1 Institut für Neurobiochemie, 4 Abteilung für Pneumologie der Medizinischen Fakultät der Otto-von-Guericke-Universität Magdeburg, 39120 Magdeburg, Germany; 2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow; and 3 Research Centre of Molecular Diagnostics and Therapy, 113149 Moscow, Russia
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ABSTRACT |
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Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH2 terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca2+ imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca2+ response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca2+ level nor PAR-2-elicited Ca2+ response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca2+ response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.
calcium signaling; cathepsin G; trypsin; protease-activated receptor-2
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INTRODUCTION |
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ASTHMA IS A COMPLEX INFLAMMATORY disease of the lung characterized by the presence of tissue edema, mucus hypersecretion, bronchospasm, and airway hyperresponsivness. The inflammatory response in the asthmatic lung is characterized by infiltration of the airway wall with mast cells, lymphocytes, and eosinophils. Activation of these cells results in the release of a number of chemical mediators, e.g., leukotrienes, cytokines, and proteases, which are responsible for the symptoms of disease and further on lead to epithelial shedding and airway remodeling (33). Recent investigations have implicated serine proteases as mediators in the pathology of numerous allergic and inflammatory conditions. These effects may be mediated in part by protease-activated receptors (PARs), which can be activated by extracellular serine proteases. However, the underlying molecular mechanisms are still largely unknown (3, 5).
PARs belong to the superfamily of G protein-coupled receptors that are activated by a specific proteolytic cleavage of their extracellular NH2-terminal domain (9, 17, 31, 34). Cleavage of the receptor unmasks a new sequence at the NH2 terminus that acts as a so-called tethered ligand, which binds and activates the receptor itself. Molecular cloning has identified four PARs. PAR-1, PAR-3, and PAR-4 are designated thrombin receptors, because they are mainly activated by the serine protease thrombin. PAR-2 is activated by trypsin and in addition by mast cell-derived tryptase (12). Besides the proteolytic activation, PARs have been shown to be directly activated by short synthetic peptides on the basis of the sequence of the tethered ligand domain. These peptides are therefore useful pharmacological tools for identification and characterization of receptor function.
PARs play important roles in response to injury in several tissues, notably in inflammation and repair (16, 22). In particular, agonists of PAR-2 have widespread proinflammatory effects. Trypsin, tryptase, and activating peptides cause nitric oxide-dependent vasodilatation (20), induce extravasation of plasma proteins and infiltration of leukocytes (27, 28), and stimulate the secretion of proinflammatory neuropeptides (21). Airway epithelium plays an active role in inflammation through the production of a variety of lipid mediators, cytokines, chemokines, growth factors, and extracellular matrix components. PAR-2 expression, which colocalizes immunohistochemically with trypsin- (ogen) was detected for mouse, rat, guinea pig, and human airway epithelium (4, 7). Activation of PAR-2 in airway epithelium caused relaxation in mouse and rat airway preparations by the release of a cyclooxygenase product, most probably prostaglandin E2, and thus may have a protective role in the airways (4). On the other hand, PAR-2 activation by trypsin or tc-LIGRLO, a highly specific PAR-2-activating peptide, mediates the release of matrix metalloproteinase (MMP)-9 and eosinophil survival-promoting factor [granulocyte-monocyte colony-stimulating factor (GM-CSF)] from human airway epithelial cells (29, 30), thus being a critical element in tissue remodeling and eosinophil invasion in asthma and other inflammatory conditions.
On the basis of these results, it became apparent that PAR-2 plays an important role in lung physiology and pathophysiology. Due to the unique activation mechanism of the PARs by proteases, which can cleave within the extracellular NH2 terminus of the PARs, these may be receptor-activating or -inactivating proteases or may even inhibit activation by preceding proteolysis, depending on the cleavage site. Using different airway epithelial cell lines, a virus-transfected human bronchial epithelial (HBE) cell line and A549 cells, we found that, in both cell lines, trypsin and SLIGKV induced a Ca2+ response with similar characteristics. Furthermore, we investigated the influence of thermolysin, the prototype of the M4 family of metalloproteases (8). This family M4 comprises several proteases generated by bacteria involved in lung pathology, such as pseudolysin from Pseudomonas aeruginosa or extracellular proteinase A from Legionella pneumophila. Their substrate specificity requires an aromatic amino acid residue in the adjacent COOH-terminal (P1') position of the cleavage site (8).
We found that thermolysin behaved on HBE cells as both an activating and activation-inhibiting protease, whereas on A549 cells thermolysin was only an inactivating protease. Thus different epithelial cell types display contrasting physiological responses to thermolysin.
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MATERIALS AND METHODS |
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Materials. Thrombin (human), trypsin, thermolysin, hydrocortisone, 3,5,3'-triiodothyronine (T3), epidermal growth factor (EGF), trypsin inhibitor (type I-S: from soybean), and cholera toxin were from Sigma (Deisenhofen, Germany). Pituitary extract was from GIBCO-BRL (Eggenstein, Germany), and insulin-transferrin-sodium selenite (ITS) solution was from Roche Diagnostics (Mannheim, Germany). The cell culture medium Dulbecco's modified Eagle's medium (DMEM)-Ham's nutrient mixture F-12 (1:1), DMEM, fetal calf serum (FCS), and antibiotics (gentamicin, kanamycin, penicillin, and streptomycin) were obtained from Biochrom KG (Berlin, Germany). Fura 2-AM was purchased from Molecular Probes (MoBiTec, Göttingen, Germany). The synthetic thrombin receptor agonist peptide (TRag, Ala-parafluorPhe-Arg-Cha-homoArg-Tyr-NH2) and human PAR-2- activating peptide (SLIGKV) were from Neosystems Laboratoire (Strasbourg, France); human PAR-3- and PAR-4-activating peptides (TFRGAP and GYPGQV, respectively) were from BACHEM (Heidelberg, Germany).
Cell culture. The HBE cell line, originally established by Yankaskas et al. (36) by transfection of HBE cells with the genes E6 and E7 of the human papilloma virus HPV-18, was kindly provided by Dr. T. Meyer and Prof. Dr. L. Pott (Institut für Physiologie, Ruhr-Universität Bochum, Germany). For long-term storage, HBE cells were frozen in liquid nitrogen. After thawing, HBE cells were resuspended in DMEM-Ham's F-12 (1:1) culture medium supplemented with gentamicin (50 µg/ml), kanamycin (50 µg/ml), ITS (10 µg/ml), hydrocortisone (1 µM), pituitary extract (3.75 µg/ml), EGF (25 ng/ml), T3 (30 nM), cholera toxin (10 ng/ml), and 10% FCS. A549 cells were cultured in DMEM supplemented with 10% FCS and 100 µg/ml penicillin and streptomycin. Cells were kept at 37°C in a humidified atmosphere of 10% CO2.
For the experiments, the cells were grown on round coverslips (22-mm diameter) placed in petri dishes (60-mm diameter) for 3-7 days reaching 50-80% confluence, corresponding approximately to 1 × 106 cells/dish.Cytosolic Ca2+ measurements. The cytosolic Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura 2-AM. For dye loading, the cells grown on a coverslip were placed in 1 ml of HEPES-buffered saline (HBS) [buffer composition in mM: 145 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 25 glucose, 20 HEPES, pH 7.4 adjusted with Tris(hydroxymethyl)-aminomethane] for 30 min at 37°C, supplemented with 2 µM fura 2-AM. Loaded cells were transferred into a perfusion chamber with a bath volume of ~0.2 ml and mounted on an inverted microscope (Zeiss, Axiovert 135, Jena, Germany). During the experiments, the cells were continuously superfused with medium heated to 37°C. The perfusion system was combined with a six-port valve (Thomachrom, type RH 0112) from Reichelt (Heidelberg, Germany) to allow the switch between solutions containing different agents to be tested.
Single cell fluorescence measurements of [Ca2+]i were performed by using an imaging system from T.I.L.L. Photonics (Munich, Germany). Cells were excited alternately at 340 and 380 nm for 25-75 ms at each wavelength with a rate of 0.33 Hz, and the resultant emission was collected above 510 nm. Images were stored on a personal computer, and subsequently the changes in fluorescence ratio (F340 nm/F380 nm) were determined from selected regions of interest covering a single cell.Characterization of PAR-2 gene expression. Total RNA was isolated and treated with DNAse from cultured HBE and A549 cells with the total RNA isolation kit (RNeasy) from Qiagen (Hilden, Germany). RNA was reverse transcribed, and cDNA was amplified by using primers based on human PAR-1 (accession number M62424, position 473-1,009), PAR-2 (accession number XM003671, position 307-759), PAR-3 (accession number U92971, position 151-663), PAR-4 (accession number XM008930, position 836-1,377), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PAR-1 primers (sense, 5'-GATCAGCTATTACTTTTCCGGCA-3', antisense 5'-TAATGCGCAATCAGGAGGACG-3'), PAR-2 primers (sense, 5'-GAACGAAGAAGAAGCACC-3', antisense, 5'-GGAACAGAAAGACTCCAATG-3'), PAR-3 primers (sense, 5'-TCCCCTTTTCTGCCTTGGAAG-3', antisense, 5'-AAACTGTTCCCCACACCAGTCCAC-3'), PAR-4 primers (sense, 5'-AACCTCTATGGTGCCTACGTGC-3', antisense, 5'-CCAAGCCCAGCTAATTTTTG-3'), GAPDH primers (sense, 5'-ACCACCTGGTGCTCAGTGTAGCCC-3', antisense, 5'-TTCAAAATCAAGTGGGGCGATGCT-3') were chosen to amplify a 536-bp fragment for PAR-1, 452-bp fragment for PAR-2, 512-bp for PAR-3, 541-bp for PAR-4, and 600-bp for the internal control GAPDH. The mRNA derived from a human PAR-1, PAR-2, and PAR-3 clone were used as a positive control. PCR conditions were denaturation 15 min at 95°C, 30 cycles at 94°C for 30 s, 55°C for 90 s, 72°C for 60 s, and elongation at 72°C for 10 min. Because the primers used are not flanking an intron, a PCR was performed with the total RNA to exclude any contamination of the RNA with the genomic DNA. The RNA-PCR (40 cycles) carried out was always negative, indicating that the resulting PCR fragments were not amplified from genomic DNA. PCR products were analyzed by Tris-borate-EDTA agarose (1%) gel electrophoresis with ethidium bromide. Documentation was done by using a still video system (Eagle Eye; Stratagene, Heidelberg, Germany).
Immunocytochemistry.
HBE cells grown on coverslips were washed three times in
phosphate-buffered saline (PBS) and subsequently fixed at 20°C in methanol for 15 min. After fixation, the cells were washed three times
in PBS and permeabilized with 1% Triton X-100 in PBS for 15 min. Again
the cells were washed in PBS. Before addition of the anti-PAR-2
antiserum, the cells were incubated for 30 min in PBS supplemented with
1% BSA. Cells were incubated in primary antibody solution 20 µg/ml
overnight at 4°C. The affinity-purified goat polyclonal antibody to
PAR-2 was purchased from Santa Cruz Biotechnology (Heidelberg,
Germany). After incubation with the primary antibody, the cells were
washed three times in PBS, then incubated in 4 µg/ml of donkey
anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz
Biotechnology) for 5 h at room temperature. After secondary
antibody incubation, the cells were washed three times in PBS and
incubated in diaminobenzidine (DAB) for 10 min. After the DAB
staining, cells were rinsed with H2O for 5 min and counterstained with hematoxylin for 1 min, rinsed again with
H2O, dehydrated, and then mounted in Entellan (Merck,
Darmstadt, Germany). Observations of the cultured cells were performed
on a Zeiss LSM 510 laser-scanning confocal microscope.
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RESULTS |
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RT-PCR and immunocytochemistry.
The RT-PCR methodology was used to detect the mRNA expression of all
four PARs in the HBE and A549 cell lines. Therefore, total mRNA was
extracted. As shown in Fig.
1A, cultured HBE and A549
cells express only PAR-2 mRNA. We did not see a PCR fragment corresponding to PAR-1, PAR-3, and PAR-4 (data not shown). As an
internal control for the RT-PCR conditions, the mRNA for GAPDH was
detected by primers specific for this gene product.
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Characteristics of PAR-2-evoked Ca2+
response.
After identifying the presence of PAR-2 mRNA and protein in HBE and
A549 cells, we continuously monitored [Ca2+]i
in fura 2-AM-loaded epithelial cells, stimulated with either the serine
protease trypsin or the PAR-2-activating peptide (SLIGKV). A brief (60 s) addition of trypsin (50 nM) or SLIGKV (100 µM) induced a transient
change in [Ca2+]i in both cell lines, as
shown in Fig. 2, A and
B, respectively. On the other hand, long-term stimulation
with PAR-2 agonists elicited a prolonged, biphasic change of
[Ca2+]i. Figure 2C shows a
representative example for such a response evoked by trypsin in HBE
(left) and A549 cells (right). Similar responses
were observed for long-term treatment by using the agonist peptide
SLIGKV (data not shown). From these experiments, two things became
apparent: 1) the amplitude of the initial Ca2+
transient induced by 50 nM trypsin or 100 µM SLIGKV was significantly smaller in A549 compared with HBE cells and 2) the
Ca2+ response persisted as long as the agonists were
present. We further characterized the initial transient increase of
[Ca2+]i in HBE cells by analyzing the
concentration dependence of the amplitude (peak change of the ratio
above basal level) in the presence and absence of extracellular
Ca2+. Omission of Ca2+ from HBS gives a
nominally Ca2+-free solution. Previously, we found that
such nominally Ca2+-free conditions are sufficient to
inhibit Ca2+ influx as well as prolonged Ca2+
signaling in HBE cells (data not shown). Analysis of the
concentration-effect curves clearly demonstrated for HBE cells that
1) the amplitude of the initial transient did not depend on
external Ca2+, 2) the PAR-2-activating peptide
(SLIGKV) was about 10,000 times less effective, and 3) the
maximal amplitude evoked by the peptide was lower than the
Ca2+ response elicited by proteolytic activation of the
receptor (Fig. 3). In the absence of
extracellular Ca2+, emptying of intracellular
Ca2+ stores by cyclopiazonic acid (10 µM), an inhibitor
of the Ca2+-ATPase of the sarco-/endoplasmic reticulum,
completely abolished a subsequent Ca2+ response elicited by
trypsin (not shown).
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Effect of cathepsin G and thermolysin on PAR-2-mediated
Ca2+ response.
Due to the unique activation mechanism of PARs, proteases that cleave
the receptor at a site apart from the tethered ligand domain can act as
activation-inhibiting proteases. Previous experiments in our laboratory
revealed that the proteases cathepsin G and thermolysin could cleave
PAR-1 in rat astrocytes, leaving the cells unresponsive to a subsequent
stimulation by thrombin (25, 26). Similarly, in HBE and
A549 cells we tested whether these proteases could influence the
signaling mediated by activation of PAR-2. Superfusion of epithelial
cells with 100 nM cathepsin G neither influenced the resting
Ca2+ level nor altered the trypsin-evoked Ca2+
signal in HBE and A549 cells (data not shown). In contrast, as shown in
Fig. 7, thermolysin (0.05-5 U/ml)
induced a transient, concentration-dependent rise of
[Ca2+]i in HBE cells (Fig. 7, B
and C, left). In the same way it abolished the
response to a subsequent exposure to 50 nM trypsin, whereas the
response to a subsequent addition of SLIGKV (100 µM) was not altered
(Fig. 7D).
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DISCUSSION |
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Recently, PAR-2 activation has been shown to cause relaxation of airway preparations from human, mouse, rat, and guinea pig by the release of a cyclooxygenase product from the epithelium (4), which demonstrates a protective role for PAR-2 in airway epithelial cells. In addition, trypsin and PAR-2-activating peptides were shown to induce the release of MMP-9 and eosinophil survival-promoting factor (GM-CSF) in A549, a human airway epithelial cell line and primary cultures of small airway epithelial cells (29, 30). Thus it is important to understand the signaling events leading to these physiological consequences of PAR-2 activation, which are still elusive.
In this study, we clearly demonstrate the functional expression of PAR-2 in HBE and A549 cells, two cell lines of human airway epithelium origin. The failure of thrombin to induce a Ca2+ signal indicates that the lung epithelial cells tested do not express other members of the PAR family coupled to intracellular Ca2+ signals. In this respect, the epithelial cells strongly differ from isolated astrocytes, which were shown by us to express all four PAR subtypes (26, 32). This is consistent with the ubiquitous but distinct distribution of all four PARs in the whole brain, as our group demonstrated recently in a study that systematically investigated the occurrence of PARs in the brain (23). The study also showed that activation of different PARs in astrocytes may evoke different physiological signaling and lead to distinct downstream events with respect to either proliferation or cytotoxicity (32), providing useful insights into the role of PARs in the processes of protection and degeneration of neuronal tissue.
PAR-2 activation in the epithelial cells elicited a Ca2+ response, which was partially PTX sensitive in A549 cells, but completely PTX resistant in HBE cells. The Ca2+ rise is transduced via PLC activation to InsP3-mediated release of Ca2+ from intracellular stores. The lower efficacy of the soluble peptide ligand compared with that of the tethered ligand that we have observed here is a well-documented characteristic of PARs (18, 31). The fact that the amplitude of the PAR-2-evoked Ca2+ response showed no dependence on extracellular Ca2+ confirms that the initial PAR-2 activation is exclusively coupled to the release of Ca2+ from internal stores.
Immunocytochemical studies revealed an abundant PAR-2 expression in the lung and the airways, especially the epithelium and the smooth muscle (7), but the physiological functions and the endogenous ligands that activate PAR-2 are still poorly understood. Trypsin immunoreactivity has been identified in airway epithelium (4), and a trypsin-like protease was purified from human airways (35). It can be speculated that this airway-derived, trypsin-like protease may be able to activate PAR-2. On the other hand, tryptase from mast cells, which was shown to activate PAR-2 in human vascular endothelial cells and human lung fibroblasts (1, 12), might be an important endogenous protease able to stimulate PAR-2 activation in bronchial epithelial cells. This is of special interest because mast cells infiltrate the inflamed airway wall. Moreover, recent biological and immunological investigations have implicated tryptase released from mast cells as a mediator in the pathology of numerous allergic and inflammatory conditions, such as asthma (2) or neurogenic inflammation processes (21).
A consequence of PAR-2 activation might also be the induction of an inflammatory response in the airways. This suggestion seems to contradict the protective effects observed for epithelial PAR-2 in bronchial rings (4). However, initial inflammatory reactions have a protective function. Only persistent inflammation leads to pathological reactions. Therefore, it could be possible that the consequence of PAR-2 activation, whether it is cytoprotective or deleterious, strongly depends on the protease concentration or the duration of the exposure to protease. Indeed, such behavior was found for thrombin-induced PAR-1 activation in the hippocampus (22). That study showed that low doses of thrombin increased cell survival after experimental ischemia in organotypic hippocampal slice culture. The protease thrombin by itself applied at higher concentrations even potentiated cell death after ischemia.
A physiologically controlled activation of PAR-2 might evoke mechanisms that are protective for the lung. On the other hand, uncontrolled activation of PAR-2, via other proteases, probably causes the induction of pathophysiological reactions leading to cell death. Therefore, it is of special interest to investigate the capacity of other proteases to activate PAR-2 or to inhibit activation. Two airborne dust mite antigens, the serine proteases Der p3 and Der p9, were found to activate PAR-2 in lung epithelial cells (24). It is documented that in addition to trypsin, tryptase and the lung human airway trypsin-like protease can activate PAR-2 (11). For PAR-1 in astrocytes we found that treatment with thermolysin, which is known to cleave the corresponding tethered ligand, generated a thrombin-insensitive receptor, whereas the response to the activating peptide was not affected (25). From our data in that study, we could establish a novel model for PAR-1 inactivation, which implies that, after proteolytic or nonproteolytic agonist activation of the receptor, an additional, still unidentified protease physiologically terminates the signal. However, in contrast to PAR-1, little has been reported so far in the literature about proteases inhibiting activation of PAR-2 (19). Therefore, we tested the serine protease cathepsin G and the thermophilic bacterial protease thermolysin on PAR-2 in airway epithelial cells. Prior addition of cathepsin G failed to alter the trypsin-induced Ca2+ response in the epithelial cell lines.
Thermolysin is the prototype of a family of zinc metalloproteases comprising only secreted eubacterial endopeptidases from both gram-positive and gram-negative bacteria (8). Using thermolysin, we obtained apparently contradictory results. In HBE as well as in A549 cells, prior addition of thermolysin leaves the cells unresponsive to a subsequent challenge with trypsin but not the PAR-2-activating peptide SLIGKV. So far, these results indicate that, similar to PAR-1 (25, 26), thermolysin cleaves the receptor in such a way that the tethered ligand domain is destroyed.
However, thermolysin seems to play a dual role in lung epithelia. Thermolysin has also been found here to have the potency to evoke a concentration-dependent increase of [Ca2+]i via activation of PAR-2. The latter was seen only in HBE cells, not A549 cells. This finding strongly suggests that either there is a structural difference between PAR-2 expressed in HBE and in A549 cells, or thermolysin activates a protein, expressed only in HBE cells, that helps as an agonist for PAR-2. In the literature, examples for both possibilities have been reported. Recently, a polymorphic PAR-2 was detected, displaying a reduced sensitivity to trypsin and differential responses to PAR agonist peptides (6). This polymorphic form of human PAR-2 was identified by a phenylalanine to serine mutation at residue 240 within extracellular loop 2. Interestingly, extracellular loop 2 is suggested to be important for the interaction with the tethered ligand and therefore governing agonist specificity and receptor signaling (14, 15). This report demonstrates that minor changes in the amino acid sequence of PARs can lead to significant changes in the receptor structure, causing a dramatic switch in the sensitivity toward proteases and/or peptide agonists. On the other hand, elegant studies coexpressing human PAR-3 and PAR-4 in fibroblasts, derived from PAR-1 knockout mice, strongly suggested a coactivation mechanism, whereby the activation of PAR-3 by thrombin promotes the activation of PAR-4 (13). Similarly, one could postulate that in HBE cells thermolysin could activate a "sleeping" agonist activating PAR-2. In the future, it will be an interesting task to investigate the cellular consequences of the physiological polymorphism observed here to better understand the role of PARs in the physiology and pathophysiology of the airway epithelium.
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ACKNOWLEDGEMENTS |
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We thank M. Schubert and K. Christoph for technical assistance in cultivating HBE cells.
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FOOTNOTES |
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This study was supported by grants from the Deutsche Forschungsgemeinschaft (Re 563/11-1), International Association for the promotion of cooperation with scientists from the New Independent States of the former Soviet Union (97-1504), Land Sachsen-Anhalt, and Fonds der Chemischen Industrie.
Address for reprint requests and other correspondence: G. Reiser, Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Medical School, Leipziger Str. 44, D-39120 Magdeburg, Germany (E-mail: georg.reiser{at}medizin.uni-magdeburg.de).
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.
First published January 11, 2002;10.1152/ajplung.00392.2001
Received 5 October 2001; accepted in final form 7 January 2002.
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