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INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Pharmacology, University of Melbourne, Parkville, Australia (H.T.A., R.W.B., T.M.C.); Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia (D.P.F.); and Sackler Institute of Pulmonary Pharmacology, King's College, London, United Kingdom (J.D.M.)
Received October 13, 2005; accepted January 3, 2006.
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Abstract |
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; Asokananthan et al., 2002; De Campo and Henry, 2005), which when activated by either trypsin or the mouse PAR2 peptide SLIGRL, release prostaglandin E2 (PGE2) (Lan et al., 2001; Asokananthan et al., 2002; De Campo and Henry, 2005) to cause airway smooth muscle relaxation (Cocks et al., 1999; Chow et al., 2000; Lan et al., 2001; De Campo and Henry, 2005) and inhibition of immune cell trafficking (Moffatt et al., 2002; De Campo and Henry, 2005). However, some effects of PAR2 peptides have been claimed to be independent of PAR2 activation (Moffatt, 2004), including mediator release from mast cells (Stenton et al., 2002) and contractions in endothelium-intact umbilical vein tissue (Saifeddine et al., 1998). In mice, which are commonly used to model airway inflammatory disease, precise roles for PAR2 in airway physiology and pathology remain controversial. Although some studies have shown clear anti-inflammatory protective effects, including a range of PGE2-dependent mechanisms (Cocks et al., 1999; Cocks and Moffatt, 2000; De Campo and Henry, 2005), others have reported proinflammatory actions of PAR2 in the airways (Schmidlin et al., 2002; Ebeling et al., 2005; Su et al., 2005). One of the main roles for epithelial PAR2 seems to be activation of mucosal chloride (Cl–) secretion. This has been shown to occur in dog pancreatic duct epithelial cells (Nguyen et al., 1999), rat jejunum (Vergnolle et al., 1998), porcine ileum (Green et al., 2000), mouse distal colon (Cuffe et al., 2002), and monolayers of human cultured bronchial epithelial cells (Danahay et al., 2001). Such an increase in Cl– secretion would maintain the protective layer underneath the mucus and as such aid in the efficient removal of inhaled antigens and has been proposed as a possible protective role of epithelial PAR2 (Cocks and Moffatt, 2000). In the mouse trachea, Kunzelmann et al. (2005) reported that PAR2 located on the basolateral surface of epithelial cells couple to basolateral K+ channels and mucosal Cl– channels to mediate net Cl– secretion into the lumen of the airways. They also suggested that these basolateral PAR2 as well as apical PAR2 are coupled to the production of PGE2 to mediate smooth muscle relaxation. As in our previous study (Cocks et al., 1999), Kunzelmann et al. (2005) similarly reported that PAR2 are predominantly expressed on the apical surface of mouse tracheal epithelial cells. Activation of these apically expressed PAR2, however, with either trypsin or SLIGRL, did not have any effect on ion conductance (Kunzelmann et al., 2005). In the present study we found an increase in Cl– conductance to SLIGRL in the mouse trachea that was not PGE2-dependent and was not mimicked by trypsin or affected by prior exposure to high concentrations of trypsin. In addition, the supposedly inactive PAR2 peptide LSIGRL (Lindner et al., 2000; Fiorucci et al., 2001; Stenton et al., 2002) caused a similar increase in Cl– conductance to SLIGRL. Furthermore, the increase in Cl– conductance across the epithelium in response to SLIGRL was blocked by three structurally different NK1R antagonists, SR 140333, GR205171, and GR82334. Thus, results from our study indicate that PAR2-like peptides directly activate apical NK1R coupled to Cl– secretion and as such may need to be considered when roles for PAR2 are claimed, particularly in inflammatory diseases of the airways. |
Materials and Methods |
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Epithelial Ion Conductance Studies: Ussing Chamber Assay. Male BALB/c mice (30 g) were killed by CO2 asphyxiation, and their tracheae were rapidly dissected, cut into sheets via an incision through the dorsal part of the tracheal cartilage rings, and mounted between two plastic sheets with an aperture of 0.03 cm2. The preparations were then mounted in Lucite Ussing chambers, allowing separate perfusion of each side of the preparations with Krebs' solution (143.1 mM Na+, 5.9 mM K+, 2.5 mM Ca2+, 1.2 mM Mg2+, 127.8 mM Cl–, 25.0 mM HCO–3, 1.2 mM SO2–4, 1.2 mM H2PO–4, and 11.0 mM glucose) heated, and continuously bubbled with 95% O2/5% CO2 via gas lift to maintain pH 7.4 and temperature at 37°C. Electrodes were placed in each side of the Ussing chambers and connected to an amplifier (DVC 1000; World Precision Instruments, Sarasota, FL) The preparations were then clamped at a potential difference of 0 V to record short-circuit current (ISC), a measure of the net ionic flux across the preparation. Once mounted, the preparation was allowed to equilibrate (>20 min) until ISC was stable, and any antagonists to be used were added for at least 15 min before challenge with agonists.
Mouse Tracheal and Rat Aorta Smooth Muscle Contractility Studies. Mouse tracheae (isolated as described above) and thoracic aortae from male Sprague-Dawley rats (200–300 g) were cut into 3-mm rings and mounted on stainless steel hooks in Krebs' solution-filled organ baths maintained at 37°C and continuously bubbled with 95% O2/5% CO2. One hook was attached to a micrometer-driven anchor in the bath, whereas the other hook was attached to a force transducer for isometric recording of aortic and trachealis muscle tone. After a 30-min equilibration period, 0.5 and 1.0 g of tension were applied to the trachea and aorta, respectively, and the preparations then were allowed to return to a steady plateau (>20 min) before the maximal contractile capacity of the preparation (Fmax) was determined by contracting the trachea with 10 µM acetylcholine and the aorta with high K+ (120 mM) isotonic Krebs' solution. After Fmax had been obtained, the preparations were washed repeatedly and allowed to equilibrate at baseline passive tension for a further 30 min in the presence of 0.3 µM nifedipine to reduce spontaneous phasic contractile activity. The preparations were then contracted with titrated concentrations of carbachol (trachea) or phenylephrine (aorta) until a stable, active tension equivalent to approximately 40 to 50% Fmax was reached and allowed to stabilize before cumulative additions of agonists were added to the bath.
Intracellular Ca2+ Measurement in A549 Cells. Ca2+ fluorescence was measured with a FLUOstar plate reader (BMG LabTechnologies, Mornington, VIC, Australia). A549 human epithelial cells (American Type Culture Collection, Manassas, VA) were grown to 
80% confluence and harvested with nonenzymatic cell dissociation solution (Sigma Chemical) and rinsed with Dulbecco's modified Eagle's medium with HEPES (Gibco, Grand Island, NY). Pelleted cells were resuspended in a Krebs-HEPES buffer with 1 mM probenecid (Sigma Chemical) at pH 7.4. Cells were then loaded with 2 µM Fura-2/acetoxymethyl ester (Molecular Probes, Eugene, OR) with 0.01% Pluronic F127 to facilitate more even dispersion of the dye for 30 min at 37°C and 5% CO2. After this incubation, cells were rinsed twice and resuspended in the Krebs-HEPES buffer and left for 20 min at room temperature for de-esterification. For experiments testing responses in the presence of any antagonists, cells were preincubated with the specific antagonist at room temperature for the final 10 min of de-esterification. At the completion of this procedure, all cells were placed on ice until use to reduce leakage of dye. Cells were plated into black 96-well plates (BMG LabTechnologies) at a density of 2 x 106 cells · ml–1. Microplates were then inserted into the preincubated plate reader (37°C). Estimation of [Ca2+]i was achieved by exposure of the cells to rapidly alternating 340- and 380-nm wavelengths and measuring the resulting 510-nm emission from the Fura-2/acetoxymethyl ester. After a 30-s baseline reading, test compounds were injected via the plate reader's prefitted injectors into each specified well.
Statistics. All responses are expressed as means ± S.E.M. ISC responses are expressed as a ratio of the surface area of the preparation (microamperes per square centimeter; assuming no edge damage). In the tracheal and aortic smooth muscle contractility studies, relaxant responses are expressed as a percentage of carbachol- or phenylephrine-induced tone. Where possible, pEC50 values were determined by nonlinear regression analysis using Prism 4.0a (GraphPad Software, San Diego, CA). Statistical comparisons were performed with either paired or nonpaired Student's t tests or analysis of variance (ANOVA) as appropriate (Snedecor and Cochran, 1989).
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Results |
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; Chow et al., 2000; Lan et al., 2001). Similar relaxant responses produced by the PAR2 peptide agonist, the isolated N-terminal hexapeptide of rodent PAR2 SLIGRL (0.1–30 µM), were prevented if the preparation was initially desensitized to trypsin (3 U · ml–1) (Fig. 1). However, in the Ussing chamber assay, using an identical protocol, trypsin failed to either elicit a response or prevent ISC responses to SLIGRL (Fig. 1) when added to either side of the preparation (not shown) at concentrations 10-fold in excess of those required for 50% relaxation. SLIGRL was only active if applied to the mucosal surface of the preparations (Fig. 2). A 33-fold higher concentration of trypsin (100 U · ml–1) caused a small, slowly developing increase in ISC (Fig. 1), again only if applied to the mucosal surface (Fig. 2). However, at the plateau of this response, the addition of SLIGRL elicited a further, sharp increase in ISC that was not different in magnitude to the response to SLIGRL alone, suggesting that trypsin and SLIGRL do not act via the same mechanism (Fig. 1).
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) with EC50 of 0.5 ± 0.1 U · ml–1 and pEC50 of 5.6 ± 0.1, respectively. Two novel PAR2 peptide agonists, SLIGRT (pEC50 5.9 ± 0.2) and SLCGRL (pEC50 5.3 ± 0.2), were approximately 6- and 20-fold less potent, respectively, than SLIGRL (pEC50 6.4 ± 0.1) in causing endothelium-dependent relaxation of the rat aorta preparation (Fig. 3), which has previously been shown to relax in response to SLIGRL via PAR2 in an endothelium-dependent manner (Hwa et al., 1996; Saifeddine et al., 1998). In addition, we confirmed that a recently reported stable PAR2 peptidomimetic agonist, 2-furoyl-LIGRL, was more potent (pEC50 7.3 ± 0.1) than SLIGRL in this assay (Kawabata et al., 2004). By contrast, another novel PAR2 peptide agonist, SLSGRT, and the commonly used partial scramble peptide for SLIGRL, LSIGRL (Lindner et al., 2000; Fiorucci et al., 2001; Stenton et al., 2002), were both inactive in the aorta up to 100 µM (Fig. 3). Thus, an estimated rank order of potency for these peptides in the rat aortic endothelium-dependent relaxation assay was 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL, with LSIGRL and SLSGRT inactive. Also in the aorta, responses to 2-furoyl-LIGRL, SLIGRL, SLIGRT, and SLCGRL were abolished if preparations were desensitized to trypsin (3 U · ml–1; data not shown), suggesting that all peptides activate PAR2. In the Ussing chamber mouse tracheal assay, however, SLCGRL, SLIGRT, and LSIGRL, but not SLSGRT, induced ISC increases over concentration ranges similar to those of SLIGRL. 2-Furoyl-LIGRL, however, seemed to be more potent than any of the other active peptides but with a maximal increase in ISC of only 9.0 ± 3.0 µA · cm–2 at a concentration of 3 µM (Fig. 3). Therefore, an apparent rank order of potency of the peptides was 2-furoyl > SLCGRL > SLIGRT = SLIGRL > LSIGRL and SLSGRT inactive (Fig. 3), with 2-furoyl-LIGRL appearing to act as a partial agonist.
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Involvement of Cl– Conductance and Prostanoids in ISC Responses to SLIGRL. Replacement of Cl– with gluconate abolished ISC responses to SLIGRL (Fig. 4). In addition, of glibenclamide (100 µM) and DIDS (100 µM), inhibitors of cystic fibrosis transmembrane conductance regulator and Ca2+-activated Cl– channels, respectively, only glibenclamide inhibited the response to SLIGRL by approximately 50%. In addition, combined glibencamide and DIDS reduced the response to SLIGRL by 80% (Fig. 4). Indomethacin (3 µM) had no significant effect on the ISC response curve to SLIGRL (Fig. 5).
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10 pM and was maximum at approximately 1 nM (Fig. 5). The selective NK1R antagonist SR 140333 (0.1 µM) abolished the first phase of the substance P curve and caused an estimated approximate 1000-fold shift to the right in the second component (Fig. 5). SR 140333 (0.1 µM) had no effect on the increase in ISC to a submaximal concentration (10 µM) of ATP (97 ± 18 µA · cm–2 control; 95 ± 18 µA · cm–2 SR 140333; n = 4). Two further NK1R-selective antagonists, GR205171 (10 nM) and GR82334 (0.1 µM), similarly blocked the ISC curve to SLIGRL (Fig. 5).
Capsaicin (10 µM) added to both sides of the Ussing tracheal preparation caused an increase in ISC of 32.9 ± 7.3 µA · cm–2 (n = 4), which declined to baseline levels after 30 min, at which time it had no inhibitory effect on the ISC curve to SLIGRL; if anything, it seemed to be potentiated, although not significantly (Fig. 5). In addition, the addition of the CGRP antagonist CGRP8–37 (3 µM) for 20 min to both sides of the preparation had no effect on the increase in ISC to luminal SLIGRL (Fig. 5). Like SLIGRL, substance P failed to cause any increase in ISC when added to the contraluminal surface of the tracheal preparation (data not shown). In addition, treatment of the luminal side with the mast cell-degranulating agent, compound 48/80 (10 µM for 20 min), had no effect on ISC nor did it inhibit increases in ISC to SLIGRL (data not shown). Surprisingly, perhaps, indomethacin (3 µM) caused a significant 10-fold rightward shift in the concentration-dependent ISC curve to substance P (Fig. 5). As for SLIGRL (Fig. 5), similar indomethacin treatment failed to have any effect on the increases in ISC to ATP (data not shown).
In tension assays in the mouse trachea, the NK1R antagonist SR 140333 (100 nM) had no inhibitory effect on the relaxation curve to SLIGRL, whereas the biphasic response curve to substance P was abolished by SR 140333 (Fig. 6). In addition, in rat thoracic aorta, in which substance P failed to cause any mechanical responses (either endothelium-dependent relaxation or contraction; data not shown), SR 140333 had no effect on the relaxation curve to SLIGRL and LSIGRL was inactive (Fig. 6).
Ca2+ Measurement in A549 Cells. Substance P (100 nM) caused little if any increase in intracellular Ca2+ in A549 cells, and the response to SLIGRL was unaffected by SR 140333 but was inhibited after desensitization with trypsin (Fig. 7), confirming the above tension data in the rat thoracic aorta. Clearly, SR 140333 had no inhibitory effect at PAR2.
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Discussion |
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), any claims for roles of PAR2 in inflammatory airway disease that are based solely on data from experiments using SLIGRL or other similar PAR2-activating peptides may need to be reconsidered in light of our study.
Unlike Kunzelmann et al. (2005), we found no change in ISC to the PAR2 activators trypsin and SLIGRL when they were applied to the basolateral surface of mouse tracheal epithelial cells. In addition, contrary to our findings here, Kunzelmann et al. (2005) found no response to apically applied trypsin or SLIGRL, even though, as we reported earlier (Cocks et al., 1999), they found that PAR2 was predominantly expressed on the apical surface of mouse tracheal epithelial cells. Both the inability of Kunzelmann et al. (2005) to demonstrate responses to apically applied SLIGRL and our inability to similarly show responses to basolaterally applied SLIGRL most likely reflect a lack of NK1R expressed on the mucosal surface of airway epithelial cells in the strain of mice used by Kunzelmann et al. (2005) and the inability of basolateral PAR2 to couple to Cl– secretion in the strain of mouse (BALB/c) used in our study.
The increase in ISC to SLIGRL in the trachea was most likely due to NK1 R-mediated stimulation of luminal Cl– secretion as it was inhibited by removal of Cl– from the bathing Krebs' solution, by inhibitors of cystic fibrosis transmembrane conductance regulator (glibenclamide) (Schultz et al., 1999) and Ca2+-activated (DIDS) (Kidd and Thorn, 2000) Cl– channels, and by three potent, selective and structurally dissimilar NK1R antagonists (Emonds-Alt et al., 1993; Maggi et al., 1994; Gardner et al., 1996; Moriarty et al., 2001). In addition, the increase in ISC to SLIGRL only occurred when the peptide was applied to the luminal surface of the trachea and was unaffected by indomethacin. Together, these results indicate that SLIGRL causes an increase in luminal Cl– secretion in the mouse trachea via activation of NK1R located on the apical surfaces of epithelial cells and that the resultant increase in ISC is not due to concomitant PAR2-dependent abluminal PGE2 release (Asokananthan et al., 2002), which has been shown previously to mediate epithelium-dependent relaxation in this tissue (Cocks et al., 1999; Lan et al., 2001; De Campo and Henry, 2005).
Fortner et al. (2001b) have claimed that substance P causes similar increases in luminal Cl– conductance in the mouse airway but that this activity is linked in some way to smooth muscle relaxation, which the same group showed was due to concomitant abluminal release of PGE2 from the epithelium (Fortner et al., 2001). A possible simple explanation for this rather confusing concept is that some of the PGE2 released contraluminally into the wall of the airway activates basolateral EP receptors to increase luminal ion conductance. This idea is supported by our current finding that part of the increase in ion conductance to luminal substance P was inhibited by indomethacin, which abolished the concomitant relaxation and which, like that to ATP, is epithelium-dependent (Kao et al., 1999). The lack of effect of indomethacin on increases in ISC to SLIGRL observed here is most likely due to the relatively weak efficacy of SLIGRL at luminal NK1R, such that levels of PGE2 released abluminally are insufficient to stimulate basolateral epithelial EP receptors to increase ISC. Likewise, the failure of indomethacin to block relatively large increases in ISC to ATP indicates poor coupling of the purinoceptor involved in PGE2 synthesis. This possibility is supported by ATP only being able to cause weak epithelium-dependent relaxations in the mouse trachea which, like those for SLIGRL and substance P, are abolished by indomethacin (J. D. Moffatt and T. M. Cocks, unpublished data).
Because SLIGRL, 2-furoyl-LIGRL, LSIGRL, and SLCGRL are all cationic with arginine at position 5, it is possible that these and other peptides increase ISC indirectly via nonreceptor-dependent degranulation of mast cells (Stenton et al., 2002). That the response is blocked by three different NK1R antagonists, however, still implies a role for NK1R, which is possible as rat colonic mast cells have been shown to release substantial amounts of substance P in response to anti-IgE, which was inhibited by SR 140333 (Moriarty et al., 2001). However, such a nonspecific action on mast cells is unlikely to explain the current findings for two reasons. First, we found that the mast cell degranulating agent compound 48/80 failed to cause an increase in ISC or affect subsequent ISC responses to SLIGRL (H. T. Abey and T. M. Cocks, unpublished data). The second reason that mast cells are unlikely to be involved in the increase in ISC to PAR2 peptides is that capsaicin, which failed to affect ISC increases to SLIGRL in the present study, has been shown to deplete mast cells of substance P (Moriarty et al., 2001), most likely via activation of vanilloid VR1 receptors (Biro et al., 1998). Furthermore, another novel R5 cationic peptide, SLSGRT, failed to cause either an increase in ISC in the mouse trachea or smooth muscle relaxation in both the mouse trachea and rat thoracic aorta. That single amino acid substitutions can markedly modify the binding characteristics of these peptides further argues against a nonspecific action of these molecules such as their gross charge.
Substance P and capsaicin both caused increases in ISC in the mouse trachea. Therefore, it is possible that the similar responses to PAR2 peptides in this preparation were due to their ability to cause release of substance P from sensory nerves, either directly through activation of PAR2 on sensory nerves (Fiorucci et al., 2001; Kawabata et al., 2001; Su et al., 2005) or indirectly via a mechanism similar to that reported for mast cells (Stenton et al., 2002). This seems unlikely, however, because sensory neuropeptide depletion with prolonged exposure to capsaicin had no inhibitory effect on the ISC response to SLIGRL. In addition, any role for sensory nerves in the ISC response to SLIGRL was further ruled out by the lack of effect of an antagonist (CGRP8–37) of the other major sensory neurotransmitter in the airways, CGRP (Barnes, 1998). Rather, combined with the different rank order of potencies of 2-furoyl-LIGRL, SLIGRL, LSIGRL, SLIGRT, and SLCGRL for ion secretion compared with that for endothelium-dependent nitric oxide production in the rat aorta (Hwa et al., 1996), the lack of activity of LSIGRL in both the mouse tracheal and rat aortic smooth muscle assays, and the selectivity of SR 140333 for substance P in the ISC and airway relaxation assays, our data suggest that SLIGRL directly activates NK1R located on the epithelium.
The biphasic concentration-ISC curve to substance P may reflect two molecular isoforms of NK1R that have been shown in molecular recombination studies to be determined by the type of G-protein to which each NK1R form couples (Holst et al., 2001). In addition, the high potency of substance P in this assay may reflect high affinity with both isoforms of the receptor. If all of the PAR2 peptides activated the same isoform of NK1R to increase ISC, then some structure activity can be deduced. Thus, their rough rank order of potency (2-furoyl-LIGRL > SLCGRL > SLIGRL = SLIGRT > LSIGRL with SLSGRL inactive) indicates that reversal of the first two amino acids diminishes NK1 R activity, S3 for I3 substitution abolishes it, C3 for I3 substitution appears to increase it, T6 for L6 substitution has little effect, and substitution of 2-furoyl for S1 increases potency but markedly reduces efficacy. This apparent weak partial agonist activity of 2-furoyl-LIGRL at NK1R in the mouse trachea compared with its potent, full agonist profile at PAR2 in the rat aorta makes it an attractive alternative to SLIGRL for further research into the biology of PAR2, particularly if complicating effects of NK1R are possible.
The results presented here allow a closer examination of some recent studies in which PAR2 has been suggested to mediate effects in the airways but in which the pharmacological data presented are not always consistent with classic PAR2 pharmacology (Moffatt, 2004). In some studies, the nonspecific mechanism of action may account for the findings rather than PAR2 per se. For example, high micromolar concentrations of PAR2-activating peptides have been shown to increase ISC in human airway epithelial cells (Danahay et al., 2001), act as a mitogen for airway smooth muscle (Chambers et al., 2000), and activate eosinophils (Miike et al., 2001). Although trypsin may also elicit a response in these studies, it is often not practically possible to perform cross-desensitization studies to establish that trypsin and peptide activators act via a common mechanism. Furthermore, concentrations of trypsin that are supramaximal for PAR2 activation, as we found here in the ISC response to trypsin, are often required to elicit a response. Therefore, in the absence of PAR2 antagonists and the ability to cross-desensitize with trypsin, PAR2 knockout animals provide the only certain information regarding the role of this receptor in airway inflammation. Thus, Schmidlin et al. (2002) showed that the early but not the late phase of immune cell trafficking into the airway lumen after ovalbumin challenge in ovalbumin-sensitized wild-type mice was reduced and increased in PAR2 knockout and PAR2 overexpressing transgenic mice, respectively, suggesting that PAR2 was indeed involved in this early-phase allergic response in the airways. However, unexplained effects of PAR2 deletion on the production of IgE in response to sensitization (Schmidlin et al., 2002) may account for this difference.
In conclusion, roles for PAR2 in airway barrier defense, inflammation, and other processes will only be unequivocally resolved with the development of more selective PAR2 ligands (agonists and antagonists). Although reported PAR2-binding peptides are typically selective for PAR2 over PAR1, they may not be selective for PAR2 over other GPCRs (e.g., NK1), and, as such, receptor specificity beyond simple PAR2 subtypes clearly needs to be considered when one draws conclusions regarding the roles of PAR2 in airway inflammation.
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Footnotes |
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ABBREVIATIONS: PAR2, protease-activated receptor(s)-2; PGE2, prostaglandin E2; SR 140333, nolpitantium; GR205171, 3(S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine; GR82334, D-Pro(9)-[spiro-gamma-lactam]-Leu(10)-Trp(11)-physalaemin-(1-11); DIDS, 4,4-diisothio-cyanostilbene-2,2-disulfonic acid; NK1R, neurokinin-1 receptor(s); CGRP, calcitonin gene-related peptide; ISC, short-circuit current; ANOVA, analysis of variance.
Address correspondence to: Dr. Thomas M. Cocks, Department of Pharmacology, The University of Melbourne, Victoria, 3010, Australia. E-mail: thomasmc{at}unimelb.edu.au
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, 0.1 µM) on relaxations to SLIGRL (
) or substance P (
) in smooth muscle relaxation assays of mouse trachea (A and B) and rat aorta (C). LSIGRL (
) was inactive in both preparations. Responses are expressed as percentage of relaxations of 
, n = 8, n = 7, respectively), SLIGRT (
, n = 9, n = 13, respectively), and 2-furoyl-LIGRL (
, 10 nM; and 
, 0.1 µM, respectively), indomethacin (
4), SR 140333 (n = 4), indomethacin (n 