Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca2+ or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [S-35]GTP gamma S binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca2+ after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor. (c) 2006 Elsevier Inc. All rights reserved.

Inverse agonism of the antipsychotic drugs at the D-2 dopamine receptor

The antipsychotic drugs had been assumed to act as antagonists at D-2 dopamine receptors but recently these drugs have been shown to possess inverse agonist properties at this receptor. Inverse agonism may be demonstrated from the ability of these drugs to potentiate forskolin-stimulated cAMP accumulation or to suppress agonist-independent [S-35]GTPgammaS binding. The antipsychotic drugs tested generally appear as full inverse agonists in these assays regardless of chemical or therapeutic class. The mechanism of inverse agonism of the antipsychotic drugs is still unclear but may involve stabilisation of the ground state of the D-2 receptor. (C) 2003 Elsevier Science B.V All rights reserved.

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

PSNCBAM-1 has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated [35S]GTPγS binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneurone-Purkinje cell (IN-PC) synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused non-competitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist CP55940 than for WIN55,212-2 (WIN55). In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency, but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pre-treatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency, but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist AM251 increased mIPSC frequency beyond control, this effect was reversed by PSNCBAM-1. PSNCBAM-1 pre-treatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependency associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian CNS. PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.

The bile acid receptor TGR5 does not interact with β-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid, DCA, taurolithocholic acid, TLCA) and the selective agonists oleanolic acid (OA) and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide (CCDC) stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, assessed by confocal microscopy. DCA, TLCA and OA did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, determined by bioluminescence resonance energy transfer. CCDC stimulated a low level of TGR5 interaction with β-arrestin2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of extracellular signal regulated kinase (ERK1/2). BRET analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin

(1) Stimulation of the vanilloid receptor-1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. (2) Recently, iodo-resiniferatoxin (I-RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I-RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). (3) We have demonstrated that I-RTX inhibited capsaicin-induced mobilization of intracellular Ca(2+) in rat trigeminal neurons (IC(50) 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC(50) 0.071 nM). (4) Furthermore, I-RTX significantly inhibited both capsaicin-induced CGRP release from slices of rat dorsal spinal cord (IC(50) 0.27 nM) and contraction of isolated guinea-pig and rat urinary bladder (pK(B) of 10.68 and 9.63, respectively), whilst I-RTX failed to alter the response to high KCl or SP. (5) Finally, in vivo I-RTX significantly inhibited acetic acid-induced writhing in mice (ED(50) 0.42 micro mol kg(-1)) and plasma extravasation in mouse urinary bladder (ED(50) 0.41 micro mol kg(-1)). (6) In in vitro and in vivo TRPV1 activated responses I-RTX was approximately 3 log units and approximately 20 times more potent than capsazepine, respectively. This high affinity antagonist, I-RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 micro g/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Expression and function of proteinase-activated receptor 2 in human bronchial smooth muscle

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 (PAR2) to induce alterations in contraction of airway smooth muscle that have been implicated in asthma in experimental animals. Although tryptase inhibitors are under development for treatment of asthma, little is known about the localization and function of PAR2 in human airways. We detected PAR2 expression in primary cultures of human airway smooth muscle cells using reverse transcriptase/polymerase chain reaction (RT-PCR) and immunofluorescence. The PAR2 agonists trypsin, tryptase, and an activating peptide (SLIGKV-NH2) stimulated calcium mobilization in these cells. PAR2 agonists strongly desensitized responses to a second challenge of trypsin and SLIGKV-NH2, but not to thrombin, indicating that they activate a receptor distinct from the thrombin receptors. Immunoreactive PAR2 was detected in smooth muscle, epithelium, glands, and endothelium of human bronchi. Trypsin, SLIGKV-NH2, and tryptase stimulated contraction of isolated human bronchi. Contraction was increased by removal of the epithelium and diminished by indomethacin. Thus, PAR2 is expressed by human bronchial smooth muscle where its activation mobilizes intracellular Ca2+ and induces contraction. These results are consistent with the hypothesis that PAR2 agonists, including tryptase, induce bronchoconstriction of human airway by stimulating smooth muscle contraction. PAR2 antagonists may be useful drugs to prevent bronchoconstriction.