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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka, Japan (A.K., S.K., T.I., N.K., F.S., R.K.); Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada (M.D.H.); and Tokyo New Drug Research Laboratories II, Pharmaceutical Division, Kowa Company Limited, Tokyo, Japan (T.K., N.S.)
Received for publication
March 12, 2004
Accepted
June 15, 2004.
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Abstract |
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; Hollenberg and Compton, 2002). PARs are activated by proteolytic unmasking of the tethered ligand present in the N-terminal extracellular domain and, except for PAR-3, also by direct binding of exogenously applied receptor-activating peptides as short as 5 to 6 amino acids, based on the tethered ligand sequence. Among the four PAR family members, the physiological and/or pathophysiological functions of PAR-2, a receptor for trypsin, tryptase, and coagulation factors VIIa and Xa, and PAR-1, the first thrombin receptor, activated by thrombin and trypsin, have been well studied (Macfarlane et al., 2001). Both PAR-2 and PAR-1 are widely distributed in mammalian tissues, particularly throughout the alimentary tract. PAR-2 and/or PAR-1 modulate multiple gastric mucosal functions (Kawabata et al., 2001b, 2004b; Kawabata, 2002), salivary and pancreatic exocrine secretion (Bohm et al., 1996; Nguyen et al., 1999; Kawabata et al., 2000a,b), and alimentary smooth muscle motility (Corvera et al., 1997; Cocks et al., 1999b; Kawabata et al., 2001c). These two receptors also play a role in the central and peripheral nervous systems (Steinhoff et al., 2000; Kawabata et al., 2001a, 2002a,b; Vergnolle et al., 2001; Asfaha et al., 2002; Noorbakhsh et al., 2003), the cardiovascular system (Hollenberg et al., 1996; Cicala et al., 2001a; Kawabata et al., 2003), and so on.
In the respiratory system, PARs, particularly PAR-2, are abundantly expressed in airway epithelial cells in humans, mice, rats, and guinea pigs. Upon activation, PARs can enhance the formation of prostanoids, especially prostaglandin E2, resulting in tracheal and bronchial smooth muscle relaxation (Cocks et al., 1999a; Chow et al., 2000; Ricciardolo et al., 2000; Lan et al., 2001). In contrast, activation of PAR-1 and/or PAR-2 present in airway smooth muscle cells can also cause contractile responses (Cocks et al., 1999a; Ricciardolo et al., 2000; Schmidlin et al., 2001). Although a protective role for epithelial PAR-2 in the trachea or bronchus has been suggested (Cocks et al., 1999a; Cicala et al., 2001b; Moffatt et al., 2002), there is also much evidence for a pro-inflammatory role for PAR-2 in the respiratory system (Vliagoftis et al., 2000; Sun et al., 2001; Schmidlin et al., 2002). Clinical evidence suggests that PAR-2 is up-regulated in the respiratory epithelium of patients with asthma (Knight et al., 2001). Since PAR-2 antagonists have yet to be discovered, the potential physiological functions of PAR-2 have been assessed in large part with the use of receptor-selective activating peptides along with their scrambled sequence inactive controls. As an alternative, the actions of such peptides in PAR-deficient animals have proved revealing (Vergnolle et al., 2001; Ferrell et al., 2003; Kawabata et al., 2004a). To date, the exact signal transduction pathways responsible for epithelial PAR-2-triggered prostanoid-dependent airway relaxation have yet to be evaluated in any depth. To evaluate more critically a role for PAR-2 in regulating airway tone, we characterized the relaxation of tracheal and bronchial smooth muscle preparations caused by trypsin, an endogenous PAR-2 activator; Ser-Leu-Ile-Gly-Arg-Leu-amide (SLI-GRL-NH2), a mouse/rat-derived PAR-2-activating peptide; and 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide (2f-LIGRL-NH2), a novel potent PAR-2-activating peptide (Kawabata et al., 2004a). We used both ddY mice and either wild-type or PAR-2-knockout mice with a C57BL/6 genetic background, and evaluated the involvement of the cyclooxygenase isoforms, cyclooxygenase (COX)-1 and COX-2, the MEK-ERK pathway, and the p38 MAP kinase pathway in the epithelial PAR-2-mediated relaxation of mouse airway tissues.
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Materials and Methods |
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Animals. Male ddY mice (22–28 g) were obtained from Japan SLC Inc. (Shizuoka, Japan). The PAR-2-knockout mice of C57BL/6 background were generated as described previously (Ferrell et al., 2003). The PAR-2-deficient strain was maintained by backcrossing heterozygous (PAR-2+/-) males with C57BL/6 females at each generation. The genotype of the mice was confirmed by Southern blot analysis and polymerase chain reaction analysis of DNA obtained from tail biopsy. Homozygous (PAR-2-/-) and wild-type (PAR-2+/+) female mice generated from male and female PAR-2+/- mice at backcross generation 8 were used at 8 to 12 weeks of age for the experiments. All animals were used with approval by the Kinki University School of Pharmaceutical Science's Committee for the Care and Use of Laboratory Animals.
Relaxation Bioassay in Isolated Mouse Tracheal and Bronchial Smooth Muscle. Mice were sacrificed by abdominal exsanguination under urethane (1.5 g/kg i.p.) anesthesia, and the airway was removed. Ring segments of the trachea and main bronchus (4 and 2 mm in length, respectively) were prepared in an ice-cooled Krebs-Henseleit solution (composition: 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM KH2PO4,10 mM glucose), and suspended in organ baths containing 2 ml of Krebs-Henseleit solution, pH 7.4, maintained at 37°C, and gassed with 95% O2/5% CO2. The tracheal and bronchial segments were allowed to equilibrate for 1 h under a resting tension of 5 and 2.5 milliNewton, respectively, and isometric tension was recorded through a force-displacement transducer (UL-10GR; Minebea Co., Ltd., Tokyo, Japan). The integrity of the ring segment was first monitored by measuring the contractile response to cumulative concentrations of carbachol at 0.2 to 10 µM. The relaxation responses for agonists including trypsin, PAR-activating peptides, or prostaglandin E2 applied in cumulative or single concentrations were monitored in the ring preparations that had been precontracted with 1 µM carbachol. The relaxation responses were expressed as a percentage (percentage papaverine) of the relaxation to 100 µM papaverine.
Inhibition Experiments. The nonselective COX inhibitor indomethacin at 10 µM, the specific COX-1 inhibitor SC-560 at 0.3 µM, and the specific COX-2 inhibitors NS-398 at 10 µM and nimesulide at 1 µM were added to the tissue bath 30 min before precontraction with carbachol followed by application of relaxants. It has been reported that the IC50 values of SC-560, NS-398, and nimesulide are 0.0048, 28.9 to 125, and 12.5 µM for COX-1, and 1.4, 0.04 to 5.6, and 0.4 µM for COX-2, respectively (Miralpeix et al., 1997; Kato et al., 2001). The Ca2+-independent phospholipase A2 (iPLA2) inhibitor BEL at 10 µM, the cytosolic Ca2+-dependent phospholipase A2 (cPLA2) inhibitor AACOCF3 at 30 µM, and the diacylglycerol lipase inhibitor RHC-80267 at 20 µM were applied 30, 30, and 20 min, respectively, before carbachol. The nonspecific tyrosine kinase inhibitor genistein at 30 µM, the MEK inhibitor PD98059 at 10 µM, the p38 MAP kinase inhibitor SB203580 at 10 µM, and the protein kinase C inhibitor GF109203X at 1 µM were added to the bath 30, 30, 30, and 15 min, respectively, before carbachol application. It has been reported that the IC50 values are 7 µM for BEL (Takuma and Ichida, 1997), 1 µM for AACOCF3 (Choudhury et al., 2000), 5 µM for RHC-80267 (Moriyama et al., 1999), 15 µM for genistein (Tremblay et al., 1992), 0.75 to 3 µM for PD98059 (Choudhury et al., 2000; Newton et al., 2000), 0.6 µM for SB203580 (Newton et al., 2000), and 0.01 to 0.4 µM for GF109203X (Le Panse et al., 1994). In the preliminary experiments, these inhibitors did not affect the active tension induced by 1 µM carbachol.
Desensitization Experiments. For PAR-2 and/or PAR-1 desensitization, SLIGRL-NH2 at 100 µM and/or TFLLR-NH2 at 100 µM were applied twice, respectively, to the tracheal or bronchial tissues precontracted with carbachol, followed by cumulative application of trypsin. Complete desensitization of each PAR was confirmed by observing no response to the second application of the corresponding PAR agonist.
Statistics. Relaxation responses are represented as mean ± S.E.M., and EC50 values are shown with 95% confidence intervals in parentheses. Statistical significance was analyzed by Student's t test for two-group data and by Tukey's test for multiple comparisons. Significance was set at the P < 0.05 level.
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SLIGRL-NH2 in the trachea, and trypsin < 2f-LIGRL-NH2 < SLIGRL-NH2 TFLLR-NH2 in the bronchus (Table 1).
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Effects of Inhibitors of COX Isoforms, PLA2 Isoforms, and Diacylglycerol Lipase on the Tracheal and Bronchial Relaxation Caused by Receptor-Activating Peptides for PAR-2 and PAR-1 in ddY Mice. The relaxation responses to the PAR-2-activating peptide SLIGRL-NH2 or the PAR-1-activating peptide TFLLR-NH2 at 100 µM in the trachea and bronchus from ddY mice were almost completely abolished by the nonspecific COX inhibitor indomethacin at 10 µM, by the COX-1-selective inhibitor SC-560 at 0.3 µM, and by the COX-2-selective inhibitor NS-398 at 10 µM or nimesulide at 1 µM (Fig. 2). Similarly, the relaxant effect of trypsin was also blocked by SC-560 or nimesulide (data not shown). The iPLA2 inhibitor BEL at 10 µM partially (about 40–60% inhibition) blocked the relaxant effects of SLIGRL-NH2 and TFLLR-NH2 in the tracheal, but not bronchial, rings (Fig. 3). Neither the cPLA2 inhibitor AACOCF3 at 30 µM nor the diacylglycerol lipase inhibitor RHC-80267 at 20 µM significantly altered the tracheal and bronchial relaxation caused by the PAR-2- or PAR-1-activating peptide (Fig. 3).
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Effects of Inhibitors of Tyrosine Kinase, MEK, p38 MAP Kinase, and Protein Kinase C on the Tracheal and Bronchial Relaxation Caused by the PAR-2- and PAR-1-Activating Peptides in ddY Mice. The MEK inhibitor PD98059 at 10 µM nearly completely blocked the tracheal and bronchial relaxation evoked by SLIGRL-NH2 or TFLLR-NH2 at 100 µM in ddY mice (Fig. 4). The p38 MAP kinase inhibitor SB203580 at 10 µM also exerted almost complete and partial inhibition in the trachea and bronchus, respectively (Fig. 4). In contrast, the tyrosine kinase inhibitor genistein at 30 µM or the protein kinase C inhibitor GF109203X at 1 µM exhibited no significant inhibitory effects in the trachea or bronchus (Fig. 4).
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Effects of Inhibitors of iPLA2, MEK, and p38 MAP Kinase on the Tracheal or Bronchial Relaxation Caused by Prostaglandin E2 in ddY Mice. To clarify whether activation of iPLA2, MEK, and p38 MAP kinase after PAR-2 or PAR-1 stimulation is downstream or upstream of prostaglandin E2 formation, we examined effects of their inhibitors, BEL, PD98059, and SB203580, respectively, on airway smooth muscle relaxation caused by prostaglandin E2 in ddY mice. Prostaglandin E2 at 25 nM elicited relaxation responses in the tracheal and bronchial strips from the mice. Neither PD98059 nor SB203580 blocked the relaxant effects of prostaglandin E2 in the trachea and/or bronchus (Fig. 5), indicating an involvement of MEK and p38 MAP kinase upstream of prostaglandin E2 formation. In contrast, BEL significantly (P < 0.05) reduced the prostaglandin E2-evoked relaxation by approximately 30% in the tracheal preparation; the relaxation responses caused by prostaglandin E2 in the absence and presence of BEL were 47.8 ± 3.1 and 33.7 ± 3.6 (n = 6).
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Effects of Desensitization of PAR-2 and/or PAR-1 on the Trypsin-Evoked Tracheal and Bronchial Relaxation in ddY Mice. To examine whether PAR-2 and/or PAR-1 mediate trypsin-evoked airway relaxation, we performed desensitization experiments. The trypsin-evoked relaxation response was reduced slightly by desensitization of PAR-2 or PAR-1 and more dramatically by desensitization of both PAR-2 and PAR-1 in ddY mouse bronchus (Fig. 6a and Fig. 6b, bottom). The EC50 value of trypsin was significantly elevated by desensitization of both PAR-2 and PAR-1 in the bronchus, as compared with the control and that after desensitization of PAR-2 or PAR-1 alone (Table 2). In the tracheal preparation from ddY mice, desensitization of PAR-2 and/or PAR-1 tended to suppress the relaxant effect of trypsin at low, but not high, concentrations (Fig. 6b, top). However, there were no statistically significant differences in the apparent EC50 values (Table 2).
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Concentration-Related Relaxant Effects of Trypsin and PAR-2-Activating Peptides in the Tracheal and Bronchial Rings Isolated from Wild-Type and PAR-2-Knockout Mice of C57BL/6 Background. SLIGRL-NH2 or TFLLR-NH2 at 100 µM produced relaxation responses in the bronchial rings isolated from wild-type (PAR-2+/+) C57BL/6 mice (Fig. 7a), which were inhibited by indomethacin 10 µM (data not shown). The relaxation responses to 100 µM SLIGRL-NH2, but not TFLLR-NH2, disappeared in the bronchus from PAR-2-knockout (PAR-2-/-) mice (Fig. 7a). As observed in ddY mice, SLIGRL-NH2 produced concentration-dependent relaxation in both trachea and bronchus from wild-type C57BL/6 mice (Fig. 7b), the potency being much greater in the latter preparation than the former (Table 3). In contrast, the potency of 2f-LIGRL-NH2 was a little greater in the trachea than in the bronchus (Fig. 7b; Table 3). Since SLIGRL-NH2, but not 2f-LIGRL-NH2, is degraded by aminopeptidase (Kawabata et al., 2004a), the relaxant effects of SLIGRL-NH2 were also tested in the presence of amastatin, an aminopeptidase inhibitor. The concentration-relaxation curves for SLIGRL-NH2 in the trachea and bronchus were left-shifted by pretreatment with amastatin (Fig. 7b), whereas the potency was still greater in the latter preparation than in the former (Table 3). Thus, the discrepancy that the potencies of SLIGRL-NH2 and 2f-LIGRL-NH2 are "bronchus > trachea" and "trachea > bronchus," respectively, in C57BL/6 mice (Table 3), as in ddY mice (see Fig. 1; Table 1) cannot be explained by tissue differences in the expression levels of aminopeptidase. Of importance is that neither SLIGRL-NH2 nor 2f-LIGRL-NH2 caused a relaxant responses either in the trachea or the bronchus of PAR-2-knockout mice of C57BL/6 background (Fig. 7).
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Trypsin evoked tracheal and bronchial relaxation in wild-type C57BL/6 mice, the potency being 100-fold greater in the bronchial preparation than in the trachea (Fig. 7b; Table 3), in agreement with the data in ddY mice (see Fig. 1 and Table 1). The concentration-response curve for trypsin in C57BL/6 mouse trachea was not biphasic (Fig. 7b), in contrast to that in ddY mouse trachea (see Fig. 1), PAR-2-independent relaxant effects of trypsin were also observed in the trachea and bronchus from PAR-2-knockout mice, but at much higher enzyme concentrations (Fig. 7b; Table 3).
As shown in ddY mouse preparations (Figs. 2 and 4), the tracheal and bronchial relaxation responses to activation of PAR-2 and PAR-1 by SLIGRL-NH2 at 100 µM and TFLLR-NH2 at 100 µM, respectively, were completely or partially blocked by SC-560 at 0.3 µM, NS-398 at 10 µM, PD98059 at 10 µM, or SB203580 at 10 µM in wild-type C57BL/6 mouse preparations (P < 0.05). The SLIGRL-NH2-induced relaxation responses (percentage papaverine) were: 71.1 ± 8.2, 3.0 ± 2.1 and 17.0 ± 8.5 in the trachea and 81.1 ± 4.3, 46.4 ± 5.3, and 9.5 ± 1.45 in the bronchus (n = 4), when pretreated with vehicle (control), SC-560, and NS-398, respectively; and 84.1 ± 5.1 (n = 4), 14.6 ± 6.6 (n = 4), and 38.7 ± 11.0 (n = 4) in the trachea and 76.3 ± 4.1 (n = 5), 30.2 ± 7.0 (n = 4), and 45.3 ± 6.5 (n = 14) in the bronchus, when pretreated with vehicle (control), PD98059, and SB203580, respectively. The TFLLR-NH2-evoked responses (percentage papaverine) were: 54.0 ± 12.0, 0.8 ± 0.8, and 0.0 ± 0.0 (n = 4) in the trachea and 75.0 ± 8.2, 13.0 ± 6.1, and 0.0 ± 0.0 (n = 4) in the bronchus, when pretreated with vehicle (control), SC-560, and NS-398, respectively; and 72.9 ± 4.4, 16.9 ± 6.6, and 34.0 ± 10.2 (n = 4) in the trachea and 73.2 ± 4.8 (n = 5), 17.7 ± 3.4 (n = 4), and 11.9 ± 5.0 (n = 14) in the bronchus, when pretreated with vehicle (control), PD98059, and SB203580, respectively.
Effects of Desensitization of PAR-2 and/or PAR-1 on the Trypsin-Evoked Tracheal and Bronchial Relaxation in Wild-Type C57BL/6 Mice. In wild-type C57BL/6 mouse trachea and bronchus, desensitization of PAR-2 by two applications of SLIGRL-NH2 at 100 µM reduced the relaxant effect of trypsin, particularly at low concentrations, in the bronchus (Fig. 8a and 8b, bottom) and trachea (Fig. 8b, top), although statistically significant difference in EC50 values was detected in the bronchus, but not the trachea (Table 4). Interestingly, the difference between relaxant effects of trypsin in the control and PAR-2-desensitized preparations (Fig. 8b) was similar to that between the responses in the wild-type and PAR-2-knockout mice (see Fig. 7b). In contrast, PAR-1 desensitization by two applications of TFLLR-NH2 at 100 µM failed to reduce the relaxant effect of trypsin (Fig. 8b; Table 4). Desensitization of both PAR-2 and PAR-1 caused strong suppression of the trypsin-induced relaxation in either the tracheal or the bronchial strip (Fig. 8, a and b; Table 4).
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It has been reported that COX-2, but not COX-1, is involved in the PAR-2 agonist SLIGRL-NH2-evoked relaxation in the trachea from CBA/CaH mice (Lan et al., 2001) and that PAR-2 and COX-2 proteins are colocalized in basal epithelial cells in the mouse airway (Lan et al., 2004). However, our present data clearly indicate involvement of both COX isoforms in either tracheal or bronchial relaxation caused by SLIGRL-NH2 as well as the PAR-1 agonist TFLLR-NH2 in ddY mice by using the potent and highly selective inhibitors, SC-560, for COX-1, and NS-398 or nimesulide, for COX-2. The differences between our data and those of Lan et al. (2001) may possibly be due to differences in the strain and age of mice and the effectiveness of the COX inhibitors used. Nonetheless, our results suggest that both constitutively expressed COX-1 and COX-2 are necessary for prostanoid production following activation of PAR-2 and PAR-1, as described previously in the PAR-1-mediated chloride secretion in intestinal epithelial cells (Buresi et al., 2002). It is likely that prostanoids formed by one of the COX isoforms might enhance activity of the other COX isoform, as described recently (Yamada et al., 2004). Although the inhibitors for COX-1 and COX-2 were used at very specific concentrations, we cannot completely rule out the possibility that they affected prostanoid formation through unknown mechanisms other than inhibition of COX-1 and COX-2. In contrast, the pathways for production of arachidonic acid, a substrate for COX, following PAR-2 or PAR-1 stimulation could not be clearly identified by the present study. The finding that BEL produced 40 to 60% inhibition of the tracheal relaxation caused by activation of PAR-2 or PAR-1 may be in agreement with evidence for involvement of iPLA2 in PAR-2-mediated contraction of rat urinary bladder (Kubota et al., 2003). However, the inhibitory effect could be a result of nonspecific actions of BEL, because BEL also caused about 30% inhibition of the prostaglandin E2-evoked tracheal relaxation. Neither cPLA2 nor diacylglycerol lipase appears to contribute to the PAR-2- and PAR-1-mediated airway relaxation in ddY mice, although the former enzyme is involved in PAR-1-mediated intestinal chloride secretion (Buresi et al., 2002). It is most interesting that PD98059 and SB203580, but not genistein or GF109203X, almost completely inhibited the tracheal and bronchial relaxant effects of PAR-2- and PAR-1-activating peptides, suggesting the involvement of the MEK-ERK pathway and the p38 MAP kinase pathway, but not tyrosine kinase or protein kinase C. Both pathways are considered upstream of prostaglandin formation, as suggested by the lack of the effects of those inhibitors on the relaxation responses to prostaglandin E2, known to be responsible for the PAR-mediated tracheal relaxation in mice (Lan et al., 2001). Since the prostanoid-dependent airway relaxation following PAR-2 and PAR-1 stimulation occurs within several seconds and peaks within a few minutes, the underlying mechanisms should involve acute nontranscriptional signals mediated by the MEK-ERK pathway and p38 MAP kinase. There is evidence that activation of either or both of the two pathways participates in smooth muscle contraction (Zheng et al., 1998; Bolla et al., 2002; Harnett and Biancani, 2003). Our evidence that both the MEK and p38 MAP kinase are upstream of facilitation of prostanoid formation through nontranscriptional mechanisms may be in keeping with the previous report suggesting the involvement of MEK in PAR-1-mediated prostanoid-dependent gastric smooth muscle contraction (Zheng et al., 1998). In certain vascular tissues, p38 MAP kinase can be downstream of the constrictor action of an arachidonate metabolite like thromboxane (Bolla et al., 2002).
It is of note that trypsin-evoked tracheal and bronchial relaxation in mice is mediated only in part by PAR-2, as indicated by the data from PAR-2-knockout mice and the results from receptor desensitization experiments. Since the latter experiments also suggest minor or no involvement of PAR-1 in the effect of trypsin, mechanisms other than PAR-2 and PAR-1 should also be considered. The trypsin-evoked residual airway relaxation independent of PAR-2 and PAR-1 might be explained, in part, by activation of PAR-4 that is sensitive to trypsin, because PAR-4, upon activation, also causes small but significant relaxation in mouse trachea (Lan et al., 2001). Particularly, the biphasic concentration-relaxation curve for trypsin in ddY mouse trachea (see Figs. 1 and 6b) may imply involvement of multiple receptors. To our surprise, we observed a significant difference in trypsin responsiveness between the trachea and main bronchus isolated from the same animals, the apparent EC50 values being 1.93 and 0.0501 µM (approximately 960 and 25 U/ml) in ddY mice and 0.303 and 0.0130 µM (approximately 150 and 7 U/ml) in C57BL/6 wild-type mice, respectively. It has been reported that CBA/CaH mouse trachea relaxed in response to trypsin at a high concentration, 100 U/ml (Lan et al., 2001), and that the BALB/c mouse bronchus was greatly dilated by trypsin even at 0.3 U/ml (Cocks et al., 1999a). The ddY mouse trachea was even less sensitive to trypsin than the C57BL/6 mouse trachea (see Figs. 1 and 7b). In rats, trypsin at 10 U/ml is incapable of inducing relaxation in the trachea, main bronchus, or intrapulmonary bronchus, although a PAR-2-activating peptide causes clear responses in these preparations (Chow et al., 2000). In contrast, guinea pig trachea and main bronchus exhibit similar relaxation responses to trypsin at 0.001 to 0.1 µM (Ricciardolo et al., 2000). The physiological/pathophysiological meaning of regional tissue differences in addition to species difference, as described above, is an open question. It is possible that the expression levels of trypsin-inhibitory serpins might be different between the trachea and bronchus in the mice, leading to distinct trypsin sensitivity. On the other hand, the differences of sensitivities to PAR-2-activating peptides between the trachea and bronchus are even more complicated, since the potencies of SLIGRL-NH2 and 2f-LIGRL-NH2 were bronchus > trachea and trachea > bronchus, respectively, in either ddY or C57BL/6 mice. Although the former peptide but not the latter is sensitive to aminopeptidase degradation (Kawabata et al., 2004a), our data from experiments using the aminopeptidase inhibitor amastatin show that the distinct peptide sensitivity in the trachea and bronchus cannot be explained by tissue differences in expression levels of aminopeptidase. The possible involvement of other degradative enzymes including carboxypeptidase remains to be investigated. In general, regulation of the tracheal contractility by PARs might not directly contribute to respiratory functions, compared with PAR modulation of the bronchial contractility. However, given the presence of exogenous agonist proteinases from environmental origins, such as mite allergens (Sun et al., 2001), information about characteristics of epithelial PARs in the trachea, as evidenced by the present smooth muscle assay, may be important to understand the pro- and/or anti-inflammatory roles for PARs in pathological conditions. The hypothesis that the trachea is more abundant in trypsin-inhibitory serpins in the epithelium than in the bronchus, as described above, may be reasonable, because the tracheal epithelium would be first exposed to exogenous proteinases that inhaled organisms might have. There were also some strain differences between airway relaxation responses to PAR agonists in ddY mice and C57BL/6 mice. In particular, trypsin caused biphasic relaxation in ddY mouse trachea, but relatively simple responses in C57BL/6 mouse trachea. However, our data from desensitization experiments and evidence from PAR-2-knockout mice clearly show that the relaxation responses to trypsin in C57BL/6 mouse trachea also contain two distinct components that are PAR-2-dependent and -independent.
In conclusion, our study suggests novel aspects of PAR-2- and PAR-1-triggered signaling pathways involved in mouse airway relaxation, including MEK and p38 MAP kinase, and provides unequivocal evidence for the involvement of PAR-2 in the tracheal and bronchial relaxation caused by PAR-2-activating peptides and, in part, for the responses to trypsin. Furthermore, mouse trachea and bronchus appear to show complex distinct sensitivities to PAR-2 agonists including trypsin and PAR-2-activating peptides.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAR, proteinase-activated receptor; AACOCF3, arachidonyl trifluoromethyl ketone; BEL, bromoenol lactone; COX, cyclooxygenase; cPLA2, cytosolic Ca2+-dependent phospholipase A2; 2f-LIGRL-NH2, 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide; iPLA2, Ca2+-independent phospholipase A2; SLIGRL-NH2, Ser-Leu-Ile-Gly-Arg-Leu-amide; TFLLR-NH2, Thr-Phe-Leu-Leu-Arg-amide; MEK, mitogen-activated protein kinase kinase; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide; PD98059, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl) imidazole; GF109203X, 3-(1-(3-(dimethylamino)propyl)-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; RHC-80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane.
Address correspondence to: Atsufumi Kawabata, Division of Physiology and Pathophysiology, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan. E-mail: kawabata{at}phar.kindai.ac.jp
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