Contribution of transient receptor potential vanilloid subfamily 1 to endothelin-1-induced thermal hyperalgesia

Neuroscience 154 (2008) 1067–1076

CONTRIBUTION OF TRANSIENT RECEPTOR POTENTIAL VANILLOID SUBFAMILY 1 TO ENDOTHELIN-1-INDUCED THERMAL HYPERALGESIA T. KAWAMATA,* W. JI, J. YAMAMOTO, Y. NIIYAMA, S. FURUSE AND A. NAMIKI

Tissue damage associated with inflammation, ischemia, infection, injury, or tumor invasion produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit or exacerbate pain. Endothelin-1 (ET-1) is a candidate mediator that is generated by a number of cell types, including inflammatory cells and some tumor cell lines (Gandhi et al., 1994). Increasing evidence indicates that ET-1 has a role in peripheral pain signaling in animals and human. In rodents, s.c. administration of ET-1 resulted in pain-like flinching behavior (Zhou et al., 2002), mechanical hyperalgesia (Motta et al., 2006) and thermal hyperalgesia (Menendez et al., 2003). In humans, intradermal ET-1 causes wheal and flare responses (Ferreira et al., 1999). ET-1 injected into the brachial artery also induced severe pain and prolonged, touch-evoked allodynia (Dahlof et al., 1990). While ET-1 exerts important biological actions mediated by two receptor subtypes, endothelin type A receptor (ETA) and endothelin type B receptor (ETB) (Gandhi et al., 1994), the cell body of sensory neurons expresses only ETA(Peters et al., 2004; Pomonis et al., 2001). Accordingly, selective ETA antagonists attenuated tactile allodynia in a diabetic model of neuropathic pain (Jarvis et al., 2000), thermal and mechanical hyperalgesia in complete Freud’s adjuvant-induced inflammation (Baamonde et al., 2004) and bone cancer-related pain (Peters et al., 2004; Wacnik et al., 2001). These findings suggest that the activation of ETA by ET-1 is involved in peripheral nociceptive transmission. However, the mechanisms of the nociceptive action of ET-1 are not fully understood. We previously reported that ET-1 enhances capsaicinevoked intracellular calcium response in dorsal root ganglion (DRG) neurons through the activation of ETA in a protein kinase C (PKC) – dependent manner, suggesting the interaction of ET-1 with transient receptor potential vanilloid subfamily 1 (TRPV1), a capsaicin receptor that is an integrator of noxious stimuli, including capsaicin, proton and noxious heat stimuli (Yamamoto et al., 2006). In addition, recent studies have shown that in whole-cell patch clamp recording of human embryonic kidney 293 (HEK293) cells expressing TRPV1 and ETA, ET-1 potentiated TRPV1 activity (Plant et al., 2006, 2007). These findings suggest the involvement of TRPV1 in ET-1-induced thermal hyperalgesia, because TRPV1 plays an important role in inflammatory-induced thermal hyperalgesia (Caterina et al., 2000). However, it has remained unknown whether TRPV1 activity contributes to ET-1-induced thermal hyperalgesia. In the current study, we performed a behavioral study using TRPV1-deficient mice in order to clarify the contri-

Department of Anesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo, 060-8543, Japan

Abstract—Endothelin-1 (ET-1) plays an important role in peripheral pain processing. However, the mechanisms of the nociceptive action of ET-1 have not been fully elucidated. In this study, we investigated the contribution of transient receptor potential vanilloid subfamily 1 (TRPV1) to ET-1-induced thermal hyperalgesia. Intraplantar ET-1-induced thermal hyperalgesia was examined by assessing the paw withdrawal latency to noxious heat stimuli. In electrophysiological study, whole-cell patch-clamp recordings were performed to investigate the interaction of ET-1 and TRPV1 using human embryonic kidney 293 (HEK293) cells expressing endothelin type A receptor (ETA) and TRPV1. Intraplantar ET-1 (3, 10 and 30 pmol) produced thermal hyperalgesia in a dose-dependent manner. Thermal hyperalgesia was attenuated by the inhibition of ETA and protein kinase C (PKC) but not that of ETB. ET-1-induced thermal hyperalgesia was significantly attenuated in TRPV1deficient mice compared with that in wild-type mice. In voltage-clamp experiments, 10 nM capsaicin evoked small inward currents in HEK293 cells expressing TRPV1 and ETA. In the presence of ET-1, capsaicin produced much larger current responses (P<0.05). Mutation at PKC-specific TRPV1 phosphorylation sites (S800A/S502A) and PKC inhibitors inhibited the potentiating effect of ET-1. In addition, ET-1 decreased the temperature threshold for TRPV1 activation in a PKC-dependent manner (from 41.0ⴞ0.4 °C to 32.6ⴞ0.6 °C). In addition, Western blot analysis was also performed to confirm ET-1-induced phosphorylation of TRPV1. Incubation of ET-1 and intraplantar ET-1 evoked phosphorylation of TRPV1 in HEK293 cells expressing TRPV1 and ETA and the skin, respectively. These results suggest that the sensitization of TRPV1 activity through an ETA-PKC pathway contributes to ET-1-induced thermal hyperalgesia. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: inflammation, hyperalgesia, dorsal root ganglion, capsaicin, pain.

*Corresponding author. Tel: ⫹81-11-611-2111x3568; fax: ⫹81-11-6319683. E-mail address: [email protected] (T. Kawamata). Abbreviations: BIM, bisindolylmaleimide I; CalpC, calphostin C; DMSO, dimethyl sulfoxide; DRG, dorsal root ganglion; ETA, endothelin type A receptor; ETB, endothelin type B receptor; ET-1, endothelin-1; HEK293, human embryonic kidney 293; MOR, mu opioid receptor; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PWL, paw withdrawal latency; TRPV1, transient receptor potential vanilloid subfamily 1; TTX-R, tetrodotoxinresistant; WT, wild-type.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.04.010

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bution of TRPV1 to ET-1-induced thermal hyperalgesia and re-examined the interaction TRPV1 and ET-1 using HEK293 cells expressing TRPV1 and ETA.

EXPERIMENTAL PROCEDURES Animals Male C57BL/6-strain mice (wild-type mice, WT mice) (weighing 20 – 25 g, Japan SLC; Hamamatsu, Shizuoka, Japan) and male TRPV1deficient mice (TRPV1 KO mice) (weighing 20 –25 g, Jackson Laboratory, Bar Harbor, MA, USA) were used for behavioral analyses. Each mouse was used in only one experiment. The mice were housed in a temperature-controlled (21⫾1 °C) room with a 12-h light/dark cycle and given free access to food and water. All procedures were approved by the Sapporo Medical University Animal Care Committee and were consistent with the ethical guidelines of the National Institutes of Health on animal care and the International Association for the Study of Pain. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Drugs and chemicals Synthetic ET-1 (98% pure peptide content), BQ-123 (an ETA antagonist) and BQ-788 (an ETB antagonist) were obtained from American Peptides (Sunnyvale, CA, USA). Bisindolylmaleimide I (BIM; a PKC inhibitor) and calphostin C (CalpC; a PKC inhibitor) were obtained from Calbiochem (San Diego, CA, USA). Capsaicin and phorbol-12-myristate-13-acetate (PMA) were obtained from Sigma (St. Louis, MO, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO) and preserved. Before the experiments, all drugs were diluted in phosphate-buffered saline, (PBS) adjusted to pH 7.4. The final concentration of DMSO was ⱕ3%.

Behavioral analyses Thermal nociceptive testing was conducted using an analgesimeter (Plantar test 7370, Ugo Basile, Italy). Before beginning experiments, the mice were placed in plastic chambers (10⫻ 6⫻6 cm) and habituated to the test apparatus for 30 min prior to testing and thereby acclimated to both the testing environment and the experiments. The mice were unrestrained, and radiant heat was applied to the plantar surface of left hind paw until it was actively withdrawn by the animal. The paw withdrawal latency (PWL) was taken as an index of the thermal nociceptive threshold, whereas a decrease in this parameter was considered to be indicative of thermal hyperalgesia. Intensity of the light beam was adjusted so that the basal PWL was 9 –11 s. Cutoff time was 20 s in order to avoid tissue damage. In order to examine the effects of ET-1 on thermal nociception, ET-1 (3, 10 and 30 pmol/10 ␮l) was intraplantarly injected into the left hind paw of each mouse after 30 min of habituation. In some mice, an ETA antagonist (BQ-123, 3 nmol/10 ␮l), an ETB antagonist (BQ-788, 10 nmol/10 ␮l), a PKC inhibitor (BIM, 3 nmol/10 ␮l) or vehicle (3% DMSO, 10 ␮l) was intraplantarly injected 15 min before ET-1 injection. The doses of antagonists were determined on the basis of previous studies (Piovezan et al., 1998, 2000).

Mammalian cell culture HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 ␮g streptomycin). Cells were transiently transfected with 1 ␮g of rat TRPV1 cDNA (given generously by Dr. David Julius, UCSF, San Francisco, USA) or 1 ␮g of rat TRPV1 mutant S502A/S800A cDNA with 0.7 ␮g of rat ETA cDNA (given generously by Dr. Soichi Miwa, Hokkaido University, Japan) using Lipofectamine 2000 Reagent (Invitrogen, Grand Island, NY, USA). TRPV1 cDNA and TRPV1 mutant S502A/S800A

cDNA were ligated into the pEGFP mammalian expression vector (Clontech, Mountain View, CA, USA), and ETA cDNA was ligated into the pDsRed-monomer mammalian expression vector (Clontech). TRPV1 mutant S502A/S800A is insensitive to PKC-dependent phosphorylation (Numazaki et al., 2002). Genes cloned into the multicloning site in the vectors are expressed as fusions to the N-terminal region of EGFP or DsRed-monomer. TRPV1 mutant S502A/S800A was generated by using oligonucleotide-directed mutagenesis. The transfected cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 for 24 – 48 h before experiments. Expressions of TRPV1, TRPV1 mutant and ETA were confirmed by the expression of EGFP fluorescence and dsRED fluorescence, respectively, using fluorescence microscopy.

Electrophysiology Whole-cell patch-clamp recordings were performed with a holding potential of ⫺60 mV at 24 – 48 h after transfection of cDNA to HEK293 cells. To prevent extracellular Ca2⫹-dependent desensitization of TRPV1 (Numazaki et al., 2003), Ca2⫹-free bath solution was used. The bath solution contained (in mM) 140 NaCl, 5 EGTA, 1.6 MgCl2, 4 KCl, 10 Hepes, and 4 glucose, neutralized to pH 7.4 with NaOH. Electrodes were filled with pipette solution containing (in mM) 135 KCl, 1.6 MgCl2, 2 EGTA, 2.5 MgATP, 0.2 Li2GTP, and 10 Hepes, neutralized to pH 7.4 with KOH, and had resistances of 2–5 M⍀. The patch electrodes were pulled from thin-walled borosilicate glass capillaries (OD, 1.5 mm) using a puller (P-97; Sutter Instruments, Novato, CA, USA). In some experiments, a PKC inhibitor, CalpC or BIM, was included in the pipette solution. All patch-clamp experiments were performed at room temperature (22–25 °C) unless otherwise noted. When examining heat-evoked current responses, the bath temperature was increased by using a preheated solution at the rate of 1.5– 2.0 °C/s. When the heat-activated currents started to become inactivated, the preheated solution was changed to a room temperature solution. Chamber temperature was monitored with a thermocouple (accuracy⫾0.1 °C) placed within 100 ␮m of the patch-clamped cell. Various solutions were applied to the chamber by gravity at a flow rate of 5 ml/min. Threshold temperature for activation was defined as the point where two lines approximating the stable baseline current and the clearly increasing temperaturedependent current cross in the temperature-response profile (Sugiura et al., 2002; Moriyama et al., 2005). Whole-cell recording data were sampled at 10 kHz and filtered at 5 kHz for analysis (Axopatch 200B amplifier with pCLAMP software, Axon Instruments, Union City, CA, USA). According to previous studies, the concentrations of inhibitors used in patch-clamp experiments were as follows: CalpC, 1 ␮M; BIM, 1 ␮M; BQ-123, 1 ␮M (Zhou et al., 2002; Dai et al., 2004; Bhave et al., 2003).

Antibody generation Because a serine residue, S800, is predicted to be a substrate only for PKC-dependent phosphorylation, we generated an anti-phospho TRPV1 antibody that recognizes phospho-S800, as previously reported (Mandadi et al., 2006). First, peptide LRDA[pS]TRDRHA was synthesized. Synthetic peptides coupled to keyhole limpet hemocyanin were emulsified with Freund’s complete adjuvant. For immunization, emulsions containing a given amount of antigen peptide were injected into rabbits at 2-week intervals. From antisera sampled at 2 weeks after the sixth injection, antibodies specific for the phosphorylated form of this antigen were purified by HPLC after absorption with a peptide of the same sequence but lacking phosphorylation.

Western blot analysis The mice were deeply anesthetized with ketamine and killed by decapitation 20 min after intraplantar drug injection. The skin of

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Fig. 1. ET-1-induced thermal hyperalgesia in WT mice. (A) Time courses of ET-1-induced thermal hyperalgesia. ET-1 was s.c. administered in a volume of 10 ␮L. Data are presented as means⫾S.D., n⫽6 – 8 in each treatment group. * Statistical significance compared with the withdrawal latency at 0 min (basal withdrawal latency); one-way analysis of variance for repeated measures followed by Dunnett’s test. (B) Effects of pretreatment with BQ-123 (an ETA antagonist), BQ-788 (an ETB antagonist) and BIM (a PKC inhibitor) on ET-1 (10 pmol)-induced thermal hyperalgesia in WT mice. Vehicle and inhibitors were s.c. administered 15 min before ET-1 injection. All drugs were administered in a volume of 10 ␮L. Data are presented as means⫾S.D., n⫽6 – 8 in each treatment group. * Statistical significance compared with the withdrawal latency at 0 min (basal withdrawal latency); one-way analysis of variance for repeated measures followed by Dunnett’s test. # Statistical significance compared with vehicle⫹ET-1; two-way analysis of variance for repeated measures followed by the Turkey-Kramer test.

the injected hind paw was rapidly removed and homogenized in the presence of 0.01 mM PBS containing protease inhibitor cocktail (Sigma) and Na3VO4 on ice. HEK293 cells transfected with TRPV1 cDNA or TRPV1 mutant cDNA and ETA cDNA were incubated with or without 1 ␮M PMA or 10 nM ET-1 in 35-mm dishes for 10 min at 37 °C, washed with PBS, resuspended with lysis buffer containing protease inhibitor cocktail and Na3VO4, and homogenized. The crude homogenates from HEK293 cells and skin were centrifuged at 15,000⫻g for 10 min at 4 °C. Supernatants of the homogenates were collected, and the protein concentration was determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin. Equal amounts of protein (0.2 mg) were loaded onto a 10 –20% polyacrylamide gel and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). Then the membranes were incubated overnight at 4 °C with anti-phospho TRPV1 antibody (1 ␮g/ml) or anti-TRPV1 antibody (1:100; Calbiochem) in PBS containing 10% skim milk. After several washes, the membranes were incubated with anti-rabbit IgG antibody conjugated with HRP in PBS containing 10% skim milk. Immunoreaction was visualized with an ECL plus chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA). The reaction product was visualized using an image analyzer (LAS-3000mini, Fuji Film, Tokyo, Japan). Quantification by densitometry of TRPV1 and pTRPV1 bands was done using Multi Gauge Ver2.2 (Fuji Film, Tokyo, Japan), and the results were presented as the band density ratio of pTRPV1/TRPV1 from four separate experiments.

Statistical analysis All results are expressed as means⫾S.D. An unpaired t-test and a one-way analysis of variance for repeated measures followed by Dunnett’s test were used for within-group comparison. A two-way analysis of variance for repeated measures followed by the Turkey-Kramer test was used for between-group comparison. The concentration of capsaicin required to produce a 50% (EC50) maximal effect was derived mathematically using nonlinear regression, and EC50 values were compared using an F test. A difference was accepted as significant if the probability was ⬍5% (P⬍0.05).

RESULTS Intraplantar ET-1-induced thermal hyperalgesia in WT mice Intraplantar ET-1 evoked thermal hyperalgesia as previously reported (Menendez et al., 2003). Although the vehicle did not affect the withdrawal latency, ET-1 (3, 10 and 30 pmol) decreased the withdrawal latencies in a dosedependent manner (P⬍0.05) (Fig. 1A, Fig. 2B). Peak effects were observed 10 –20 min after ET-1 injection, and the decreased withdrawal latencies returned to the basal withdrawal latencies within 60 min after injection. In order to clarify the involvement of receptor subtypes, the vehicle, ETA antagonist, BQ-123 (3 nmol) or ETB antagonist, BQ788 (10 nmol) was intraplantarly injected 15 min before ET-1 (10 pmol) injection (Fig. 1B). Pretreatment with BQ123 but not that with the vehicle completely inhibited ET-1 (10 pmol) -induced thermal hyperalgesia without changing the basal withdrawal latencies. On the other hand, BQ-788 enhanced the peak effect of ET-1. Since ETA is a Gq/11coupled metabotropic receptor, we next examined the contribution of PKC to ET-1-induced thermal hyperalgesia. Pretreatment with a PKC inhibitor, BIM (3 nmol), inhibited ET-1(10 pmol) -induced thermal hyperalgesia without changing the basal withdrawal latencies, indicating that ET-1-induced thermal hyperalgesia develops through the activation of PKC. Involvement of TRPV1 in ET-1-induced thermal hyperalgesia In order to determine the involvement of TRPV1 in ET-1induced thermal hyperalgesia, we performed behavioral analysis using TRPV1 KO mice. The basal withdrawal latencies were 9.7⫾0.4 and 9.8⫾0.6 s in WT mice and TRPV1 KO mice, respectively, showing no significant dif-

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Fig. 2. ET-1-induced thermal hyperalgesia in TRPV1 KO mice. (A) Comparison of time courses of ET-1 (10 pmol) -induced thermal hyperalgesia between KO mice and WT mice. ET-1 was s.c. administered in a volume of 10 ␮L. Data are presented as means⫾S.D., n⫽6 – 8 in each treatment group. * Statistical significance compared with the withdrawal latency at 0 min (basal withdrawal latency); one-way analysis of variance for repeated measures followed by Dunnett’s test. (B) Dose-response curve of ET-1-induced thermal hyperalgesia in TRPV1 KO mice and WT mice. Data are presented as means⫾S.D., n⫽6 – 8 in each treatment group. # Statistical significance between WT mice and TRPV1 KO mice; two-way analysis of variance for repeated measures. (C) Effects of pretreatment with BQ-123 (an ETA antagonist) and BIM (a PKC inhibitor) on ET-1 (30 pmol)-induced thermal hyperalgesia in TRPV1 KO mice. Vehicle and inhibitors were s.c. administered 15 min before ET-1 injection. All drugs were administered in a volume of 10 ␮L. Data are presented as means⫾S.D., n⫽6 – 8 in each treatment group. * Statistical significance compared with the withdrawal latency at 0 min (basal withdrawal latency); one-way analysis of variance for repeated measures followed by Dunnett’s test. KO mice⫽TRPV1-deficient mice.

ference. ET-1 (10 pmol) -induced thermal hyperalgesia was significantly attenuated in TRPV1 KO mice compared with that in WT mice (Fig. 2A, P⫽0.0105). A clear rightward shifting in the dose-response curve was observed for TRPV1 KO mice (Fig. 2B). Thus, TRPV1 is involved in ET-1-induced thermal hyperalgesia. Although ET-1-induced thermal hyperalgesia was attenuated in TRPV1 KO mice, 30 nmol of ET-1 produced apparent thermal hyperalgesia that was less pronounced compared with that in WT mice (Fig. 2B). Next, we examined the involvement of ETA and PKC in thermal hyperalgesia observed in TRPV1 KO mice. Pretreatment with either BQ-123 (3 nmol) or BIM (3 nmol), but not that with the vehicle, completely inhibited ET-1induced thermal hyperalgesia without changing the basal withdrawal latencies. These results indicate that not only TRPV1 but also other molecules contribute to high-dose ET-1-induced thermal hyperalgesia in an ETA/PKC-dependent manner.

Phosphorylation of TRPV1 in WT mice Because PKC phosphorylates TRPV1 and potentiates or sensitizes TRPV1 activity (Tominaga et al., 2001; Moriyama et al., 2003), we examined whether intraplantar ET-1 (10 pmol) can modify phosphorylation of TRPV1 in the injected skin, using an antibody specific for phosphorylated TRPV1 at S800, a PKC-specific TRPV1 phosphorylation site. Although the level of phosphorylation of TRPV1 protein was low in the lysate from the untreated skin of WT mice, the bands became denser upon ET-1 treatment but not upon vehicle treatment (Fig. 3A and 3B). These results indicate that intraplantar ET-1 induced the phosphorylation of TRPV1. ET-1 evoked current responses through the activation of TRPV1 in HEK293 cells expressing TRPV1 and ETA The results of the behavioral study suggest that ET-1 activates or sensitizes TRPV1 through the activation of

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Fig. 3. Western blot analysis of phosphorylated TRPV1 at S800 (pTRPV1) in the skin with ET-1 injection of WT mice using anti-phosphoTRPV1S800 antibody. (A) Upper and lower panels are representative gel traces of pTRPV1 and TRPV1, respectively. (B) Statistical summary of densitometric analysis of Western blot analysis. Data are presented as the band density ratio of pTRPV1/TRPV1. Data are presented as means⫾S.D., n⫽6 in each treatment group. * Statistical significance compared with no treatment; one-way analysis of variance for repeated measures followed by Dunnett’s test. KO mice⫽TRPV1-deficient mice.

PKC, resulting in thermal hyperalgesia. Next, we investigated the functional interaction of TRPV1 with ET-1 using HEK293 cells expressing both TRPV1 and ETA. In voltageclamp experiments, 30 nM or 100 nM of ET-1 alone evoked inward current responses, which lasted for a long time even after the washout of ET-1, in HEK293 cells expressing TRPV1 and ETA (Fig. 4). ET-1 (30 and 100 nM) -induced currents were not observed in HEK293 cells ex-

Fig. 4. ET-1-induced currents in HEK293 cells expressing TRPV1 and ETA. Cells were perfused for 100 s with a solution containing ET-1. Holding potential⫽⫺60 mV.

pressing ETA alone (data not shown). This indicates that 100 nM of ET-1 evoked current responses through the activation of TRPV1. On the other hand, 10 nM of ET-1 alone did not evoke obvious current responses (Fig. 4). To simply examine the interaction of ET-1 and TRPV1 activity, we used 10 nM of ET-1 in the following experiments. ET-1 potentiated TRPV1 response to capsaicin through PKC-dependent phosphorylation in HEK293 cells expressing TRPV1 and ETA Ten nanomoles of capsaicin evoked small inward currents in HEK293 cells expressing TRPV1 (Fig. 5A). In the absence of extracellular Ca2⫹, no change was observed in the magnitude of responses evoked by repetitive applications of capsaicin (Fig. 5C, Cont). After pretreatment with 10 nM of ET-1 for 100 s, the same dose of capsaicin produced much larger current responses (4.96⫾1.09-fold, n⫽8 for ET-1; 1.02⫾0.08-fold, n⫽7 for control; P⬍0.05)

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Fig. 5. ET-1 potentiates or sensitizes capsaicin-activated currents in a PKC-dependent manner in HEK293 cells and phosphorylates TRPV1. (A) A representative trace of increase in capsaicin (10 nM) -activated current in transfected HEK293 cells expressing TRPV1 and ETA. Cells were perfused for 100 s with a solution containing ET-1 (10 nM) before capsaicin application (10 nM, 10 s). Holding potential⫽⫺60 mV. (B) Capsaicin dose-response curve for TRPV1 activation in the absence (closed circles) and presence (open circles) of 10 nM ET-1. Currents were normalized to the currents maximally activated by 1 ␮M capsaicin in the absence of ET-1. Values were obtained from 22 different cells. (C) PKC-dependent pathway is involved in ET-1-induced potentiation of capsaicin-activated currents. Currents were normalized to values first induced by capsaicin application in the absence of ET-1. In some experiments, CalpC at 1 ␮M or BIM at 1 ␮M was included in the pipette solution. Cont, preperfusion with bath solution without ET-1 before reapplication of capsaicin; ET-1, preperfusion with bath solution with ET-1 (10 nM, 10 min); wt, WT TRPV1; S502A/S800A, cells expressing a TRPV1 mutant lacking sites for PKC-dependent phosphorylation. Number in parenthesis indicates the number of cells tested. *, # Statistical significance compared with Cont and ET-1, respectively; one-way analysis of variance for repeated measures followed by the Turkey-Kramer test. (D) Western blot analysis of phosphorylated TRPV1 at S800 (pTRPV1) in HEK293 cells using anti-phosphoTRPV1S800 antibody. Upper, middle and lower panels are representative bands of pTRPV1 at S800, TRPV1 and actin, respectively. Arrows indicate the expected protein bands. NT, no treatment; PMA, PMA (1 ␮M, 10 min) stimulation; ET-1, ET-1 (10 nM, 10 min) stimulation; wt, WT TRPV1; S502A/S800A, cells expressing a TRPV1 mutant lacking sites for PKC-dependent phosphorylation. (E) Statistical summary of densitometric analysis of Western blot analysis. Data are presented as the band density ratio of pTRPV1/TRPV1 from four separate experiments. ET-1, ET-1 (10 nM, 10 min) stimulation. * Statistical significance compared with vehicle; unpaired t-test.

(Fig. 5A and 5C). ET-1-induced sensitization of TRPV1 was not observed in HEK293 cells transfected with only TRPV1 cDNA. Lack of sensitization by ET-1 in HEK293 cells transfected with only TRPV1 cDNA was observed even when high concentrations of ET-1 (30 and 100 nM) were used (data not shown). When TRPV1 currents were measured in single cells by serially applying a range of concentrations of capsaicin in the absence or presence of

ET-1, the dose-response curves clearly demonstrated that ET-1 significantly enhanced capsaicin action on TRPV1 by lowering EC50 values (EC50 from 420 nM to 150 nM for capsaicin-activated currents; P⬍0.05) without altering maximal responses (normalized to the maximal current produced by the application of 1 ␮M of capsaicin without ET-1) (Fig. 5B). We previously reported that inhibition of PKC activity suppressed the enhancing effect of ET-1 on

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capsaicin-evoked intracellular Ca2⫹ elevation in DRG neurons (Yamamoto et al., 2006). Therefore, next, the involvement of PKC was examined. When either CalpC (1 ␮M) or BIM (1 ␮M), highly potent and specific PKC inhibitors, was included in the pipette solution, the sensitizing effect of ET-1 on capsaicin-activated currents was almost completely inhibited (BIM, 0.92⫾0.05-fold; n⫽8; P⬍0.05 vs. ET-1; Cal C, 0.98⫾0.07-fold; n⫽7; P⬍0.05 vs. ET-1) (Fig. 5C). To further confirm the involvement of PKC-dependent phosphorylation, the effect of ET-1 was examined using cells expressing a TRPV1 mutant, S502A/S800A, that is insensitive to PKC-dependent phosphorylation. No potentiation of capsaicin-activated currents was observed in ET-1-pretreated cells expressing S502A/S800A (1.04⫾ 0.06-fold; n⫽7; P⬍0.05 vs. ET-1) (Fig. 5C), further indicating the involvement of PKC-dependent phosphorylation. In addition, we examined using an antibody specific for phosphorylated TRPV1 at S800 whether ET-1-evoked ETA activation can modify phosphorylation of TRPV1. Although phosphorylation of TRPV1 protein was either undetectable or present at a low level in the lysate from the untreated cells, the bands became denser upon PMA (1 ␮M) treatment, whereas no such bands were detected in the lysate from cells expressing a TRPV1 mutant (S800A/S502A) lacking the substrate for PKC-dependent phosphorylation (Fig. 5D), indicating that the antibody we used in this study detects the phosphorylation of TRPV1 at S800 by PKC. ET-1 treatment (10 nM, 10 min) evoked phosphorylation of

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TRPV1 (Fig. 5D, 5E), indicating direct phosphorylation of TRPV1 by ETA activation. These findings suggest that ET-1-evoked sensitization of TRPV1 responsiveness develops through the activation of PKC. ET-1 lowered the temperature threshold for TRPV1 activation in HEK293 cells expressing TRPV1 and ETA Potentiating effects of ET-1 on heat-evoked responses were also examined. For this analysis, heat-evoked current responses were compared between different cells rather than within the same cell, because repetitive heatevoked currents show significant desensitization even in the absence of extracellular Ca2⫹ (Tominaga et al., 1998). Without ET-1 pretreatment, heat-evoked currents in HEK293 cells expressing TRPV1 and ETA developed at 42.0⫾0.42 °C (n⫽13) with an extremely steep temperature dependence (Fig. 6A). On the other hand, pretreatment with 10 nM ET-1 significantly lowered the threshold temperature for TRPV1 activation to 32.6⫾0.63 °C (n⫽9; P⬍0.05 vs. ET-1) (Fig. 6B and 6C). The effects of ET-1 on the threshold temperature for TRPV1 activation were also completely inhibited (41.3⫾0.72 °C, n⫽6; P⬍0.05 vs. ET-1) when BIM was included in the pipette solution (Fig. 6C). Thus, these data clearly show that the threshold temperature for TRPV1 activation is lowered by extracellular ET-1.

Fig. 6. ET-1 reduces temperature threshold for TRPV1 activation in the presence of ET-1 in HEK293 cells. (A, B) Representative temperatureresponse profiles of heat-activated currents in the absence (A) and presence (B) of ET-1 (10 nM). Dashed arrows show the threshold temperature for heat activation of TRPV1. Holding potential⫽⫺60 mV. (C) PKC-dependent pathway is involved in ET-1-induced reduction of the threshold temperature for TRPV1 activation. In some experiments, BIM at 1 ␮M was included in the pipette solution. Cont, perfusion with bath solution without ET-1; ET-1, perfusion with bath solution with ET-1. Number in parentheses indicates the number of cells tested. *, # Statistical significance compared with Cont and ET-1, respectively; one-way analysis of variance for repeated measures followed by the Turkey-Kramer test. Threshold was defined as the temperature at which a clear current increase was observed in the temperature-response profile.

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DISCUSSION Our major finding in the behavioral study was that the activation of PKC and TRPV1 as well as that of ETA contributed to ET-1-induced thermal hyperalgesia. In addition, using a specific antibody, we obtained in vivo and in vitro evidence that ET-1 phosphorylated TRPV1. In an electrophysiological study, ET-1 enhanced TRPV1 responsiveness to capsaicin and decreased the temperature threshold of TRPV1 through PKC activation. Although a recent study using HEK293 cells expressing ETA and TRPV1 or TRPV1 S800A showed that ET-1 enhanced TRPV1 responsiveness to capsaicin, proton and heat (Plant et al., 2007), our electrophysiological study provided additional information on the dose-response relationship for the interaction of ET-1 and TRPV1, and the change in the threshold temperature for TRPV1 activation, the effect of TRPV1 mutant S502A/S800A. It is important to clarify the effect of TRPV1 mutant S502A/S800A because not only S800 but also S502 is a substrate for PKC-dependent phosphorylation of TRPV1 (Numazaki et al., 2002). The results of our study indicate that the sensitization of TRPV1 activity through an ETA–PKC pathway contributes to ET1-induced thermal hyperalgesia. Involvement of TRPV1 and PKC in ET-1-induced thermal hyperalgesia Although previous studies showed that ETA activation was involved in ET-1-induced thermal hyperalgesia (Menendez et al., 2003), we found that not only ETA activation but also PKC and TRPV1 activations were involved in ET-1-induced thermal hyperalgesia. Since ETA is a Gq/11-coupled metabotropic receptor, increased activity of ETA can activate PKC. Accordingly, inhibition of PKC activity reversed ET-1-induced thermal hyperalgesia in our study. Intraplantar PMA, an activator of PKC, produced thermal hyperalgesia that was markedly reduced in TRPV1 KO mice compared with that in WT mice (Bölcskei et al., 2005), indicating that TRPV1 activation is involved in PKC-induced thermal hyperalgesia. Activation of PKC (especially PKC␧) can induce phosphorylation of TRPV1, resulting in sensitization or potentiation of TRPV1 activity (Numazaki et al., 2002; Mandadi et al., 2006). Therefore, we examined the contribution of TRPV1 to ET-1-induced thermal hyperalgesia. As expected, ET-1 induced PKC-sensitive phosphorylation of TRPV1 expressed within the skin, and ET-1induced thermal hyperalgesia was attenuated in TRPV1 KO mice compared with that in WT mice. This is the first evidence for TRPV1 phosphorylation in vivo, although further study is necessary in order to clarify the localization of phosphorylated TRPV1 because TRPV1 is expressed in not only neurons and but also keratinocytes (Ständer et al., 2004). Thermal hyperalgesia was attenuated but was not completely inhibited in TRPV1 KO mice when a higher dose of ET-1 was administered. The residual thermal hyperalgesia observed in TRPV1 KO mice was completely inhibited by ETA or PKC inhibition. These results indicate that not only TRPV1 but also other molecules were required for ET1-induced thermal hyperalgesia when higher doses of ET-1

were administered. A previous study showed that ET-1 induced hyperpolarizing shifts in the voltage-dependent activation of tetrodotoxin-resistant (TTX-R) sodium channels through the activation of ETA in cultured DRG neurons, indicating a possible mechanism for ET-1-induced excitation of nociceptors (Zhou et al., 2002). In addition, the activation of TTX-R sodium channels is enhanced in a PKC-dependent manner (Ikeda et al., 2005; Baker, 2005; Khasar et al., 1999). Therefore, TTX-R sodium channels may also be involved in high-dose ET-1-induced thermal hyperalgesia. The inhibition of ETA activity completely reversed ET1-induced thermal hyperalgesia in WT mice, while ETB inhibition enhanced it. ET-1 acts on two receptor subtypes, ETA and ETB. It has been reported that ETB is expressed in keratinocytes and that the activation of ETB results in anti-nociceptive effects through opioid release from keratinocytes (Khodorova et al., 2003). Most capsaicin-sensitive DRG neurons are mu opioid receptor (MOR) –positive (Rau et al., 2005; Yamamoto et al., 2008), and more than 80% of cultured DRG cells are both MOR- and TRPV1immunoreactive (Vetter et al., 2006). Accordingly, the primary neurons expressing TRPV1 can receive not only excitatory but also indirect inhibitory control by ET-1. Thus, the balance between ETA and ETB activations would control pain at the tissue level. Indeed, inhibition of ETB enhanced ET-1-induced pain-like behavior (Piovezan et al., 2000; Khodorova et al., 2003). On the other hand, it has been reported that the development of mechanical hyperalgesia is associated with reduction of ETB expression in peripheral nerves in a rat model of chronic diabetes (BertiMattera et al., 2006). In addition, ET-1-induced mechanical hyperalgesia is reversed by ETB antagonism (Menendez et al., 2003; Baamonde et al., 2004). Thus, effects of ETB activation on nociceptive processing appear to be complex. Functional interaction of TRPV1 with ET-1 in HEK293 cells expressing both TRPV1 and ETA In the present study, TRPV1 currents evoked by capsaicin were potentiated or sensitized by ET-1 in transfected HEK293 cells, although our data were not obtained under normal physiological conditions because we used calciumfree solution in order to avoid desensitization of TRPV1. Although activation of ETA by ET-1 results in intracellular Ca2⫹ mobilization (Yamamoto et al., 2006), it is not a likely mechanism for the capsaicin-evoked current increase observed in our experiments, because cytosolic-free Ca2⫹ is buffered with the 5 mM of EGTA included in the pipette solution. Activation of ETA by ET-1 evoked phosphorylation of TRPV1 protein. Lack of potentiating effects of ET-1 in cells treated with PKC inhibitors and in cells expressing a TRPV1 mutant lacking substrates for PKC-dependent phosphorylation indicates that a PKC-dependent pathway is predominantly involved in ET-1-mediated TRPV1 sensitization. We examined the effects of ET-1 on the temperature threshold for TRPV1 activation in order to further understand the contribution of TRPV1 to ET-1-induced thermal hyperalgesia. Our results showed that the thresh-

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old temperature for TRPV1 activation was lowered by extracellular ET-1 in a PKC-dependent manner. In addition, higher concentrations of ET-1 (30 and 100 nM) alone evoked current responses in HEK293 cells expressing TRPV1 and ETA at room temperature (22–25 °C). ET-1 (30 and 100 nM) -induced current was not observed in HEK293 cells expressing ETA alone. Therefore, higher concentrations of ET-1 might lower the threshold temperature for TRPV1 activation to room temperature, evoking TRPV1-dependent currents. We did not examine whether ET-1-induced sensitization of TRPV1 observed in transfected HEK293 cells occurred in sensory neurons. However, we previously showed that ET-1 enhanced an increase in capsaicinevoked [Ca2⫹]i through ETA receptors in a PKC-dependent manner. ET-1 enhanced an increase in capsaicin-evoked [Ca2⫹]i through ETA in a PKC-dependent manner. ET-1induced potentiation of capsaicin response was observed in 45.5% of the neurons that responded to 10 nM capsaicin (Yamamoto et al., 2006), indicating colocalization of TRPV1 and ETA. In addition, a recent immunohistochemical study showed the colocalization of TRPV1 and ETA in DRG neurons (Plant et al., 2006). Therefore, taken together with our electrophysiological results, theses findings suggest that ET-1 sensitizes TRPV1 in peripheral terminals through activation of ETAand PKC. Thus, neuronal interaction of ET-1 and TRPV1 through an ETA/PKC pathway plays an important role in ET-1-induced thermal hyperalgesia.

CONCLUSION Our results indicate that the sensitization of TRPV1 activity through an ETA–PKC pathway contributes to ET-1-induced thermal hyperalgesia. Therefore, a strategy to target TRPV1 may be more effective to reduce pain associated with ET-1. Acknowledgments—T.K. and J.Y. contributed equally to this work. Support was provided by a Grant-in-Aid for Scientific Research (19659403 and 19791076) to T. Kawamata and Y. Niiyama, respectively.

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(Accepted 7 April 2008) (Available online 12 April 2008)