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¹ Departments of Surgery and Physiology, University of California, San Francisco, 521 Parnassus Avenue, San Francisco, CA 94143-0660, USA
² Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada
³ Instituto Potosino de Investigacion Cientifica y Tecnologica San Luis Potosi, SLP, Mexico
⁴ Gastrointestinal Diseases Research Unit, Division of Gastroenterology, Queen's University, Kingston, Ontario, Canada

	Abstract

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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) C and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR₂ agonists promoted translocation of PKC 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 Cß, 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 PKC and PKA prevented sensitization of TRPV1 Ca²⁺ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR₂ activates PKC 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.

(Received 13 April 2006; accepted after revision 20 June 2006; first published online 22 June 2006)
Corresponding author N. W. Bunnett: University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, CA 94143-0660, USA. Email: nigelb{at}itsa.ucsf.edu

	Introduction

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Protease-activated receptor 2 (PAR₂) is widely expressed in the nervous system where it mediates the actions of proteases on diverse neuronal processes (reviewed in Ossovskaya & Bunnett, 2004). Proteases from the circulation (coagulation factors VIIa, Xa; Camerer et al. 2000), inflammatory cells (tryptase; Corvera et al. 1997; Molino et al. 1997), epithelial cells and neurons (trypsins I, II, IV; Cottrell et al. 2004) can cleave PAR₂ to expose a tethered ligand domain that binds to and activates the cleaved receptor. Activated PAR₂ controls neurogenic inflammation, pain and neuronal excitability. PAR₂ is expressed by primary spinal afferent neurons, where activation stimulates release of substance P and calcitonin gene-related peptide in peripheral tissues to cause neurogenic inflammation (Steinhoff et al. 2000; Cenac et al. 2002, 2003; Nguyen et al. 2003). Similar mechanisms mediate the effects of PAR₂ agonists on airway constriction (Ricciardolo et al. 2000) and gastric mucus secretion (Kawabata et al. 2001b). PAR₂ agonists also cause thermal and mechanical somatic hyperalgesia (Kawabata et al. 2001a; Vergnolle et al. 2001), and excite mesenteric sensory neurons to induce visceral hyperalgesia (Hoogerwerf et al. 2001; Coelho et al. 2002; Kirkup et al. 2003). However, the molecular mechanisms by which PAR₂ regulates neuronal functions are incompletely understood.

PAR₂-induced thermal hyperalgesia depends on sensitization of transient potential receptor vanilloid 1 (TRPV1) (Amadesi et al. 2004; Dai et al. 2004). TRPV1 is a non-selective cation channel expressed by nociceptive neurons, that mediates inflammatory and thermal hyperalgesia (Caterina et al. 1997, 2000; Davis et al. 2000). Exogenous (capsaicin, ethanol) and endogenous (protons pH < 6.0, heat > 43°C, anandamide) factors directly activate TRPV1 (Caterina et al. 1997; Zygmunt et al. 1999; Trevisani et al. 2002). In addition, PAR₂ agonists (Amadesi et al. 2004; Dai et al. 2004) and other inflammatory agents, including bradykinin, ATP, prostaglandin E₂ (PGE₂) and nerve growth factor (NGF) (Lopshire & Nicol, 1997; Chuang et al. 2001; Tominaga et al. 2001; Vellani et al. 2001) indirectly sensitize TRPV1, causing hyperalgesia. The mechanisms of this sensitization include activation of protein kinase (PK) C and PKA, which phosphorylate TRPV1 to modify channel gating (Lopshire & Nicol, 1998; Premkumar & Ahern, 2000; Bhave et al. 2002; Mohapatra & Nau, 2003). The PKC isozyme, which plays a major role in mechanical and thermal hyperalgesia (Khasar et al. 1999), phosphorylates TRPV1 (Numazaki et al. 2002) to mediate bradykinin-induced sensitization of TRPV1 currents (Cesare et al. 1999). PKA, a mediator of injury-induced hyperalgesia (Malmberg et al. 1997), also phosphorylates TRPV1 to regulate its desensitization (Bhave et al. 2002; Mohapatra & Nau, 2003), and thereby mediates PGE₂-induced sensitization of TRPV1 (Lopshire & Nicol, 1998; Rathee et al. 2002). Additional mechanisms of TRPV1 sensitization include altered interactions of TRPV1 with the endogenous inhibitor phosphatidylinositol-4,5-bisphosphate (Chuang et al. 2001; Prescott & Julius, 2003), and PKC- and Src kinase-dependent trafficking of TRPV1 to the plasma membrane (Morenilla-Palao et al. 2004; Zhang et al. 2005).

The purpose of the present investigation was to define the mechanisms by which PAR₂ sensitizes TRPV1 to induce thermal hyperalgesia. Although PKC contributes to PAR₂-induced sensitization of TRPV1 currents (Dai et al. 2004), the role of PKC in PAR₂-induced thermal hyperalgesia has not been examined, and the contributions of other second messenger kinases, such as PKA, are unknown. Our objectives were to determine whether (1) primary spinal afferent neurons that express PAR₂ also contain PKC and PKA, using immunofluorescence and confocal microscopy; (2) PAR₂ agonists activate and cause membrane translocation of PKC and PKA in neurons and cell lines, using microscopy, subcellular fractionation and Western blotting; (3) PKC and PKA mediate PAR₂-induced sensitization of TRPV1 in cell lines, by measuring TRPV1 Ca²⁺ signals and currents; and (4) PKC and PKA mediate PAR₂-induced sensitization of TRPV1-dependent thermal hyperalgesia.

	Methods

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Animals

Sprague-Dawley rats (male, 200–250 g) and C57Bl6 mice (male, 6–8 weeks) were obtained from Charles River Laboratories (CA, USA, and Canada). The study was approved by Institutional Animal Care and Use Committees of the University of California, San Francisco and the University of Calgary. At the end of the experiments animals were humanely killed using sodium pentobarbital (200 mg kg^–1 I.P.) and bilateral thoracotomy.

Agonists and antagonists

PAR₂-activating peptide (PAR₂-AP, SLIGRL-NH₂), corresponding to the tethered ligand of rat and mouse PAR₂, and the inactive reverse peptide sequence (PAR₂-RP), which was used as the control, were obtained from Sigma Genosys (The Woodlands, TX, USA). Bovine pancreatic trypsin was from Worthington Biochemical Corporation (Lakewood, NJ, USA). The selective PKC translocation inhibitor peptide (EAVSLKPT, PKCI), inactive scrambled sequence (PKCI-sc), the PKA inhibitor H-89 (N-[2-((p-bromo-cinnamyl)amino)ethyl]-5-isoquinolinesulphonamide), cell-permeable myristoylated PKA inhibitor 14–22 amide (PKAI_14–22, Myr-GRTGRRNAI-NH₂), and the PKA activator forskolin were from Calbiochem (La Jolla, CA, USA). The PKC activators phorbol 12-myristate 13-acetate (PMA) and phorbol dibutyrate, and indomethacin, a cyclo-oxygenase inhibitor, were from Sigma (St Louis, MO, USA). The PKA inhibitor WIPTIDE (TTYADFIASGRTGRRNAI-NH₂) was from Peninsula Laboratories (San Carlos, CA, USA). The PKCI conjugated to TAT protein to promote membrane permeability (Schwarze et al. 1999) (TAT-PKCI, YGRKKRRQRRRC-disulphide bond-CEAVSLKPT-COOH) and the inactive scrambled sequence (TAT-PKCI-sc) were from SynPep Corp (Dublin, CA, USA). The selection of inhibitors and doses used in this study was based on previous studies (Aley et al. 2000, 2001; Hu et al. 2002; Rathee et al. 2002).

Antibodies

Rabbit anti-human PKC, rabbit anti-human PKA catalytic subunit (PKA_C), mouse anti-human PAR₂ (SAM11) and mouse anti-EGFP (enhanced green fluorescent protein) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Goat anti-rabbit and anti-mouse IgG conjugated to FITC, Rhodamine Red X or horseradish peroxidase were from Jackson ImmunoResearch (West Grove, PA, USA). In control experiments, primary antibodies were incubated before use with peptides used for immunization (1 µM, 24 h, 4°C).

Cell lines

Human embryonic kidney (HEK) 293 cells were maintained in modified Eagle's with Earle's BSS medium (MEM), 10% fetal bovine serum (FBS) and 100 U ml^–1 penicillin, 0.1 mg ml^–1 streptomycin. HEK 293 cells stably expressing human TRPV1 (HEK-TRPV1, a gift from Dr J. Davis, GSK Harlow, UK) were generated and maintained in MEM, 10% FBS and geneticin (400 mg l^–1) (Hayes et al. 2000). HEK-FLP cells (Invitrogen, Carlsbad, CA, USA) stably expressing human PAR₂ with N-terminal Flag and C-terminal T7 epitopes (HEK-PAR₂) were generated and maintained as described (Jacob et al. 2005). HEK 293 cells were transiently transfected with 1 µg of EGFP-human PKC (a gift from Dr Daria Mochly Rosen, Stanford University) using Lipofectamine 2000. (Invitrogen) to generate HEK-PKC-EGFP cells (Schechtman et al. 2004). These cells were studied 48 h after transfection.

Neuronal culture

Rats were anaesthetized with sodium pentobarbital (200 mg kg^–1 I.P.). Dorsal root ganglia (DRG) from the thoracic and lumbar spinal cord were removed and minced in cold Hank's balanced salt solution (HBSS) and incubated for 60–90 min at 37°C in Dulbecco's modified Eagle's medium (DMEM) containing 0.5 mg ml^–1 of trypsin, 1 mg ml^–1 of collagenase type IA and 0.1 mg ml^–1 of DNase type IV (Sigma) (Steinhoff et al. 2000). Soybean trypsin inhibitor (Sigma) was added to neutralize trypsin. Neurons were pelleted, suspended in DMEM containing 10% FBS, 10% horse serum, 100 U ml^–1 penicillin, 0.1 mg ml^–1 streptomycin, 2 mM glutamine and 2.5 µg ml^–1 DNase type IV, plated on glass coverslips coated with Matrigel (BD Biosciences, Bedford, MA, USA), and cultured for 2–3 days.

Localization of PKC and PKA_C in cultured cells

HEK 293 cells and cultured DRG neurons were incubated in HBSS, 0.1% BSA, 20 mM Hepes, pH 7.4 at 37°C and treated with test substances. Cells were fixed with 4% paraformaldehyde, 100 mM PBS, pH 7.4 for 20 min at 4°C. Cultured cells were incubated with PBS containing 5–10% normal goat serum and 0.1–0.3% Triton X-100 for 30 min, and incubated with primary antibodies to PKC (1 : 250) or PKA_C (1: 400) for 16 h at 4°C. After washing, cells were incubated with goat anti-rabbit IgG conjugated to FITC (1 : 300) for 2 h at room temperature. Cells expressing PKC-EGFP were similarly treated and fixed.

Localization of PKC, PKA_C and PAR₂ in DRG sections

Rats (male, 200 g) were anaesthetized with sodium pentobarbital (200 mg kg^–1 I.P.) and transcardially perfused with PBS containing 100 U of heparin, followed by 4% paraformaldehyde in 100 mM PBS, pH 7.4. DRG (L4–L6) were immersion fixed for 4 h at 4°C. DRG were incubated in 20% sucrose in PBS for 24 h at 4°C, embedded in OCT compound (Miles, Elkhart, IN, USA), and frozen sections of 16 µm were prepared. Sections were incubated with PBS containing 5–10% normal goat serum and 0.1–0.3% Triton X-100 for 30 min, and incubated with primary antibodies: PKC (1 : 750), PKA_C (1 : 750), PAR₂ (1 : 250) for 16 h at 4°C. After washing, sections were incubated with goat anti-rabbit IgG conjugated to FITC or goat anti-mouse IgG conjugated to Rhodamine Red X (1 : 200) for 2 h at room temperature. Slides were washed and mounted in Prolong (Molecular Probes, Eugene, OR, USA).

Confocal microscopy

Specimens were observed using Zeiss Axiovert and Bio-Rad MRC1000 confocal microscopes with Zeiss Plan Apo x40 (NA 1.4) or x100 (NA 1.3) objectives. Images were collected at zoom of 1–2, iris of < 3 µm and typically 5–10 optical sections were taken at intervals of 0.5 µm. Images were coloured to represent the appropriate fluorophores, and processed using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA) to adjust contrast and brightness. Images of stained and control slides were collected and processed identically. To quantify the effects of agonists on the subcellular distribution of PKC and PKA_C in cultured neurons, the fluorescence intensity of the cells was measured by selecting a straight line across the neuronal soma and using the plot profile function of ImageJ software (version 10.2, NIH image). The averaged fluorescence intensities between peripheral and central regions of the cell were compared. The number of neurons showing redistribution of these kinases between the cytosol and periphery of the cells was also determined and expressed as a percentage of the total number of neurons analysed.

Subcellular fractionation and Western blotting

HEK 293 cells were maintained overnight in MEM and 0.1% BSA. Cells were incubated in HBSS, 0.1% BSA, 20 mM Hepes, pH 7.4 and treated at 37°C for 0–10 min with PAR₂ agonists. Cells were washed with ice-cold PBS, scraped into 150 µl of homogenization buffer (20 mM Tris-HCl, pH 7.4 and protease inhibitors) and disrupted with 20 strokes in a glass homogenizer. The lysate was centrifuged at 500 g for 10 min, and the resulting supernatant was centrifuged at 100 000 g for 1 h. The supernatant was collected as the cytosolic fraction. The pellet containing the membrane fraction was treated with lysis buffer (1% SDS, 20 mM Tris-HCl, pH 7.4 and protease inhibitor cocktail, Roche Diagnostics, Indianapolis, IN, USA). Both cytosolic and membrane fractions were boiled for 5 min and clarified by centrifugation at 15 000 g for 15 min. Protein concentration was measured by BCA assay. Samples (5 µg protein) were separated by SDS-PAGE (8% for PKC, 12% for PKA_C). Proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4°C with antibodies to PKC (1 : 10 000), PKA_C (1 : 10 000) or EGFP (1 : 10 000) in PBS containing 5% milk powder, 2% BSA, 0.1% Tween-20. Membranes were washed and incubated with goat anti-rabbit or anti-mouse horseradish peroxidase (1 : 10 000) for 1 h at room temperature. Immunoreactive proteins were detected by Enhanced Chemiluminescence (Pierce, Rockford, IL, USA). To ensure equal loading, membranes were stained with amido-black to detect all proteins.

Measurement of intracellular calcium concentration ([Ca²⁺]_i)

The experimental procedure has been previously described (Amadesi et al. 2004). HEK 293 cells were incubated in HBSS, 0.1% BSA, 20 mM Hepes, pH 7.4, containing 2.5 µM of fura-2/AM (Invitrogen) for 30–45 min at 37°C. Coverslips were mounted in an open chamber (350 µl) at 37°C. Fluorescence of individual cells was measured at 340 nm and 380 nm excitation and 510 nm emission using a Zeiss Axiovert microscope, an ICCD video camera (Stanford Photonics, Stanford, CA, USA) and a video microscopy acquisition program (Axon Instruments, Inc., Union City, CA, USA). Test substances were directly added to the chamber (50 µl injection). Each coverslip received only one treatment with PAR₂-AP or RP followed by capsaicin. In some experiments cells were pretreated for 30 min at 37°C with inhibitors before challenging with test substances. The magnitude of capsaicin responses was calculated as the increase above baseline. Results were first calculated as the 340 nm/380 nm emission ratio, which is proportional to the [Ca²⁺]_i, and then expressed as a percentage of the potentiation observed in cells pretreated with PAR₂-AP and antagonist vehicle (100%).

Measurement of intracellular cAMP

HEK 293 cells were maintained overnight in MEM-0.1% BSA. HEK 293 cells and DRG neurons in culture were incubated in HBSS, 0.1% BSA, 20 mM Hepes, pH 7.4 and treated with PAR₂ agonists for 0–5 min at 37°C. Intracellular cAMP levels were measured using the cAMP enzyme immunoassay system following the non-acetylation protocol according to the manufacturer's directions (Amersham Bioscience, Bucks, UK).

Electrophysiology

The experimental procedure has been previously described (Amadesi et al. 2004) with slight modifications. Membrane currents of HEK 293 cells were recorded using an Axopatch 1D amplifier (Axon Instruments). Patch pipette resistance ranged from 1 to 3 M, and the holding potential was –60 mV. The pipette solution was (mM): KCl 140, MgCl₂ 1, EGTA 5, Hepes 5, and ATPNa₂ 5. The external solution was (mM): NaCl 140, CaCl₂ 2, KCl 4, MgCl₂ 1, glucose 11, Hepes 5 and CsCl 3. The pH was adjusted with either KOH (pipette solution, pH 7.2) or NaOH (external solution, pH 7.4). In these standard solutions the input resistance of the HEK 293 cells was 1–10 G. Whole-cell currents were recorded using Axotape software and analysed using Axograph software (Axon Instruments). Membrane potentials were corrected for the liquid junction potential (pipette –11 mV). The recording chamber was continuously superfused with the external solution at approximately 2 ml min^–1. Rapid application of agonists in the external solution was made using an eight-barrelled fast-flow device (Barajas-Lopez et al. 1994). Experiments were at room temperature (23°C). Control TRPV1 responses were obtained by repeated application of capsaicin (300 nM) for 20 s. Only neurons that did not exhibit desensitization to two consecutive capsaicin applications were selected for the study. PAR₂-AP (100 µM) or, in a small series of experiments, the PKC agonist phorbol dibutyrate (300 nM) was then applied for 2 min and TRPV1 responses were examined at 30 s to 18 min following PAR₂ activation. Results were expressed as a percentage of the control TRPV1 current. In some experiments, PKC translocation inhibitor peptide or H89 was added to the pipette solution.

Paw withdrawal latency

Test substances were administered by intraplantar injection in mice under light halothane (5%) anaesthesia (Vergnolle et al. 2001). PAR₂-AP, PAR₂-RP, PKC and PKA inhibitors were dissolved in physiological saline (0.9% NaCl); capsaicin was dissolved in 80% physiological saline, 10% ethanol, and 10% Tween 80. To allow the entry of the membrane-impermeant inhibitors into cells, injection of PKCI, PKCI-sc and WIPTIDE (all 2.5 µl paw^–1) was preceded by injection of distilled water (2.5 µl paw^–1) to cause a transient hyposmotic permeabilization. Mice received an intraplantar injection of PAR₂-AP and capsaicin (5 µl paw^–1) 15–30 min later (final volume 10 µl paw^–1). The latency of paw withdrawal to a radiant heat stimulus was measured before and after the intraplantar injections of test substances, using a plantar test apparatus (Ugo Basile, Milan, Italy). Thermal hyperalgesia was defined as a decrease in the withdrawal latency compared to the basal measurement.

Statistical analysis

Results are expressed as mean ± S.E.M. Comparisons between groups were made by ANOVA and the Bonferroni test, Student's t test, or a paired Student's t test to evaluate differences between the same cells. Comparisons between proportions were made using Primer of Biostatistics software (3.1 version, Macintosh version, McGraw Hill, 1992) and the ‘standard error of proportion’ and the ‘compare two proportions’ functions. Differences were considered significant when P 0.05.

	Results

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DRG neurons that express PAR₂ also contain PKC and PKA_C

PAR₂ is present in DRG neurons that express substance P, calcitonin gene-related peptide and TRPV1, and which therefore mediate neurogenic inflammation and nociception (Steinhoff et al. 2000; Amadesi et al. 2004). To determine if PAR₂-expressing neurons also contain the potential regulatory kinases PKC and PKA_C, we used immunofluorescence. Immunoreactive PAR₂ was present in small- and medium-diameter neurons that are typically considered nociceptive neurons (Fig. 1A and D). These neurons also contained immunoreactive PKC and PKA_C (Fig. 1B, C, E and F, arrows). Staining for PKC and PKA_C was abolished by preabsorption of the antibodies with the peptides used for immunization, suggesting specificity (Fig. 1G and H). Since PAR₂ and TRPV1 are coexpressed in nociceptive neurons (Amadesi et al. 2004), PKC and PKA_C are appropriately localized to mediate PAR₂-induced regulation of TRPV1.

View larger version (121K): [in this window] [in a new window]   Figure 1.  Localization of immunoreactive PAR2, PKC and PKAC in DRG neurons A and D, PAR2; B, PKC; E, PKAC; C and F, merge. G, PKC preabsorption control; H, PKAC preabsorption control. Neurons expressing PAR2 also expressed PKC and PKAC (arrows). Scale bar = 20 µm.

PAR₂ agonists activate PKC in HEK 293 cells and DRG neurons

PKC phosphorylates and sensitizes TRPV1 (Numazaki et al. 2002). We investigated the effects of PAR₂ agonists on translocation of PKC from the cytosol to the plasma membrane, since membrane translocation of PKC is an index of activation of this kinase (Dorn & Mochly Rosen, 2002). We first studied trafficking of PKC-EGFP expressed in HEK 293 cells, which can be detected with high sensitivity and specificity. PAR₂ agonists sensitize TRPV1 similarly in HEK 293 cells and DRG neurons (Amadesi et al. 2004). Thus, HEK 293 cells, which naturally express PAR₂, are a useful model system to study regulation of TRPV1. In unstimulated HEK 293 cells, PKC-EGFP was detected in the cytosol (Fig. 2A). PMA (1 µM), a PKC activator, induced a prominent redistribution of PKC-EGFP from the cytosol to the plasma membrane at 5 min (Fig. 2A). In a similar manner, PAR₂-AP (100 µM) induced trafficking of PKC-EGFP to the membrane within 0.5 min and 1 min (Fig. 2A), and after 15 min PKC-EGFP returned to the cytosol (results not shown). PAR₂-RP (100 µM, 1 min) did not affect the subcellular distribution of PKC-EGFP (Fig. 2A). PMA and PAR₂-AP also stimulated membrane translocation of immunoreactive PKC in HEK-TRPV1 cells (results not shown). Thus, EGFP does not interfere with the redistribution of PKC to the plasma membrane. Of note, PAR₂-AP also caused translocation of PKC-EGFP to the nucleus after 1 min (Fig. 2A). However, we did not detect nuclear translocation of endogenous PKC after exposure to PAR₂-AP (not shown). Thus, the physiological relevance of this redistribution is unknown.

View larger version (74K): [in this window] [in a new window]   Figure 2.  Effects of PAR2 agonist on the subcellular distribution of PKC determined by confocal microscopy A, HEK 293 cells expressing PKC-EGFP (enhanced green fluorescent protein). In unstimulated cells (non-treated, NT), PKC-EGFP was cytosolic and in vesicles (arrows). PMA (phorbol 12-myristate 13-acetate) induced translocation to the plasma membrane at 5 min (arrowheads). PAR2-AP induced translocation to the plasma membrane at 1 min (arrowheads). PAR2-RP did not affect the subcellular location of PKC-EGFP, which remained in the cytosol and vesicles (arrows). Scale bar = 10 µm. B, rat DRG neurons in culture. In unstimulated neurons (NT, non-treated), PKC was cytosolic and in vesicles (arrows). PMA induced translocation of PKC to the plasma membrane at 5 min (arrowheads). PAR2-AP also induced translocation of PKC to the plasma membrane at 0.5 and 1 min (arrowheads). PKC was in the cytosol and vesicles of cells treated with PAR2-RP (arrows). Control shows staining with preabsorbed PKC antibody. Scale bar = 10 µm. C, fluorescence intensity (in arbitrary units) measured in a line bisecting the neuronal soma (e.g. see dashed line in B). In untreated cells (NT) or cells incubated with PAR2-RP (1 min), the signal was mostly cytosolic. In cells treated with PMA (5 min) or PAR2-AP (1 min), the signal in the cytosol diminished and was more prominent at the plasma membrane. Each trace is an average of 4–6 cells. D, the effect of PMA, PAR2-AP and PAR2-RP on the percentage of the total number of observed neurons expressing PKC with PKC either in the cytosol or at the plasma membrane of the soma. The proportion of cells with PKC at the plasma membrane was increased after treatment with PMA and PAR2-AP. Observations from n > 100 cells.

To confirm that PAR₂ activation induces membrane translocation of PKC in HEK 293 cells, and to determine the kinetics of this process, we quantified PKC in cytosolic and membrane fractions by Western blotting. In unstimulated HEK-PKC-EGFP cells, PKC-EGFP was predominantly in cytosolic fractions (Fig. 3A and B). After incubation with PAR₂-AP (100 µM), the level of PKC-EGFP increased in membrane fractions and diminished in cytosolic fractions within 0.5–1 min (Fig. 3A and B). PAR₂-AP also caused translocation of endogenous immunoreactive PKC from cytosolic to membrane fractions of HEK-TRPV1 cells within 1 min (Fig. 3C and D). PKC-EGFP was still detected in membrane fractions 10 min after PAR₂ activation, when endogenous PKC had returned to the cytosol. The more persistent presence of PKC-EGFP in membrane fractions may reflect the higher sensitivity of detection of PKC-EGFP, or be due to the over-expression of PKC, or be related to an effect of EGFP on association of PKC with the membrane. Trypsin (10 nM) caused a similar membrane translocation of PKC-EGFP and immunoreactive PKC in HEK 293 cells (not shown). We did not quantify membrane translocation of PKC in DRG neurons by Western blotting, due to the limited number of cells and the low levels of PKC expression. Together, our results show that agonists of PAR₂ induce redistribution of PKC to the plasma membrane in both HEK 293 cells and DRG neurons, suggesting activation of PKC.

View larger version (17K): [in this window] [in a new window]   Figure 3.  Effects of PAR2 agonist on the subcellular distribution of PKC in HEK 293 cells determined by subcellular fractionation and Western blotting A, Western blot, and B, densitometric analysis of PKC-EGFP (enhanced green fluorescent protein) in cytosolic (c) and membrane (m) fractions of HEK-PKC-EGFP cells. PAR2-AP increased PKC-EGFP in membrane fractions and decreased PKC-EGFP in cytosolic fractions. C, Western blot, and D, densitometric analysis of immunoreactive PKC in cytosolic and membrane fractions of HEK-TRPV1 cells. PAR2-AP increased PKC in membrane fractions and decreased PKC in cytosolic fractions. *P < 0.05 compared to 0 min, n = 4 experiments.

TRPV1 has been proposed as a target for the cAMP/PKA cascade, and PKA phosphorylates TRPV1 to regulate its desensitization (Bhave et al. 2002; Rathee et al. 2002). Exposure of sensory neurons to forskolin, which activates the cAMP/PKA cascade, induces translocation of the PKA_C to the cell periphery, which is indicative of activation (Rathee et al. 2002). To determine if PAR₂ agonists activate PKA, we examined their effects on the subcellular distribution of PKA_C in DRG neurons. In most unstimulated neurons, immunoreactive PKA_C was cytosolic or present in vesicles that were uniformly distributed throughout the cytoplasm (Fig. 4A). This uniform cytosolic distribution of PKA_C was confirmed by measuring the pixel intensity (Fig. 4B). Indeed, PKA_C was detected in the vicinity of the plasma membrane in only 22 ± 5% of unstimulated neurons (of n = 270 total neurons) (Fig. 4C). Forskolin (10 µM, 0.5 min), which activates PKA, stimulated the redistribution of PKA_C from the central to the peripheral region of the cell (Fig. 4A). This redistribution was confirmed by measurement of the pixel intensity of immunoreactive PKA_c (Fig. 4B). This peripheral staining of PKA_c was detected in 92 ± 2% of neurons (n = 141) (Fig. 4C). PAR₂-AP (100 µM) also stimulated the redistribution of PKA_C from the central to the peripheral regions of the soma, and this effect was observed in 70 ± 4% of neurons at 1 min (n = 143), and 76 ± 3% of neurons at 5 min (n = 205) after stimulation (Fig. 4A–C). After 10–15 min, PKA_C returned to cytosolic vesicles (not shown). In contrast, PAR₂-RP had no effect on the subcellular distribution of immunoreactive PKA_C, with only 15 ± 4% of neurons showing localization of PKA_C close to the plasma membrane at 5 min (n = 106) (Fig. 4A–C).

View larger version (89K): [in this window] [in a new window]   Figure 4.  Effects of PAR2 agonist on the subcellular distribution of PKAC determined by confocal microscopy in rat DRG neurons in culture A, unstimulated neuron (non-treated, NT), PKAC (PKA catalytic subunit) was in vesicles and uniformly distributed throughout the cytosol (arrows). Forskolin (FSK) induced redistribution of PKAC from the central to the peripheral region of the soma at 0.5 min (arrows). PAR2-AP also induced redistribution of PKAC to the peripheral region of the cell at 1 min and 5 min (arrows). PKAC was uniformly distributed in the cytosol and in vesicles of cells treated with PAR2-RP (arrows). Control shows staining with preabsorbed PKAC antibody. Scale bar = 10 µm. B, fluorescence intensity (in arbitrary units) measured in a line bisecting the neuronal soma (e.g. see dashed line in A). In untreated cells (NT) or cells incubated with PAR2-RP (5 min), the signal was mostly cytosolic. In cells treated with FSK (0.5 min) or PAR2-AP (5 min), the signal in the cytosol diminished and was more prominent in superficial regions of the soma. Each trace is an average of 4–6 cells. C, the effects of FSK, PAR2-AP and PAR2-RP on the percentage of the total number of observed neurons expressing PKAC with PKAC either in the cytosol or near the plasma membrane of the soma. The proportion of cells with PKA in a superficial region of the cytosol was increased after treatment with FSK and PAR2-AP. Observations from n > 100 cells.

View larger version (17K): [in this window] [in a new window]   Figure 5.  Effects of PAR2 agonists on the subcellular distribution of PKAC (catalytic subunit) in HEK 293 cells and on cAMP levels A, Western blot, and B, densitometric analysis of PKAC in cytosolic (c) and membrane (m) fractions of HEK-TRPV1 cells. PAR2-AP caused an increase in PKAC in membrane fractions and a decrease in cytosolic fractions. *P < 0.05 compared to 0 min, n = 4 experiments. C, effects of PAR2 agonists on cAMP level in HEK-PAR2 cells (left) and DRG neurons (right). Try. = trypsin. PAR2-AP and trypsin caused an increase in cAMP levels after 1 and 5 min. n = 3 experiments. *P < 0.05 compared to non-treated cells (NT), ***P < 0.05 compared to PAR2-RP.

q/11

PAR₂ agonists sensitize TRPV1-mediated Ca²⁺ responses and TRPV1 currents by PKC- and PKA-dependent mechanisms

We have previously reported that PAR₂ agonists sensitize TRPV1 Ca²⁺ signals and currents in HEK 293 cells and DRG neurons by mechanisms that require activation of phospholipase Cß and PKC (Amadesi et al. 2004). We now used selective inhibitors of PKC and of PKA to determine their contributions to PAR₂-induced sensitization of TRPV1. HEK-TRPV1 cells were used for these experiments in view of the similar effects of PAR₂ agonists on activation of PKC and PKA and sensitization of TRPV1 in both HEK 293 cells and DRG neurons (Amadesi et al. 2004).

Exposure of HEK-TRPV1 cells to PAR₂-AP (100 µM, 5 min) potentiated the effects of capsaicin (10 nM) on [Ca²⁺]_i by 70% (n = 131 cells), indicative of TRPV1 sensitization (Fig. 6A and B). In contrast, PAR₂-RP (100 µM, 5 min) had no effect. Pretreatment with TAT-PKCI (10 µM) prevented this sensitization (Fig. 6B). Thus, the effect of capsaicin in cells treated with TAT-PKCI and PAR₂-AP was 56 ± 7% (n = 116 cells) of the responses in cells treated with PAR₂-AP and vehicle (100%). Pretreatment with inactive TAT-PKCI-sc (10 µM) had no effect on PAR₂-AP-induced sensitization of TRPV1 (n = 110 cells). Pretreatment with two selective inhibitors of PKA, H-89 (10 µM) and the cell-permeable PKAI_14–22 (0.1 µM), also prevented PAR₂-AP-mediated sensitization of TRPV1 (Fig. 6B). The effect of capsaicin in cells treated with H-89 and PAR₂-AP was 62 ± 11%, and in cells treated with PKAI_14–22 and PAR₂-AP was 53 ± 4% of the responses of cells treated with PAR₂-AP and vehicle (100%) (both n > 100 cells). Together, these results suggest that activation of PAR₂ sensitizes TRPV1-mediated increases in [Ca²⁺]_i by PKC-and PKA-dependent mechanisms.

View larger version (20K): [in this window] [in a new window]   Figure 6.  Effects of PAR2 agonists on capsaicin-induced Ca2+ signalling in HEK-TRPV1 cells Cells were exposed to PAR2-AP or PAR2-RP for 5 min, and then challenged with capsaicin. A, changes in [Ca2+]i, with each line the average trace from n = 31–35 cells. B, the effects of antagonists on PAR2-induced sensitization of capsaicin responses (100%). Veh = vehicle control. *P < 0.05 compared to PAR2-AP/vehicle cells. n = 70–80 cells from 3 experiments.

View larger version (15K): [in this window] [in a new window]   Figure 7.  PKC and PKA antagonists inhibit the PAR2-mediated sensitization of TRPV1 currents in HEK-TRPV1 cells A, whole-cell inward currents induced by capsaicin (300 nM) before (left trace) and 3 min after (right trace) application of PAR2-AP for 2 min. B, summary of mean TRPV1 currents over time following activation of PAR2 with PAR2-AP (100 µM; n 5 for each time point). PAR2-AP sensitized the capsaicin-induced current in a time-dependent manner; reaching its maximum value 3 min after PAR2-AP treatment (P < 0.01; n = 10). Membrane current values are expressed as percentage of the control current recorded prior to PAR2-AP application (second control bar). In five control cells (first control bar), capsaicin was applied for 20 s and re-applied 3 min later (second control bar), demonstrating the consistency of the capsaicin response. In another five control cells, capsaicin was only applied once before application of PAR2-AP. C, TRPV1 sensitization by PAR2-AP (Veh = vehicle control n = 10, at 3 min) was prevented by PKC (PKCI, 200 µM, n = 5) and PKA (H89, 3 µM, n = 5) inhibitors. *P < 0.05.

PAR₂ agonists sensitize TRPV1 to induce thermal hyperalgesia by PKC- and PKA-dependent mechanisms

We have previously reported that the co-administration of non-algesic doses of PAR₂-AP and capsaicin induces thermal hyperalgesia (Amadesi et al. 2004). Antagonism or deletion of TRPV1 prevents this response, indicating that PAR₂ sensitizes TRPV1 to cause thermal hyperalgesia. Using the same experimental approach, we now sought to investigate the role of PKC and PKA in PAR₂-induced sensitization of TRPV1-dependent thermal hyperalgesia. By measuring the paw withdrawal latency to radiant heat in mice, we found that co-injection of non-algesic doses of PAR₂-AP (1 ng paw^–1) and capsaicin (1 µg paw^–1) caused hyperalgesia, as indicated by a decrease in the paw withdrawal latency (Fig. 8A and B). This hyperalgesia was detected within 15 min and sustained for at least 120 min. Thus, as previously reported (Amadesi et al. 2004), PAR₂-AP can sensitize TRPV1-induced thermal hyperalgesia. To determine the contribution of PKC and PKA to this effect, we administered selective inhibitors (Aley et al. 2000). Intraplantar injection of PKCI (1 µg paw^–1), a selective inhibitor of PKC translocation, alone did not affect the basal withdrawal latency (Fig. 8A). However, PKCI strongly inhibited the hyperalgesic effect of PAR₂-AP and capsaicin at 15 and 30 min, and abolished hyperalgesia at 60 and 120 min (Fig. 8A). The inactive PKCI-sc (1 µg paw^–1) had no effect on the response to co-injection of PAR₂-AP and capsaicin (Fig. 8A). The intraplantar injection of WIPTIDE (1 µg paw^–1), a selective PKA inhibitor, alone had no significant effect on the basal withdrawal latency (Fig. 8B). However, WIPTIDE abolished the hyperalgesic effect of PAR₂-AP and capsaicin at 30–120 min (Fig. 8B). Thus, PAR₂ sensitizes TRPV1 to induce thermal hyperalgesia by a mechanism that requires activation of both PKC and PKA.

View larger version (22K): [in this window] [in a new window]   Figure 8.  Mechanisms of PAR2-induced potentiation of TRPV1-mediated thermal hyperalgesia Compounds were injected into the paws of mice, and hyperalgesia was measured as a significant decrease in withdrawal latency in response to a thermal stimulus, compared to basal (time 0). A, effects of the PKC inhibitor (PKCl) alone or on PAR2-AP and capsaicin-induced hyperalgesia. B, effects of the PKA inhibitor (WIPTIDE) alone or on PAR2-AP and capsaicin-induced hyperalgesia. Controls include use of inactive PKCl-sc or the WIPTIDE vehicle. n = 8 mice per group, *P < 0.05 compared to basal values.

	Discussion

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Several observations from the current investigation suggest that PAR₂ agonists activate PKC and PKA. We found that small- to medium-diameter neurons, which are known to express PAR₂ and TRPV1 (Amadesi et al. 2004), also contain PKC and PKA. By a combination of confocal microscopy, subcellular fractionation and Western blotting, we observed that PAR₂ agonists promoted translocation of PKC and the catalytic subunit of PKA, which represents the active subunit of the PKA heterotetramer, from the cytosol to the plasma membrane in both DRG neurons and HEK 293 cells. PMA and forskolin, established activators of PKC and PKA, had similar effects on the subcellular localization of these kinases. Thus, translocation of kinases from the cytosol to the plasma membrane can be used in our system as an indicator of kinase activation. Our results show that PKC and PKA_C are not only appropriately colocalized with PAR₂ and TRPV1 in nociceptive neurons, but that PAR₂ activates these kinases. Our findings are consistent with other reports of the localization of PAR₂, PKC and PKA_C in primary sensory neurons. PAR₂ is expressed by > 50% of small- to medium-diameter neurons of rat DRG (Steinhoff et al. 2000), and PKC is present in 90% (Khasar et al. 1999) and PKA_C in 50% (Rathee et al. 2002) of these neurons. We found that PAR₂ agonists caused redistribution of kinases in 60% of cells expressing PKC and in 80% of cells expressing PKA.

It is well established that PAR₂ couples to G_q/11, resulting in activation of phospholipase Cß and generation of 1,4,5-inositol trisphosphate and diacylglycerol, which would be expected to mobilize intracellular Ca²⁺ ions and activate PKC (Ossovskaya & Bunnett, 2004). Therefore, PAR₂-induced activation of PKC is consistent with the known signalling pathway of this receptor. Moreover, our results are in line with observations that agonists of PAR₂ induce activation and membrane translocation of PKC in prostate stromal cells (Myatt & Hill, 2005). Given the predominant coupling of PAR₂ to G_q/11, the observation that PAR₂ agonists caused membrane translocation of PKA_C was unanticipated. However, we observed that PAR₂ agonists stimulated generation of cAMP in DRG neurons and HEK 293 cells, suggesting that PAR₂ activates the cAMP/PKA cascade. Our results are in agreement with the observations that PAR₂ agonists elevate cAMP levels in keratinocytes (Scott et al. 2003), and that PAR₂ activation causes release of von Willebrand factor in endothelial cells by a PKA-dependent mechanism (Cleator et al. 2006). Thus, PAR₂, like PAR₁, may couple to different heterotrimeric G proteins and thereby regulate multiple signalling pathways, including mobilization of intracellular Ca²⁺ ions and generation of cAMP (Ossovskaya & Bunnett, 2004). Alternatively, PAR₂ agonists could induce release of mediators that act in an autocrine manner to generate cAMP and activate PKA. One possible mediator is PGE₂, since PAR₂ agonists promote PGE₂ release from epithelial cells (Kong et al. 1997) and PGE₂ sensitizes TRPV1 through a PKA-dependent mechanism (Lopshire & Nicol, 1998; Rathee et al. 2002). However, we found that indomethacin did not prevent the effects of PAR₂ agonists on cAMP generation. Further studies are thus required to determine the mechanism by which PAR₂ agonists activate PKA in neurons.

Upon translocation to the plasma membrane, activated PKC and PKA may phosphorylate and thereby sensitize ion channels that participate in nociception, such as TRPV1. Several observations from our investigation support this possibility. Firstly, we observed that PAR₂ agonists sensitized capsaicin-induced increases in [Ca²⁺]_i and capsaicin currents in HEK-TRPV1 cells, and we and others have previously reported similar sensitization in DRG neurons (Amadesi et al. 2004; Dai et al. 2004). Secondly, the co-injection of non-algesic doses of PAR₂ and capsaicin induced sustained thermal hyperalgesia, which we have shown to depend on sensitization of TRPV1 (Amadesi et al. 2004). Finally, selective antagonists of PKC and PKA, including those that prevent membrane translocation of these kinases, prevented the PAR₂-induced sensitization of TRPV1-dependent Ca²⁺ signalling, TRPV1 currents and TRPV1 thermal hyperalgesia. These results are supported by the observation that PAR2 agonists phosphorylate TRPV1 in HEK 293 cells (Amadesi et al. 2004). Our observation that PKC makes an important contribution to PAR₂-induced sensitization of TRPV1 is in agreement with the report that PKC antagonists suppress the effects of PAR₂ agonists on TRPV1 Ca²⁺ signalling and TRPV1 currents (Amadesi et al. 2004; Dai et al. 2004). However, our results extend this report by showing that PAR₂ activates a specific PKC isozyme, and by demonstrating a major role for both PKC and PKA in PAR₂-induced thermal hyperalgesia.

The precise signal transduction pathway by which PAR₂ activates PKC and PKA to sensitize TRPV1 remains to be elucidated. PKC and PKA can both directly phosphorylate TRPV1 (Bhave et al. 2002; Numazaki et al. 2002; Mohapatra & Nau, 2003). However, after activation of PAR₂, we do not know if PKC and PKA regulate TRPV1 in an additive or synergistic manner, or whether one pathway is upstream of the other. When administered separately, we observed that PKC and PKA inhibitors almost completely prevented PAR₂-induced sensitization of TRPV1 Ca²⁺ signals and currents. Moreover, PKC and PKA inhibitors both prevented PAR₂-induced thermal hyperalgesia at later times (> 30 min). Together, these results suggest the activation of tandem and possibly redundant PKC and PKA pathways. However, at earlier times (< 30 min), PKC and PKA inhibitors, when given separately, did not prevent PAR₂-induced thermal hyperalgesia. Thus, the relative contributions of PKC and PKA may vary with time after activating PAR₂. This possibility is supported by the identification of a novel cAMP/PKC signalling pathway that is responsible for the long-lasting latent susceptibility to hyperalgesia induced by an acute inflammatory stimulus (Parada et al. 2005) where an upstream release of cAMP and activation of the guanine exchange factor Epac, but not of PKA, are required to activate downstream PKC (Hucho et al. 2005). Although our observations that PKA inhibitors prevented the PAR₂-induced sensitization of TRPV1 suggest a distinct role for PKA, in addition to the contribution of PKC, further experiments are required to define the relative roles of these kinases at different times after activation of PAR₂.

In addition to the direct phosphorylation of TRPV1 by PKC and PKA, other mechanisms may account for PAR₂-induced sensitization of this channel. PAR₂ agonists may also activate other second messenger kinases, such as protein kinase D, a substrate for PKC (Rey et al. 2004; Wang et al. 2004) that can also phosphorylate and sensitize TRPV1 (Rey et al. 2004; Wang et al. 2004). Furthermore, PAR₂ agonists could regulate the subcellular distribution of TRPV1 rather than affecting gating of the channel. Indeed, phorbol esters, agonists of the metabotropic glutamate receptor and nerve growth factor sensitize neurons by promoting trafficking of TRPV1 to the plasma membrane (Morenilla-Palao et al. 2004; Zhang et al. 2005). These possibilities remain to be examined, and the existence of other mechanisms cannot be excluded.

Further studies are required to define the physiological relevance of PAR₂-induced hyperalgesia. In the present investigation, we studied the effects of a synthetic agonist, PAR₂-AP, on thermal hyperalgesia. However, we have previously reported that proteases such as tryptase and trypsin similarly cause hyperalgesia by activating PAR₂ (Vergnolle et al. 2001). Agents that degranulate mast cells also induce hyperalgesia by a PAR₂-dependent mechanism (Vergnolle et al. 2001). Although tryptase may mediate these effects, experiments with tryptase inhibitors are required to confirm this possibility. Extrapancreatic trypsins, such as trypsin IV, can activate PAR₂ (Cottrell et al. 2004), but their role in pain transmission remains to be defined. In the present study, we observed that PAR₂-AP-induced thermal hyperalgesia was sustained for 2 h, and we have previously reported detectable hyperalgesia 24 h after PAR₂ activation (Vergnolle et al. 2001). Although our results suggest that PKC and PKA account for hyperalgesia at up to 120 min after activation, different mechanisms may mediate hyperalgesia at later times. Proteases activate PAR₂ in a catalytic and irreversible manner; once the receptor is cleaved the exposed tethered ligand is always available to activate PAR₂. Receptor phosphorylation by G protein receptor kinases and interaction with ß-arrestins serve to uncouple receptors from heterotrimeric G proteins and desensitize G protein signalling (Bohm et al. 1996; Dery et al. 1999). However, ß-arrestins also mediate receptor endocytosis and are scaffolds for mitogen-activated protein kinases, which permits internalized PAR₂ to continue to signal through this pathway (DeFea et al. 2000). The contribution of ß-arrestins and mitogen-activated protein kinases to sustained hyperalgesia remains to be defined. Eventually, PAR₂ is targeted for degradation in lysosomes by a ubiquitin-mediated mechanism, which irrevocably terminates signalling (Jacob et al. 2005). In cell lines, resensitization of responses to proteases requires synthesis of new receptors and mobilization of prominent stores of PAR₂ from the Golgi apparatus. We have shown that PAR₂-AP causes redistribution of ß-arrestin1-EGFP from the cytosol to the plasma membrane, followed by translocation of PAR₂-EGFP and ß-arrestin1-EGFP from the plasma membrane to endosomes of DRG neurons (S. Amadesi & N. W. Bunnett, unpublished observations). However, the mechanisms of resensitization of PAR₂ in neurons, which may be required for the sustained hyperalgesic effects of PAR₂ agonists, are completely unknown.

Together, our results further clarify the signalling pathways by which PAR₂ regulates TRPV1 to induce hyperalgesia. We used a combination of experimental approaches, including localization of kinases in DRG, analysis of the subcellular distribution of PKC and PKA_C, measurement of TRPV1 mediated Ca²⁺ signalling and currents, and behavioural studies in conscious animals, to reveal a major role of PKC and PKA in PAR₂ signalling and PAR₂-induced sensitization of TRPV1. An understanding of the mechanisms by which proteases that activate PAR₂ sensitize ion channels such as TRPV1 is important for the development of novel therapies to treat pain.

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