Research Paper

A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons Kalina Szteyna, Matthew P. Rowana, Ruben Gomeza, Junhui Dub, Susan M. Carltonb, Nathaniel A. Jeskea,c,d,*

Abstract Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-kinase anchoring protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity after group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP-dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature. Keywords: AKAP, Glutamate, TRPV1, Skin-nerve prep, Peripheral

1. Introduction Chronic pain is a debilitating symptom of many disease states, which is now being studied as its own separate and unique pathological event. Although central nervous system changes have been shown to contribute to this pathology,46,53 increasingly more studies highlight the contributions of peripheral neurons.7,21 Despite this newfound focus, few have identified signaling mechanisms that peripherally support the perpetual nature of persistent somatosensitivity. This represents an opportunity to identify molecular coordinators of signaling events that may support chronic pain. Glutamate serves as the primary excitatory neurotransmitter throughout the body, both in central and peripheral nervous systems. Growing evidence indicates that glutamate plays a significant role in afferent neuronal sensitization after injury and/or inflammation. Indeed, electrical stimulation of peripheral afferents, peripheral application of capsaicin (CAP), and circumstances of chemical and arthritic inflammation stimulate glutamate accumulation in the periphery.18,37,40 This accumulated glutamate is capable Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. a

Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, b Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA, Departments of c Pharmacology and, d Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA *Corresponding author. Address: Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78299, USA. Tel: (210) 567-3466; fax: (210) 567-2995. E-mail address: [email protected] (N. A. Jeske). PAIN 156 (2015) 2364–2372 © 2015 International Association for the Study of Pain http://dx.doi.org/10.1097/j.pain.0000000000000295

2364

·

K. Szteyn et al. 156 (2015) 2364–2372

of activating both ionotropic and metabotropic receptors throughout the body, although only certain isoforms are expressed by peripheral sensory neurons.17 Of these isoforms, those belonging to group I, which include mGluR1 and mGluR5, display significant physiological relevance in peripheral pain sensitization. Previous studies have identified signaling pathways correlated with peripheral somatosensitivities after group I mGluR activation. Indeed, the mGluR5 G-protein-coupled receptor signals through a classic Gaq/11-coupled pathway to stimulate phospholipase C (PLC).5 mGluR5 activation stimulates protein kinases A (PKA) and C (PKC) to reduce the activation threshold for transient receptor potential type V1 (TRPV1),16,29 contributing to thermal sensitivity and peripheral pain behaviors. TRPV1, a nonselective cationpermeable ligand-gated ion channel activated by heat (.43˚C), protons (pH ,5.9), endogenous eicosanoids, and exogenous vanilloids, is dynamically modified through scaffolding functions coordinated by A-kinase anchoring protein 79/150 (AKAP).30,31,56 Furthermore, PLC hydrolysis of AKAP150 anchorage to the plasma membrane stimulates AKAP150 association with TRPV1.32 Without this PLC-induced association, TRPV1 modification by PKA/PKC would not be possible, thereby preventing receptor sensitization from occurring. Therefore, we hypothesize that receptors coupled to Gaq-signaling pathways should stimulate AKAP150 association with TRPV1 to coordinate peripheral glutamate-induced somatosensitization through mGluR5. Herein, we investigated the role of AKAP150 in glutamateinduced peripheral sensitization. Experimental findings indicate that AKAP serves an integral role as a signal coordinator between mGluR activation and somatosensitization through TRPV1. Importantly, this biochemical coordination of peripheral signaling events may support an inflammatory positive feedback system to maintain sensory afferent sensitivity to subthreshold stimulation.5,7,11,14,15,18,20,21,24,28,30–32,34,35,37,39,40,44,46,48,50,51,53,54,56 PAIN®

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

November 2015

·

Volume 156

·

Number 11

2. Materials and methods 2.1. Reagents (S)-3,5-dihydroxyphenylglycine (DHPG), 3-((2-methyl-1,3-thiazol4-yl)ethynyl)pyridine hydrochloride (MTEP), and fenobam were purchased from Tocris/R&D Systems (Minneapolis, MN). Capsaicin and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. 2.2. Animals Procedures using animals were approved by either the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio or the University of Texas Medical Branch at Galveston and were conducted in accordance with policies for the ethical treatment of animals established by the National Institutes of Health (NIH) and International Association for the Study of Pain. Male SpragueDawley rats 175 to 200 g in weight (Charles River Laboratories, Wilmington, MA) and AKAP wild-type (WT)/knockout (KO) mice with a C57/Bl6 background49 were used for behavior analyses and for dorsal root ganglion (DRG) and trigeminal ganglion (TG) dissection. Dorsal root ganglia (L4-L6) and TG were removed bilaterally from male rodents and dissociated by collagenase treatment (30 minutes, Worthington, Lakewood, NJ), followed by trypsin treatment (15 minutes, Sigma, St. Louis, MO). Cells were centrifuged and resuspended between each treatment with Pasteur pipettes. Cells were centrifuged, aspirated, resuspended in DMEM (Gibco, Grand Island, NY) with 10% FBS (Gibco), 250 ng/mL NGF (Harlan, Indianapolis, IN), 1% 5-fluorodeoxyuridine (Sigma), 1% penicillin/streptomycin (Gibco), and 1% L-glutamine (Sigma), and then placed onto plates coated with poly-D-lysine or onto coverslips coated with poly-D-lysine and laminin. Cultures were maintained at 37˚C, 5% CO2, and grown in 10-cm plates for 5 to 7 days for phosphorylation experiments.30 Rat TG cultures used for co-immunoprecipitation (Co-IP) were maintained at 37˚C for 5 to 7 days before the experiment. Rat tissue was preferred over mouse TG tissue because rat TGs are larger and contain more neurons, thereby requiring significantly fewer animals, following the guidelines released by the NIH Office of Laboratory Animal Welfare. Mouse DRG cultures for calcium imaging were grown 18 to 36 hours. AKAP150 WT and KO mice were originally created and characterized in the laboratory of John D. Scott49 and maintained at the University of Texas Health Science Center at San Antonio. Genotyping was performed as previously described,32 with males aged 4 to 9 weeks used for experiments. All WT and KO animals used for experiments were littermates. 2.3. Calcium imaging Dorsal root ganglion neurons were isolated from male AKAP150 WT and KO mice. Before measurements, cells were incubated for 30 minutes at 37˚, 5% CO2, with Fura2-AM (2 mM, Invitrogen Life Technologies, Grand Island, NY) in the presence of 0.05% Pluronic (Invitrogen Life Technologies). Experiments were performed with an inverted Nikon Eclipse TE2000-U microscope, equipped with 203/0.8 NA Fluor objective. Cultured neurons were excited at the 340 and 380 nm wavelength from a Lambda LS Fluorescent light source (Sutter Instruments, Novato, CA), while a Hamamatsu digital CCD camera detected emissions at 510 nm. Collected data were analyzed with MetaFluor Software (Molecular Devices, Sunnyvale, CA). Ratiometric 340/380 nm values were converted into nano mole of Ca21 according to the formula [Ca21] 5 199 3 (R 2 0.46)/(1.45 2 R), where R 5 340/380 ratio, and all

www.painjournalonline.com

2365

other values are specific to the experimental setup and were determined with the use of a Fura2-AM calibration kit (Invitrogen Life Technologies). Experiments were performed at room temperature with the use of a gravity-feed perfusion system, which allowed for continuous exchange of bath solution. Recordings were performed in standard extracellular solution (SES) that contained the following (in milli mole): 140 NaCl, 5 KCl, 2 MgCl2, 2CaCl2, 10 HEPES, 10 glucose, pH 7.4 [NaOH]. Coverslips of cultured DRG neurons were treated with DHPG (100 mM29) through bath application, and CAP (100 nM) was applied for 30 seconds through pipette application. Standard extracellular solution also served as a vehicle for CAP (100 nM, Sigma-Aldrich) and DHPG (100 mM, Tocris). The micropipette, which delivered drug-containing solutions, was precisely placed in the optic field that includes our cells of interests. Both bath and pipette solution were under automatically controlled pressurized system (PM2000 Cell Microinjector; MicroData Instruments Inc, South Plainfield, NJ) that, in pairing with a pneumatic pump, supported precise, fast, and consistent fluid flow and exchange. Data were analyzed by oneway analysis of variance, with Bonferroni post hoc correction as needed. 2.4. Electrophysiological recordings: in vitro skin-nerve preparation The preparation used to record from nociceptors has been previously described in detail.12,27 Briefly, male C57/Bl6 WT and KO mice (10-13 weeks of age) were killed with an overdose of CO2. The glabrous skin was dissected from each hind paw starting at the ankle and moving to the tips of the toes. The medial and lateral plantar nerves were also dissected free and kept intact with the glabrous skin. The preparation was placed corium side up in a 2-chambered tissue bath. The nerves were drawn into one chamber, and the skin was laid out and pinned to the gel floor of the other chamber. In the recording chamber, the nerves were desheathed and carefully teased apart so that small nerve bundles could be obtained. These were laid across the gold wire recording electrode. A glass rod was pressed into the glabrous skin to find receptive fields of units in the nerve bundles. Once the receptive field of a unit was located, the conduction velocity (CV) was measured and a sequence of testing was performed as described below. 2.5. Thermal and chemical testing procedures For thermal stimulation, radiant heat was applied to each receptive field by a feedback-controlled lamp (made in house) beneath the organ bath. The beam was focused through the bottom of the bath onto the epidermal surface of the skin. A thermocouple in the corium above the light beam measured intracutaneous temperature. A standard heat ramp starting from an adapting temperature of 34˚C and rising to 51˚C in 10 seconds was applied to each unit from the epidermal side. To investigate unit responses to chemicals, a small plastic ring (4.5 mm diameter) was placed over the receptive field of each unit. The synthetic interstitial fluid in the ring was replaced with a drug dissolved in synthetic interstitial fluid (buffered to pH 7.40 6 0.05). As with any in vivo intraplantar (i.pl.) injections, drugs must diffuse through the plantar connective tissue, dermis, and epidermis to reach the nociceptor nerve terminals; thus, drug concentrations were considerably diluted by the time they reach their target sites. Drug concentrations for DHPG (1 and 3 mM) and

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

2366

·

PAIN®

K. Szteyn et al. 156 (2015) 2364–2372

CAP (1 mM) were similar to those previously used for activating primary sensory neurons.3,5,57 To determine the percent of units responding to a drug application, only those units whose discharge rate increased more than the mean 1 2 SDs (calculated using the background discharge rate for the population) were considered “responders.” 2.6. Behavior Paw withdrawal latency to a thermal stimulus was measured with a plantar test apparatus (IITC, Woodland Hills, CA) as previously described using the Hargreaves’ method.26 Briefly, animals were placed in plastic boxes on a warm glass surface. After a 30minute habituation period, the plantar surface of the hind paw was exposed to a beam of light that gradually heated the glass floor. The latency to withdraw the paw from the glass surface was measured, and the intensity of the light and basal temperature of the glass floor were adjusted so that baseline withdrawal latencies for rats and mice were close to 10 seconds. Cutoff was set at 25 seconds to avoid tissue damage from repeated testing. Measurements were taken in duplicate at least 30 seconds apart, and the average was used for statistical analysis. Data are presented as mean 6 SEM of paw withdrawal latencies or as mean 6 SEM of the individual changes from individual preinjection baseline values. Observers were blinded to the treatment conditions and genotypes of the animals. All injections were given i.pl. in 50 mL (rat) or 10 mL (mouse) volumes through a 28-gauge needle inserted through the lateral footpad just under the skin to minimize tissue damage. Drug doses were taken from previous work by Aley et al.1 and Bhave et al.5 (for MTEP). Drug stocks were dissolved in phosphatebuffered saline (PBS). For rat priming experiments, rats were tested for baseline responses before injections and then injected with carrageenan (50 mL of a 0.1% solution in PBS) or vehicle (PBS) on day 1. On days 2, 3, and 4, rats were injected with MTEP (400 mg) or vehicle (PBS). Rats were tested again on day 5 for baseline responses and then injected with PGE2 (100 ng) followed by repeated thermal testing at 15 minutes, 30 minutes, 2 hours, and 4 hours after PGE2 injection. For mouse priming experiments, WT and AKAP150 KO mice were tested for baseline responses before injections and then injected with carrageenan (10 mL of

Figure 1. DHPG sensitization of thermal sensitivity in AKAP wild-type (WT) and knockout (KO) mice. AKAP WT and KO mice were monitored for baseline paw withdrawal latency responses to a thermal stimulus before experimentation. AKAP WT and KO mice were injected intraplantarly with DHPG (50 nmol in 10 mL PBS) or vehicle (Veh) as indicated and tested 10 minutes later for paw withdrawal latency to a thermal stimulus. n 5 6 mice per treatment paradigm, **P , 0.01, *P , 0.05, 2-way ANOVA with Bonferroni correction. ANOVA, analysis of variance; NS, not significant; PBS, phosphate-buffered saline.

a 0.5% solution in PBS) on day 1. Thermal withdrawal latency was measured again on day 5 (after carrageenan) before injection with PGE2 (100 ng) and repeated thermal testing at 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours after PGE2 injection. 2.7. Co-immunoprecipitation and Western blotting Trigeminal ganglion cultures in 10-cm plates were treated and prepared for homogenization and isolation of crude plasma membrane fractions, as described previously.30 Crude plasma membrane homogenates are quantified for protein concentration by Bradford analysis.9 Equal samples underwent Co-IP (100 mg total protein) or gross homogenate Western blot analysis (10 mg total protein). Samples isolated for Co-IP were diluted to 500 mL with homogenization buffer,30 incubated with antibodies specific to AKAP150 (1 mg, Santa Cruz Biotechnology, Santa Cruz, CA30), pulled down with protein agarose A (Sigma-Aldrich), and resolved

Figure 2. Carrageenan priming of PGE2-induced thermal allodynia requires mGluR5 and AKAP. (A) Rats were injected intraplantarly (i.pl.) with vehicle (Veh) or carrageenan (Cg, 5 mL of 1% solution in 50 mL phosphate-buffered saline [PBS] final) on day 1 (see Materials and methods section for full protocol). Rats were then injected with Veh or MTEP (400 mg in 50 mL PBS) on days 2, 3, and 4. Rats were tested for baseline paw withdrawal latency (PWL) on day 5, then injected with PGE2 (indicated by arrow, 100 ng in 50 mL PBS, i.pl.), and tested again for PWL to a thermal stimulus at 15 minutes, 30 minutes, 2 hours, and 4 hours. Paw withdrawal latency displayed as change from baseline, n 5 6 rats per treatment paradigm, **P , 0.01, *P , 0.05, 2-way ANOVA with Bonferroni correction. (B) Mice were tested for baseline PWL to a thermal stimulus before i.pl. injections (Pre-Cg). Mice were then injected with Cg (5 mL of 1% solution in 10 mL PBS final, i.pl.) on day 1. Mice were tested for baseline PWL on day 5 (Post-Cg), then injected with PGE2 (indicated by arrow, 50 ng in 10 mL, i.pl.), and tested for PWL at 15 minutes, 30 minutes, 2 hours, and 4 hours. Paw withdrawal latency displayed as change from baseline illustrated, n 5 6 mice per group, ***P , 0.005, 2-way ANOVA with Bonferroni correction. ANOVA, analysis of variance.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

November 2015

·

Volume 156

·

Number 11

by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to polyvinyldifluoride (PVDF; EMD Millipore, Billerica, MA), blocked in 5% nonfat milk in Trisbuffered saline with 0.1% Tween-20 nonionic detergent, and incubated for 18 hours with the primary antibody anti-TRPV1 (Santa Cruz Biotechnology8,55) or anti-AKAP150. Anti-rabbit secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ) were applied to rinsed blots and incubated at room temperature for 1 hour, and then blots were rinsed again. Blots were incubated with enhanced chemiluminescence solution (GE Healthcare Life Sciences), exposed to X-ray film, and developed for analysis. Films were scanned, and immunoreactive bands were quantified using NIH Image 1.62 shareware.

3. Results 3.1. mGluR1/5 and AKAP150 support acute pain In Figure 1, AKAP150 WT and KO littermate male mice were i.pl. injected in the hind paw with DHPG before stimulating the same hind paw with radiant heat using the Hargreaves’ method26 to monitor timed latency to remove the injected hind paw from a thermal stimulus. Mice were injected with vehicle or 50 nmol DHPG (10 mL injection volume) and tested for paw withdrawal latency to a thermal stimulus 30 minutes after injection, as performed previously.5 In Figure 1, AKAP150 WT mice

Figure 3. mGluR sensitization of heat response in nociceptors from AKAP wild-type (WT) and knockout (KO) mice. (A) After a 10-second heat stimulation, the mean discharge rate is higher in KO compared with WT; however, the increase is not significant. (B) The 10-second heat ramp, ascending from 34˚C to 51˚C, demonstrates that the mean threshold to heat activation is significantly lower in KO compared with WT mice. *P , 0.05, Mann–Whitney U test. (C) Traces of unfiltered raw data show unit activity a few seconds before and after a 10-second heat stimulus is applied to the receptive field of a nociceptor. In the KO mice, there is no change in heat-induced activity after a 2-minute treatment with 1 or 3 mM DHPG. (D) In contrast, a unit from WT shows an enhanced discharge rate to heat after 3 mM DHPG. The population data are summarized in (E and F) showing that a 2-minute application of 1 or 3 mM DHPG to nociceptors in KO has no effect on heat-induced discharge rate (E) or threshold (F), but both are modified in WT. *P , 0.05, Kruskal–Wallis followed by Dunn’s post hoc test.

www.painjournalonline.com

2367

experienced a significant increase in sensitivity to a thermal stimulus after DHPG treatment, as paw withdrawal latency was dramatically reduced. Importantly, no change was observed in AKAP150 KO mice after DHPG injection, indicating that AKAP150 plays a physiological role in the sensitized thermal response after mGluR5 agonist administration. 3.2. Persistent thermal sensitivity requires mGluR1/5 and AKAP150 Next, we evaluated whether acute thermal behavior observed in AKAP150 KO animals would translate to persistent pain models. Studies published by the Levine group identify a behavioral protocol by which acute pain events become persistent in nature.7,41,42 In this protocol, the peripheral injection of an inflammatory algogen, such as carrageenan, several days before another inflammatory event led to a significant temporal extension of mechanical sensitivity and nociceptive behavior. However, rodents that underwent this treatment protocol were not studied for thermal sensitivity. Given the importance of TRPV1 to chronic pain28,34,39,44,51 and the inclusive importance of IB4(1) neurons to hyperalgesic priming,33 we sought to determine whether the hyperalgesic priming protocol could affect behavioral measures of TRPV1 responsiveness, including thermal sensitivity. We used the Hargreaves’ method for measuring sensitivity to a thermal stimulus, following the carrageenan preinjection PGE2-timed injection protocol.42 As demonstrated in Figure 2A, we observed a significant difference in paw withdrawal latency between

Figure 4. mGluR sensitization of capsaicin (CAP) response in nociceptors from AKAP wild-type (WT) and knockout (KO) mice. (A and B) Traces of unfiltered raw data demonstrate that repeated 2-minute exposures of receptive fields to 10 mM DHPG have no effect on discharge rate in WT and KO. (C) The response to CAP alone is not different in KO vs WT mice. However, 10 mM DHPG greatly enhances 1 mM CAP-induced activity in WT (A and C) compared with KO (B and C), *P , 0.05, Kruskal–Wallis followed by Dunn’s post hoc test. For KO, the mean discharge rate after DHPG 1 CAP is not different from that after CAP alone in KO; however, for WT, the mean discharge rate after DHPG 1 CAP is significantly higher compared with that after CAP alone in WT (P , 0.05, Kruskal–Wallis test followed by Dunn’s post hoc test). The percent of CAP responders is similar in KO compared with WT; however, deletion of AKAP (KO) significantly reduces the percent of nociceptors enhanced by DHPG-CAP (D) *P , 0.05, Fisher exact test.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

2368

·

K. Szteyn et al. 156 (2015) 2364–2372

animals receiving vehicle vs carrageenan preinjection in the hind paw 2 hours after PGE2 injection. Furthermore, antagonism of mGluR5 with MTEP reversed priming effects after PGE2 injection, indicating an important role for mGluR5 in persistent pain. Next, AKAP150 WT and KO littermates underwent a similar protocol, receiving carrageenan-priming injections before PGE2 injections. In Figure 2B, AKAP150 WT mice displayed persistent hypersensitive behavior in response to a thermal stimulus as rats previously did (Fig. 2A). However, AKAP150 KO mice demonstrated little persistent response to carrageenan priming, returning to baseline readings 2 hours after an initial PGE2 response. Taken together, these data indicate that functional AKAP150 expression is necessary to maintain persistent thermal hypersensitivity. 3.3. mGluR1/5 sensitizes peripheral TRPV1 responses in an AKAP-dependent manner Next, we used an electrophysiological approach to monitor agonist-activated mGluR1/5 sensitization of TRPV1 in peripheral primary afferent neurons responsible for innervating the tissues tested in previous behavior experiments. We monitored action potential generation of peripheral nerves using a skin-nerve preparation approach following previously published protocols.12,27 In Figures 3 and 4, glabrous skin preparations were generated from AKAP150 WT and KO littermate mice for in vitro measurements of peripheral neuron depolarization by TRPV1 agonists CAP (1 mM) and heat (51˚C). A total of 170 nociceptors were recorded from WT mice and 119 from KO. For WT, units consisted of 129 C fibers with a median CV of 0.68 (range 5 0.381.18 m/s) and 41 Ad fibers with a median CV of 2.64 (range 5

PAIN®

1.21-8.43 m/s). For KO, units consisted of 82 C fibers with a median CV of 0.62 (range 5 0.45-1.10 m/s) and 37 Ad fibers with a median CV of 2.15 (range 5 1.23-9.43 m/s). All units had their receptive fields on the glabrous skin of the hind paw. The mean background discharge rate for units from each strain was not different (WT 5 0.10 6 0.04; KO 5 0.13 6 0.03 imp/s). However, deletion of AKAP150 resulted in a change in the response of units to a noxious heat stimulus. The thermal threshold of activation was significantly lower in nociceptors from KO compared with WT (40.61 6 0.59˚C vs 43.01 6 0.43˚C, Fig. 3B). Furthermore, the discharge rate was elevated in KO (3.19 6 0.64 imp/s) compared with WT (1.66 6 0.18 imp/s); however, this difference did not reach significance (P 5 0.33, Fig. 3A). DHPG applied at 1 or 3 mM before heat stimulation produced no change from predrug levels in discharge rate or threshold to activation in units from KO mice (Fig. 3C-F). The 1 mM dose also had no effect on heat responses in WT. In contrast, 3 mM DHPG sensitized WT units, significantly elevating the discharge rate (150 6 26%) and lowering the mean threshold for activation compared with predrug levels (94 6 1%, P , 0.05). Changing from a natural stimulus (heat) to a chemical stimulus (CAP) to activate TRPV1, it was clear that DHPG enhanced TRPV1 activation in WT mice compared with KO mice (Fig. 4A and B). DHPG had no effect on discharge rate in WT or KO when applied alone, but when combined with CAP, DHPG sensitized WT units, producing significantly enhanced discharge rates when compared with KO (0.90 6 0.1 imp/s vs 0.69 6 0.13 imp/s, Fig. 4C). Furthermore, WT responses to DHPG 1 CAP were significantly enhanced compared with WT responses to CAP alone (P , 0.05, Kruskal–Wallis test followed by Dunn’s post hoc

Figure 5. AKAP plays a role in DHPG-enhanced Ca21 responses after sequential capsaicin (CAP) treatment. Dorsal root ganglion neurons were isolated from WT and AKAP KO mice. Changes in intracellular Ca21 levels were assessed by measurements with Fura-2 calcium indicator. (A) Representative tracings of single-cell measurements. Starting with the establishment of baseline, cells were treated with CAP (100 nM) for 30 seconds and washed with constant perfusion of SES buffer for 5 minutes, followed by administration of the second CAP treatment. In DHPG groups, cells were washed with SES buffer for 3 minutes and DHPG (100 mM) was administered for 2 minutes before the second CAP treatment. (B) The net change of intracellular Ca21 concentration was calculated by subtraction of established basal concentration from peak concentration after CAP administration, n 5 28 to 76 neurons per group. In case of the second peak, concentration at the end of wash was taken as base value. (C) The percentage of desensitization was determined by comparison of responses after the first and second CAP application. The first Ca21 increase was normalized to 100%, and the second response was calculated as its percentage, and the difference between the 2 illustrated the rate of desensitization for each group. ***P , 0.001, as determined by one-way ANOVA, with Bonferroni correction. ANOVA, analysis of variance; KO, knockout; NS, no significance; SES, standard extracellular solution; WT, wild type.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

November 2015

·

Volume 156

·

Number 11

test). Units recorded from KO animals responded to CAP, but these responses were not enhanced by DHPG (Fig. 4C). To ensure that deletion of AKAP150 did not change the number of CAP-sensitive units in the KO mice, the percentage of responders was calculated and there was no significant difference in the number of units in KO and WT responding to CAP (57% vs 62%, Fig. 4D, x2); however, the percent of units responding to DHGP 1 CAP was significantly higher in WT compared with KO (69% vs 50%, Fig. 4D, x2). Taken together, data presented in Figures 3 and 4 demonstrate that mGluR activation increases CAP- and heat-induced neuronal excitability in an AKAP150-dependent manner. 3.4. mGluR5 sensitization of TRPV1 is AKAP150- and PLC-dependent Next, we sought to identify AKAP150 as a functional mediator of mGluR1/5 modulation of TRPV1 sensitivity to agonist activation. Dorsal root ganglion neurons from AKAP150 WT and KO mice were prepared for real-time single-cell calcium imaging to analyze mGluR5 effects on CAP responses of TRPV1. For this work, we followed a previously published protocol detailing the effects of mGluR5 agonists on pharmacological desensitization of TRPV1 in sensory neurons.29 We report similar findings in Figure 5, as pretreatment with DHPG (2 minutes, 100 mM) before the second application of CAP significantly reduces pharmacological desensitization of TRPV1. Importantly, this relationship was lost in AKAP150 KO neurons, indicating an important role for the scaffolding protein in downstream signal transduction pathway(s) after mGluR5 activation.

www.painjournalonline.com

2369

As mGluR1/5 activation drives Gaq-coupled PLC activity,47 we next sought to determine whether PLC inhibition would reverse DHPG-induced reduction in TRPV1 pharmacological desensitization. In Figure 6, DRG neurons were prepared for real-time calcium imaging as in Figure 5 but co-treated between successive CAP applications with DHPG (2 minutes, 100 mM) and either U73122 (PLC inhibitor, 1 mM) or U73343 (negative control, inactive analog for U73122, 1 mM).32 After PLC inhibition, DHPG treatment is no longer able to reduce TRPV1 pharmacological desensitization, indicating that the Gaq-coupled signaling pathway is primarily responsible for the mGluR5 effects on TRPV1 responses in sensory neurons.5 3.5. mGluR1/5 activation increases AKAP150/ TRPV1 association We previously demonstrated that direct PLC activation increases TRPV1 association with AKAP150.32 This is important because AKAP150 scaffolds PKA and PKC to coordinate posttranslational modification and subsequent sensitization of TRPV1. However, no one has demonstrated this phenomenon through receptormediated activation of PLC, as occurs through Gaq-coupled metabotropic receptors such as mGluR5.47 In Figure 7, cultured sensory neurons were treated with either vehicle and DHPG (100 mM29) or fenobam (1 mM38) and DHPG for 5 minutes each, and TRPV1 association with AKAP150 was determined by Co-IP. As shown in Figure 7A and B, DHPG treatment induces a 2-fold increase in AKAP association with TRPV1 in a manner sensitive to mGluR5 antagonism. To confirm the PLC-dependent nature of this receptor-activated association, we pretreated cultures with the PLC inhibitor U73122 (1 mM, 5 minutes) or its negative control

Figure 6. DHPG reversal of TRPV1 pharmacological desensitization is reversed by inhibition of PLC. Dorsal root ganglion neurons were isolated from WT mice. Changes in intracellular Ca21 levels were assessed by measurements with Fura-2 calcium indicator. (A) Representative tracings of single-cell measurements. Starting with the establishment of baseline, cells were treated with capsaicin (CAP) (100 nM) for 30 seconds and washed with constant perfusion of SES buffer for 5 minutes, followed by administration of the second CAP treatment. In DHPG treatment groups, cells were washed with SES buffer for 3 minutes and vehicle, DHPG (100 mM) and vehicle, DHPG and U73122 (1 mM), or DHPG and U73343 (1 mM) was administered for 2 minutes before the second CAP treatment. (B) The net change of intracellular Ca21 concentration was calculated by subtraction of established basal concentration from peak concentration after CAP administration, n 5 77 to 107 neurons per group. In case of the second peak, concentration at the end of wash was taken as base value. (C) The percentage of desensitization was determined by comparison of responses after the first and second CAP application. The first Ca21 increase was normalized to 100%, and the second response was calculated as its percentage, and the difference between the 2 illustrated the rate of desensitization for each group. ***P , 0.001, as determined by 2-way ANOVA, with Bonferroni correction. ANOVA, analysis of variance; NS, no significance; SES, standard extracellular solution; WT, wild type.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

2370

·

K. Szteyn et al. 156 (2015) 2364–2372

PAIN®

Figure 7. mGluR1/5 activation stimulates AKAP association with TRP receptors. (A) Cultured rat TG neurons were treated with DHPG (100 mM, 5 minutes), and AKAP:TRPV1 association was determined by co-immunoprecipitation (Co-IP). Increased Co-IP is inhibited by pretreatment with the mGluR5 negative allosteric modulator, fenobam (1 mM, 5 minutes before DHPG). (B) Quantified Co-IP results from (A), normalized to total AKAP IP. Results representative of 3 independent trials, *P , 0.05, **P , 0.01, 2-way ANOVA with Bonferroni correction. (C) Cultured rat TG neurons were pretreated with PLC inhibitor U73122 (1 mM, 5 minutes) or negative control U73433 (1 mM, 5 minutes) before DHPG (100 mM, 5 minutes). AKAP:TRPV1 association was determined by Co-IP. (D) Quantified Co-IP results from (C), normalized to total AKAP IP. Results representative of 3 independent trials, *P , 0.05, **P , 0.01, 2-way ANOVA with Bonferroni correction. For (A and C), molecular weights of immunoreactive proteins in kilodaltons are indicated to the left of representative Western blots. ANOVA, analysis of variance; TG, trigeminal ganglion. These symbols of significance are represented in the artwork in panels B and D. This statement was added at the end of the figure legend to reduce space by repeating the asterisk symbols and their respective meanings for each panel.

U73343 (1 mM, 5 minutes) before administering DHPG (5 minutes) as in panel A. Co-immunoprecipitation results in Figure 7C and D confirm that PLC inhibition summarily prevents DHPG-stimulated AKAP association with TRPV1. Furthermore, U73343 has no effect, thus identifying AKAP as an important coordinating scaffolding protein that manages mGluR5 sensitization of TRPV1.

4. Discussion Glutamate serves as the most abundant excitatory neurotransmitter in the human body. In addition to this, it also plays an important role in peripheral inflammation18,37,40 and participates in afferent sensitization to somatosensation.5,50 Previous work has identified that glutamate increases peripheral thermal sensitivity through both PKC36 and PKA activities29; yet, it is unclear how these signaling processes are coordinated. This is especially important given that both PKA and PKC increase peripheral thermal sensitivity through posttranslational modifications of the ligand-gated ion channel TRPV1.4,6 Here, we identify that AKAP150 (AKAP), a scaffolding protein responsible for organizing PKA- and PKC-mediated phosphorylation of TRPV1,30,31,56 coordinates mGluR5 sensitization of TRPV1 in peripheral sensory neurons. AKAP scaffolding supports mGluR5sensitive acute and persistent pain models. Also, AKAP is required for peripheral mGluR5 sensitization of sensory neurons by either thermal or chemical stimuli. In addition, mGluR5 activation reduces TRPV1 pharmacological desensitization in a manner that requires AKAP expression but is sensitive to PLC inhibition. Furthermore, mGLuR5 stimulation of AKAP association with TRPV1 in sensory neurons is sensitive to PLC inhibition. Taken together, these results indicate that mGluR5-stimulated AKAP association with TRPV1 coordinates inflammatory thermal sensitization on an acute time scale that can become more persistent in nature.

mGluR5 belongs to the group I family of metabotropic glutamate receptors, of which both subtypes (mGluR1 and mGluR5) are expressed by peripheral sensory neurons.5 Both receptor subtypes couple to Gaq-mediated PLC signaling pathways in multiple models, including central nervous system neurons25 and transfected cells.43 Additional work in peripheral sensory neurons indicates that PLC mediates mGluR5-induced sensitization of TRPV1 responses.29 Herein, we similarly demonstrate a significant importance for PLC in mGluR5 modulation of TRPV1 responses in cultured sensory neurons (Fig. 6). Co-immunoprecipitation experiments reveal that mGluR5 activation stimulates AKAP association with TRPV1 in a PLC-dependent manner (Fig. 7). Although this result confirms previous biochemical findings,19,32 it also serves to identify that activation of peripherally expressed Gaq-coupled receptors can stimulate scaffolded coordination of phosphorylation of certain TRP channels, including TRPV1. Taken together, these data identify AKAP as a target for drug development that may reduce TRPV1 sensitization linked to inflammatory thermal hyperalgesia.13 Thermal hyperalgesia can be measured using multiple behavioral models. In this study, we used the acute Hargreaves’ thermal sensitivity behavioral model (Fig. 1, Ref. 26) and the Levine hyperalgesic priming behavioral model (Fig. 2, Ref. 1). Following the acute behavioral paradigm, we found AKAP expression to be of significant importance to mGluR5 sensitization of thermal behavior. However, we modified the Levine model for mechanosensitivity to measure for persistent thermal sensitivity and found that carrageenan-induced hyperalgesic priming was sensitive to mGluR5 inhibition by MTEP. Importantly, use of AKAP KO animals in the same model revealed that AKAP expression is required for persistent thermal sensitivity. Together, these data identify an important role for peripheral glutamate in persistent pain that may involve AKAP.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

November 2015

·

Volume 156

·

Number 11

A prominent feature of mechanical hyperalgesic priming in the Levine behavioral model is the extended length of time that the behavior persists (.24 hours, Ref. 1). Using the same model, we were only able to observe significant priming effects for thermal sensitivity at 2 hours in rats (Fig. 2A) and at 0.5 and 2 hours in WT mice (Fig. 2B). This difference could be explained by a number of factors. First, several reports have identified that PGE2 effects on thermal sensitivity are short lived, losing significance less than 2 hours after exposure.2,45 Second, peripheral glutamate release after afferent depolarization18 might affect receptors/channels sensitive to thermal activation differently than those activated by mechanical stimulation. Third, central sensitization could account for extended mechanical somatosensitivities52 not observed here. However, an autocrine feed-forward loop22 could also explain the findings presented here and may significantly contribute to persistent peripheral somatosensitivities. Autocrine feed-forward plasticity often arises in neuronal signaling paradigms when depolarized neurons release neurotransmitters capable of acting on receptors expressed by the depolarized neuron. In this vein, glutamate released from depolarized peripheral afferents could act on metabotropic glutamate receptors, including receptors belonging to the group I family, thereby resulting in TRPV1 phosphorylation and sensitization. This sensitized ligand-gated ion channel then has a reduced threshold for activation,4 increasing the likelihood for neuronal depolarization, and subsequent repeated release of glutamate. Indeed, this feed-forward scenario has been shown to be sensitive to peptidergic inhibition of AKAP/TRPV1 association,10,23 identifying a unique protein–protein interaction that could support persistent thermal hyperalgesia. In conclusion, behavioral, functional, and biochemical data presented here demonstrate that mGluR5 stimulates AKAP150 association with TRPV1 in a model of thermal hyperalgesic priming, suggesting an innovative direction for future therapeutic interventions.

Conflict of interest statement The authors have no conflicts of interest to declare. This work was supported by funding from the National Institutes of Health NINDS, NS082746 (N.A.J.), NS027910, and DA027460 (S.M.C.). Article history: Received 5 May 2015 Received in revised form 23 June 2015 Accepted 2 July 2015 Available online 13 July 2015

References [1] Aley KO, Messing RO, Mochly-Rosen D, Levine JD. Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci 2000;20:4680–5. [2] Bastos LC, Tonussi CR. PGE(2)-induced lasting nociception to heat: evidences for a selective involvement of A-delta fibres in the hyperpathic component of hyperalgesia. Eur J Pain 2010;14:113–19. [3] Bedi SS, Yang Q, Crook RJ, Du J, Wu Z, Fishman HM, Grill RJ, Carlton SM, Walters ET. Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury. J Neurosci 2010;30:14870–82. [4] Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW IV. Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl Acad Sci U S A 2003;100:12480–5. [5] Bhave G, Karim F, Carlton SM, Gereau RW IV. Peripheral group I metabotropic glutamate receptors modulate nociception in mice. Nat Neurosci 2001;4:417–23.

www.painjournalonline.com

2371

[6] Bhave G, Zhu W, Wang H, Brasier DJ, Oxford GS, Gereau RW IV. cAMPdependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. Neuron 2002;35:721–31. [7] Bogen O, Alessandri-Haber N, Chu C, Gear RW, Levine JD. Generation of a pain memory in the primary afferent nociceptor triggered by PKCepsilon activation of CPEB. J Neurosci 2012;32:2018–26. [8] Botto L, Bernabo N, Palestini P, Barboni B. Bicarbonate induces membrane reorganization and CBR1 and TRPV1 endocannabinoid receptor migration in lipid microdomains in capacitating boar spermatozoa. J Membr Biol 2010;238:33–41. [9] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [10] Btesh J, Fischer MJ, Stott K, McNaughton PA. Mapping the binding site of TRPV1 on AKAP79: implications for inflammatory hyperalgesia. J Neurosci 2013;33:9184–93. [11] Carlton SM, Coggeshall RE. Peripheral capsaicin receptors increase in the inflamed rat hindpaw: a possible mechanism for peripheral sensitization. Neurosci Lett 2001;310:53–6. [12] Carlton SM, Zhou S, Du J, Hargett GL, Ji G, Coggeshall RE. Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel. PAIN 2004;110:616–27. [13] Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. [14] Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, PetersenZeitz KR, Koltzenburg M, Basbaum AI, Julius D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288: 306–13. [15] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. [16] Chung MK, Lee J, Joseph J, Saloman J, Ro JY. Peripheral group I metabotropic glutamate receptor activation leads to muscle mechanical hyperalgesia through TRPV1 phosphorylation in the rat. J Pain 2015;16: 67–76. [17] Conn PJ, Pin JP. Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 1997;37:205–37. [18] deGroot J, Zhou S, Carlton SM. Peripheral glutamate release in the hindpaw following low and high intensity sciatic stimulation. Neuroreport 2000;11:497–502. [19] Dell’Acqua ML, Faux MC, Thorburn J, Thorburn A, Scott JD. Membranetargeting sequences on AKAP79 bind phosphatidylinositol-4, 5bisphosphate. EMBO J 1998;17:2246–60. [20] Doly S, Fischer J, Salio C, Conrath M. The vanilloid receptor-1 is expressed in rat spinal dorsal horn astrocytes. Neurosci Lett 2004;357: 123–6. [21] Ferrari LF, Bogen O, Levine JD. Role of nociceptor alphaCaMKII in transition from acute to chronic pain (hyperalgesic priming) in male and female rats. J Neurosci 2013;33:11002–11. [22] Ferrari LF, Levine E, Levine JD. Role of a novel nociceptor autocrine mechanism in chronic pain. Eur J Neurosci 2013;37:1705–13. [23] Fischer MJ, Btesh J, McNaughton PA. Disrupting sensitization of transient receptor potential vanilloid subtype 1 inhibits inflammatory hyperalgesia. J Neurosci 2013;33:7407–14. [24] Guo A, Vulchanova L, Wang J, Li X, Elde R. Immunocytochemical localization of the vanilloid receptor 1 (VR1): relationship to neuropeptides, the P2X3 purinoceptor and IB4 binding sites. Eur J Neurosci 1999;11:946–58. [25] Hannan AJ, Blakemore C, Katsnelson A, Vitalis T, Huber KM, Bear M, Roder J, Kim D, Shin HS, Kind PC. PLC-beta1, activated via mGluRs, mediates activity-dependent differentiation in cerebral cortex. Nat Neurosci 2001;4:282–8. [26] Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. PAIN 1988;32:77–88. [27] Hogan D, Baker AL, Moron JA, Carlton SM. Systemic morphine treatment induces changes in firing patterns and responses of nociceptive afferent fibers in mouse glabrous skin. PAIN 2013;154:2297–309. [28] Honore P, Wismer CT, Mikusa J, Zhu CZ, Zhong C, Gauvin DM, Gomtsyan A, El Kouhen R, Lee CH, Marsh K, Sullivan JP, Faltynek CR, Jarvis MF. A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)urea], a novel transient receptor potential type V1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. J Pharmacol Exp Ther 2005;314:410–21. [29] Hu HJ, Bhave G, Gereau RW IV. Prostaglandin and protein kinase Adependent modulation of vanilloid receptor function by metabotropic glutamate receptor 5: potential mechanism for thermal hyperalgesia. J Neurosci 2002;22:7444–52.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.

2372

·

K. Szteyn et al. 156 (2015) 2364–2372

[30] Jeske NA, Diogenes A, Ruparel NB, Fehrenbacher JC, Henry M, Akopian AN, Hargreaves KM. A-kinase anchoring protein mediates TRPV1 thermal hyperalgesia through PKA phosphorylation of TRPV1. PAIN 2008;138:604–16. [31] Jeske NA, Patwardhan AM, Ruparel NB, Akopian AN, Shapiro MS, Henry MA. A-kinase anchoring protein 150 controls protein kinase C-mediated phosphorylation and sensitization of TRPV1. PAIN 2009; 146:301–7. [32] Jeske NA, Por ED, Belugin S, Chaudhury S, Berg KA, Akopian AN, Henry MA, Gomez R. A-kinase anchoring protein 150 mediates transient receptor potential family V type 1 sensitivity to phosphatidylinositol-4,5bisphosphate. J Neurosci 2011;31:8681–8. [33] Joseph EK, Levine JD. Hyperalgesic priming is restricted to isolectin B4positive nociceptors. Neuroscience 2010;169:431–5. [34] Kanai Y, Nakazato E, Fujiuchi A, Hara T, Imai A. Involvement of an increased spinal TRPV1 sensitization through its up-regulation in mechanical allodynia of CCI rats. Neuropharmacology 2005;49: 977–84. [35] Maione S, Starowicz K, Cristino L, Guida F, Palazzo E, Luongo L, Rossi F, Marabese I, de Novellis V, Di Marzo V. Functional interaction between TRPV1 and mu-opioid receptors in the descending antinociceptive pathway activates glutamate transmission and induces analgesia. J Neurophysiol 2009;101:2411–22. [36] Marcon R, Luiz AP, Werner MF, Freitas CS, Baggio CH, Nascimento FP, Soldi C, Pizzolatti MG, Santos AR. Evidence of TRPV1 receptor and PKC signaling pathway in the antinociceptive effect of amyrin octanoate. Brain Res 2009;1295:76–88. [37] McNearney T, Speegle D, Lawand N, Lisse J, Westlund KN. Excitatory amino acid profiles of synovial fluid from patients with arthritis. J Rheumatol 2000;27:739–45. [38] Montana MC, Cavallone LF, Stubbert KK, Stefanescu AD, Kharasch ED, Gereau RW IV. The metabotropic glutamate receptor subtype 5 antagonist fenobam is analgesic and has improved in vivo selectivity compared with the prototypical antagonist 2-methyl-6-(phenylethynyl)pyridine. J Pharmacol Exp Ther 2009;330:834–43. [39] Niiyama Y, Kawamata T, Yamamoto J, Omote K, Namiki A. Bone cancer increases transient receptor potential vanilloid subfamily 1 expression within distinct subpopulations of dorsal root ganglion neurons. Neuroscience 2007;148:560–72. [40] Omote K, Kawamata T, Kawamata M, Namiki A. Formalin-induced release of excitatory amino acids in the skin of the rat hindpaw. Brain Res 1998;787:161–4. [41] Parada CA, Reichling DB, Levine JD. Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCepsilon second messenger pathways. PAIN 2005;113:185–90. [42] Parada CA, Yeh JJ, Reichling DB, Levine JD. Transient attenuation of protein kinase cepsilon can terminate a chronic hyperalgesic state in the rat. Neuroscience 2003;120:219–26.

PAIN®

[43] Pin JP, Joly C, Heinemann SF, Bockaert J. Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. EMBO J 1994;13:342–8. [44] Pomonis JD, Harrison JE, Mark L, Bristol DR, Valenzano KJ, Walker K. N-(4Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine -1(2H)-carboxamide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: II. In vivo characterization in rat models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 2003;306:387–93. [45] Rowan MP, Ruparel NB, Patwardhan AM, Berg KA, Clarke WP, Hargreaves KM. Peripheral delta opioid receptors require priming for functional competence in vivo. Eur J Pharmacol 2009;602:283–7. [46] Simone DA, Sorkin LS, Oh U, Chung JM, Owens C, LaMotte RH, Willis WD. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. J Neurophysiol 1991;66:228–46. [47] Swanson CJ, Bures M, Johnson MP, Linden AM, Monn JA, Schoepp DD. Metabotropic glutamate receptors as novel targets for anxiety and stress disorders. Nat Rev Drug Discov 2005;4:131–44. [48] Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE, Basbaum AI, Julius D. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21:531–43. [49] Tunquist BJ, Hoshi N, Guire ES, Zhang F, Mullendorff K, Langeberg LK, Raber J, Scott JD. Loss of AKAP150 perturbs distinct neuronal processes in mice. Proc Natl Acad Sci U S A 2008;105:12557–62. [50] Walker K, Reeve A, Bowes M, Winter J, Wotherspoon G, Davis A, Schmid P, Gasparini F, Kuhn R, Urban L. mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia. Neuropharmacology 2001;40:10–19. [51] Walker KM, Urban L, Medhurst SJ, Patel S, Panesar M, Fox AJ, McIntyre P. The VR1 antagonist capsazepine reverses mechanical hyperalgesia in models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 2003;304:56–62. [52] Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. PAIN 2011;152(3 suppl):S2–15. [53] Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science 2000;288:1765–9. [54] Young MR, Fleetwood-Walker SM, Mitchell R, Dickinson T. The involvement of metabotropic glutamate receptors and their intracellular signalling pathways in sustained nociceptive transmission in rat dorsal horn neurons. Neuropharmacology 1995;34:1033–41. [55] Zhang D, Spielmann A, Wang L, Ding G, Huang F, Gu Q, Schwarz W. Mastcell degranulation induced by physical stimuli involves the activation of transient-receptor-potential channel TRPV2. Physiol Res 2012;61:113–24. [56] Zhang X, Li L, McNaughton PA. Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/ 150. Neuron 2008;59:450–61. [57] Zhou S, Komak S, Du J, Carlton SM. Metabotropic glutamate 1alpha receptors on peripheral primary afferent fibers: their role in nociception. Brain Res 2001;913:18–26.

Copyright Ó 2015 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.