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Protease-activated receptor-2 (PAR2) is involved in airway inflammation and airway hyperresponsiveness; both are the prominent features of asthma. Transient receptor potential vanilloid receptor 1 (TRPV1) is expressed in pulmonary sensory nerves, functions as a thermal and chemical transducer, and contributes to neurogenic inflammation. By using the cell-attached single-channel recordings, this study was carried out to investigate the effect of PAR2 activation on single TRPV1 channel activities in isolated pulmonary sensory neurons. Our immunohistochemical study demonstrated the expression of PAR2 in rat vagal pulmonary sensory neurons. Our patchclamp study further showed that intracellular application of capsaicin (0.75 µM) induced single-channel current that exhibited outward rectification in these neurons. The probability of the channel being open (Po) was significantly increased after the cells were pretreated with PAR2-activating peptide (100 µM, 2 min). Pretreatment with trypsin (0.1 µM, 2 min) also increased the single-channel Po and the effect was completely inhibited by soybean trypsin inhibitor (0.5 µM, 3 min). In addition, the effect of PAR2 activation was abolished by either U73122 (1 µM, 4 min), a phospholipase C inhibitor, or chelerythrine (10 µM, 4 min), a protein kinase C inhibitor. In conclusion, our data demonstrated that activation of PAR2 upregulated single-channel activities of TRPV1 and the effect was mediated through the protein kinase C-dependent transduction pathway.
Protease activated receptor-2 (PAR2) belongs to a family of G-protein coupled receptors named PAR that are activated by specific proteases (Déry et al. 1998; Macfarlane et al. 2001; Ossovskaya & Bunnett, 2004). PAR2 activation occurs after proteolytic cleavage of its extracellular N-terminal domain by proteases derived from circulation (coagulation factors), inflammation cells (mast cell tryptase), epithelial and neuronal tissues (trypsins), revealing a tethered ligand domain that binds to and activates the cleaved receptor (Macfarlane et al. 2001; Ossovskaya & Bunnett, 2004). PAR2 can also be activated by short synthetic peptides that mimic the sequence of the tethered ligands (e.g. SLIGRL and SLIGKV for human and rodent PAR2, respectively) (Vergnolle et al. 2001). Recent studies have suggested that PAR2 plays an important role in a variety of physiological/pathophysiological processes such as inflammation, pain, itch, repair, and cell survival (Steinhoff et al. 2000; Vergnolle et al. 2001; Ossovskaya & Bunnett, 2004; Shimada et al. 2006; Ramachandran & Hollenberg, 2008). In respiratory system, PAR2 is distributed in various cells in the lung and airways including epithelial cells, airway smooth muscles, endothelial cells, fibroblasts, as well as inflammatory cells such as mast cells, neutrophils, and macrophages (Howells et al. 1997; D'Andrea et al. 1998; Akers et al. 2000; Chambers et al. 2001; Reed & Kita, 2004). It has been recently known that activation of PAR2 by endogenous or exogenous agonists contributes to airway inflammation and airway hyperresponsiveness, the hallmarks of airway inflammatory diseases such as asthma (Ricciardolo et al. 2000; Chambers et al. 2001; Schmidlin et al. 2002; Barrios et al. 2003; Ebeling et al. 2005).
The afferent activities arising from sensory terminals located in the lung and airways are conducted mainly by vagus nerves and their branches (Coleridge & Coleridge, 1984). Cell bodies of these sensory nerves reside in nodose and jugular ganglia. The majority of vagal bronchopulmonary afferents are non-myelinated (C-) fibers that innervate the entire respiratory tract ranging from larynx, trachea to lung parenchyma. The importance of these C-fiber afferents in regulating the respiratory and cardiovascular functions under both normal and abnormal conditions has been well documented (Coleridge & Coleridge, 1984; Lee & Pisarri, 2001; Lee & Undem, 2005). The bronchopulmonary C-fibers are generally known to possess polymodel sensitivity, and the expression of transient receptor potential vanilloid receptor 1 (TRPV1), a Ca2+ permeant non-selective cation channel, on the sensory terminal is one of the most prominent features of these C-fiber afferents (Jia & Lee, 2007). Because capsaicin, the major pungent ingredient of hot peppers and a derivative of vanillyl amide, is a potent and selective activator of the TRPV1 receptor, it has been used as a common tool to study the physiological properties and functions of the bronchopulmonary C-fibers. A recent study from our laboratory has demonstrated that PAR2 activation upregulates the capsaicin-induced pulmonary chemoreflexes in vivo and whole-cell responses in isolated pulmonary sensory neurons (Gu & Lee, 2006). However, how the activation of PAR2 regulates the capsaicin-induced single TRPV1 channel activities and kinetics in these sensory neurons was not known. The present study was carried out to answer this question.
The procedures described below were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Young Sprague-Dawley rats (4–6 weeks old; n = 15) were anesthetized with isoflurane inhalation (1% in O2) via a nose cone connected to a vaporizing machine (AB Bickford Inc. NY). A small mid-line incision was made on the ventral neck skin to expose the trachea. The fluorescent tracer DiI (0.2 mg/ml, 0.05 ml) was instilled into the lungs via a 30-gauge needle inserted into the lumen of the trachea; the incision was then closed. All animals recovered undisturbed for 7–10 days until they were killed for the study of immunohistochemistry or cell culture of pulmonary sensory neurons.
Rats (150–250 g; n = 3) were killed after isoflurane inhalation. Nodose and jugular ganglia were dissected and placed in 4% paraformaldehyde overnight at 4°C. The ganglia were then incubated in 15% sucrose in PBS (0.15 M NaCl in 0.01 M sodium phosphate buffer pH 7.2) overnight at 4°C. The tissue was embedded in optimal cutting temperature compound (Richard-Allan Scientific, Kalamazoo, MI) and sectioned at 8 µm. The sections were incubated in 10% normal goat serum in 0.02 M PBS for 1 h at room temperature before exposure to the mouse monoclonal antibody for PAR2 (SAM11; Santa Cruz Biotechnology, Inc. Santa Cruz, CA) that diluted in PBS containing 10% normal goat serum and 0.1% Triton X-100. The preparations were incubated for 24 h with the primary antibody at 4°C followed by 3 × 10-min washes with PBS and then incubated with FITC-labeled goat anti-mouse secondary IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for another 2 h at room temperature followed by 3 × 10-min washes with PBS. The preparations were mounted with coverslips in Vectorshield (Vector Laboratories, Burlingame, CA). Fluorescent labeling was examined and photographed by using a Nikon Eclipse TE2000-U fluorescent microscope.
Rats (n = 12) were killed after isoflurane inhalation. Nodose and jugular ganglia were extracted under a dissecting microscope and placed in ice-cold Dulbecco’s minimal essential medium/F-12 (DMEM/F12) solution. Each ganglion was desheathed, cut into ~10 pieces, placed in 0.125% type IV collagenase, and incubated for 1 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 × g, 5 min) and supernatant-aspirated. The cell pellet was resuspended in 0.05% trypsin in Hanks’ balanced salt solution for 5 min and centrifuged (150 × g, 5 min); the pellet was then resuspended in a modified DMEM/F12 solution (DMEM/F12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µM MEM nonessential amino acids) and gently triturated with a small bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 × g, 8 min) through a layer of 15% bovine serum albumin to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/F12 solution, plated onto poly-L-lysinecoated glass coverslips, and then incubated at 37°C in 5% CO2 in air. Isolated neurons were used within 48 h of culture.
The coverslip containing the attached cells was placed in the center of a small recording chamber (0.2 ml). Single-channel recording in cell-attached patches was performed by using Axopatch 200B/pCLAMP 9.0. The extracellular bath solution (ECS) contained (in mM): potassium gluconate 140, KCl 2.5, MgCl2 1; HEPES 5, EGTA 1.5; pH adjusted with NaOH to 7.4. The pipette filing solution contains (in mM): sodium gluconate 140, NaCl 10, MgCl2 1, HEPES 5, EGTA 1.5; pH adjusted with NaOH to 7.4. Capsaicin (0.75 µM) was applied in pipette solution. Other chemicals were administered by a pressure-driven drug delivery system (ALA-VM8; ALA Scientific Instruments, Westbury, NY), with its tip positioned to ensure that the cell was fully within the stream of the injectate. The experiments were performed at room temperature (~22°C). Membrane potential was held at +60 mV except mentioned otherwise. For the analysis of amplitudes and open probabilities (Po), the data were filtered at 2.5 kHz (−3 db, 4-pole Bessel) and digitized at 5 kHz. Data are presented as means ± SEM.
Recordings were made in pulmonary sensory neurons selected based upon the following criteria: 1) labeled with DiI as indicated by fluorescence intensity; 2) cell diameter <35 µm; and 3) responding to 0.75 µM capsaicin. These neurons presumably give rise to pulmonary C-fiber afferents as proposed in our recent studies (Gu et al. 2008). Although neurons from rat nodose and jugular ganglia were isolated and studied separately, data from the neurons of these two different origins (nodose: 13; jugular: 11) were pooled for group analysis because no difference was found between responses of the neurons obtained from these two ganglia.
DiI was purchased from Molecular Probes (Eugene, OR), and PAR2-AP (SLIGRL-NH2) was from Bachem (King of Prussia, PA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Stock solution of capsaicin (1 mM) was prepared in a vehicle of 10% Tween 80, 10% ethanol, and 80% ECS; those of PAR2-AP (10 mM), trypsin (0.3 mM), and soybean trypsin inhibitor (SBTI; 0.1 mM) were in ECS; and U73122 (3 mM) and chelerythrine (20 mM) were in dimethyl sulfoxide. These stock solutions were divided into small aliquots and kept at −80°C. The solutions of these chemicals at desired concentrations were prepared daily by dilution with ECS before use. No detectable effect of the vehicles of these chemical agents was found in our preliminary experiments.
Data were analyzed by a one-way analysis of variance (ANOVA). When the ANOVA showed a significant interaction, pair-wise comparisons were made with a post hoc analysis (Fisher’s least significant difference). Results were considered significant when P < 0.05. Data are means ± SE.
The expression of TRPV1 channels in vagal pulmonary sensory neurons has been well documented (Geppetti et al. 2006; Jia & Lee, 2007). To determine whether PAR2 is also expressed in these neurons, we localized the proteins by immunofluorescence in tissue sections of nodose and jugular ganglia. The fluorescent tracer DiI was used to identify the neurons that specifically innervating the lung and airways. We found that 28.3% (299 out of 1055) of nodose ganglion neurons and 19.6% (168 out of 855) of jugular ganglion neurons were labeled with DiI. Immunoreactive PAR2 was widely detected in all size of vagal sensory neurons including the pulmonary neurons (Fig. 1).
Single-channel currents were recorded in the cell-attached configuration with 0.75 µM capsaicin contained in the recording pipette (Fig. 2A & B). All recordings were carried out in the Ca2+-free (1.5 mM EGTA, 1 mM Mg2+) extracellular solution in order to avoid Ca2+-induced desensitization and tachyphylaxis (Koplas et al. 1997; Premkumar et al. 2002). When capsaicin-induced channel activity was recorded from −60 to +60 mV, it showed that both single channel Po and conductance were reduced at hyperpolarized potentials (Fig. 2C–E), which was in agreement with the properties of capsaicin-induced single TRPV1 channel activities in other native or heterologous expression cell systems (Premkumar et al. 2002).
Extracellular exposure to PAR2-AP (100 µM, 2 min) did not significantly affect the amplitude of the capsaicin-induced single channel current, however, it drastically increased the single-channel Po in cell-attached patches from vagal pulmonary sensory neurons. For example, the capsaicin-induced single-channel Po increased from 0.07 ± 0.02 at control to 0.18 ± 0.04 after PAR2-AP (n = 6, P < 0.05) (Fig. 3). Similarly, pretreatment with trypsin (0.1 µM, 2 min) also significantly potentiated the single-channel Po in cell-attached patches (at control: 0.01 ± 0.00; after trypsin: 0.14 ± 0.05; n = 5, P < 0.05) (Fig. 4A–C). The potentiating effect of trypsin was completely abolished after the pre-incubation with SBTI (0.5 µM, 3 min; n = 4, P > 0.05) (Fig. 4D–F).
In a number of different cell systems, PAR2 has been reported to be coupled to PKC activation via G proteins (Gq/11) and the phosphatidylinositol pathway (Macfarlane et al. 2001; Amadesi et al. 2004; Dai et al. 2004). In the present study, the cell-attached single-channel recordings allowed us to investigate the involvement of the PLC-PKC pathways because various intracellular second messengers and kinases are kept intact only in this recording configuration but not in the single-channel recordings from inside-out or outside-out patches. As shown in Fig. 5, pre-incubation with U73122 (1 µM, 4 min), an inhibitor of PLC, completely prevented the potentiation of capsaicin-induced single-channel activities by PAR2-AP pretreatment (100 µM, 2 min); the single-channel Po was 0.04 ± 0.02 at control and 0.04 ± 0.02 after pretreatment with both U73122 and PAR2-AP (n = 4, P > 0.05) (Fig. 5A–C). Similarly, after the pre-incubation with chelerythrine (10 µM, 4 min), a general PKC inhibitor, the potentiating effect of PAR2-AP (100 µM, 2 min) was also completely abolished (0.03 ± 0.00 at control; 0.03 ± 0.00 after pretreatment with chelerythrine and PAR2-AP; n = 4, P > 0.05) (Fig. 5D–F).
As the first cloned member of TRPV ion channel family, TRPV1 is a six transmembranedomain, Ca2+ permeable cation channel protein (Caterina et al. 1997). The function of TRPV1 as a transducer for multiple physiological and environmental stimuli has been well recognized. It is activated not only by vanilloid molecules including capsaicin, but also by protons, hyperthermia, anandamide, and certain lipooxygenase metabolites of arachidonic acid (Caterina & Julius, 2001; Pingle et al. 2007). TRPV1 is abundantly expressed in the C-fiber afferents innervating the lung and airways, which also contain sensory neuropetides such as tachykinins. Increasing evidence from recent studies has collectively suggested that TRPV1 may play an important role in the manifestation of various symptoms of airway hypersensitivity (irritation, chest tightness, breathlessness, cough, etc) associated with airway inflammatory reactions (Geppetti et al. 2006; Lee & Gu, 2009). For example, upregulation of TRPV1 could contribute to the proinflammatory role of nerve growth factor released from mast cells during asthma exacerbations (Bonini et al. 1996; Shu & Mendell, 1999). In addition, a number of endogenous inflammatory mediators, such as bradykinin and prostaglandin E2, can sensitize TRPV1 during tissue inflammation, which leads to nociceptor hypersensitivity and hyperalgesia (Chuang et al. 1999; Ho et al. 2000; Gu et al. 2003).
Expression of PAR2 has been demonstrated in a variety of cells in the lung and airways (Reed & Kita, 2004), including TRPV1-positive sensory neurons as demonstrated in the present study. Mast cell tryptase, trypsin and trypsin-like proteases, and coagulation factors VIIa and Xa are considered as the endogenous agonists of PAR2 (Reed & Kita, 2004; Sokolova & Reiser, 2007). PAR2 can also be activated by certain airborne allergens such as house dust mite Der P1, P3 and P9 (Sun et al. 2001; Asokananthan et al. 2002; Ramachandran et al. 2008). In addition, tissue kallikreins, a large family of secreted serine proteases with tryptic or chymotryptic activity, are recently proposed as physiological regulators of PAR2 (Oikonomopoulou et al. 2006). Compelling evidence indicates that PAR2 plays a critical role in the pathogenesis of airway inflammation and airway hyperresponsiveness. The elevated levels of both the endogenous agonists and the expression of PAR2 have been reported from patients and animals under airway inflammatory conditions (Knight et al. 2001; Sokolova & Reiser, 2007). Activation of PAR2 in the lung induces airway constriction, lung inflammation, and protein-rich pulmonary edema. These effects are inhibited by either perineural capsaicin treatment of both vagi or the combination of neurokinin-1 (NK1), NK2 and CGRP receptor antagonists, indicating the involvement of centrally mediated reflex and local release of neuropeptides from activation of TRPV1-containing C-fiber afferents (Ricciardolo et al. 2000; Barrios et al. 2003; Su et al. 2005). Indeed, our recent study showed that PAR2 activation upregulates the excitability of rat pulmonary sensory neurons and potentiate the capsaicin-induced pulmonary chemoreflex responses (Gu & Lee, 2006). In the present study, we have further demonstrated that activation of PAR2 significantly potentiates single-channel activities of TRPV1 in these sensory neurons. In addition, it has been recently reported that PAR2 activation exaggerates the TRPV1-dependent tussive response in guinea pigs (Gatti et al. 2006). Therefore, activation of PAR2 represents another important pathway in sensitization of TRPV1 under airway inflammatory conditions.
In addition to its sensitizing effect on TRPV1, PAR2 has been known to regulate the activity of afferent neurons by other mechanisms. For example, PAR2 activation has been reported to induce the sensitization of other TRP channels such as TRPV4 and TRPA1, as well as the suppression of delayed rectifier potassium currents (Dai et al. 2007; Grant et al. 2007; Kayssi et al. 2007). Furthermore, PAR2 activation is often associated with release of various proinflammatory mediators including prostanoids such as prostaglandin E2, and cytokines such as interleukin (IL)-6 and IL-8 (Kauffman et al. 2000; Lan et al. 2001; Reed & Kita, 2004). These mediators are known to have potential regulatory effects on the bronchopulmonary sensory neurons (Jia & Lee, 2007).
The signaling mechanisms of PAR2 are not fully understood. In a number of cell systems, PAR2 has been reported to be coupled to Gq/11, resulting in activation of PLC and generation of 1,4,5-inositol trisphosphate and diacylglycerol, which would be expected to mobilize intracellular Ca2+ and activate PKC (Macfarlane et al. 2001; Amadesi et al. 2004; Dai et al. 2004). By using a combination of confocal microscopy, subcellular fractionation and Western blotting, Amadesi et al. (2006) demonstrated that, in both dorsal root ganglion neurons and HEK 293 cells, activation of PAR2 promoted the translocation of PKCϵ from the cytosol to the plasma membrane. Indeed, activation of PKC with phorbol 12-myristate 13-acetate (PMA) has been demonstrated to mimic the effect of PAR2 agonist and potentiate the capsaicin-evoked Ca2+transient in rat dorsal root ganglion neurons (Amadesi et al. 2004). In the present study, the sensitization of the TRPV1 single-channel activity by PAR2 was completely abolished by either U73122 or chelerythrine, indicating the effect is predominantly mediated through the PLC-PKC-dependent transduction pathway. Although the significantly elevated open probabilities of TRPV1 after PAR2 activation, as observed in this study, are presumably resulted from the PKC-induced phosphorylation of the single TRPV1 channels, PKC is also known to induce a rapid delivery of functional TRPV1 channels to the neuronal surface and therefore lead to the increased sensitivity of TRPV1 (Morenilla-Palao et al. 2004). In addition, involvements of PKC as well as PKA, PKD, and ERK1/2 have recently been proposed in PAR2-induced sensitization of various TRP channels in dorsal root ganglion neurons (Amadesi et al. 2006; Dai et al. 2007; Grant et al. 2007). Whether those additional protein kinase other than PKC are also involved in the sensitization of TRPV1 in bronchopulmonary sensory neurons remains to be determined.
Under airway inflammatory conditions including asthma, the elevated levels of PAR2 agonist such as trypsin as well as the increased PAR2 expression may lead to the activation of this receptor, which may then upregulate the TRPV1 channel sensitivities in bronchopulmonary sensory terminals. The latter will, through both central reflex pathways and by local/axon reflexes, evoke increased cardiopulmonary reflex responses such as airway constriction, mucous secretion, cough, tachypnea and hypotension. Considering the important role that TRPV1 plays in the manifestation of these cardiopulmonary symptoms associated with inflammatory reactions, antagonisms of PAR2, TRPV1, and PLC-PKC intracellular signaling may represent effective therapeutic approaches for the treatment of airway inflammatory diseases.
We thank Michelle E. Lim for technical assistance. We thank Dr. Louis S. Premkumar (Southern Illinois University) for the valuable advice with single-channel data analysis. This study was partially supported by AHA Scientist Development Grant 0835320N (Q Gu), NIH AI076714 (Q Gu), and NIH HL058686 (LY Lee).