PAR-2 agonists activate trigeminal nociceptors and induce... : PAIN (original) (raw)
1. Introduction
Nearly 50 years ago, Armstrong and colleagues demonstrated that inflammatory exudates from inflamed human skin demonstrate a protease-like activity that was associated with a burning pain sensation (Armstrong et al., 1953, 1957). This was confirmed in subsequent studies where fluid collected from patients or cats contained increased amount of proteases capable of inducing biological effects (Chapman and Wolff, 1959; Chapman et al., 1960). In the 1990s, molecular studies revealed a unique way by which these proteases regulate biological systems via activation of certain G-protein coupled receptors (GPCRs), namely, the protease activated receptors (Vu et al., 1991; Nystedt et al., 1994; Xu et al., 1998). Recently, several groups have determined that proteases such as trypsin and tryptase regulate the development of neurogenic inflammation and inflammatory pain in animal studies (Steinhoff et al., 2000; Hoogerwerf et al., 2001; Vergnolle et al., 2001). Moreover, it appears that one of the protease activated receptors, PAR-2, mediates this component of protease-induced inflammation and pain. PAR-2 can be activated by proteases such as trypsin, epithelial trypsin IV, mast cell tryptase, neutrophil proteinase 3, tissue factor/factor VIIa/factor Xa, and membrane-tethered serine protease-1 (Coughlin and Camerer, 2003; Cottrell et al., 2004). Studies performed in human volunteers or patients have further confirmed the role of PAR-2 in the generation of pain (Steinhoff et al., 2003). Trigeminal nociceptors express PAR-2 and activation of them in the parotid gland is associated with increased c-fos activity in the nucleus caudalis (Kawabata et al., 2004; Thai Dinh et al., 2005). However, no study to our knowledge has directly addressed PAR-2 activation of TG nociceptors and thus their role in the generation of orofacial pain.
Several studies indicate that peripheral opioid receptors undergo rapid changes in functional competence after injury. Although peripheral opioid receptors do not appear active under most basal states, inflammation or injection of inflammatory mediators significantly increases the efficacy of peripherally administered opioids for producing antihyperalgesic effects (Joris et al., 1987; Stein et al., 1989; Stein et al., 2003). In addition, clinical trials indicate that peripherally administered opioids are efficacious in the treatment of human inflammatory pain (Stein et al., 1991; Khoury et al., 1992; Dionne et al., 2001). Recently, we demonstrated that bradykinin activation of the B2 receptor leads to rapid heterologous regulation of the delta opioid receptor (DOR) present on TG nociceptors for signaling via inhibitory pathways and that the priming effect of this inflammatory mediator requires activation of the PKC pathway (Patwardhan et al., 2005). Although PAR-2 also couples to the PLC–PKC pathway (Schmidlin and Bunnett, 2001) in DRG neurons, it is not known whether this receptor induces functional competence of the DOR in TG nociceptors.
In the present study, we addressed three major hypotheses. Firstly, that agonists of PAR-2 activate TG nociceptors, secondly, they do so in a PLC–PKC dependent fashion and thirdly that activation of PAR2 rapidly evokes functional competence in DOR to inhibit TG nociceptors.
2. Materials and methods
2.1. Animals
Adult male Sprague–Dawley (Charles River, Wilmington, MA, USA) rats weighing 250–300g were used in this study. All animal study protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to the International Association for the Study of Pain (IASP) and federal guidelines. Animals were housed for one week prior to the experiment with food and water available ad lib.
2.2. Compounds
[D-Pen2,5]-Enkephalin (DPDPE), porcine pancreatic trypsin IX-S, bradykinin (all from Sigma–Aldrich, St. Louis, MO, USA), Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SL-NH2, PAR-2 activating peptide), Ruthenium Red (RR) (both from Tocris, Ellisville, MO, USA), Leu-Arg-Gly-Ile-Leu-Ser-NH2 (LR-NH2, PAR-2 reverse peptide, custom synthesized from Bachem California Inc., Torrance, CA, USA) were all made up in stock solutions of ddH2O on the day of the experiment. To test the interference of ddH2O in the assay, additional experiments were performed dissolving the peptides directly into the experimental buffer and they demonstrated similar effect. Bisindolylmaleimide (BIS, Calbiochem, San Diego, CA, USA) was dissolved in DMSO and diluted in buffer (final 0.05% DMSO). Prostaglandin E2 (PGE2) (Cayman Chem, Ann Arbor, MI, USA) was made in stock solutions with EtOH and diluted by buffer on the day of the experiment (final 0.01% EtOH). All control experiments contained appropriate vehicles which were without effect in these assays.
2.3. Rat trigeminal ganglia (TG) primary culture
TGs were quickly removed after decapitation and placed in ice-cold balanced Hanks’ solution calcium- and magnesium-free (HBBS, Gibco, Carlsbad, CA) and washed twice with HBBS. Ganglia were then treated with 5mg/ml collagenase (Worthlington Biomedical, Lakewood, NJ, USA) for 30min and 0.1% trypsin (Sigma) for 15min prior to homogenization. The TG were then treated with 10U of DNase I (Roche, Indianapolis, IN, USA), centrifuged at 2000rpm for 2min and resuspended in Dulbecco's modified Eagle's media (DMEM, Gibco), that also contained 1× pen-strep (Gibco), 1× glutamine (Gibco) 3μg/mL of 5-FDU and 7μg/mL uridine (Sigma), 10% fetal calf serum (Gibco) and 100ng/ml nerve growth factor (NGF, Harlan, Indianapolis, IN, USA). The tissue was gently triturated and cells from six ganglia were plated on one 24-well poly-d-lysine-coated plate (BD Biosciences, Bedford, MA, USA) yielding ˜8000 cells per well. For the calcium imaging and electrophysiology experiments, cells were plated on poly-d-lysine/laminin-coated coverslips. The media were replaced at the end of 24h and then 48h later. All the experiments were performed on day 5–6 of cultures.
2.4. siRNA design and transfection
siRNA was designed against the target sequence CCTACGTGCTCATGATCAA (ORF position 782, PAR-2 mRNA sequence, accession # U61373) using the software Dharmacon siDESIGN Center™ (Dharmacon, Inc., Lafayette, CO, USA). The Alexa-Fluor-488 conjugated siRNA was custom synthesized from Qiagen (Qiagen Inc., Valencia, CA, USA). The transfection protocol was modified from a previously published method (Vasko et al., 2005). On days 2 and 4, the cultured TG neurons were transfected with either HiPerFect (Qiagen) alone (0.28μl/100μl of F-12 media, GIBCO) or HiPerFect mixed with either scrambled siRNA #1 (Ambion Inc., Austin, TX, USA) or PAR-2 siRNA (175ng/ml). The cells that showed green fluorescence were considered as transfected cells. Transfection efficiency of approximately 75–80% was routinely obtained using this method.
2.5. RNA isolation
Total RNA was isolated from cultured TG neurons treated with siRNA against PAR2, scramble siRNA or vehicle using the guanidinium thiocyanate method as described previously (Chomczynski and Sacchi, 1987). The isolated RNA was treated with DNase I (Ambion) and samples were used as templates for the real-time RT-PCR experiments.
2.6. Quantitative real-time RT-PCR (qRT-PCR)
Total RNA (100ng) was used as template in a one-step RT-PCR protocol. Amplification of target sequences was detected by a sequence detector ABI 7700 (Applied Biosystems, Foster City, CA) utilizing TaqMan Gene Expression Assays on Demand (assay # Rn00588089_m1, Applied Biosystems, Foster City, CA) using specific primers and probes for the PAR-2 gene. The reactions were run in triplicates of 25μl, containing the respective TaqMan Gene Expression Assay on Demand and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). For each individual gene expression assay, the endogenous control, 18S ribosomal subunit, gene expression assay (assay # Hs99999901_s1, Applied Biosystems) was also run in triplicate. The comparative delta–delta Ct (ddCt) was utilized to normalize the data based on the endogenous reference, and to express it as the relative fold change after the exclusion criteria were verified by comparing primer efficiencies (Livak and Schmittgen, 2001). Data were acquired from three independently cultured and treated plates of cells.
2.7. CGRP release assay
All culture experiments were performed on day 5–6, at 37°C, using modified Hanks (Gibco) buffer (10.9mM Hepes, 4.2mM sodium bicarbonate, 10mM dextrose and 0.1% bovine serum albumin were added to 1× Hanks). After two initial washes, a 15min baseline sample was collected. In experiments evaluating the direct effect of PAR-2 agonists on TG neurons, the cells were exposed to either vehicle or SL-NH2 (1–100μM) or LR-NH2 (100μM) or trypsin (0.1–600 nM) for 15min and samples were collected. In the experiments evaluating signaling involved downstream of PAR-2 activation, cells were pretreated with either vehicle or BIS (500nM) or RR (10μM) for 15min and then exposed to either SL-NH2 (100μM) or trypsin (100nM) for 15min and samples were collected. In experiments evaluating the effect of PAR-2 activation on DOR function, cells were pretreated with either vehicle/SL-NH2 (100μM, 15min), exposed to either vehicle or DPDPE (1μM, 15min) and then stimulated with the combination of BK(10μM)/PGE2(1μM) for 15min. All supernatants were collected for analysis of iCGRP content by radioimmunoassay (RIA). The basal release was typically 6–8fmol per well. All experiments were repeated at least three times.
2.8. cAMP accumulation
The experimental protocol was identical to that of the CGRP release assay except for the fact that all these experiments included the phosphodiesterase inhibitor, rolipram (10μM). Following extraction on ice for 20min, the EtoH extract was transferred to RIA tubes, evaporated under a gentle stream of air and reconstituted with RIA buffer. cAMP content was measured using RIA as previously described (Berg et al., 1994a,b). All experiments were repeated at least three times.
2.9. iCGRP RIA
A previously used (Garry et al., 1994) primary antibody against CGRP (final dilution 1:1,000,000, kindly donated by Dr. M. J. Iadarola, NIH) was added in the tubes containing superfusate either from cultured rat TGs or acutely isolated rat TGs and incubated at 4°C for 24h. Then 100μl of [I125]-Tyr0-CGRP28–37 (˜20,000 CPM) and 50μl of goat anti-rabbit antisera coupled to ferric beads (Qiagen, Valencia, CA, USA) were added to these tubes. The tubes were incubated for another 24h at 4°C. The assay was stopped using immunomagnetic separation of bound from free tracer. All compounds used in experiments were tested for interference with the RIA. The minimum detectable levels for CGRP for this assay are approximately 3fmol and the 50% displacement is at 28fmol.
2.10. Inositol phosphate (IP accumulation)
Cells were labeled with 2μCi/well [H3]-myo-inositol for 24h prior to experiments. Measurements of total [H3]-IP (ie., IP1, IP2 and IP3) accumulation were made essentially as described previously (Berg et al., 1994a,b).
2.11. Calcium imaging
Cultured TG neurons were loaded with 1μM of the cell permeable calcium sensitive dye, FURA 2AM (Molecular Probes, Eugene, OR), in presence of 0.01% Pluronic (Molecular Probes) for 30min at 37°C. Coverslips containing cells were placed in a chamber with constant infusion of external buffer (SES) of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 10 Hepes, pH 7.4. at 37°C. Fluorescence was detected by a Nikon Eclipse TE 2000-U microscope fitted with a 20× Fluor/NA 0.75 objective. Fluorescence images of 340 and 380 excitation wavelengths were collected for 200ms, each 5s throughout the experiment, analyzed and the 340/380 ratio calculated by the MethaFluor Software (MethaMorph, Web Universal Imaging Corporation, Downingtown, PA). SL-NH2 (100μM), trypsin (100nM) or vehicle were locally delivered for 3min to the cells, after which responsive cells were allowed to recover for approximately 1–2min. In order to identify a major subclass of nociceptors, capsaicin (30nM at 40s) was applied to the cells at the end of the experiment. The net change in internal Ca2+ was calculated by subtracting the basal Ca2+ levels (mean value collected for 30s prior to agonist addition) from the peak Ca2+ levels achieved after exposure to the agonists. Ratiometric data were converted to [Ca2+]i (in μM) by using the equation [Ca2+]i=K* (_R_−_R_min)/(R_max−_R), where R is the 340/380nm fluorescence ratio. _R_min, _R_max and K* (0.35, 2.44 and 1.43μM, respectively) were measured according to a previously described method (Gamper and Shapiro, 2003). A cell was considered responsive if the calcium influx was more than 25nM. All experiments were repeated at least on four independent cultures.
2.12. Electrophysiology
Whole-cell patch configuration was used for current- or voltage-clamp recordings from individual TG neurons using protocol similar to that described previously (Liu et al., 1997). In voltage-clamp (_V_h=−60mV) or current (_I_h=0pA)-clamp recordings, cells were exposed SL-NH2 (100μM), trypsin (100nM), or vehicle to record ISL-NH2/trypsin. Recordings were made at 22–24°C from the somata of neurons (15–45pF) using an Axopatch200B amplifier and pCLAMP9.0 software (Axon Instruments, Union City, CA). Cell diameters were calculated using _d_=✓[100×_C_m/π], where d (μm) is cell diameter and _C_m (pF) is membrane capacitance. Data were filtered at 0.5kHz (voltage-clamp) or 2.5kHz (current-clamp) and sampled at 2 or 10kHz, respectively. Borosilicate pipettes (Sutter, Novato, CA) were polished to resistances of 2–4Mω in standard pipette solution. Access resistance (_R_s) was compensated (by 40–80%) when appropriate to achieve 5–10Mω. All compensations were performed in the voltage-clamp configuration. Data were rejected when _R_s changed >20% during recording; leak currents were >50pA; or input resistance was <300Mω. Currents were considered positive when their amplitudes were 5-fold bigger than displayed noise (in root mean square). Standard external solution (SES) contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 d-glucose and 10 Hepes, pH 7.4. The standard pipette solution (SIS) for the whole-cell configurations contained (in mM): 140 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA, 10 d-glucose, 10 Hepes, 0.2 Na-GTP and 2.5 Mg-ATP, pH 7.3. Drugs were applied using a fast, pressure-driven and computer controlled 8-channel system (ValveLink8; AutoMate Scientific, San Francisco, CA). Multiple cells from at least three independently cultured neurons were used for the measurements.
2.13. Immunohistochemistry
The cultured TG neurons grown on coverslips were fixed with 4% formalin, permeabilized with 0.2% Triton X-100, and blocked with 10% normal goat serum. The coverslips were then incubated with a previously characterized (Uehara et al., 2003; Dai et al., 2004) mouse monoclonal antibody against PAR-2 (1:100, SAM-11, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) overnight at 4°C. For experiments evaluating the co-expression of PAR-2 with TRPV1, coverslips were incubated with a previously used (Price et al., 2003) guinea pig antibody against TRPV1 (1:3000, Neuromics, Bloomington, MN) overnight at 4°C. The immunoreactivity was detected using appropriate Alexa-488 or Alexa-594 conjugated secondary antibodies (1:300, Molecular Probes, Eugene, OR, USA). Images were acquired using a Nikon Eclipse 90 microscope with a Nikon C1si confocal scanner (Melville, NY, USA). Images were analyzed using Metamorph software (Version 4.5 r6, Universal Imaging Inc.).
2.14. Behavioral assay
After habituation in the test chamber, rats were injected with either vehicle (0.9% saline) or SL-NH2 (50μg) or trypsin (1%) in the right vibrissal pad in 50μl volume and the number of flinches (nocifensive behavior) was counted over 5min period by observers blinded to treatment allocation. The control experiments included injection of LR-NH2 (50μg) or boiled trypsin (1%).
2.15. Doppler blood flow measurements
The changes in the vibrissal pad blood flow after SL-NH2 or trypsin injection were measured using a laser Doppler blood flow meter device (Moor Instruments Ltd., Delaware, USA). Briefly, animals were anesthetized with a ketamine hydrochloride/xylazine hydrochloride solution (1ml/kg, i.p., Sigma) and a 27 gauge needle connected to a syringe with a thin tube was carefully inserted into the right vibrissal pad to minimize the trauma. Then probe #1 was placed and secured right over the area where the needle was inserted. The probe was in light contact with the underlying skin. The flux was measured in arbitrary units (AU, range 1–1000). The basal flux (preinjection) was around 200–300AU in most rats. After attaining a stable basal flux reading, the area under the probe infiltrated with either veh (0.9% saline) or SL-NH2 (50μg) or trypsin (1%) in 50μl volume. The positive control included injection of capsaicin (10μg). The data were acquired with MoorSoft for Windows/moorLAB ver 1.31 (Moor Instruments Ltd). The change in the blood flow was calculated using peak blood flow measurements normalized to the basal measurements.
2.16. Data analysis
All experiments were conducted with _n_=6 or more wells to determine the experimental observation and then repeated at least three times to conduct the statistical analysis. Data are presented as means±SEM. The CGRP data are presented as percent of basal release and cAMP accumulation data are presented as percent of peak stimulus (PGE2). Data were analyzed using GraphPad Prism software version 4 (GraphPad software Inc., San Diego, CA, USA). The DPDPE treated groups (with our without SL-NH2 pretreatment) were compared to respective (with or without SL-NH2 pretreatment) control BK/PGE2 groups. Multifactor experimental data were analyzed using two-way ANOVA, single factor, multiple treatment data were analyzed using one-way ANOVA and individual groups were compared using a Bonferroni post hoc test whereas experiments examining difference between two groups were analyzed by using Student's t test. The statistical significance was tested at 0.05.
3. Results
3.1. Trigeminal nociceptors express PAR-2
Firstly we evaluated the expression of PAR-2 with the nociceptor marker TRPV1 in trigeminal neurons. Immunohistochemistry results showed that a substantial proportion (approximately 78%) of cultured TG neurons expressed PAR-2 protein. Many of the PAR-2 expressing neurons also co-expressed TRPV1 (Fig. 1).
Co-expression of PAR-2 with TRPV1 in TG nociceptors. Cultured TG neurons were fixed and double immunohistochemistry was performed using antibodies against PAR-2 and TRPV1. The white horizontal arrows denote examples of cells that denote both PAR-2 immunoreactivity (PAR-2 ir) and TRPV1 immunoreactivity (TRPV1-ir). The yellow vertical arrow denotes an example of a cell that expresses only PAR-2 but not TRPV1.
3.2. The PAR-2 agonist SL-NH2 activates TG nociceptors in a PLC–PKC dependent pathway and evokes nociception
Our initial experiments tested the hypotheses that an agonist of PAR-2 activates TG nociceptors and that this occurs via the PLC–PKC signaling pathway (Fig. 2). We employed three independent methods to assess this question. In cultured TG neurons, the vehicle treatment did not change the amount of iCGRP release compared to the basal release. In the same cultures, the application of SL-NH2 (1–100μM, 15min) resulted in a concentration-dependent and significant increase in iCGRP release (p<0.001, Fig. 2A). However, the application of the reverse peptide LR-NH2 (100μM) did not alter iCGRP release compared to vehicle treatment (Fig. 2A). The SL-NH2 (100μM)-evoked iCGRP release was almost completely reversed by pretreatment with a pan PKC inhibitor, BIS (500nM), whereas BIS by itself did not have any effect on basal iCGRP release (Fig. 2A). Because PAR2 signals via the PLC–PKC pathway in DRG neurons (Amadesi et al., 2004) and the present data demonstrate PAR2 activation of PKC dependent pathways in TG neurons, we next evaluated whether SL-NH2 activates PLC as assessed by IP accumulation. The results showed that SL-NH2 application resulted in a significant increase in IP accumulation (p<0.001, Fig. 2B). Cultured TG neurons are a heterogeneous population that consists of nociceptive and nonnociceptive neurons. Therefore, we next determined whether SL-NH2-induced activation of TG neurons occurs in the nociceptive subpopulation. We employed single cell level calcium imaging to address this question. The results showed that SL-NH2 induces calcium influx in TG neurons that also were capsaicin sensitive (Fig. 2C). Further quantification of responsive cells from multiple independent experiments demonstrated that SL-NH2-induced calcium influx occurs in 68.6±10.3% (_n_=156) of cultured TG neurons and that 56.4±9.3% of this population were also capsaicin sensitive. Next, we evaluated whether activation of PAR2 induces an inward current in TG neurons, since this is a direct measure of neuronal excitation. Indeed, SL-NH2 induced an inward current in TG neurons (Fig. 2D). This response was observed in 44% (8 of 18) of the tested cells. The average size of SL-NH2 (100μM)-induced current was 149pA. Also, in a current-clamp recording, application of SL-NH2 (100μM) generated action potentials in 27% of tested neurons (6/22) (Fig. 2E). The in vivo relevance of PAR-2 activation of trigeminal nociceptors was verified using an orofacial model of pain with observers blinded to the treatment allocation. The injection of SL-NH2 (50μg) into the right vibrissal pad of the rat evoked nocifensive behavior that was significantly greater (p<0.05) than that observed after vehicle injection (Fig. 2F). However, the injection of the same dose of the control peptide LR-NH2 (50μg) did not result in significant nocifensive behavior (Fig. 2F). Since we noted that all the SL-NH2 injected animals developed a local edema post injection, we evaluated the increase in blood flow subsequent to PAR-2 agonist injection in the injected area. The laser Doppler measurements showed that vehicle injection resulted in a small increase in blood flow (Fig. 2G). However, in the same animal, capsaicin injection resulted in a substantial increase in blood flow (positive control, Fig. 2G). The injection of SL-NH2 also resulted in an increase in blood flow (Fig. 2H) and measurements from multiple animals showed that this increase was significantly different than vehicle injected animals (p<0.05, Fig. 2I).
Peptide PAR-2 agonist SL-NH2 activates TG nociceptors in a PLC–PKC dependent pathway and evokes nociception. (A) Cultured TG neurons were exposed to either vehicle or LR-NH2 (100 μM) or increasing concentration of SL-NH2 (1–100 μM) for 15 min. In experiments evaluating the effect of PKC inhibitor on SL-NH2 evoked release, neurons were pretreated with BIS (500 nM) for 15 min and then exposed to SL-NH2 (100 μM) for 15 more minutes. The supernatant was analyzed for the amount of iCGRP released. Data are presented as means ± SEM and analyzed using one-way ANOVA with Bonferroni post hoc test (n = 6–18 wells per condition, ***p < 0.001). (B) Cultured TG neurons were exposed to SL-NH2 (100 μM) and amount of IP accumulated was measured as described in methods. Data were analyzed with two tailed t test (n = 6 wells per condition, ***p < 0.001). (C) A representative tracer showing cultured TG neurons preincubated with a calcium sensitive fura 2-AM that were exposed to SL-NH2 (100 μM) for 3 min and then pulsed with capsaicin (30 nM) for 45 s (n = 156 cells from four independent cultures). (D) A representative tracer showing voltage-clamp recording of a TG neuron that was exposed to SL-NH2 (100 μM) and evoked inward current was measured (8/18 tested cells). (E) A representative tracer showing current-clamp recording of a TG neuron that was exposed to SL-NH2 (100 μM) and evoked changes in membrane potential were observed (6/22 tested cells). (F) After acclimatization to the testing chamber, animals were injected with vehicle or SL-NH2 (50 μg) or LR-NH2 (50 μg) in the right vibrissal pad and number of flinches was observed for 5 min period. Data were analyzed with one way ANOVA with Bonferroni post hoc test (n = 5–10 animals per group, *p < 0.05). (G) A representative tracer showing Doppler recording of an anesthetized animal that was first injected with vehicle and then with capsaicin (10 μg, positive control) in the right vibrissal pad. (H) A representative tracer showing Doppler recording of an anesthetized animal that was injected with SL-NH2 (50 μg) in the right vibrissal pad. (I) Comparison of change in blood flow evoked by SL-NH2 (50 μg) application to that evoked by vehicle application. Data were analyzed with t test (n = 6 animals per group, *p < 0.05).
3.3. The enzyme PAR-2 activator, trypsin, activates TG nociceptors in a PLC–PKC and ruthenium red-sensitive manner and evokes nociception
Because proteases such as trypsin are possible endogenous activators of PAR-2, we next evaluated whether trypsin also activates TG nociceptors and if so, whether this is mediated by activation of the PKC pathway (Fig. 3). The application of trypsin (0.1–600nM) evoked a significant iCGRP release in a concentration-dependent fashion, with a maximal response occurring at 100nM (EC50=30nM, Fig. 3A). The trypsin (100nM)-evoked iCGRP release was significantly inhibited by pretreatment with BIS (500nM) and a nonselective channel blocker ruthenium red (10μM) (Fig. 3B, p<0.001 for both). The application of trypsin (100nM) induced a calcium influx in a major subpopulation of nociceptive neurons as determined by their response to capsaicin (Fig. 3C). Pooled data from multiple independent experiments indicated that approximately 75.6±3.4% of TG neurons responded to trypsin (_n_=126), and that 74.8±2% of this population is capsaicin sensitive. In patch-clamp experiments, the application of trypsin (100nM) induced an inward current in cultured TG neurons (Fig. 3D). Such responses were observed in 40% (8 of 20) of cells tested. The average size of trypsin evoked current was 235pA. In a current-clamp configuration, the application of trypsin resulted in the generation of action potentials in approximately 30% of tested neurons (Fig. 3E). Because the enzyme PAR-2 activator, trypsin, excited TG nociceptors, we further verified the in vivo relevance of this in a rat model of orofacial nociception using observers blinded to treatment allocation. The injection of trypsin (1%) into the right vibrissal pad of the rat evoked nocifensive behavior that was significantly greater (p<0.05) than that observed after vehicle injection (Fig. 3F). In addition, as observed with SL-NH2 application, trypsin (1%) increased the blood flow in the injected area (Fig. 3H) that was significantly different than that induced by vehicle injection (p<0.05, Fig. 3I).
Enzyme PAR-2 activator, trypsin, activates TG nociceptors in a PLC–PKC and RR-sensitive manner and evokes nociception. (A) Cultured TG neurons were exposed to increasing concentration of trypsin (0.1–600 nM) for 15 min and supernatant was analyzed for amount of iCGRP released. Data were analyzed with ANOVA with Bonferroni post hoc test (n = 8–12 wells per condition). Trypsin-evoked release at concentrations (10–600 nM) was significantly different from basal release (p < 0.05 or 0.001). (B) Cultured TG neurons were pretreated with vehicle or BIS (500 nM) or RR (10 μM) for 15 min and then exposed to vehicle or trypsin (100 nM) for 15 min. The supernatant was analyzed for amount of iCGRP released. Data were analyzed with ANOVA with Bonferroni post hoc test (n = 12–28 wells per condition, *p < 0.001). (C) A representative tracer showing cultured TG neurons preincubated with a calcium sensitive fura 2.AM that were exposed to trypsin (100 nM) for 3 min and then pulsed with capsaicin (30 nM) for 45 s (n = 126 cells from four independent cultures). (D) A representative tracer showing voltage-clamp recording of a TG neuron that was exposed to trypsin (100 nM) and evoked inward current was measured (8/20 cells tested). (E) A representative tracer showing current-clamp recording of a TG neuron that was exposed to trypsin (100 nM) and evoked changes in membrane potential were observed (7/22 cells tested). (F) After acclimatization to the testing chamber, animals were injected with vehicle or trypsin (1%) or boiled trypsin (1%) in the right vibrissal pad and number of flinches was observed for 5 min period. Data were analyzed with one-way ANOVA with Bonferroni post hoc test (n = 6–10 animals per group, *p < 0.05). (G) A representative tracer showing Doppler recording of an anesthetized animal that was injected with trypsin (1%) in the right vibrissal pad. (H) Comparison of change in blood flow evoked by trypsin (1%) application to that evoked by vehicle application. Data were analyzed with t test (n = 6 animals per group, *p < 0.05).
3.4. RNA interference confirms the specificity of SL-NH2 and trypsin in trigeminal cultures
We next evaluated whether PAR-2 is the target for trypsin and SL-NH2 in trigeminal cultures. We used custom synthesized, HPLC purified siRNA directed against the PAR-2 mRNA sequence. We validated the knock down of PAR-2 at the mRNA as well as protein levels. The real-time RT-PCR data showed that PAR-2 mRNA message was substantially and significantly (p<0.05) reduced in cultures transfected with siRNA against PAR-2 but not in mock transfected cultures or in cultures transfected with scrambled siRNA (Fig. 4A). Similarly, immunohistochemistry performed using a monoclonal antibody against PAR-2 showed that PAR-2 protein was selectively absent in the cells transfected with PAR-2 siRNA as identified by the presence of green Alexa-488 fluorescence (Fig. 4B). However, untransfected cells in the same field demonstrated expression of PAR-2. Next, calcium imaging experiments were performed on cultures transfected similarly. When PAR-2 siRNA treated cultures were exposed to SL-NH2 (100μM), most of the transfected cells did not respond to the agonist application. However, a significant proportion of untransfected cells in the same field responded to SL-NH2 application. A representative field is shown in Fig. 4C. Further analysis of data generated from multiple independently transfected cultures showed that the mean total calcium influx was diminished by ˜74% in PAR-2 siRNA transfected cells compared to the mock transfected cells or cells transfected with scrambled siRNA (p<0.001, Fig. 4D). Importantly, in cultures treated with PAR-2 siRNA, the mean calcium influx in response to SL-NH2 application in untransfected cells was similar to that of mock transfected cells. This within group control further confirms the specificity of the PAR-2 knock down. Similar results were observed when the transfected cells were exposed to trypsin (100nM). The trypsin response was substantially reduced only in cells that showed transfection with PAR-2 siRNA (p<0.001, Fig. 4E). These data support the hypothesis that the majority of actions of trypsin in trigeminal nociceptors are mediated via activation of PAR-2.
RNA interference demonstrates the selectivity of trypsin and SL-NH2 in trigeminal cultures. (A) Cultured TG neurons were transfected with HiPerFect alone (control), scrambled siRNA (scrambled) or with Alexa-Fluor 488 conjugated siRNA against PAR-2. The RNA isolated from these samples was used for real-time RT-PCR experiments using primers specific for PAR-2 gene and the internal control (18S). The expression was normalized to the housekeeping gene 18S. Data were analyzed with one-way ANOVA with Bonferroni post hoc test (n = 3 cultures, *p < 0.05). (B) Similarly transfected neurons were fixed and immunohistochemistry was performed using an antibody against PAR-2 protein. The horizontal white arrow denotes an example of transfected cell that did not show PAR-2 immunoreactivity. The vertical yellow arrow denotes an example of an untransfected cell that showed PAR-2 immunoreactivity. (C) Calcium imaging was performed with similarly transfected neurons. The left panel shows transfected cells as visualized by Alexa-Fluor 488. The middle and right panels show cells before and after SL-NH2 (100 μM) application. The horizontal arrows denote examples of untransfected cells that responded to SL-NH2 application. (D) In similarly transfected cells, the amount of calcium influx caused by the application of SL-NH2 (100 μM) was quantified. The data were pooled from four independent experiments. The total number of cells from all the experiments that responded to the SL-NH2 application is shown in each bar. The data were analyzed with one-way ANOVA with Bonferroni post hoc test (n = 4 independent experiments, ***p < 0.001). (E) In similarly transfected cells, the amount of calcium influx caused by the application of trypsin (100 nM) was quantified. The data were pooled from four independent experiments. The total number of cells from all the experiments that responded to the trypsin application is shown in each bar. The data were analyzed with one-way ANOVA with Bonferroni post hoc test (n = 4 independent experiments, ***p < 0.001).
3.5. Activation of PAR-2 by SL-NH2 rapidly induces functional competence in the DOR to inhibit TG nociceptors
Recently, we demonstrated that activation of the PLC–PKC pathway by bradykinin induces a rapid functional competence in the DOR to inhibit TG nociceptor function (Patwardhan et al., 2005). Here, we evaluated whether activation of PAR-2, which also signals via the PLC–PKC pathway, leads to DOR competence. Confirming our previous findings, the activation of DOR by DPDPE (1μM) does not modulate BK(10μM)/PGE2(1μM) evoked iCGRP release or PGE2 (1μM) evoked cAMP accumulation from these neurons. However, pretreatment with SL-NH2 (100μM at 15min) induced DOR competence such that DPDPE significantly inhibited both the stimulated iCGRP release and cAMP accumulation by 31% and 39% compared to control BK/PGE2 evoked iCGRP release or PGE2 evoked cAMP accumulation, respectively (Figs. 5A and B).
Activation of PAR-2 by SL-NH2 rapidly induces functional competence in the DOR to inhibit TG nocieptors. (A) Cultured TG neurons were treated with either vehicle or SL-NH2 (100 μM) for 15 min, aspirated and then DPDPE (1 μM) modulation of BK (10 μM)/PGE2 (1 μM) evoked iCGRP release was observed. Data are analyzed with two-way ANOVA with Bonferroni post hoc test (n = 8–10 wells per condition, *p < 0.05). (B) In a similar paradigm, DPDPE (1 μM) modulation of PGE2 (1 μM) stimulated cAMP accumulation was observed (n = 6 wells per condition, *p < 0.05).
4. Discussion
The results from this study demonstrate that PAR-2 is expressed by trigeminal TRPV1 positive nociceptors and that PAR-2 agonists activate these nociceptors in a PLC–PKC dependent pathway as shown by single cell, multicell, and in vivo behavioral assays. These results were replicated using two structurally distinct PAR-2 agonists, SL-NH2 and trypsin. The activation of trigeminal nociceptors by trypsin was significantly inhibited by blockade of cation channels by RR. In addition, the peripheral injection of both SL-NH2 and trypsin rapidly evokes nocifensive behavior and results in increased blood flow in an orofacial pain model. The specificity of PAR-2 agonists was confirmed by the diminished efficacy of these agonists in cultures in which PAR-2 was knocked down. Activation of PAR-2 by SL-NH2 induces functional competence in the DOR and thus mimics the priming effect previously demonstrated with activation of B2 receptors.
Several previous studies have demonstrated that activation of the PAR-2 in the DRG innervated tissue leads to nociception and neurogenic inflammation (Steinhoff et al., 2000; Hoogerwerf et al., 2001; Vergnolle et al., 2001). However, no study to date has evaluated the pharmacology of PAR-2 activation in the trigeminal sensory system and whether this receptor heterologously regulates other GPCRs such as opioid receptors.
The present study demonstrates that both SL-NH2 and trypsin activate trigeminal nociceptors in a PKC-dependent fashion. Multiple mechanisms could be involved in the trypsin-evoked activation of TG nociceptors as measured by iCGRP release. Previous studies have demonstrated that trypsin can evoke release of calcium from internal stores in DRG nociceptors (Amadesi et al., 2004). Indeed, thapsigargin, which releases calcium from intracellular stores (Thastrup et al., 1990), is capable of evoking iCGRP release in TG neurons (our unpublished observations). Secondly, it is known that PKC can phosphorylate TRP channels such as TRPV1 (Amadesi et al., 2004; Cesare et al., 1999) leading to reduction of this channel threshold to temperatures as low as 37°C (Ding-Pfennigdorff et al., 2004). Thus, PAR-2 activation of PKC might lead to nociceptor sensitization via TRPV1, a hypothesis consistent with our findings of ruthenium red blockade of trypsin-evoked CGRP release in TG neurons. Thirdly, as demonstrated by previous studies with bradykinin (Shin et al., 2002; Bandell et al., 2004), activation of PAR-2 can lead to generation of diacylglycerol (DAG) or hydroperoxy eicosatetraenoic acid (HPETE) and thus can open DAG/HPETE activated channels such as TRPA1 and TRPV1 and lead to the release of iCGRP. Indeed, our data support all of these possibilities since SL-NH2 and trypsin-evoked release is significantly reduced by application of a PKC inhibitor and by RR.
In the in vitro RNAi experiments, we demonstrated that both SL-NH2 and trypsin evoked calcium influx was substantially diminished in PAR-2 siRNA treated cultures. This demonstrates for the first time that the major target of trypsin in trigeminal nociceptors is PAR-2. The in vivo application of both SL-NH2 and trypsin resulted in nocifensive behavior and increase in blood flow. The lack of such effect by LR-NH2 (inactive at PAR-2) and boiled trypsin (enzymatically inactive) denotes the specificity of such response. Although the direct activation of PAR-2 on trigeminal afferents probably plays a significant role in this effect, the involvement of nonneuronal cells in the vicinity of these trigeminal afferents that also express PAR-2 cannot be discounted.
In this study, we used two distinct agonists of PAR2, including the synthetic peptide SL-NH2 and an enzyme activator, trypsin. Although extrapancreatic sources of trypsin are currently under evaluation, it should be noted that trypsin IV is found in the epithelial cells (Cottrell et al., 2004). Moreover, several other trypsin-like serine proteases (e.g., mast cell tryptase and coagulation factors) can be released in the vicinity of nociceptive trigeminal afferents during inflammation and thus PAR-2 might play a pivotal role in the generation and maintenance of inflammatory pain in the orofacial region.
Inflammation leads to increased efficacy of peripherally administered opioid agonists as measured by behavioral assays (Joris et al., 1987; Stein et al., 1989; Stein et al., 2003). Activation of the PKC pathway by the inflammatory mediator BK leads to a rapid functional competence in DOR for inhibiting trigeminal nociceptor activity (Patwardhan et al., 2005). As shown in the present study, the activation of PAR-2 also leads to a rapid functional competence in the DOR. Thus, there is now evidence for heterologous regulation of DOR signaling by multiple inflammatory mediators which activate GPCRs that signal via the PKC pathway. Since PAR-2, B2 and several other peripheral GPCRs signal via this pathway (Moriyama et al., 2003; Sugiuar et al., 2004), it is intriguing to speculate the presence of a redundancy of inflammatory mediators that converge on the PKC pathway leading to the initiation and maintenance of a functional inhibitory competence of DOR during inflammation. Since opioids have little to no peripheral antinociceptive activity under basal conditions, this signaling pathway may have considerable significance in regulating the development of peripheral opioid analgesia.
In summary, functional PAR-2 responses are evident in a large proportion of trigeminal nociceptors and activation of PAR-2 leads to generation of orofacial pain. Further, PAR-2 signaling can lead to functional competence in DOR for inhibiting trigeminal nociceptors.
Acknowledgements
We thank Jaime Cerecero, Gabriela Helesic and Teresa Sanchez for technical assistance. We thank Dr. Michael Henry for his help in acquiring confocal images. This work was supported by NIDA P01 Grant DA016179 (KMH).
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Keywords:
PAR, Protease activated receptor; Pain; Delta opioid receptor; Trypsin; Trigeminal; siRNA; RNAi
© 2006 Lippincott Williams & Wilkins, Inc.