-Opioid Receptor Activation Modulates Transient Receptor Potential Vanilloid 1 (TRPV1) Currents in Sensory Neurons in A Model of Inflammatory Pain

Current therapy for inflammatory pain includes the peripheral application of opioid receptor agonists. Activation of opioid receptors modulates voltage-gated ion channels, but it is unclear whether opioids can also influence ligand-gated ion channels [e.g., the transient receptor potential vanilloid type 1 (TRPV1)]. TRPV1 channels are involved in the development of thermal hypersensitivity associated with tissue inflammation. In this study, we investigated -opioid receptor and TRPV1 expression in primary afferent neurons in the dorsal root ganglion (DRG) in complete Freund’s adjuvant (CFA)-induced paw inflammation. In addition, the present study examined whether the activity of TRPV1 in DRG neurons can be inhibited by -opioid receptor ( -receptor) ligands and whether this inhibition is increased after CFA inflammation. Immunohistochemistry demonstrated colocalization of TRPV1 and -receptors in DRG neurons. CFA-induced inflammation increased significantly the number of TRPV1and -receptor-positive DRG neurons, as well as TRPV1 binding sites. In whole-cell patch clamp studies, opioids significantly decreased capsaicin-induced TRPV1 currents in a naloxoneand pertussis toxinsensitive manner. The inhibitory effect of morphine on TRPV1 was abolished by forskolin and 8-bromo-cAMP. During inflammation, an increase in TRPV1 is apparently rivaled by an increase of -receptors. However, in single dissociated DRG neurons, the inhibitory effects of morphine are not different between animals with and without CFA inflammation. In in vivo experiments, we found that locally applied morphine reduced capsaicin-induced thermal allodynia. In summary, our results indicate that -receptor activation can inhibit the activity of TRPV1 via Gi/o proteins and the cAMP pathway. These observations demonstrate an important new mechanism underlying the analgesic efficacy of peripherally acting -receptor ligands in inflammatory pain. The treatment of patients with inflammatory pain includes opioids acting outside the central nervous system (Stein et al., 2003). All three types of opioid receptors are synthesized and expressed in the cell bodies of dorsal root ganglion (DRG) neurons. These opioid receptors are intra-axonally transported into the neuronal processes and are detectable on peripheral Cand A-nerve terminals. High-voltage activated calcium currents can be reduced by opioid receptoractivated inhibitory G-proteins (Gi), as suggested by experiments in cultured DRG neurons (Schroeder and McCleskey, 1993). In addition, opioids suppress tetrodotoxin-resistant sodium-selective and nonselective cation currents, which are mainly expressed in nociceptors (Ingram and Williams, 1994; Gold and Levine, 1996). The nonselective ion channel TRPV1 is also predominantly expressed in nociceptive sensory neurons (Caterina et al., 1997) and can be sensitized and/or up-regulated during conditions associated with tissue damage (Tominaga et al., 1998). TRPV1 seems to be essential for the development of thermal hypersensitivity associated with tissue inflammation but not that associated with nerve injury (Caterina and Julius, 2001). Inflammation-induced therThis work was supported by Deutsche Forschungsgemeinschaft (DFG) grant KFO 100/2 and a European Society of Anaesthesiology research grant. Parts of this work have been published previously in abstract form [EndresBecker J, Heppenstall PA, Mousa S, Labuz D, Schäfer M, Stein C, Zöllner C. Opioids modulate the transient receptor potential vanilloid 1 (TRPV1) ion channel. International Narcotic Research Conference; July 10–15, 2005; Annapolis, MD]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.026740. ABBREVIATIONS: DRG, dorsal root ganglion; TRPV1, transient receptor potential vanilloid type 1; -receptor, -opioid receptor; PKA, protein kinase A; PCR, polymerase chain reaction; CFA, complete Freund’s adjuvant; RTX, resiniferatoxin ; PBS, phosphate-buffered saline; MEM, minimal essential medium; PTX, pertussis toxin; DAMGO, [D-Ala,N-Me-Phe,Gly-ol]-enkephalin; FSK, forskolin; 8-Br-cAMP, 8-bromo-cAMP; PWL, paw withdrawal latency; ANOVA, analysis of variance. 0026-895X/07/7101-12–18$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 1 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 26740/3159178 Mol Pharmacol 71:12–18, 2007 Printed in U.S.A. 12 at A PE T Jornals on O cber 4, 2017 m oharm .aspeurnals.org D ow nladed from mal hypersensitivity may result from the actions of heat, low pH, and other inflammatory mediators on TRPV1 (Caterina and Julius, 2001). However, other mechanisms, such as upregulation of TRPV1 expression, may also come into play. Because peripheral -receptor agonists are particularly effective in inflammatory hyperalgesia, we hypothesized that opioids can modulate the activity of TRPV1. To elucidate possible TRPV1– -receptor interactions, we investigated Gprotein and second messenger pathways. After opioids bind to their receptors, dissociation of the heterotrimeric G protein complex into G and G subunits can subsequently lead to the inhibition of adenylyl cyclase isoforms. As a result, formation of cAMP is inhibited, and protein kinase A (PKA) cannot be activated (Law et al., 2000). TRPV1 can be phosphorylated and subsequently regulated by several kinases, including PKA (De Petrocellis et al., 2001; Bhave et al., 2002; Mohapatra and Nau, 2003), and protein kinase C (Bhave et al., 2003). In this study, we investigated the activity of TRPV1 after -receptor activation and its modulation by the cAMP/PKA pathway in single DRG neurons using patch clamp technique. We examined expression and colocalization of -receptors and TRPV1 in DRG using immunohistochemistry, real-time PCR, and radioligand binding in animals with and without painful hindpaw inflammation, a model that resembles postoperative pain, arthritis and other types of inflammatory pain (Machelska et al., 2003). Materials and Methods Animals. Experiments were performed in individually housed male Wistar rats (180–200 g). Control animals were treated with intraplantar saline (0.15 ml) injections. Inflammation was induced by intraplantar CFA (0.15 ml; Calbiochem, San Diego, CA) administered into the right hindpaw. All intraplantar injections were performed under brief isoflurane (Willy Rüsch GmbH, Böblingen, Germany) anesthesia. The animal protocol was approved by the state animal care and use committee (“Landesamt für Arbeitsschutz, Gesundheit und Technische Sicherheit Berlin”) and the guidelines on ethical standards for investigations of experimental pain in animals were followed (Zimmermann, 1983). TRPV1 Light Cycler PCR Experiments. Real time-PCR was performed as described previously (Puehler et al., 2004). The following primers for TRPV1 were used (Ji et al., 2002): 5 -AAA CTC CAC CCC ACG CTG AA-3 and 5 -GTC GGT TCA AGG GTT CCA CG-3 , corresponding to bases 1031 and 1321 (GenBank accession number NM_031982). A mathematical model was used to determine the relative quantification of a target gene compared with a reference gene (Pfaffl, 2001). Ribosomal protein L19 (RPL-19) was chosen as a reference gene and the following primers were used: 5 -AAT CGC CAA TGC CAA CTC TCG-3 and 5 -TGC TCC ATG AGA ATC CGC TTG-3 corresponding to bases 1521 and 3274 (GenBank accession number X82202). Target and reference genes were quantified using triplicate samples. Light cycler PCR was performed with a DNA Sybr Green kit according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Amplification was carried out for 45 cycles, each consisting of 10 s at 95°C, 5 s at 57°C, and 16 s at 72°C. A temperature was determined just below the product-specific melting temperature (Tm), and this temperature step was incorporated into the PCR for detection of fluorescence (TRPV1, Tm 89°C; RPL-19, Tm 88°C). As a control for the PCR reaction, reverse transcriptase (RT)-negative samples were used. PCR products and a size marker (DNA Marker XIV; Roche Diagnostics, Mannheim, Germany) were separated by electrophoresis on a 2% agarose gel containing ethidium bromide (Sigma, Taufkirchen, Germany) and were visualized under UV light. PCR products were sequenced in forward and backward direction (AGOWA, Berlin, Germany) and were compared with the published sequences of TRPV1 (homologies 99%). TRPV1 mRNA was determined at 2, 6, 12, 24, 48, 72, and 96 h after CFA inoculation. Membrane Preparations. All chemicals and drugs were purchased from Sigma (Taufkirchen, Germany) unless indicated otherwise. Rats were killed by isoflurane anesthesia 96 h after intraplantar saline or CFA application. Ipsilateral lumbar (L4/5) DRG neurons were removed from saline-injected animals. In CFA animals, ipsilateral and contralateral (internal controls) lumbar (L4/5) DRG neurons were removed. The tissue was placed immediately on ice in ice-cold assay buffer (50 mM Tris-HCl and 1 mM EGTA, pH 7.4). Tissue was pooled from 10 rats, homogenized, centrifuged twice at 42,000g and 4°C for 20 min, and resuspended in assay buffer. TRPV1 Binding. Binding studies with the labeled TRPV1 agonist resiniferatoxin ([H]RTX) were carried out according to a modified protocol (Szallasi et al., 1999). In brief, appropriate concentrations of cell membranes (40 g) were prepared and incubated in assay buffer (5 mM KCl, 5.8 mM NaCl, 0.75 mM CaCl2, 2 mM MgCl2, 320 mM sucrose, and 10 mM HEPES) with increasing doses of [H]RTX (15–2000 pM, 43 Ci/mmol; GE Healthcare, Little Chalfont, Buckinghamshire, UK) in the absence or presence of 10 M unlabeled RTX. Because adenosine (released during sample preparation) can directly interact with TRPV1 (Puntambekar et al., 2004), we pretreated DRG membranes with adenosine deaminase (1 U/ml) for 15 min at 37°C to degrade adenosine. Membranes were incubated in a final volume of 750 l for 1 h at 30°C in assay buffer. Filters were soaked in 0.1% (w/v) polyethylenimine solution for 30 min before use. Bound and free ligand were separated by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by four washes with ice-cold buffer (50 mM Tris-HCl, pH 7.4). Bound radioactivity was determined by liquid scintillation spectrophotometry at 70% counting efficiency for [H] after overnight extraction of the filters in 3 ml of scintillation fluid (PerkinElmer Wallac, Turku, Finland). Immunohistochemistry. Immunofluorescence staining for -receptors and TRPV1 was performed as described previously (Mousa et al., 2001). Rats were deeply anesthetized with isoflurane and perfused transcardially with 100 ml of 0.1 M PBS, pH 7.4, and 300 ml of ice-cold PBS containing 4% paraformaldehyde and 0.2% picric acid (pH 6.9; fixative solution). Lumbar DRG (L4–L5) were removed, postfixed for 90 min at 4°C in the fixative solution, and cryoprotected overnight at 4°C in PBS containing 10% sucrose. The tissues were embedded in Tissue-Tek compound (Bayer Healthcare, Pittsburgh, PA) and consecutive sections (9 m) mounted onto gelatin-coated slides were prepared on a cryostat. To prevent nonspecific binding, the sections were incubated in PBS containing 0.3% Triton X-100, 1% bovine serum albumin, 4% goat serum, and 4% donkey serum (block solution). The sections were incubated overnight at 4°C with rabbit anti-receptor (Drs S. Schulz and V. Höllt, Magdeburg, Germany) in combination with guinea pig anti-TRPV1 antibody (1:1000 dilution; Neuromics, Minneapolis, MN) and then incubated with goat anti-rabbit conjugated with Texas Red (1:250 dilution; Vector Laboratories, Burlingame, CA) and donkey anti-guinea pig conjugated fluorescein isothiocyanate (1:250 dilution; Vector Laboratories). Thereafter, sections were washed with PBS, mounted in Vectashield, and viewed under a confocal laser scanning microscope (Zeiss, Jena, Germany) by an experimenter blinded to the treatment regime. Settings for excitation of fluorescein isothiocyanate (488 nm) and Texas Red (543 nm) and of photodetectors (pinhole, amplifier gain) were identical throughout the analysis. Controls included (1) preabsorption of diluted antibodies with their respective immunizing peptides and (2) omission of either the primary antisera or the secondary antibodies. These control experiments did not show staining. Quantification of Immunostaining. The method of quantification for DRG staining has been described previously (Ji et al., 1995). In brief, we stained every fourth section of DRG that was serially cut at 9 m for each animal (n 5). Square grids avoid double count of -Opioid Receptor Ligands and Inflammatory Pain 13 at A PE T Jornals on O cber 4, 2017 m oharm .aspeurnals.org D ow nladed from neurons. The cell body diameter was measured with the nucleus in the focal plane and was estimated from the average length and width determined with a calibrated micrometer. A total number of 60 immunoreactive neurons with nucleus were measured for each animal. The -receptor-ir and TRPV1-ir neurons were counted, and the proportion was calculated as percentage of total number of DRG cells. In addition, the -receptor-ir neurons, which were also immunoreactive for TRPV1, were counted, and the proportion was calculated as percentage of TRPV1-ir neurons. For neuron counting, only those immunostained neurons containing a distinct nucleus were counted for a total of 332 neurons. Data were obtained from four sections of each DRG and five rats per group. Cultures of DRG Neurons. DRG were prepared as described previously (Bolyard et al., 2000). In brief, L4/L5 DRG were removed and placed in sterile MEM at 4°C. DRG were digested with collagenase type 2 in MEM at 37°C for 50 min and 0.025% trypsin for 10 min at 37°C. After digestion, DRG were carefully dissociated by mechanical agitation and centrifuged at 500g for 5 min and at 300g for 5 min. The cells were maintained in MEM (Biochrom AG, Berlin, Germany) growth media supplemented with 10% horse serum/50 g/ml penicillin and streptomycin and plated in six-well polylysine-coated culture plates at 37°C for 2 to 4 h in a 5% CO2 atmosphere. Drug and Heat Stimulation. For drug application and heat stimulation of single neurons, a fast channel system with common outlet was used (Warner Instruments, Hamden, CT). Magnetic valves to open and close the syringes were controlled manually from a switchboard. Heat was applied using a Peltier device (ESF Electronic, Goettingen, Germany) that heated the extracellular buffer at approximately 1°C per second from 24°C to 46°C. Temperature was monitored with a thermocouple placed within the flow of buffer and close to the cells. Electrophysiology in Dissociated DRG Cells. Whole-cell measurements in the voltage-clamp configuration of the patch-clamp technique were performed 2 to 4 h after dissociation at 60 mV holding potential with an EPC-10 patch clamp amplifier and PULSE software (HEKA Elektronik, Lambrecht, Germany). For pertussis toxin (PTX) experiments, dissociated DRG cells were kept in culture for 2 h. Afterward, DRG cells were pretreated with PTX for 6 h. Borosilicate glass electrodes (Hilgenberg, Malsfeld, Germany) pulled on a horizontal puller (Sutter Instrument Company, Novato, CA) had resistances of 2 to 7 M after filling with 145 mM KCl, 1 mM MgCl2, 2 mM Na-ATP, 10 mM HEPES, 10 mM glucose, and 0.2 mM NaGTP, pH adjusted to 7.3 with KOH. The external solution consisted of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, at pH adjusted to 7.3 with NaOH. Only small sensory neurons (cell diameter 26 m) that were sensitive to the nociceptor excitant capsaicin (1 M) or heat (46°C) were included in the study, and these cells were considered responsive if the inward current magnitude was at least 100 pA. Because in preliminary experiments repeated applications of capsaicin to the same neuron produced currents of progressively smaller magnitude, Ca was removed from the extracellular buffer. We compared responses in dissociated DRG cells from animals with and without CFA inflammation. After the first capsaicin application, dissociated DRG cells were incubated with buffer or the -receptor agonists morphine (10 M) or DAMGO (10 M), and capsaicin (1 M) was applied again 60 and 360 s later. To examine whether the morphine-evoked inhibitory effect was reversible, DRG cells were treated with morphine for 60 s. Thereafter, cells were treated with buffer or morphine and capsaicin was applied again 360 and 720 s later. Temperature ramps were used to heat the extracellular buffer from room temperature to 46°C. After the first increase in temperature, DRG cells were incubated with morphine (10 M) or buffer and heat was applied again 60 and 360 s later. To investigate the involvement of -receptors and Gi/o-proteins, dissociated DRG neurons were treated with naloxone (10 M) concomitantly with morphine or pretreated for 6 h with PTX (100 ng), respectively. To study the role of cAMP pathways, dissociated DRG neurons were treated with morphine (10 M) and forskolin (FSK; 10 M), a potent stimulator of adenylyl cyclase activity. In addition, the reversibility of the morphine effect was investigated by concomitant application of the cAMP analog 8-bromo-cAMP (8-Br-cAMP) (10 M) with morphine (10 M) for 5 min. Measurement of Thermal Hyperalgesia. Nociceptive thresholds were determined by measuring the paw withdrawal latency (PWL) upon application of acute thermal stimulation (Hargreaves et al., 1988). Rats were placed in clear plastic chambers positioned on a glass surface (model 336 Analgesia Meter; IITC Life Science, Woodland Hills, CA). Radiant heat was applied to the plantar surface of a hindpaw from underneath the glass floor with a high-intensity projector lamp bulb and PWL was measured using an electronic timer. Nociceptive testing was performed twice per paw, separated by at least 30 s, and the mean value was calculated for each animal. The heat intensity was adjusted to obtain a basal PWL of approximately 9 to 10 s. A 20-s cut-off was used to prevent tissue damage. Animals were tested before and 15, 30, 45, and 60 min after intraplantar injection of capsaicin (30 g/10 l). In a separate experiment, morphine (intraplantar; 100 g/20 l) was injected 10 min after capsaicin, and PWL was measured 5 min later. For control (Ctrl), this same volume of vehicle was injected into the paw. Statistics. Statistical differences are represented as means S.E.M. Light cycler PCR experiments were made using one-way ANOVA followed by Student-Newman-Keuls test in the case of normally distributed data and ANOVA on ranks followed by KruskalWallis analysis in the case of not normally distributed data. Statistical differences in electrophysiology were determined with two-way ANOVA and Bonferroni post hoc test. Unpaired, two-tailed Student’s t test was used to assess statistical significance in immunohistochemistry, ligand binding and behavioral experiments. All ligand binding data are reported as means S.E.M. of at least four experiments, performed in duplicate. Differences were considered significant at p 0.05. All tests were performed using Prism 4 (GraphPad, San Diego, CA) or Sigma Stat 2.03 (SyStt Software, Point Richmond, CA) statistical software.