2 AP (SLIGKV). The cells were also incubated with 2.5 µM indomethacin for at least 30 min before solutions of tryptase, trypsin, or the PAR-2 AP were added. Lung-purified, skin, and lung recombinant beta -tryptases were prepared as 10-7 M aliquots in a buffer solution of 10 mM bis-Tris, pH 6.1, with 0.5 M NaCl and 60 µg/ml of heparin. These three types were tested to obtain maximal information regarding the actions of tryptase (3, 29). The controls for tryptase consisted of equivalent amounts of this heparin and buffer solution. All solutions were made up in DMEM supplemented with 20 U/ml of penicillin, 20 µg/ml of streptomycin, 2.5 µg/ml of amphotericin B, and 1% FBS. Both 5 and 10% FBS in DMEM were included as positive controls for proliferation (25). The cells were incubated in the various conditions for a total of 24 h.

The degree of proliferation was assessed by measuring [3H]thymidine incorporation. After incubation for 19 h at the various conditions to be tested, 1 µCi in 10 µl of DMEM was added to 100 µl of the test solution in all wells, and the wells were incubated for a further 5 h at 37°C (25). Cell proliferation was then arrested by freezing at -20°C. The cells were later harvested onto Canberra Packard GFC filters and dried. Microscintillant was then added to the filters, and the counts per minute were obtained on a beta-emission top counter (Packard) for 1 min/sample.

Direct cell counts. HASM cells were seeded in 12-well plates at 104 cells/cm2. After growth to confluence for 7 days in 10% FBS in DMEM, the cells were equilibrated in 1% FBS for 24 h. The cells were then incubated with 1 nM lung-purified tryptase, 1 nM skin recombinant beta -tryptase, or an equivalent amount of vehicle in 1% FBS for 2 or 4 days. The cells were also incubated with 1, 5, or 10% FBS for the same amount of time. Each treatment was added to duplicate wells, and after 2 or 4 days, the number of cells was counted manually after removal from the wells with 0.05% trypsin in 1 mM EDTA.

PGE2 assay. HASM cells were seeded in 24-well plates at a density of 104 cells/cm2 in DMEM supplemented with 10% FBS. After 7 days, the confluent cells were equilibrated for 24 h in 1% FBS and then treated with trypsin (50 U/ml), lung-purified tryptase (5 nM), the PAR-2 AP (1 mM), or bradykinin (10 µM) in serum-free medium for 30 min. These treatments were added to duplicate wells. The supernatant was then collected and stored at -80°C until the levels of PGE2 were assayed with an ELISA.

Measurement of intracellular calcium. HASM cells were grown on coverslips in petri dishes with DMEM supplemented with 10% FBS for 3-7 days. The cells were washed with a physiological saline solution (PSS) and then loaded with 4 µM fura 2-AM in PSS with 3 mg/ml of bovine serum albumin for 30 min at 37°C. The cells were then washed again in PSS and placed in a chamber mounted on an Olympus inverted microscope.

Excitation wavelengths were alternated between 340 nm and 380 nm with a Lambda xenon light source (Shutter Instrument) and a fura 2 dichroic mirror. The fluorescence emitted at 510 nm was recorded every 5 s with a charge-coupled device camera and camera controller (Hamamatsu). The ratio of fluorescence at 340 nm to that at 380 nm was determined in individual cells that were selected with Metafluor software (Universal Imaging).

The compounds added to the cells (lung-purified tryptase, lung recombinant beta -tryptase, trypsin, PAR-2 AP, and bradykinin) were prepared in PSS, warmed to 37°C, and then perfused into the chamber. For desensitization experiments, the solutions were perfused through sequentially, without any separate washes between treatments. The changes in the fluorescence ratio resulting from the various treatments are expressed as a percentage of the response to the positive control, bradykinin (5 µM).

Materials. The following compounds were obtained from the sources given: NaHCO3, glucose, NaCl, and MgSO4 · 7H2O from ICN Biochemicals (Aurora, OH); KH2PO4 from British Drug House Laboratory Chemical Group; EDTA disodium salt, CaCl2 · 2H2O and KCl from Ajax Chemicals; acetylcholine perchlorate, histamine, indomethacin, fluorescein isothiocyanate-conjugated monoclonal anti-alpha -smooth muscle actin antibody (mouse), monoclonal anti-calponin antibody (mouse), fluorescein isothiocyanate-conjugated polyclonal anti-mouse antibody, porcine trypsin (1,120 U/mg), bradykinin, N-p-tosyl-Gly-Pro-Lys p-nitroanilide, Tris · HCl, bis-Tris, and bovine lung heparin sodium salt from Sigma (St. Louis, MO); tissue culture flasks and 96-well plates from Becton Dickinson; Hanks' balanced salt solution, DMEM, penicillin, streptomycin, and amphotericin B from GIBCO BRL (Life Technologies); FBS from CSL Biosciences; tryptase purified from human lung (activity of assay substrate 4.7 µg/ml) from Cortex Biochem (San Leandro, CA); recombinant beta -tryptases derived from skin and lung (activity of assay substrate 8 and 2.3 µg/ml, respectively) from Promega; allergen extracts of D. pteronyssinus standardized mite DP (30,000 AU/ml), Timothy P. pratense (1:20 wt/vol), A. tenuis (1:10 wt/vol), cat pelt (10,000 bioequivalent AU/ml), and Aspergillus mixture (1:10 wt/vol) from Bayer Diagnostic; PAR-2 AP (SLIGKV) from Auspep; [3H]thymidine from Gene Search; Microscint-20 and GFC filters from Canberra Packard; PGE2 enzyme immunoassay kit from Cayman Chemical; and fura 2-AM from Molecular Probes (Eugene, OR).

Analysis of results. Contractile results are expressed as a percentage of the response to 1 mM ACh, and a mean value was obtained from duplicates for each treatment. An overall mean curve was calculated for all patients. Mean curves for various treatments or different patient groups were compared with a factorial ANOVA (24). Responses at individual concentrations were compared with an unpaired t-test. Proliferation results were obtained as counts per minute, and a mean value was obtained from the quadruplicate results for each treatment. PGE2 release is expressed as a percentage of the control release, and means values were obtained from duplicate results. Changes in intracellular calcium were assessed by changes in the fluorescence ratio for the various treatments and are expressed as a percentage of the change in the ratio in response to bradykinin for each individual cell tested. Overall means ± SE were calculated from all patients. Repeated-measures ANOVA with Fisher's protected least significant difference or unpaired t-tests were used to compare responses (25). For all tests, differences were considered significant at P <=  0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Contractile responses to PAR-2 activation. Contractile responses to trypsin varied between patients and sometimes between tissues from the same patient. Trypsin induced small contractions with no relaxations in 12 tissues from 7 of the 9 patients tested (Fig. 1A). The contractions at 10-3 g/ml were slow, taking up to 15-30 min to reach a maximum. In four tissues from four patients, contractions were observed to low concentrations and small relaxations in response to higher concentrations of trypsin. In the presence of indomethacin, there was an increase in the contractile responses to trypsin. There was a significant potentiation of the mean curves in the presence of indomethacin (P < 0.05; n = 8 patients including 1 who was asthmatic; Fig. 1B). Tissues from the asthmatic patient (current medication was salbutamol) also contracted in response to trypsin in the absence and presence of indomethacin.


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Fig. 1.   Effect of increasing concentrations of trypsin on human isolated airways in vitro. A: responses in tissues that contracted (excluding tissues that relaxed in response to trypsin). Values are means ± SE for 12 tissues from 7 patients. B: responses to trypsin in the presence (8 patients; ) and absence (9 patients; ) of indomethacin (2.5 µM; including tissues that relaxed to trypsin). Values are means ± SE. * Significant difference between the 2 curves, P < 0.05.

A bolus dose of the PAR-2 AP (100 µM) induced contractions in five of eight tissues tested from four patients, although the contractions were very small. The mean response in the tissues that contracted was 6 ± 2% of the response to ACh (n = 4 patients). However, there was an increase in the mean response to the PAR-2 AP in the presence of indomethacin when all tissues were compared (P < 0.05; n = 4 patients). In the presence of indomethacin, contractions were recorded in seven of eight tissues tested from four patients, with a mean response of 10 ± 2%.

In sensitized tissues, incubation with 1 µg/ml of trypsin significantly increased the contractions to histamine as shown in Fig. 2A (P < 0.05; n = 4 patients). Although there was a significant increase in the maximal response to histamine (72 ± 14 and 93 ± 18% in the absence and presence, respectively, of trypsin; P < 0.05; n = 4 patients), the pD2 values were not significantly different between the two groups of tissues (P > 0.05; n = 4 patients). There was no difference in the responses to histamine in the presence or absence of trypsin in nonsensitized tissues (n = 5 patients; Fig. 2B). Higher concentrations of trypsin (10 and 100 µg/ml; n = 3 and 4 patients, respectively) did not significantly potentiate the contractions to histamine (P > 0.05; data not shown).


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Fig. 2.   Effect of 1 µg/ml of trypsin on increasing concentrations of histamine in sensitized (4 patients; A) and nonsensitized (5 patients; B) human isolated airways in vitro. , Absence of trypsin; , presence of trypsin. Values are means ± SE. * Significant difference between the 2 curves, P < 0.05.

Identification of smooth muscle cells. On examination under a light microscope, the cells displayed typical characteristics of airway smooth muscle cells. The cells were long and thin, with central oval nuclei, and were displayed in the typical "hill-and-valley formation" (40). Immunohistochemistry confirmed the identity of the smooth muscle cells with uniform staining for smooth muscle alpha -actin and calponin (26).

Proliferative responses to PAR-2 activation. Incubation of HASM cells with lung-purified tryptase induced an increase in [3H]thymidine incorporation that was concentration related (Fig. 3). There were significant increases induced by 1, 5, and 10 nM tryptase, with a mean increase of 229 ± 45% of the control response evident at 1 nM. The lung recombinant beta -tryptase also induced significant increases in [3H]thymidine incorporation at 1, 5, and 10 nM. Significant increases in thymidine incorporation were induced by skin recombinant beta -tryptase at all concentrations tested.


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Fig. 3.   Effect of increasing concentrations of lung-purified tryptase (8 patients; solid bars), lung recombinant beta -tryptase (5 patients; hatched bars), and skin recombinant beta -tryptase (10 patients; open bars) on human airway smooth muscle cells in culture as measured by [3H]thymidine incorporation. Values are means ± SE. * Significant difference from response to control, P < 0.05.

These increases in DNA synthesis corresponded to increases in cell number as confirmed by direct cell counts. Lung-purified tryptase increased the cell number to 116 ± 3% of the control value after a 2-day incubation (n = 8 patients) and to 135 ± 10% of the control value after 4 days (n = 9 patients; Fig. 4). Skin recombinant beta -tryptase also increased the cell numbers after 2 and 4 days but was not as efficacious (Fig. 4).


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Fig. 4.   Effect of lung-purified tryptase (1 nM), skin recombinant (rec) beta -tryptase (1 nM), and 5 and 10% fetal bovine serum (FBS) on proliferation of human airway smooth muscle cells (from 9 patients) in culture as measured by direct cell counts. Cells were incubated with treatments for 2 (open bars) or 4 (solid bars) days. Values are means ± SE. * Significant increase compared with response to control, P < 0.05.

In the presence of indomethacin, there was a trend toward a decrease in DNA synthesis in response to lung-purified and skin recombinant beta -tryptases (Fig. 5); however, there was a significant difference only at 0.01 nM skin recombinant tryptase. The effect of indomethacin on the responses to lung recombinant tryptase was not investigated.


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Fig. 5.   Effect of increasing concentrations of lung-purified tryptase (4 patients; A) and skin recombinant beta -tryptase (4 patients; B) on human airway smooth muscle cells in culture in the absence (solid bars) and presence (open bars) of indomethacin (2.5 µM) as measured by [3H]thymidine incorporation. Values are means ± SE. * Significant difference from response in absence of indomethacin, P < 0.05.

Trypsin produced inconsistent changes in proliferation between patients at the various concentrations tested. However, trypsin at 5 U/ml did produce increases in [3H]thymidine incorporation in six of the eight patients tested. The mean response in the six patients was 121 ± 5% of the control response (P < 0.05). At the lower concentrations of 5 × 10-4 and 0.5 U/ml, trypsin significantly inhibited proliferation to 65 ± 4 and 74 ± 6%, respectively, of the control responses (P < 0.05; n = 8 patients). The addition of indomethacin to trypsin produced a significant increase in DNA synthesis to 142 ± 11 and 157 ± 16% of the responses to 5 × 10-4 and 0.5 U/ml of trypsin, respectively, in the absence of indomethacin (P < 0.05; n = 8 patients).

The PAR-2 AP induced significant increases in [3H]thymidine incorporation to 149 ± 33% (P < 0.05; n = 9 patients) at 100 µM as shown in Fig. 6A. At the lower concentrations tested, the responses were inconsistent, and there was a significant inhibition of DNA synthesis at 1 µM. There was no significant difference in responses to the peptide when tested in the presence and absence of indomethacin (Fig. 6B).


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Fig. 6.   Effect of increasing concentrations of protease-activated receptor (PAR)-2 activating peptide (AP) alone (9 patients; 1 mM in 3 patients; A) and in the absence (solid bars) and presence (open bars) of indomethacin (2.5 µM; 3 patients; B) on human airway smooth muscle cells in culture as measured by [3H]thymidine incorporation. Values are means ± SE. * Significant increase compared with response to control, P < 0.05. dagger  Significant decrease compared with response to control, P < 0.05.

PGE2 assay. There was no significant increase in PGE2 release after treatment with trypsin, tryptase, or the PAR-2 AP (Fig. 7). Incubation with the positive control bradykinin increased PGE2 release to 634 ± 241% of the control value (P < 0.05; n = 7 patients).


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Fig. 7.   Effect of trypsin (50 U/ml; 7 patients), lung-purified tryptase (5 nM; 4 patients), PAR-2 AP (1 mM; 4 patients), and bradykinin (10 µM; 7 patients) on PGE2 release from human airway smooth muscle cells in culture as measured by ELISA. Cells were incubated with each treatment for 30 min. Values are means ± SE. * Significant increase compared with the response to control (100%), P < 0.05.

Measurement of intracellular calcium. Trypsin (50 U/ml) induced large increases in intracellular calcium, with a mean response of 111 ± 6% of the response to bradykinin (n = 108 cells from 3 patients). The PAR-2 AP (0.1 mM) also induced increases in calcium but not in all cells that were tested. The mean response was 63 ± 4% of the response to bradykinin in the 90 cells that responded from the 195 cells (5 patients) tested. Cells that did not respond to the PAR-2 AP often still exhibited an increase in calcium in response to a subsequent treatment with trypsin. All cells tested responded to the positive control bradykinin (5 µM).

However, we were unable to detect consistent changes in the calcium levels in response to lung-purified tryptase. Of the 275 cells tested from 4 different patients, increases in the calcium levels were only detected in 29 cells after treatment with lung-purified tryptase (1 nM; mean response was 83 ± 7% of the response to bradykinin). Lung recombinant tryptase was also tested in 84 cells from 2 patients, with the increases in calcium evident only in 6 cells (mean 58 ± 9%). Most of the cells that were tested with tryptase were subsequently treated with either trypsin, the PAR-2 AP, or both, and increases in the calcium levels could then be detected with these other PAR-2 agonists.

Desensitization studies were carried out with trypsin and the PAR-2 AP. After exposure to 50 U/ml of trypsin (n = 47 cells from 2 patients), only 21 cells responded to a second exposure to trypsin (50 U/ml), and these cells produced a significantly lower response of only 52 ± 6% (P < 0.05) of the first response to trypsin (Fig. 8A). All cells responded to a subsequent treatment with bradykinin. The second response to trypsin was usually slower than the first and occurred in different cells at different times. Similarly, the PAR-2 AP also induced desensitization of the calcium responses (Fig. 8B). After treatment with the PAR-2 AP (0.1 mM; n = 22 cells from 2 patients), 17 cells responded to a second exposure to the PAR-2 AP but with a significantly lower response that was a mean of 52 ± 9% of the first response to the PAR-2 AP (P < 0.05).


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Fig. 8.   Effect of trypsin and PAR-2 AP on the intracellular calcium levels of human airway smooth muscle cells in culture as measured by changes in the ratio of fluorescence at 340 to that at 380 nm. Cells were exposed to 2 consecutive treatments with trypsin (50 U/ml; A), 2 consecutive treatments with PAR-2 AP (100 µM; B), trypsin (50 U/ml) followed by PAR-2 AP (100 µM; C), or PAR-2 AP (100 µM) followed by trypsin (50 U/ml; D). At the conclusion of each experiment, all cells were exposed to 5 µM bradykinin. Traces represent results in single cells.

After the cells had been exposed to trypsin, a subsequent treatment with the PAR-2 AP did not have any effect on intracellular calcium levels (n = 35 cells from 1 patient; Fig. 8C). After exposure to the PAR-2 AP, the cells still produced responses to trypsin (n = 57 cells from 3 patients; Fig. 8D). However, these responses were significantly lower, with a mean of 53% of the responses to trypsin alone (P < 0.05; n = 82 cells from 3 patients).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown, for the first time, that in contrast to the results reported in animal studies, activation of PAR-2 induced contractions of human isolated airways in vitro. Moreover, similar to tryptase, trypsin potentiated contractions to histamine in sensitized but not in nonsensitized patients. Lung-purified, skin, and lung recombinant tryptases, the PAR-2 AP, and trypsin all increased proliferation of HASM cells in culture. Stimulation of HASM cells in culture with PAR-2 activators did not stimulate PGE2 release. PAR-2 activation also increased the intracellular calcium levels of HASM cells, and desensitization of the responses was observed between trypsin and the PAR-2 AP.

Trypsin and the PAR-2 AP both produced small contractions in human isolated airway preparations. A previous study (21) in human isolated bronchial rings also found that the PAR-1 activator thrombin induced small contractions that were 20% of the response to a maximal concentration of carbachol, whereas even smaller contractions with the PAR-1 AP were also evident. In the present study, the contractions were potentiated when COX was inhibited by indomethacin. This indicates that PAR-2 activation may be inducing release of PGE2 from the airways, resulting in either a reduction in contractions or an induction of relaxation. Because neither trypsin nor the PAR-2 AP was found to significantly alter PGE2 release from the HASM cells, it is more likely that PGE2 was being released from the epithelium in the airway segments, and, indeed, exposure to a PAR-2 AP can induce PGE2 release from human airway epithelial cells after 48 h (39). Previous studies (11, 27) in mice have shown that activating PAR-2 induces relaxation of precontracted airways in vitro. In those studies, the relaxations were blocked by indomethacin and, in one study, were also blocked by the removal of the epithelium.

PAR-2 activation in the airways of guinea pigs also produces contractile responses as well as relaxations, but these differences appear to be related to the location of the airways in the lung (33). In vivo, intratracheal or intravenous trypsin and the synthetic peptide SLIGRL induced a bronchoconstriction in anesthetized guinea pigs. In vitro, the PAR-2 activators induced relaxation of isolated main bronchi and tracheae, whereas the intrapulmonary bronchi contracted in response to trypsin and SLIGRL. The relaxations were epithelium dependent and were abolished by inhibition of COX or nitric oxide synthase. Similarly, the contractions were increased by inhibiting these two enzymes.

In contrast to our findings in the present study, Cocks et al. (11) have reported that PAR-2 activation resulted in relaxation in human intrapulmonary airways from four patients, although the data were not shown. However, this previous study tested human airways that were 0.5-1 mm in external diameter, whereas we tested airways that were ~1-4 mm in internal diameter. This large difference in airway size may account for the conflicting results.

One of the most striking findings in the present study was the potentiation by trypsin of the histamine responses in sensitized but not in nonsensitized bronchial rings. This is identical to the situation Johnson et al. (24) have previously reported for tryptase. Not only was the potentiation confined to sensitized tissue, but in addition, with both trypsin and tryptase, higher concentrations failed to produce any effects. Berger et al. (7) have also since found that tryptase can increase the responses to histamine in human airways in vitro. Given that, in the present study, we found that trypsin could cause an increase in intracellular calcium in HASM cells, this may explain this potentiation of contraction.

In this study, we found that tryptase at nanomolar concentrations increased DNA synthesis of HASM cells in culture. This effect was not restricted to tryptase purified from the human lung because significant effects were also evident after incubation with either skin or lung recombinant beta -tryptase. However, the lung-purified tryptase was more efficacious than the recombinant forms. These differences may have been due to the varying activities of the tryptases or possibly to different isoforms of tryptase present. These increases in DNA synthesis in response to tryptase were found to correlate to increases in cell number as confirmed by direct cell counting. These nanomolar concentrations could be achieved in vivo because ~11 µg of tryptase are stored in 106 human lung mast cells (36), and the number of mast cells present in human lungs has been reported to be ~3 × 106 cells/cm3 (37). Assuming a molecular mass of 135 kDa for tryptase, 1 nM tryptase would be equivalent to 0.135 µg/ml. Therefore, even taking into account the fact that there would not be a release of tryptase from all mast cells simultaneously and that, once released, tryptase would diffuse away, it is plausible that nanomolar concentrations could be achieved in vivo. Tryptase is a known mitogen for a variety of cells, including the H292 epithelial cell line (10) and human lung parenchymal, airway, and dermal fibroblasts (2). Brown et al. (9) have found that tryptase increases proliferation of dog tracheal smooth muscle cells.

High concentrations of the PAR-2 AP were also found to induce increases in DNA synthesis that were significant but not as large as those seen in response to the tryptases. However, PAR APs are known to have low potency compared with the activating proteases. This may be due to a less efficient presentation or binding of the synthetic peptide to the receptor compared with the natural tethered ligand. Both the human PAR-2 AP SLIGKV and the murine peptide SLIGRL are proliferative in a number of cell types, including human fetal lung fibroblasts in culture where concentrations similar to those used in this study were required to induce an effect (2).

Although tryptase was mitogenic for HASM cells in our study, the effect of trypsin was not as consistent, although it did induce a significant but small increase in proliferation in most of the patients we tested. Previous studies have reported that the various activators of PAR-2 do not always exert the same proliferative effects. Although tryptase is mitogenic for keratinocytes (19), the murine PAR-2 AP inhibits human keratinocyte growth and differentiation (15). Trypsin induces proliferation of vascular smooth muscle cells (8), whereas tryptase is not mitogenic for these cells (20). Because trypsin can activate PAR-4, it is possible that this protease, which is less specific than tryptase, is acting on both PAR-4 and PAR-2 and potentially on other unidentified PARs.

When COX was inhibited, there were no significant increases in DNA synthesis in response to tryptase or the PAR-2 AP. This implies that activating PAR-2 does not induce the release of the antimitogenic COX product PGE2, and this was confirmed by the ELISA results in which we found no PGE2 release in response to PAR-2 activation of smooth muscle cells. However, there were significant increases in [3H]thymidine incorporation after incubation with trypsin and indomethacin, suggesting that perhaps some PGE2 was being released. However, no significant changes in PGE2 release were detected after incubation with trypsin for 30 min. It is possible that a longer incubation time is required to detect changes in PGE2 release in response to trypsin or that another COX product such as PGI2, which can inhibit proliferation of HASM cells (6), may be mediating the effects of trypsin.

The results of the experiments in which we examined the effects of trypsin and the PAR-2 AP on intracellular calcium concentrations demonstrated that significant desensitization occurred to these PAR-2 agonists. Thus responses to repeated application of trypsin were abolished or decreased as were those to subsequent applications of the PAR-2 AP. In addition, the responses to trypsin after PAR-2 AP application were decreased, whereas there was no response to PAR-2 AP after treatment with trypsin. In all these circumstances, the bradykinin responses were constant. This indicates that it is likely that trypsin and AP are both acting via the PAR-2 receptor, whereas bradykinin is increasing intracellular calcium via a different receptor mechanism. By contrast, however, very few cells responded to either purified or recombinant forms of tryptase at concentrations that had produced significant increases in thymidine incorporation and cell number. There are several possible explanations for this. There are relatively few studies that have reported an increase in intracellular calcium responses after tryptase, whereas there are many more reporting the effects of trypsin and the peptide. In those studies in which tryptase increased intracellular calcium levels (12, 13, 30, 35, 38), the concentrations used were 20-100 times those used in our study. However, even then the changes in calcium levels were often very small compared with those produced by trypsin or the PAR-2 AP (30, 35). Others have noted that under no circumstances can they observe a rise in intracellular calcium in response to tryptase, whereas other PAR-2 activators have produced calcium increases in the same cell (Pike R, personal communication). Tryptase has also been reported to have no effect on intracellular calcium levels in fibroblasts when tested at a concentration (7.5 nM) that produces proliferation in these cells (34). In other bioassay systems such as the rat colon, trypsin and the PAR-2 AP produced consistent responses, whereas tryptase was without effect (12). In addition, where tryptase has been reported to increase intracellular calcium, it has been obtained from noncommercial sources, unlike the tryptases used in the present study. Therefore, it is possible that differences in activity or isoforms present may influence the responses to tryptase.

In summary, the effect of PAR-2 activation in human airways is markedly different from that in airways of other species where it has been assigned a protective, anti-inflammatory role. In humans, PAR-2 activation by trypsin or the PAR-2 AP can induce contraction of the airways, and activation by trypsin or tryptase (24) potentiates the contractions to histamine in vitro. PAR-2 activation also increases proliferation and increases the levels of intracellular calcium in HASM cells. The effect of PAR-2 activation in human airways may be proinflammatory and not anti-inflammatory as has been previously suggested in animal airways (11, 27). Thus it seems that treatments designed to activate PAR-2 could be detrimental in the presence of human airway disease. In fact, PAR-2 has the potential to play an important role in airway diseases such as asthma by contributing to airway hyperresponsiveness and airway smooth muscle hyperplasia.


    ACKNOWLEDGEMENTS

We acknowledge the collaborative effort of the cardiopulmonary transplant team and pathologists at St. Vincent's Hospital. We also thank Dr. Juliette Burn and the surgical and pathology staff of the following Sydney hospitals for the supply of human lung tissue: Royal Prince Alfred, St. Vincent's, Concord, Royal North Shore, and Strathfield Private. We also thank Dr. Robert Pike for helpful discussions.


    FOOTNOTES

This work was supported by the National Health and Medical Research Council.

L. Chambers was supported by an Australian Postgraduate Award.

Address for reprint requests and other correspondence: L. S. Chambers, Dept. of Pharmacology, Univ. of Sydney, New South Wales 2006, Australia (E-mail: lindac{at}pharmacol.usyd.edu.au).

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.

Received 9 January 2001; accepted in final form 25 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 281(6):L1369-L1378
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