1 Pneumologie der 1. Medizinischen Klinik und Deutsches Herzzentrum, Technische Universität, 81675 Munich, Germany; and 2 Pulmonary and Critical Care Division, University of Pennsylvania, Philadelphia, Pennsylvania 19104-4283
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ABSTRACT |
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In a variety of
diseases, inflammation causes microvascular leakage and activates
thrombin. Evidence suggests that thrombin increases cytosolic calcium
and stimulates human airway smooth muscle (ASM) cell proliferation. The
receptor subtypes, however, that mediate the effects of thrombin on ASM
cell growth or calcium mobilization remain unknown. In this study, we
postulate that thrombin, which activates specific protease-activated
receptors (PARs), also stimulates contraction of isolated human
bronchial rings. With the use of intact human bronchial rings,
-thrombin (1-20 U/ml) increased bronchial tone to 19 ± 3%
of basal tone (P = 0.008;
n = 5 experiments) and represents 20 ± 8% of the maximum carbachol response. The
EC50 for thrombin-induced force
generation was 12.2 U/ml (95% confidence interval 9.9-15.3 U/ml)
and was not altered in bronchial rings that had the epithelium removed. In parallel experiments, a specific thrombin receptor-activating peptide (TRAP-14; 0.1-100 µmol/l) increased isometric tension to
levels (14 ± 2%; P = 0.0005;
n = 5 experiments) comparable to those
rings stimulated with thrombin. To characterize the receptors that
mediate thrombin effects on human ASM, the expression of PARs in
cultured human ASM cells was analyzed by RT-PCR analysis with specific
primers for PARs. In these cells, PAR1 (thrombin receptor), PAR2, and
PAR3 were expressed at comparable levels. In other experiments using
immunocytochemical staining with specific antibodies to PAR1 and PAR2,
we showed that ASM in bronchial rings and cultured ASM cells express
PAR1 and PAR2 proteins. Taken together, these studies suggest that
-thrombin, in a receptor-specific and dose-dependent manner, induces
contraction of bronchial rings in vitro. In addition, cultured human
ASM cells express mRNA of PAR1, PAR2, and PAR3 and express PAR1 and
PAR2 protein. Further studies are needed to determine whether
-thrombin plays a role in stimulating bronchoconstriction in
inflammatory airway diseases such as asthma and bronchiolitis obliterans.
r-hirudin; thrombin receptor-activating peptide-14; asthma; human bronchi; smooth muscle contraction
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INTRODUCTION |
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ASTHMA IS A DISEASE characterized by airway inflammation, hyperresponsiveness, bronchial smooth muscle contraction, and, in severe asthmatic patients, airway smooth muscle (ASM) hyperplasia (for a review, see Ref. 31). Although the underlying mechanisms that induce these alterations remain unknown, microvascular leakage is a prominent feature of the inflammatory response (3, 4, 18). Evidence suggests that increases in airway resistance in asthmatic patients are due in part to submucosal edema from increased vascular permeability (1, 13, 14, 22). In a variety of diseases, inflammatory responses that induce submucosal edema also activate thrombin.
Recent studies (8, 11, 17, 40) suggested that thrombin, which is activated at sites of inflammation, mediates its cellular effects through a unique receptor-ligand binding mechanism. Thrombin cleaves its receptor, releasing an inactive fragment of the receptor amino terminus and exposing a new amino terminus. This unmasked amino terminus then functions as a tethered ligand, which binds to and activates the receptor. Studies from our laboratory (15, 29, 32) and by others (39) have shown that thrombin increases cytosolic calcium and induces ASM cell proliferation. To date, however, little is known as to whether thrombin also induces bronchoconstriction in vivo, and few investigators have studied the receptor subtypes that mediate the cellular effects of thrombin. Thrombin and thrombinlike molecules activate a newly characterized family of seven-transmembrane-region receptors that are coupled to guanosine nucleotide binding proteins (for a review, see Ref. 8; see also Refs. 16, 20). This family of receptors has been termed protease-activated receptors (PARs).
In this study, we examined whether -thrombin induces
bronchoconstriction in human bronchial rings in vitro. Furthermore, we
characterized, using molecular techniques, the family of PARs that are
expressed in human ASM cells. Taken together, our studies provide
insight into a new class of potentially important bronchoconstrictor agents that may play a role in inflammatory airway disease such as
asthma, chronic bronchitis, and bronchiolitis obliterans.
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MATERIALS AND METHODS |
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Bronchial Ring Studies
Patients. Bronchial rings were obtained from 10 patients, 2 women and 8 men, who had undergone pneumonectomy or lobectomy for treatment of bronchial carcinoma. These studies were performed in accordance with the procedures approved by the University of Pennsylvania (Philadelphia, PA) Committee on Studies Involving Human Beings. The mean age of the patients was 63.3 ± 1.7 yr (range 34-76 yr). Only patients with confirmed normal preoperative lung function were included in the study. None of the patients was receiving prednisone, inhaled corticosteroids, or albuterol before the time of surgery. All patients underwent anesthesia according to the same regimen. Informed consent was obtained from all patients before lung surgery.Isolated human bronchi. Bronchi with a
diameter between 5 and 8 mm were dissected free of lung and connective
tissue and cut into 2- to 3-mm-thick rings. The tissues were then
stored overnight at 4°C in Tyrode solution and aerated with 95%
O2-5%
CO2. Subsequently, the bronchial
rings were placed in 20-ml organ baths with oxygenated Tyrode solution
containing 3 µM indomethacin at 37°C and pH 7.4. Isometric
tension was then measured with an inductive force transducer (F 30 type
372) attached to an analog-to-digital converter (type 663; Hugo Sachs,
Freiburg, Germany) (19). During an equilibration period of 90 min, the
bathing solution was changed every 15 min, and the bronchi were mounted
under an isometric load of 2 g. Concentration-response curves were then
performed by the addition of carbachol, -thrombin, or a specific
thrombin receptor-activating peptide (TRAP-14) in a cumulative dose
manner. In these studies, force generation was normalized to that
obtained with 100 µmol/l of carbachol, which was used because this
agonist concentration evokes maximal isometric tension (data not shown).
To study whether the bronchoconstrictor effect of -thrombin was
dependent on the epithelium, denudation of bronchial epithelium was
performed with cotton swabs in some experiments. This technique removed
~90% of the epithelium as confirmed by histopathology (data not
shown). In other experiments, we investigated whether TRAP-14 increased
the isometric force to similar levels compared with those induced by
-thrombin (7, 16).
Human ASM Cell Culture
Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was dissected under sterile conditions, and the trachealis muscle was isolated (30). Approximately 1 g of wet tissue was obtained, minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U/ml of collagenase, 10 mg of soybean trypsin inhibitor, and 10 U/ml of elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 125-nm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine serum. Aliquots of the cell suspension were plated at a density of 1.0 × 104 cells/cm2. Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 100 µg/ml of amphotericin B was replaced every 72 h. Details regarding the characterization of this cell line by indirect immunofluorescence to smooth muscle-specific actin and agonist-induced changes in cytosolic calcium have been previously reported by our laboratory (30).Confluent ASM cells were growth arrested by incubating the monolayers in serum-free medium consisting of Ham's F-12 medium with 5 µg/ml of insulin and 5 µg/ml of transferrin for 48 h (30, 32). Growth-arrested cells were used because the cells can be synchronized in the G0/G1 phase of the cell cycle and, at this baseline, minimally incorporate [3H]thymidine (30, 32). The cells used in this experiment were third to fifth passaged cells, <28 cumulative population doublings (30).
Characterization of PAR Family Gene Expression
To identify the family of PAR genes expressed in human ASM cells, total mRNA was extracted from human ASM cultures by using the acid-phenol technique (12). Total RNA was also extracted from dermal fibroblast cell cultures as well as from selected mouse tissues (stomach, kidney, and small intestine). The mRNAs from these cell lines served as positive controls for the specific PAR-receptor subtype. The techniques used to analyze total mRNA expression by RT-PCR analysis were similar to those previously described by our laboratory (15, 23). Briefly, 5 µg of total RNA were mixed with 0.5 µg of oligo(dT) primers (Promega), heated at 68°C for 5 min, and then placed on ice. Reaction buffers and deoxynucleotides were added to each sample. Each tube was then heated at 50°C for 2 min to allow for annealing of the oligo(dT) and mRNA. Superscript II reverse transcriptase (200 U) was added to each sample; RT was performed for 60 min at 50°C (Life Technologies). After transcription, the reverse transcriptase was heat inactivated, and 40 µl of Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH 8.0) were added to each RT reaction. For PCR analysis, 2.5 µl of cDNA were used per reaction. The following sequences were used to derive the PCR primer pairs: PAR1 (40): 5' primer, nucleotides 974-996; 3' primer, nucleotides 2195-2221; yields a 1,247-bp product; PAR2 (27): 5' primer, nucleotides 397-424; 3' primer, nucleotides 937-960; yields a 563-bp product; and PAR3 (21): 5' primer, nucleotides 47-70; 3' primer, nucleotides 1326-1349; yields a 1,302-bp product. As standards, SM22 and smooth muscleTissue Samples and Handling for Immunocytochemical Staining
Human bronchial tissue was obtained during a lobectomy for removal of a primary lung tumor performed at the Hospital of the University of Pennsylvania. Two 1-cm pieces of bronchus were washed in normal saline and then frozen in isopentane cooled in liquid nitrogen. Frozen sections were cut at 8 µm on a Reichert-Jung Cryocut 1800 cryosat and mounted on glass slides.Immunostaining
Frozen sections were thawed to room temperature and fixed in 2% formaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) in 150 mM phosphate-buffered saline (PBS) for 5 min. Sections were next rinsed three times with PBS, then incubated with 3% hydrogen peroxide in PBS for 10 min to quench endogenous peroxidase activity, followed by three more washes with PBS. Sections to be stained with anti-smooth muscle-specificThe cell cultures were stained in the same way as the sections except that after incubation with the secondary antibody, biotin-conjugated goat anti-mouse IgG, the cultures were reacted directly with streptavidin-Texas Red instead of being amplified with HRP and rhodamine-tyramide.
Observations of the sections and cultured cells were performed on a Zeiss LSM 510 laser-scanning confocal microscope.
Statistics
Contractile responses obtained from functional experiments were analyzed as independent observations. Data are presented as means ± SE. One-sample t-test was performed for each concentration-response curve to compare the mean percent change in baseline to zero. Comparison of the mean percent change in baseline between different concentration-response curves was performed with the Mann-Whitney test. Bonferroni's correction was used for multiple comparisons. Significance was considered at P < 0.05. Some data are presented as means, and 95% confidence intervals are in parentheses. All statistics were calculated by use of SigmaStat 3.01.Chemicals
Indomethacin, carbachol, TRAP-14, and all other reagents except those specified were produced by Sigma (Deisenhofen, Germany); bovine albumin-free thrombin was obtained from Calbiochem-Novabiochem (Bad Soden, Germany). Tyrode solution consisted of (in mmol/l) 136.9 NaCl, 5.4 KCl, 1.8 CaCl2, 1.05 MgCl2, 0.42 NaH2PO4, 16.6 NaHCO3, 0.05 Na2EDTA, 0.28 ascorbic acid, and 5.0 glucose. ![]() |
RESULTS |
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-Thrombin Increases Isometric Tension in Human
Bronchial Rings
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-Thrombin Stimulates Isometric Force in a
Receptor-Specific Manner
PAR Expression in Human ASM Cells
To characterize the family of PAR genes expressed in human ASM cells, total mRNA was extracted from human ASM cultures by using the acid-phenol technique (12). Total mRNA was also extracted from dermal fibroblast cell cultures as well as from selected mouse tissues (stomach, kidney, and small intestine; data not shown). The mRNAs from these cell lines served as positive controls for the specific PAR subtype.As shown in Fig. 2, cultured human ASM
cells express PAR1, PAR2, and PAR3 mRNAs. The products, which were
obtained by RT-PCR, were sequenced, and the sequences observed were
identical to those reported for PAR1, PAR2, and PAR3. Primers for SM22
and -actin, which are smooth muscle-specific proteins, also
identified RT-PCR products in which the molecular weights were
consistent with those reported for the native smooth muscle proteins.
These gels were representative of three separate experiments with
different ASM cell lines. Together, these data suggest that cultured
human ASM cells express PAR1, PAR2, and PAR3.
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Immunolocalization of PAR1 and PAR2
To determine whether PAR1 and PAR2 proteins were present on ASM in human bronchial rings and in cultured cells, monoclonal antibodies specific to PAR1 and PAR2 were used, and immunocytochemical staining was performed. Confocal microscopy allowed us to observe the immunolocalization of these proteins on the surface of human ASM, in contrast to the intracellular immunolocalization of the structural protein
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DISCUSSION |
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Thrombin, a serine protease, is generated at sites of inflammation and
has a variety of cellular effects that are distinct from its effects on
coagulation (24-26, 33-35, 38). Previous studies have
demonstrated that treatment of cultured human ASM cells with -thrombin induces several cellular responses including inositol phospholipid hydrolysis, an increase in intracellular free calcium, DNA
synthesis, and cell proliferation (for a review, see Ref. 31). Although
recent studies have described the cellular effects of
thrombin, few studies have examined whether thrombin can modulate ASM
cell function in isolated human bronchial rings (33-35). In addition,
no study has examined thrombin-receptor or PAR expression in human ASM
cells. In the present study, we demonstrated that thrombin stimulates
isometric tension in human bronchial rings. In human ASM cells, we also
showed that the thrombin-receptor (PAR1), PAR2, and PAR3 mRNAs and PAR1
and PAR2 proteins are expressed. The identification that thrombin may
directly induce ASM cell contraction may offer insight into the
mechanisms that regulate bronchomotor tone in diseases characterized by
airway inflammation and microvascular leakage.
The identification of the thrombin receptor brought questions about the existence of other receptors with the same mechanism of action and about the ability of cloned receptors to account for all the effects of thrombin in a variety of cell types including human platelets. Evidence now suggests that thrombin activates a receptor, which is a member of a larger family of PARs. The thrombin receptor (PAR1), like nearly all receptors that couple to G proteins, is composed of a single polypeptide with seven-membrane-spanning domains (36, 40). The intracellular domains of the receptor are presumably involved in G protein coupling and in receptor desensitization and clearance. The extracellular NH2 terminus contains a site for cleavage by thrombin that is located between residues Arg41 and Ser42 in the human receptor. Vu et al. (40) originally proposed that the region immediately adjacent to the COOH terminus is the cleavage site and forms a tethered ligand capable of activating the receptor, apparently by interacting with sites in the second extracellular loop and in the NH2 terminus near the first transmembrane domain. In keeping with this model, numerous studies in a variety of cells have shown that synthetic peptides beginning with the first five residues of the tethered ligand domain (SFLLR) in the human receptor can mimic the effects of thrombin (33-35). Implicit in this model is the conclusion that the role of thrombin in receptor activation is limited to cleavage on the NH2 terminus and, perhaps, facilitates docking of the tethered ligand domain in the body of the receptor. This issue, however, has not been fully resolved (for a review, see Ref. 8; see also Ref. 16).
The second known member of the PAR family was identified by Nystedt et al. (28) in 1994 and named PAR2. Human PAR2 is homologous to the human thrombin receptor at the amino acid level, with regions most notably in the second extracellular loop that are nearly identical. PAR2, like the thrombin receptor, is located on human chromosome 5 and may have arisen by gene duplication. Nystedt et al. showed that trypsin, but not thrombin, can activate PAR2 by cleaving between Arg34 and Ser35 in the murine PAR2 corresponding to Arg36 and Ser37 in the human sequence. Interestingly, mutagenesis of this site prevented PAR2 activation by trypsin. These results show that PAR2 closely resembles thrombin receptors in both structure and mechanism of action but is not necessarily activated by the same proteases. As better tools for detecting PAR2 have become available, the receptor has been shown to be expressed on keratinocytes (37), intestinal epithelium (5, 6), and at least some forms of vascular or nonvascular smooth muscle (2). In the present study, we now show that cultured human ASM cells express mRNA not only for the thrombin receptor (PAR1) but also for PAR2 and PAR3. Immunocytochemical staining reveals that bronchial ASM and cultured cells express PAR1 and PAR2. Currently, a specific antibody for PAR3 is not available. PAR3, recently described by Ishihara et al. (21), appears to mediate thrombin-triggered phosphoinositide (PI) hydrolysis and is expressed in a variety of tissues including human bone marrow and mouse megakaryocytes. The amino-terminal exodomain of the new receptor contains a possible thrombin cleavage site and a sequence strikingly identical to the thrombin-binding sequence in the leech anticoagulant hirudin. Importantly, thrombin activates the human PAR3. Compelling evidence now suggests that expression, downstream signaling events, and the physiological consequences of PAR activation are cell and tissue specific (21). The present study identifies that human ASM cells express mRNA for all three PARs and that activation of PARs increases isometric force in human bronchial rings and may suggest a new family of receptors that modulate ASM cell function.
A previous study (32) of the effects of thrombin on human ASM cells showed that thrombin potently and effectively evokes calcium transients and stimulates PI turnover. Thrombin also markedly induced ASM mitogenesis. Interestingly, the effects of thrombin on calcium mobilization and PI turnover were unaffected by pertussis toxin, which ADP-ribosylates and inactivates Gi proteins, whereas the effects of thrombin on mitogenesis were completely abrogated by pertussis toxin treatment (32). Furthermore, the pertussis toxin-sensitive G protein that mediates thrombin-induced ASM mitogenesis was not Gi because thrombin had no effect on isoproterenol-stimulated increases in cAMP levels (32). Together, these data suggested that one receptor was coupled to two different G proteins or that thrombin activated two receptors, each coupled to a different G protein. The present study demonstrated that human ASM cells express three PARs; however, only PAR1 and PAR3 have been reported to be activated by thrombin. Because specific agonists or antagonists are not currently available to discriminate between PAR1- and PAR3-mediated responses, we can only speculate that the different effects of thrombin on ASM cells may be due to activation of a different PAR. This hypothesis, however, requires further study.
Despite the evidence supporting PAR expression and function in human
ASM cells, several issues need to be addressed. In comparison to
carbachol, thrombin-induced force generation in human bronchial rings
was substantially less. There may exist several possible explanations
for this observation. Anti-proteases secreted by cells in human
bronchial ring preparations may inactivate thrombin and thus the
effects of thrombin on smooth muscle are attenuated. Possibly, agonists
that activate receptors, such as the muscarinic-receptor family,
coupled to both Gq and
Gi, which inhibit adenylyl
cyclase, may be more effective bronchoconstrictors than those coupled
to only Gq and
Go, such as thrombin.
Interestingly, in cultured human ASM cells, both carbachol and thrombin
evoke comparable increases in cytosolic calcium and PI turnover (R. A. Panettieri and R. Murray, unpublished observations).
Finally, PAR and muscarinic-receptor isotype expression in bronchial
smooth muscle ex vivo may differ from that in cultured tracheal smooth
muscle and thus may account for the differential responses. To further
address whether the action of -thrombin on basal tone of human
bronchi is stimulated by specific thrombin receptors, we also studied
the effects of TRAP-14 on isometric force generation. TRAP-14 induced
maximal increases in isometric force to levels comparable to those
observed with thrombin. These findings suggest that thrombin does
induce isometric force in human bronchial rings by activating PARs.
Unfortunately, TRAP-14 and other thrombin receptor-activating peptides
are not specific for PAR1 or PAR3 and as such are not useful in
characterizing the function of receptor isotypes. Although we show for
the first time that human bronchial smooth muscle and cultured tracheal smooth muscle cells express PAR1 and PAR2, thrombin-induced contraction of the human bronchus may have been due to activation of PARs in other
cell types in the bronchus such as epithelial or mast cells. Given the
complex nature of bronchial tissue, it is unlikely that one can
determine whether direct activation of PARs on ASM or indirect
activation of smooth muscle via release of mediators from bronchial
epithelium or mast cells is responsible for mediating thrombin-induced
bronchial ring contraction. Further studies are needed in vivo to
determine whether thrombin is an important bronchoconstrictor in
asthmatic patients.
In conclusion, we have demonstrated that thrombin and TRAP-14 increase
isometric tension in human bronchial rings. In parallel experiments, we
demonstrated that cultured human ASM cells express a family of PARs
that may mediate the effects of thrombin on cytosolic calcium
mobilization, contraction, and cell proliferation in human ASM cells.
These results suggest that -thrombin may play a role in the
pathogenesis of both increased airway resistance and the structural
changes seen in asthmatic airways. Further studies are necessary to
address whether abrogation of the effects of thrombin on ASM may have a
therapeutic value in the treatment of airway inflammation seen in
diseases such as asthma, chronic bronchitis, and bronchiolitis obliterans.
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ACKNOWLEDGEMENTS |
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We thank Mary McNichol for expert assistance in preparing the manuscript.
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FOOTNOTES |
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These studies were supported by National Heart, Lung, and Blood Institute Grant R01-HL-55301; National Aeronautics and Space Administration Grant NRA-94-OLMSA-02; and a Career Investigator Award from the American Lung Association (all to R. A. Panettieri, Jr.).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Panettieri, Jr., Pulmonary and Critical Care Division, Rm. 805 East Gates Bldg., Hospital of the Univ. of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104-4283 (E-mail: rap{at}mail.med.upenn.edu).
Received 22 April 1998; accepted in final form 10 March 1999.
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