alpha -Thrombin stimulates contraction of human bronchial rings by activation of protease-activated receptors

R. W. Hauck1, C. Schulz1, A. Schömig1, R. K. Hoffman2, and R. A. Panettieri Jr.2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha -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 alpha -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 alpha -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha -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 alpha -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 alpha -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 muscle alpha -actin primer pairs were used. PCR was run for 30 cycles at 94°C denaturation, 56°C annealing, and 72°C extension with AmpliTaq (Perkin-Elmer) DNA polymerase. Reaction products were run on 1% agarose gels. Each primer pair produced a specific size product: PAR1, 1,247 bp; PAR2, 563 bp; PAR3, 1,302 bp; SM22, 782 bp; and alpha -actin, 188 bp. Northern analysis was performed as previously described by our laboratory (15, 23). The PCR analyses were performed from triplicate experiments with three different human ASM cell lines.

Tissue 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-specific alpha -actin received an additional incubation with 0.5% Triton X-100 to facilitate entry of the anti-alpha -actin antibody to the interior of the smooth muscle cells; sections to be stained with anti-PAR antibodies were not exposed to detergent before antibody incubation. All sections were blocked with 0.5% blocking reagent [Tyramide Signal Amplification (TSA) kit, NEN Life Sciences, Boston, MA] in 150 mM NaCl and 100 mM Tris-Cl, pH 7.5 [Tris-buffered saline (TBS)] for 30 min at room temperature. Sections were incubated in primary antibody solutions diluted in 0.5% blocking reagent in TBS for 1 h at room temperature in humid chambers. The antibody to smooth muscle-specific alpha -actin was purchased from Sigma (clone 1A4; St. Louis, MO). Monoclonal antibodies to PAR1 (ATAP 2) and PAR2 (SAM 11) were a gift from Dr. Lawrence F. Brass (Department of Medicine, University of Pennsylvania) (9, 10). After incubation in primary antibody solutions, the sections were washed three times in TBS, then incubated in 2 µg/ml of goat anti-mouse IgG conjugated to biotin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. After secondary antibody incubation, the sections were washed three times in TBS, then exposed to streptavidin-horseradish peroxidase (HRP) for 30 min as per the protocol of the TSA kit. After streptavidin-HRP, the sections were washed three times with TBS, then reacted with rhodamine-tyramide for 10 min. After completion of the tyramide reaction, the sections were washed three times with TBS with 0.5% Tween 20, then two more times with 0.9% NaCl. Next, the sections were incubated with 0.5 µg/ml of 4',6-diamidino-2-phenylindole (Polysciences, Warrington, PA) for 2 min to stain the nuclei, rinsed two times with 0.9% NaCl and two times with TBS-Tween 20, and then mounted in 80% glycerol in TBS.

The 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Thrombin Increases Isometric Tension in Human Bronchial Rings

To study the bronchoconstrictor effects of alpha -thrombin, human bronchial rings were pretreated with alpha -thrombin with cumulative additions (1-20 U/ml), and isometric tension was measured. As shown in Fig. 1A and Table 1, alpha -thrombin (20 U/ml) stimulated a maximal increase in isometric force in intact bronchi to 19 ± 3% above baseline (P = 0.008). This represents ~20% (20 ± 8%) of the maximal carbachol response, with an EC50 of 12.2 U/ml (9.9-15.3 U/ml). alpha -Thrombin induced force generation that was significantly different compared with the control value (P = 0.002). In experiments using epithelium-denuded rings, alpha -thrombin induced contraction to levels comparable with those in intact epithelium (P = 0.085). The force generation of alpha -thrombin on epithelium-denuded bronchi was 15 ± 4% of the maximal carbachol response [EC50 13.3 U/ml (9.5-17.1 U/ml)].



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   A: intact () and epithelium-denuded () human bronchi contractd by alpha -thrombin. As a control, bronchi were incubated in Tyrode solution (black-diamond ) over the same time. Values are means ± SE of results from 5 different experiments and 8 control experiments. B: intact human bronchi contracted by thrombin receptor-activating peptide (TRAP-14; ). As a control, bronchi were incubated in Tyrode solution (black-diamond ) over the same time. Values are means ± SE of results from 5 different experiments and 8 control experiments.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Maximal contractile effect of different agonists on basal tone of human bronchi

alpha -Thrombin Stimulates Isometric Force in a Receptor-Specific Manner

To examine the specificity of alpha -thrombin effects on bronchoconstriction, rings were pretreated with TRAP-14 (Table 1). TRAP-14 is a synthetic thrombin receptor-activating peptide that functions as a thrombin-receptor agonist (15, 16). In a dose-dependent manner, TRAP-14 increased isometric force in intact human bronchial rings, with a maximum effect seen at 100 µmol/l (Fig. 1B). The increase in force generation was 14 ± 2% (P = 0.0005; Table 1) and was significantly different from the control value (P = 0.002). There was no difference between force generation in rings treated with alpha -thrombin compared with those treated with TRAP-14.

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 alpha -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.


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   Protease-activated receptor (PAR) expression analyzed by RT-PCR of total RNA extracted from cultured human airway smooth muscle (HASM) cells. Lane 1, smooth muscle alpha -actin (188 bp); lane 2, SM22 (782 bp); lane 3, PAR1 (1,247 bp); lane 4, PAR2 (563 bp); lane 5, PAR3 (1,302 bp). This is a representative experiment repeated on 3 occasions with different HASM cell lines.

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 alpha -actin. With the use of a monoclonal antibody specific to smooth muscle alpha -actin, ASM below the epithelium was identified in sections of the human bronchus (Fig. 3A). When bronchial sections were incubated with no primary antibody but with the secondary antibody followed by rhodamine-TSA, no specific staining was seen, although some nonspecific staining of elastin fibers can be observed (Fig. 3B). Immunostaining with the monoclonal antibody to PAR1 revealed a patchy distribution over the ASM cells and a high level of staining among the cells of the epithelium (Fig. 3C). Immunolocalization of PAR2 (Fig. 3D) showed a more even distribution over the entire surface of the ASM, but staining of the epithelium was limited to only the most superficial cells. At higher magnification, the patchy distribution of PAR1 (Fig. 3E) is recognizable as discontinuous areas of staining on the surface of the ASM, in contrast to the continuous layer of PAR2 staining along the surface of the smooth muscle cells (Fig. 3F). PAR1 and PAR2 revealed a different distribution on cultured human ASM. Immunolocalization of PAR1 showed a well-distributed punctate pattern over the entire surface of human ASM cells in culture (Fig. 3G). PAR2 had a less extensive area of distribution on the human ASM cell surface (Fig. 3H). Taken together, these data suggest that PAR1 and PAR2 proteins are present on cultured ASM cells and on ASM in the human bronchus.


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3.   Immunolocalization of PAR1 and PAR2 as shown by immunostaining of frozen sections of human bronchus (A-F) and cultures of HASM (G and H). 4',6-Diamidino-2-phenylindole-stained nuclei appear blue. A: localization of smooth muscle-specific alpha -actin (rhodamine-tyramide) in section of bronchus. B: negative control shows slight staining of elastin fibrils by rhodamine-tyramide detection system (no primary antibody was used). C: PAR1 (rhodamine-tyramide) localized to surface of HASM. D: PAR2 (rhodamine-tyramide) localized to surface of HASM. Bars in A-D, 50 µm. E: higher magnification of PAR1 (rhodamine-tyramide) localization on HASM cell surfaces. F: higher magnification of PAR2 (rhodamine-tyramide) on HASM cell surfaces. G: PAR1 (streptavidin-Texas Red) distributed on surface of HASM grown in vitro. H: PAR2 (streptavidin-Texas Red) detected on surface of HASM grown in vitro. Bars in E-H, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -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.


    ACKNOWLEDGEMENTS

We thank Mary McNichol for expert assistance in preparing the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmed, T., J. Garrigo, and I. Danta. Preventing bronchoconstriction in exercise-induced asthma with inhaled heparin. N. Engl. J. Med. 329: 90-95, 1993[Abstract/Free Full Text].

2.   Al-Ani, B., M. Saifeddine, and M. D. Hollenberg. Detection of functional receptors for the proteinase-activated-receptor-2-activated polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. Can. J. Physiol. Pharmacol. 73: 1203-1207, 1995[Medline].

3.   Barnes, P. J. New therapeutic approaches. Br. Med. Bull. 48: 231-247, 1991[Abstract].

4.   Barnes, P. J., K. F. Chung, and C. P. Page. Inflammatory mediators and asthma. Pharmacol. Rev. 40: 49-84, 1988[Medline].

5.   Böhm, S. K., L. M. Khitin, E. F. Grady, G. Aponte, D. G. Payan, and N. W. Bunnett. Mechanisms of desensitization and resensitization of proteinase-activated receptor-2. J. Biol. Chem. 271: 22003-22016, 1996[Abstract/Free Full Text].

6.   Böhm, S. K., W. Kong, D. Bromme, S. P. Smeekens, D. C. Anderson, A. Connolly, M. Kahn, N. A. Nelken, S. R. Coughlin, D. G. Payan, and N. W. Bunnett. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem. J. 314: 1009-1016, 1996[Medline].

7.   Brass, L. F., D. R. Manning, A. Williams, M. J Woolkalis, and M. Poncz. Receptor and G protein-mediated responses to thrombin in HEL cells. J. Biol. Chem. 266: 958-965, 1991[Abstract/Free Full Text].

8.   Brass, L. F., and M. Molino. Protease-activated G protein-coupled receptors on human platelets and endothelial cells. Thromb. Haemost. 78: 234-241, 1997[Medline].

9.   Brass, L. F., S. Pinzano, M. Ahuja, E. Belmonte, N. Blanchard, J. M. Stadel, and J. A. Hoxie. Changes in the structure and function of the human thrombin receptor during activation, internalization, and recycling. J. Biol. Chem. 269: 2943-2952, 1994[Abstract/Free Full Text].

10.   Brass, L. F., R. R. Vasello, Jr., E. Belmonte, M. Ahuja, K. Chichowski, and J. A. Hoxie. Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. J. Biol. Chem. 267: 13795-13798, 1992[Abstract/Free Full Text].

11.   Chen, L. B., and J. M. Buchanan. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc. Natl. Acad. Sci. USA 72: 131-135, 1995.

12.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

13.   Chung, K. F., and P. J. Barnes. PAF antagonists. Their potential therapeutic role in asthma. Drugs 35: 93-103, 1988[Medline].

14.   Chung, K. F., D. F. Rogers, P. J. Barnes, and T. W. Evans. The role of increased airway microvascular permeability and plasma exudation in asthma. Eur. Respir. J. 3: 329-337, 1990[Abstract].

15.   Cohen, M. D., V. Ciocca, and R. A. Panettieri. TGF-beta 1 modulates human airway smooth muscle cell proliferation induced by mitogens. Am. J. Respir. Cell Mol. Biol. 16: 85-90, 1997[Abstract].

16.   Coughlin, S. R. Thrombin receptor structure and function. Thromb. Haemost. 66: 184-187, 1993.

17.   Daniel, T. O., V. C. Gibbs, D. F. Milfay, M. R. Garavoy, and L. T. Williams. Thrombin stimulates c-cis gene expression in microvascular endothelial cells. J. Biol. Chem. 261: 9579-9582, 1986[Abstract/Free Full Text].

18.   Dunhill, M. S. The pathology of asthma, with special reference to the changes in the bronchial mucosa. J. Clin. Pathol. 13: 27-33, 1960.

19.   Hauck, R. W., M. Harth, C. Schulz, M. Böhm, and A. Schömig. Effects of beta 2-agonist- and dexamethasone-treatment on relaxation and regulation of beta -adrenoceptors in human bronchi and lung tissue. Br. J. Pharmacol. 121: 1523-1530, 1997[Abstract].

20.   Hollenberg, M. D., M. Saifeddine, B. Al-Ani, and A. Kawabata. Proteinase-activated receptors: structural requirements for activity, receptor cross-reactivity, and receptor selectivity of receptor-activating peptides. Can. J. Physiol. Pharmacol. 75: 832-841, 1997[Medline].

21.   Ishihara, H., A. J. Connolly, D. Zeng, M. L. Kahn, Y. W. Zheng, C. Timmons, T. Tram, and S. R. Coughlin. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386: 502-506, 1997[Medline].

22.   James, A. L., P. D. Paré, and J. C. Hogg. The mechanics of airway narrowing in asthma. Am. Rev. Respir. Dis. 139: 242-246, 1989[Medline].

23.   Krymskaya, V. P., R. Hoffman, A. Eszterhas, S. Kane, V. Ciocca, and R. A. Panettieri, Jr. EGF activates ErbB-2 and stimulates phosphatidylinositol 3-kinase in human airway smooth muscle cells. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L246-L255, 1999[Abstract/Free Full Text].

24.   Ku, D. D., and J. K. Zaleski. Receptor mechanism of thrombin-induced endothelium-dependent and endothelium-independent coronary vascular effect in dogs. J. Cardiovasc. Pharmacol. 22: 609-616, 1993[Medline].

25.   Marsen, T. A., M. S Simonson, and M. J. Dunn. Thrombin induces the preproendothelin-1 gene in endothelial cells by a protein tyrosine kinase-linked mechanism. Circ. Res. 76: 987-995, 1995[Abstract/Free Full Text].

26.   Milner, P., and G. Burnstock. Chronic sensory denervation reduces thrombin-stimulated endothelin release from aortic endothelial cells. Experientia 52: 242-244, 1996[Medline].

27.   Nystedt, S., K. Emilsson, A. K. Larsson, B. Strömbeck, and J. Sundelin. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur. J. Biochem. 232: 84-89, 1995[Abstract].

28.   Nystedt, S., K. Emilsson, C. Wahlestedt, and J. Sundelin. Molecular cloning of a potential proteinase activated receptor. Proc. Natl. Acad. Sci. USA 91: 9208-9212, 1994[Abstract/Free Full Text].

29.   Panettieri, R. A. Cellular and molecular mechanisms regulating airway smooth muscle cell proliferation and cell adhesion molecule expression. Am. J. Respir. Crit. Care Med. 158: S133-S140, 1998[Abstract/Free Full Text].

30.   Panettieri, R. A., L. R. DePalo, R. K. Murray, P. A. Yadvish, and M. I. Kotlikoff. A human airway smooth muscle cell line that retains physiological responsiveness. Am. J. Physiol. 256 (Cell Physiol. 25): C329-C335, 1989[Abstract/Free Full Text].

31.   Panettieri, R. A., and M. M. Grunstein. Airway smooth muscle hyperplasia and hypertrophy. In: Asthma, edited by P. J. Barnes, M. M. Grunstein, A. R. Leff, and A. J. Woolcock. New York: Lippincott-Raven, 1997, p. 823-842.

32.   Panettieri, R. A., I. P. Hall, C. S. Maki, and R. K. Murray. alpha -Thrombin increases cytosolic calcium and induces human airway smooth muscle cell proliferation. Am. J. Respir. Cell Mol. Biol. 13: 205-216, 1995[Abstract].

33.   Prescott, S. M., G. A. Zimmermann, and T. M. McIntyre. Human endothelial cells in culture produce platelet-activating factor when stimulated by thrombin. Proc. Natl. Acad. Sci. USA 81: 3534-3538, 1984[Abstract].

34.   Rabiet, M. J., J. L. Plantier, and E. Dejana. Thrombin-induced endothelial cell dysfunction. Br. Med. Bull. 50: 936-945, 1994[Abstract].

35.   Rabiet, M. J., L. L. Plantier, Y. Rival, Y. Genoux, M. G. Lampugnani, and E. Dejana. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler. Thromb. Vasc. Biol. 16: 488-496, 1996[Abstract/Free Full Text].

36.   Rasmussen, U. B., V. Vouret-Craviari, S. Jallat, Y. Schlesinger, G. Pages, A. Pavirani, J. P. Lecocq, J. Pouyssegur, and E. Van Obberghen-Schilling. cDNA cloning and expression of a hamster alpha -thrombin receptor coupled to Ca2+ mobilization. FEBS Lett. 288: 123-128, 1991[Medline].

37.   Santulli, R. J., C. K. Derian, A. L. Darrow, K. A. Tomko, A. J. Eckardt, M. Seiberg, R. M. Scarborough, and P. Andrade-Gordon. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc. Natl. Acad. Sci. USA 92: 9151-9155, 1995[Abstract].

38.   Schini, V. B., H. Hendrickson, D. M. Heublein, J. C. Burnett, and P. M. Vanhoutte. Thrombin enhances the release of endothelin from cultured porcine aortic cells. Eur. J. Pharmacol. 165: 333-334, 1989[Medline].

39.   Tomlinson, P. R., J. W. Wilson, and A. G. Stewart. Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture. Br. J. Pharmacol. 111: 641-647, 1994[Abstract].

40.   Vu, T.-K. H., D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057-1068, 1991[Medline].


Am J Physiol Lung Cell Mol Physiol 277(1):L22-L29
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society