- Email: [email protected]
Inhibition of ERK phosphorylation by substance P N-terminal fragment decreases capsaicin-induced nociceptive response
Neuropharmacology 61 (2011) 608e613
Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Inhibition of ERK phosphorylation by substance P N-terminal fragment decreases capsaicin-induced nociceptive response Takaaki Komatsu a, Hirokazu Mizoguchi b, Mika Sasaki a, Chikai Sakurada c, Minoru Tsuzuki c, Shinobu Sakurada b, **, Tsukasa Sakurada a, * a b c
First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Department of Biochemistry, Nihon Pharmaceutical University, 10281 Komuro Ina-Machi Kitaadachi-gun, Saitama 362-0806, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2010 Received in revised form 9 April 2011 Accepted 20 April 2011
Previous research has demonstrated that substance P N-terminal fragments produced by the action of several different enzymes in the spinal cord could reduce nociception when injected intrathecally (i.t.) into mice. The present study examined the possible involvement of spinal extracellular signal-regulated protein kinase (ERK), a mitogen-activated protein kinase (MAPK), in i.t. substance P (1e7)-induced antinociception as assayed by the capsaicin test. The i.t. injection of substance P (1e7) (20e80 nmol) into mice resulted in a dose-dependent attenuation of paw-licking/biting behavior induced by intraplantar injection of capsaicin, which was reversed by co-injection of [D-Pro2, D-Phe7]substance P (1e7), a D-isomer and antagonist of substance P (1e7). In Western blot analysis, intraplantar injection of capsaicin (400 and 1600 ng/paw) produced an increase of ERK phosphorylation in the dorsal spinal cord, whereas expression of p38 and c-Jun N-terminal kinase (JNK) phosphorylation was unchanged by capsaicin treatment. In parallel to the behavioral results, i.t. substance P (1e7) inhibited capsaicin-induced EKR phosphorylation, which was reversed by [D-Pro2, D-Phe7]substance P (1e7), a substance P (1e7) antagonist. Both nociceptive behavioral response and spinal ERK activation induced by intraplantar capsaicin were reduced by U0126, an upstream inhibitor of ERK phosphorylation. Taken together, these findings suggest that the activation of ERK, but not p38 and JNK MAPKs in the spinal cord, contributes to intraplantar capsaicininduced nociception, and that blocking ERK activation via substance P (1e7) binding sites may provide significant antinociception at the spinal cord level. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: N-Terminal fragments of substance P Capsaicin-induced nociceptive response Spinal antinociception Intrathecal injection Extracellular signal-regulated protein kinase (ERK)
1. Introduction Numerous studies have documented the presence of substance P in sensory afferent fibers in the dorsal root ganglia and the dorsal horn of the spinal cord (Pernow, 1983). Substance P is released from the spinal cord following various nociceptive stimuli (Duggan et al., 1987; Kantner et al., 1985; Schaible et al., 1990) and administrations of capsaicin through peripheral and intrathecal (i.t.) routes (Gamse et al., 1979; Go and Yaksh, 1987). Following release and binding to neurokinin1 (NK1) receptor, the biological action of substance P is terminated by enzymatic degradation (Lee et al., 1981; Matsas et al., 1984; Nyberg and Terenius, 1991). Substance P can be cleaved by several degrading enzymes such as neutral endopeptidase-24.11 * Corresponding author. Tel.: þ81 925410161; fax: þ81 925535698. ** Corresponding author. E-mail addresses: [email protected] (S. Sakurada), [email protected] (T. Sakurada). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.04.035
(EC 3.4. 24.11.: enkephalinase). Some of the substance P fragments generated by enzymatic hydrolysis are present in significant concentrations in the central nervous system (CNS), particularly in the dorsal part of spinal cord (Sakurada et al., 1985, 1991). Of several substance P fragments, substance P (1e7) was chosen for the present study, because the N-terminal peptide is the major fragment in the CNS. Substance P (1e7) is able to produce behavioral effects which are different from those produced by the C-terminal fragments and the parent compounds (Hall and Stewart, 1983). In several nociceptive assays, substance P (1e7) has been found to be antinociceptive in mice including the formalin (Goettl and Larson, 1996) and capsaicin (Komatsu et al., 2009) tests. We have recently reported that the elevated concentrations of glutamate and nitric oxide metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine were inhibited by i.t. pretreatment with substance P (1e7) (Sakurada et al., 2007). Specific binding sites for substance P (1e7) have been identified in the mouse brain and spinal cord, which are distinct from neurokinin-1 receptors (Igwe et al., 1990).
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
In the current studies there are interesting data, suggesting that substance P (1e7) binding sites possess high affinity for endomorphin-2, which are distinct from the m-opioid receptor in the spinal cord and the ventral tegmental area of rats (Botros et al., 2006, 2008). The mitogen-activated protein kinase (MAP) family including extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in the spinal transmission of pain tranduces extracellular stimulation into intracellular post-translational and transcriptional responses (Lewis et al., 1998). It is now established that ERK signaling pathway contributes to nociceptive information and central sensitization produced by noxious stimuli (Ciruela et al., 2003; Galan et al., 2003; Ji et al., 1999, 2002 Wang et al., 2004; Zhuang et al., 2005). The ERKs are expressed both in primary afferents as well as in glial cells within the dorsal spinal cord (Zhuang et al., 2005; Katsura et al., 2006; Zhao et al., 2007; Tsuda et al., 2008). Phosphorylation of spinal ERK has been reported by acute noxious stimuli of formalin and capsacin (Thomas and Hunt, 1993; Ji et al., 1999; Karim et al., 2001; Galan et al., 2002). Capsaicin activates C-fibers via the transient receptor potential vanilloid 1 (TRPV1) ion channel (formerly the VR1 receptor) (Caterina and Julius, 2001), and induces phosphorylated ERK expression (Ji et al., 1999; Karim et al., 2001) to produce primary and secondary hyperalgesia in animals (Reeh et al., 1986; Simone et al., 1991) and human (Koltzenburg et al., 1992; LaMotte et al., 1992). Spinal ERK is activated by not only shortlived pain, but also persistent inflammatory hyperalgesia induced by carrageenan and Freund’s adjuvant (Galan et al., 2002; Ji et al., 2002). In addition, inhibition of ERK phosphorylation has been found to be prevented or reduced in numerous pain models (Ji et al., 1999; Dai et al., 2002; Galan et al., 2003). Recently, we reported that phosphorylation of ERK in the dorsal spinal cord occurred in response to nociceptive behavioral responses following i.t. injection of high-dose morphine or morphine-3-glucuronide, a major metabolite of morphine (Komatsu et al., 2007, 2009). However, except for the specific inhibitor of ERK phosphorylation, the contribution of the ERK cascade to the antinociceptive changes produced by compounds remains to be elucidated. The present study was performed in order to examine the possible involvement of the MAP family including p38 and JNK as well as ERK in the spinal cord when capsaicin was injected into the hindpaw. Our findings support the hypothesis that spinal ERK signaling contributes to the pain-related behavioral response induced by intraplantar capsaicin injection. In addition, we have shown that substance P (1e7)-induced antinociception could be attributed to the inhibition of ERK phosphorylation in the spinal cord. 2. Materials and methods 2.1. Animals Male ddY mice (Shizuoka Laboratory Center, Japan) weighing 22e26 g were used in this study. All protocols were approved by the Committee of Animal Experiments in Daiichi College of Pharmaceutical Sciences and Tohoku Pharmaceutical University. These experiments conformed to the ethical guidelines of the International Association for the Study of Pain in conscious animals published (Zimmermann, 1983). The animals were housed under conditions of a 12 h lighte dark cycle, a constant temperature of 22e24 C and 55e60% relative humidity. Animals had free access to food (Clea Japan, Osaka, Japan) and drinking before the experiments. 2.2. Intrathecal injection The i.t. injection procedure was adapted from the method of Hylden and Wilcoxon (1980). Briefly, the lumbar puncture was performed using a 28 gauge needle attached to a 50-ml Hamilton microsyringe. The needle was inserted between L5 and L6, and drugs were delivered in a volume of 5 ml in conscious mice. The mice were not anesthetized during these procedures. Puncture of the dura was behaviorally indicated by a slight flick of the tail.
609
2.3. Assessment of behavioral response The capsaicin test was performed as described in detail previously (Sakurada et al., 1992). To reduce variability each mouse was acclimatized to an acrylic observation chamber (22.0 15.0 12.5 cm) for approximately 1 h before the injection of capsaicin. The mouse was injected 20 ml of a solution of capsaicin (400 ng and 1600 ng/paw) beneath the skin the plantar surface of the right hindpaw using a microsyringe with a 26-gauge needle. Licking/biting behavior induced by intraplantar injection of capsaicin was observed as an indicator of nociceptive response. The accumulated response time (in second) spent in licking/biting the capsaicin-injected paw was measured for a period of 5 min immediately after subcutaneous (s.c.) injection of capsaicin. In the paw-withdrawal test, the latencies were recorded as the time from the start of the radiant heat stimulus to withdrawal of the hindpaw. A radiant heat source was placed under one of the hindpaws, and the time to withdrawal of the stimulated paw was measured. The intensity was set so that the reaction time to paw-withdrawal under control conditions was 10e12 s. Control reaction time was determined by a total of two consecutive measurements each separated by 10 min. 2.4. Western blotting analysis To examine the spinal expression of ERK, p38 and JNK, the mice were decapitated and the whole spinal cord was taken by pressure expulsion with physiological saline. The dorsal and ventral parts of lumbar spinal cord were dissected on an icecooled glass dish. Tissue samples were homogenized in 0.1 ml of lysis buffer reagent (150 mM NaCl, 1.0% NP-40, 50 mM TriseHCl pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mM sodium vanadate and 1 mM EDTA pH 8.0) and centrifuged at 16,000 g for 30 min at 4 C. Supernatants were collected and total protein amounts were measured using the Protein Assay (BIO-RAD, Hercules, CA). An equal volume of 2 sample buffer (100 mM TriseHCl pH 6.8, 2.5% SDS, 20% glycerol, 0.006% bromophenol blue and 10% b-mercaptoethanol) was added to 30 mg of totaprotein. The samples were boiled, and then electrophoresed in a 10e15% SDS-polyacrylamide gel (BIO-RAD, Hercules, CA) and transferred to a Hybond-P membrane (Amersham Bioscience). The blotted membrane was then incubated overnight with 5% skim milk (Wako Pure Chemical Industries Ltd, Osaka, Japan) in T-PBS (PBS containing 0.1% v/v Tween 20). All antibody applications were done in T-PBS. After the membranes were washed with T-PBS, the membranes were incubated with phospho-p44/42 MAP kinase antibody (1:1000), p44/42 MAP kinase antibody (1: 1000), phospho-p38 MAP kinase antibody (1:1000) and phospho-SNAP/JNK antibody (1:1000) (Cell Signaling) for 2 h at room temperature. The membranes were extensively washed with T-PBS and incubated for 2 h with the secondary antibody (anti-rabbit IgG peroxidase-conjugated antibody 1:5000) (Amersham Bioscience). After washing, the proteins were detected using the ECL-Plus Western blotting detection system (Amersham Biosciences) and visualized with the Dolphine-Chemi Image System (Wealtec). MagicMark western protein standard (Invitrogen) was simultaneously resolved on the gel, and the molecular weight of protein was estimated. 2.5. Drugs Substance P (1e7) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). [D-Pro2, D-Phe7]substance P (1e7) was synthetized by conventional solid phase methods in our laboratory (Sakurada et al., 1991). For i.t. injections, substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7) were dissolved in sterile artificial cerebrospinal fluid (CSF) containing 126.6 mM NaCl, 10.0 mM NaHCO3, 2.5 mM KCl, 2.0 mM MgCl2, 1.3 mM CaCl2 and 1.0 mM glucose. Capsaicin (Sigma, St. Louis, MO) was initially dissolved in 100% dimethylsulfoxide (DMSO) at the concentration of 12.8 mg/20 ml as stock solution, further diluted to 400 ng/20 ml and 1600 ng/20 ml by physiological saline (0.9% wt/vol). The final concentration of DMSO for 400 ng/20 ml and 1600 ng/20 ml of capsaicin was 3.125% and 12.5%, respectively. These concentrations of DMSO were used as vehicle controls for capsaicin-injected groups. Injections of capsaicin into the hindpaw were made with a 26-gauge needle attached to a 50 ml Hamilton microsyringe. Substance P (1e7) was injected i.t. 3 min prior to intraplantar capsaicin. In the experiment with i.t. combined administration, capsaicin was injected in a volume of 5 ml into the hindpaw 3 min after i.t. co-administration of substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7). The MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4bis(2-amino-phenylthio)butadiene (U0126) was obtained from Calbiochem, Darmstadt, Germany. U0126 was initially dissolved in 100% DMSO at the concentration of 26.28 mM as stock solution, further diluted to 0.5, 1.25 or 2.5 nmol/5 ml by artificial CSF, and adjusted to 6.71% DMSO as the final concentration. To study the importance of ERK phosphorylation in capsaicin-induced nociception, U0126, an ERK upstream kinase, was injected i.t. 5 min before intraplantar injection of capsaicin. The 5 ml of 6.71% DMSO used as vehicle control for i.t. injection gave no significant effect on capsaicin-induced licking/biting response when compared to artificial CSF controls. 2.6. Analyses of data All values are expressed as means S.E.M. Statistical differences between groups were established using Dunnett’s test for multiple comparisons after analysis
610
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
of variance (ANOVA). The 5% level of statistical significance was set in all experiments.
3. Results 3.1. Intraplantar capsaicin evokes licking/biting behavioral response and hyperalgesia An injection of capsaicin (400 and 1600 ng/paw) into the plantar surface of the hindpaw evoked a spontaneous and severe licking/ biting response toward the injected paw in mice (Fig. 1A). The nociceptive response started immediately after intraplantar injection of capsaicin, reached a peak and gradually subsided to baseline within approximately 5 min. The behavioral characterization of intraplantar capsaicin confirms previously reported data (Sakurada et al., 2003). The 6.25 ng dose capsaicin produced a decrease in reaction time to paw-withdrawal from the radiant heat stimulus 15 min after intraplantar injection (Fig. 1B). A similar decrease in reaction time with response to the magnitude of the effect of capsaicin (6.25 ng) was observed 5 min post-injection, and the reaction time returned to pre-injection values at 20 and 30 min (data not shown). A higher dose (12.5 ng) of capsaicin produced a slight but significant change in withdrawal latency, whereas 25 and 100 ng capsaicin did not alter withdrawal latency. 3.2. Phosphorylation of ERK, p38 and JNK in the spinal cord by intraplantar capsaicin To investigate whether spinal ERK1 and/or ERK2, p38 and JNK are activated by intraplantar capsaicin (400 and 1600 ng/paw), we
compared the effect of capsaicin with that of vehicle control in the lumbar dorsal spinal cord extracted 5 min after intraplantar capsaicin. Western blot analysis revealed an activation of ERK in the dorsal spinal cord after intraplantar capsaicin (Fig. 1B). Both phosphorylated ERK1 (P44 MAPK) and phosphorylated ERK2 (P42 MAPK) levels were significantly increased by 1600 ng capsaicin as compared to vehicle-treated control. A lower dose of capsaicin (400 ng) induced a significant activation of ERK2 without affecting ERK1 level. In contrast to the results of ERK1 and ERK2, intraplantar capsaicin (400 and 1600 ng) did not increase phosphorylation of p38 and JNK MAPKs in the lumbar spinal cord 5 min post-injection (Fig. 2). To examine the specificity of ERK1 and ERK2 activation by intraplantar capsaicin (1600 ng), the upstream inhibitor of ERK phosphorylation, U0126 was used. In the behavioral test, i.t. U0126 (1.25e5.0 nmol, 5 min pre-capsaicin) significantly inhibited nociceptive behavioral response after intraplantar capsaicin (Fig. 3A). The results of western blot study showed that i.t. injection of U0126 at a dose of 2.5 nmol suppressed the increase of phosphorylated ERK in the dorsal spinal cord detectable at 5 min after capsaicin injection (Fig. 3B). These results further confirm that activation of the ERK signaling pathway mediates capsaicin-induced nociceptive response. 3.3. Substance P (1e7) inhibited nociceptive response and spinal ERK activation after intraplantar capsaicin Substance P (1e7) (20e80 pmol), injected i.t. 3 min prior to capsaicin, produced a dose-dependent inhibition of capsaicininduced paw-licking/biting (Fig. 4A). The inhibitory effect of substance P (1e7) (80 pmol) was reversed significantly by i.t. co-injection of [D-Pro2, D-Phe7]substance P (1e7) (8.0 nmol), a substance P (1e7) antagonist. In accordance with the behavioral results, western blot studies showed that i.t. substance P (1e7) at a dose of 80 pmol significantly suppressed the increase of phosphorylated ERKs when compared with the artificial CSF-treated controls (Fig. 4B). The inhibition of phosphorylated ERKs by substance P (1e7) was revsered significantly by i.t. [D-Pro2, D-Phe7] substance P (1e7) (8.0 nmol). Thus our results indicate that spinal ERK activation induced by intraplantar capsaicin could be mediated through substance P (1e7) binding sites in the synapse of the dorsal spinal cord. 4. Discussion
Fig. 1. Behavioral effect after intraplantar injection of capsaicin in mice. (A) The duration of licking/biting response was determined using the 5-min period starting immediately after injection of capsaicin (400 and 1600 ng/paw) into the right hindpaw. The data are given as the mean SEM for groups of 10 mice. **P < 0.01 when compared to corresponding controls (3.1% and 12.5% DMSO). (B) Effect of intraplantar capsaicin on reaction time in the paw-withdrawal test. Paw-withdrawal latencies were measured 15 min after injection of capsaicin (4.68e100 ng). The data are given as the mean SEM for groups of 10 mice. **P < 0.01 when compared to DMSO control.
In the present study, we have shown that i.t. injection of substance P (1e7) attenuated phosphorylation of spinal ERK following intraplantar injection of capsaicin. At the behavioral level, spontaneous pain-related response induced by intraplantar injection of capsaicin was reduced dose-dependently by i.t. substance P (1e7). Our data further showed that the substance P (1e7) antagonist [D-Pro2, D-Phe7]substance P (1e7), reversed the reduced actions of substance P (1e7) on nociceptive behavioral response and spinal ERK activation induced by intraplantar capsaicin. Functionally, i.t. U0126, an inhibitor of ERK1/2 phosphorylation, attenuated both spinal ERK activation and nociceptive response evoked by intraplantar capsaicin. These results for the first time suggest that spinally administered substance P (1e7) may inhibit capsaicin-induced nociceptive behavior through the ERK signaling pathway in the primary afferents. Consistent with previous studies in animals (Sakurada et al., 1992; Reeh et al., 1986; Simone et al., 1991), we observed that capsaicin could induce spontaneous pain-related behavior and hyperalgesia when locally injected the hindpaw. There were differences between licking/biting response and hyperalgesia in their time course and doses. The licking/biting behavior induced by
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
611
Fig. 3. Effect of intrathecal U0126, a MEK inhibitor, on spinal ERK activation and behavioral response induced by intraplantar injection of capsaicin. (A) Inhibition of capsaicin-induced licking/biting response by U0126 (1.25e5.0 nmol). U0126 was given intrathecally 5 min prior to intraplantar capsaicin (1600 ng/paw). The duration of licking/ biting response induced by capsaicin was determined over a 5 min period starting immediately after intraplantar injection. The data are given as the mean SEM for groups of 10 mice. **P < 0.01, *P < 0.05, when compared to vehicle controls. (B) Western blots of the dorsal spinal homogenates for phospho-ERK1/2 and total ERK1/2. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (1600 ng/paw). Total ERK1/2 was used as a loading control. U0126 (2.5 nmol) was given intrathecally 5 min prior to intraplantar capsaicin. This western blot is representative of four independent experiments.
Fig. 2. Expression of phospho-ERK, phospho-p38 and phospho-SAPK/JNK in the dorsal spinal cord following intraplantar injection of capsaicin. Representative western blots (top) and quantitative data (bottom) for the expression of phospho-ERK (A), phosphop38 (B) and phospho-SAPK/JNK (C) in spinal cord membranes. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (400 and 1600 ng/ paw). Total ERK, p38 and total SAPK/JNK were used as a loading control, respectively. The fold changes for the density of phospho-ERK, phospho-p38 and phospho-SAPK/JNK bands are calculated after normalization with vehicle controls. Phospho-ERK, phosphop38 and phospho-SAPK/JNK levels in vehicle control group were set at 1 for quantification. Data are presented as mean SEM of four mice in each group.
capsaicin was of shorter duration than hyperalgesia to heat, and hyperalgesic doses of capsaicin were not sufficient to induce he licking/biting behavior. Hence, it seems apparent that the mechanisms underlying pain-related behavior and hyperalgesia induced by capsaicin differ. It is of importance to note that capsaicin (30 and
100 mg/100 ml) injected subcutaneously into the rostral part of the back failed to induce significant scratching, suggestive of itch-associated response (Kuraishi et al., 1995). Moreover, licking/ biting behavior induced by intraplantar capsaicin at the dose we used (1600 ng/20 mg) was found to be inhibited dose-dependently by i.p. injection of morphine (Sakurada et al., 1994), suggesting that capsaicin-induced licking/biting may be a pronociceptive behavioral response, but not due to itch. Capsaicin stimulates C-fiber nociceptors TRPV1 receptors (Caterina and Julius, 2001), leading to nociceptive behavioral response, neurogenic oedema or thermal hyperalgesia (Reeh et al., 1986; LaMotte et al., 1992). Several lines of evidence have indicated that intense C-fiber stimuli induce phosphorylated ERK expression in dorsal horn neurons, and inhibitors of ERK phosphorylation are able to inhibit thermal hyperalgesia (Ji et al., 1999; Dai et al., 2002; Kawasaki et al., 2006). It seems therefore that phosphorylation of ERK may be a good marker of activity in the spinal neurons mediating thermal hyperalgesia. In this study, intraplantar injection of capsaicin increased the spinal phosphorylated ERK expression. The i.t. injection of U0126 significantly inhibited phosphorylated ERK expression in the dorsal spinal cord. In the behavioral experiment, the MEK inhibitor markedly prevented capsaicin-induced paw-licking/biting response. These results suggest that activation of spinal ERK may contribute to elicitation of pain-related licking/biting behavior. Recent studies indicate that the signaling pathway of p38 MAPK and JNK in dorsal root ganglia and spinal cord is involved in pain models such as inflammatory pain and neuropathic pain. However, the present study shows that activation of TRPV1 by capsaicin could not lead to activation of spinal p38 MAPK and JNK. It seems thus that capsaicin-induced nociceptive response may be related to spinal ERK activation rather than p38 MAPK and JNK, which are stress-induced protein kinases and participate injury responses and cell death (Widmann et al., 1999; Ji and Woolf, 2001).
612
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
substance P (1e7) may be readily cleaved into inactive shorter fragments by spinal enzymatic process. The central sensitization produced by noxious stimuli is in part mediated by activation of NMDA receptors in the dorsal horn neurons (Coderre, 1993; Dubner and Ruda, 1992; Meller and Gebhart, 1993). Previously, pharmacological i.t. treatments of NMDA receptor antagonists have been found to attenuate capsaicininduced nociception (Sakurada et al., 1998). In vitro studies show that glutamate signaling via NMDA and mGluR1 receptors is the essential component of C-fiber transmission required to initiate ERK phosphorylation of mouse dorsal horn neurons (Adwanikar et al., 2004; Karim et al., 2001; Lever et al., 2003). In the behavioral experiments, i.t. NMDA-induced nociceptive behavior could be reduced by i.t. co-administration of substance P (1e7) (Hornfeldt et al., 1994), suggesting the possible synaptic inhibition of substance P (1e7). Collectively, it is inferred that i.t. SP (1e7) may induce antinociception through the synaptic transmission in the dorsal spinal cord, reducing nociceptive transmitter release presynaptically and acting at the postsynaptic substance P (1e7) binding sites. Indeed, it has been shown that substance (1e7) decreases the basal release of glutamate from the rat spinal cord (Skilling et al.,1990) and the elevated release of glutamate evoked by i.t. injection of highdose morphine (Sakurada et al., 2007). Therefore, we propose that spinal ERK activation induced by intraplantar capsaicin may be inhibited by substance P (1e7) through the decreased release of glutamate and postsynaptic action on substance P (1e7) binding sites in the spinal cord. In conclusion, our data show that activation of the ERK signaling pathway in the dorsal spinal cord contributes to the spontaneous nociceptive behavior induced by intraplantar capsaicin. Inhibition of ERK with i.t. substance P (1e7) attenuated capsaicin-induced nociceptive response, which was reversed by [D-Pro2, D-Phe7] substance P (1e7). This implies that inhibition of the ERK pathway has potential for therapeutics to reduce pain hypersensitivity. Fig. 4. Effects of intrathecal substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7) on licking/biting response and ERK activation induced by intraplantar capsaicin. (A) Substance P (1e7) was injected intrathecally 3 min prior to intraplantar capsaicin (1600 ng/paw). [D-Pro2, D-Phe7]substance P (1e7) was co-injected intrathecally with substance P (1e7) in a total volume of 5 ml. The duration of licking/biting response induced by capsaicin (1600 ng/paw) was determined over a 5 min period starting immediately after intraplantar injection. The data are given as the mean SEM for groups of 10 mice. **P < 0.01, *P < 0.05, when compared to artificial CSF (A-CSF) controls. #P < 0.05, when compared to A-CSF plus SP(1e7) (80 pmol). (B) Representative western blots (top) and quantitative data (bottom) for the expression of phospho-ERK1/2 and total ERK1/2. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (1600 ng/paw). Total ERK1/2 was used as a loading control. The fold change for the density of phospho-ERK bands is calculated after normalization with controls. Phospho-ERK1/2 levels in vehicle control group were set at 1 for quantification. Data are presented as mean SEM of four mice in each group. Statistically significant difference compared with artificial CSF (A-CSF) controls is indicated by **P < 0.01, *P < 0.05.
Substance P (1e7) has been found to inhibit pain-related behavior induced by i.t. injection of substance P (Sakurada et al., 1988) and nociceptive behavioral responses as assayed by the tail-flick and formalin tests (Goettl and Larson, 1996; Sakurada et al., 2002). Recently, we reported that spontaneous pain-related behavior evoked by i.t. high-dose morphine was attenuated by i.t. pretreatment with substance P (1e7) in rats (Sakurada et al., 2007). In agreement with these previous reports, the present study has revealed that substance P (1e7), injected i.t. 3 min prior to capsaicin, produced a dose-dependent reduction on the nociceptive behavior evoked by intraplantar capsaicin. Antinociception observed 3 min after i.t. substance P (1e7) (40 pmol) in the present study was more effective than that observed 5 min after i.t. substance P (1e7) at the same dose (Komatsu et al., 2007). These results indicate that
Acknowledgments This work was supported by The Science Research Promotion Fund from The Promotion and Mutual Aid Corporation for Private Schools of Japan, a Grant-in-Aid for Scientific Research (C) (KAKENHI 16590058 and 17590065) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for High Technology Research Program from the Ministry of Education, Culture, Sports, Science and Technology Japan. References Adwanikar, H., Karim, F., Gereau, R.W., 2004. Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation. Pain 111, 125e135. Botros, M., Hallberg, M., Johansson, T., Zhou, Q., Lindeberg, G., Frandberg, P.-A., Tomboly, C., Toth, G., Le Greves, P., Nyberg, F., 2006. Endomorphin-1 and endomorphin-2 differently interact with specific binding sites for substance P (SP) aminoterimnal SP (1-7) in the rat spinal cord. Peptides 27, 753e759. Botros, M., Johansson, T., Zhou, Q., Lindeberg, G., Tomboly, C., Toth, G., Le Greves, P., Nyberg, F., Hallberg, M., 2008. Endomorphins interact with the substance P (SP) aminoterminal SP (1-7) binding in the ventral tegmenrtal area of the rat brain. Peptides 29, 1820e1824. Caterina, M.J., Julius, D., 2001. The vanilloid receptor: a molecular gateway to the pain pathway. Annu. Rev. Neurosci. 24, 487e517. Ciruela, A., Dixon, A.K., Bramwell, S., Gonzalez, M.I., Pinnock, R.D., Lee, K., 2003. Identification of MEK1 as a novel target for the treatment of neuropathic pain. Br. J. Pharmacol. 138, 751e756. Coderre, T.J., 1993. The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats. Mol. Neurobiol. 7, 229e246. Dai, Y., Iwata, K., Fukuoka, T., Kondo, E., Tokunaga, A., Yamanaka, H., Tachibana, T., Liu, Y., Noguchi, K., 2002. Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J. Neurosci. 22, 7737e7745.
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613 Dubner, R., Ruda, M.A., 1992. Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci. 15, 96e103. Duggan, A.W., Morton, C.R., Zhao, Z.Q., Hendry, I.A., 1987. Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes. Brain Res. 403, 345e349. Galan, A., Lopez-Garcia, J.A., Cervero, F., Laird, J.M.A., 2002. Activation of spinal extracellular signaling-regulated kinase-1 and -2 by intraplantar carrageenan in rodents. Neurosci. Lett. 322, 37e40. Galan, A., Cervero, F., Laird, J.M.A., 2003. Extracellular signaling-regulated kinase-1 and -2 (ERK1/2) mediate referred hyperalgesia in a murine model of visceral pain. Neuropharmacology 116, 126e134. Gamse, R., Molnar, A., Lembeck, F., 1979. Substance P release from spinal cord slices by capsaicin. Life Sci. 25, 629e636. Go, V.L.W., Yaksh, T.L., 1987. Release of substance P form the cat spinal cord. J. Physiol. 391, 141e167. Goettl, V.M., Larson, A.A., 1996. Nitric oxide mediate long-term hyperalgesic and antinociceptive effects of the N-terminus of substance P in the formalin assay in mice. Pain 67, 435e441. Hall, M.E., Stewart, J.M., 1983. Substance P and behavior: opposite effects of N-terminal and C-terminal fragments. Peptides 4, 763e768. Hornfeldt, C., Sun, X., Larson, A.A., 1994. The NH2-terminus of substance P modulates NMDA-induced activity in the mouse spinal cord. J. Neurosci. 14, 3364e3369. Hylden, J.L.K., Wilcoxon, G.L., 1980. Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 17, 313e316. Igwe, O.J., Kim, D.C., Seybold, V.S., Larson, A.A., 1990. Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes. J. Neurosci. 10, 3653e3663. Ji, R.R., Woolf, C.J., 2001. Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol. Dis. 8, 1e10. Ji, R.R., Baba, H., Brenner, G.J., Woolf, C.J., 1999. Nociceptive specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat. Neurosci. 2, 1114e1119. Ji, R.R., Befort, K., Brenner, G.J., Woolf, C.J., 2002. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J. Neurosci. 22, 478e485. Kantner, R.M., Kirby, M.L., Goldstein, B.D., 1985. Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus. Brain Res. 338, 196e199. Karim, F., Wang, C.C., Gereau, R.W., 2001. Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice. J. Neurosci. 21, 3771e3779. Katsura, H., Obata, K., Mizushima, T., Sakurai, J., Kobayashi, K., Yamanaka, H., Dai, Y., Fukuoka, T., Sakagami, M., Noguchi, K., 2006. Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury. J. Neurosci. 26, 8680e8690. Kawasaki, Y., Kohno, T., Ji, R.R., 2006. Different effects of opioid and cannabinoid receptor agonists on C-fiber-induced extracellular signal-regulated kinase activation in dorsal horn neurons in normal and spinal nerve-ligated rats. J. Pharmacol. Exp. Ther. 316, 601e607. Koltzenburg, M., Lundberg, L.E., Torebjork, H.E., 1992. Dynamic and static components of mechanical hyperalgesia in human hairy skin. Pain 51, 207e219. Komatsu, T., Sakurada, C., Sasaki, M., Sanai, K., Tsuzuki, M., Bagetta, G., Sakurada, S., Sakurada, T., 2007. Extracellular signal-regulated kinase (ERK) and nitric oxide synthase mediate intrathecal morphine- induced nociceptive behavior. Neuropharmacology 52, 1237e1243. Komatsu, T., Sasaki, M., Sanai, K., Kuwahata, H., Sakurada, C., Tsuzuki, M., Iwata, Y., Sakurada, S., Sakurada, T., 2009. Intrathecal substance P auguments morphineinduced antinociception: possible relevance in the production of substance P N-terminal fragments. Peptides 30, 1689e1696. Kuraishi, Y., Nagasawa, T., Hayashi, K., Satoh, M., 1995. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. Eur. J. Pharmacol. 275, 229e233. LaMotte, R.H., Lundberg, L.E., Torebjork, H.E., 1992. Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin. J. Physiol. 448, 749e764. Lee, C.M., Sandberg, B.E.B., Hanley, M.R., Iversen, L.L., 1981. Purification and characterization of a membrane-bound substance P degrading enzyme from human brain. Eur. J. Biochem. 114, 315e327. Lever, I.J., Pezet, S., McMahon, S.B., Malcangio, M., 2003. The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn. Mol. Cell Neurosci. 24, 259e270.
613
Lewis, T.S., Shapiro, P.S., Ahn, N.G., 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74, 49e139. Matsas, R., Kenny, A.J., Turner, A.J., 1984. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues by endopeptidase-24.11. J. Biochem. 223, 433e440. Meller, S.T., Gebhart, G.F., 1993. Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52, 127e136. Nyberg, F., Terenius, L., 1991. Enzymatic inactivation of neuropeptides. In: Henriksen, J.H. (Ed.), Degradation of Bioactive Substances, Physiology and Pathophysiology. CRC Press Inc., New York, pp. 189e200. Pernow, B., 1983. Substance P. Pharmacol. Rev. 35, 85e141. Reeh, P.W., Kocher, L., Jung, S., 1986. Does neurogenic inflammation alter the sensitivity of unmyelinated nociceptors in the rats? Brain Res. 384, 42e50. Sakurada, T., Le Greves, P., Terenius, L., 1985. Measurement of substance P metabolites in rat CNS. J. Neurochem. 44, 718e722. Sakurada, T., Kuwahara, H., Takahashi, K., Sakurada, S., Kisara, K., Terenius, L., 1988. Substance P (1-7) antagonizes substance P-induced aversive behaviour in mice. Neurosci. Lett. 95, 281e285. Sakurada, T., Yamada, T., Tan-No, K., Manome, Y., Sakurada, S., Kisara, K., Ohba, M., 1991. Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord. J. Pharmacol. Exp. Ther. 259, 205e210. Sakurada, T., Katsumata, K., Tan-No, K., Sakurada, S., Kisara, K., 1992. The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord. Neuropharmacology 12, 1279e1285. Sakurada, T., Yogo, H., Katsumata, K., Tan-No, K., Sakurada, S., Kisara, K., Ohba, M., 1994. Differential antinociceptive effects of senside, a NK-receptor antagonist, and morphine in the capsaicin test. Brain Res. 649, 319e322. Sakurada, T., Wako, K., Sugiyama, A., Sakurada, C., Tan-No, K., Kisara, K., 1998. Involvement of spinal NMDA receptors in capsaicin-induced nociception. Pharmacol. Biochem. Behav. 59, 339e345. Sakurada, C., Watanabe, C., Sakurada, T., 2002. Occurrence of substance P (1-7) in the metabolism of substance P and its antinociceptive activity at the mouse spinal cord level. Methods Find. Exp. Clin. Pharmacol. 24, 39e44. Sakurada, T., Matsumura, T., Moriyama, T., Sakurada, C., Ueno, S., Sakurada, S., 2003. Differential effects of intraplantar capsazepine and ruthenium red on capsaicininduced desensitization in mice. Pharmacol. Biochem. Behav. 75, 115e121. Sakurada, T., Komatsu, T., Kuwahata, H., Watanabe, C., Orito, T., Sakurada, C., Tsuzuki, M., Sakurada, S., 2007. Intrathecal substance P (1-7) prevents morphine-evoked spontaneous pain behavior via spinal NMDA-NO cascade. Biochem. Pharmacol. 74, 758e767. Schaible, H.G., Jarrott, B., Hope, P.J., Duggan, A.W., 1990. Release of immunoreactive substance P in the spinal cord during development of acute arthritis in the knee joint of the cat: a study with antibody microprobes. Brain Res. 529, 214e223. Simone, D.A., Sorkin, L.S., Oh, U., Chung, J.M., Owens, C., LaMotte, R.H., Willis, W.D., 1991. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. J. Neurophysiol. 66, 228e246. Skilling, S.R., Smullin, D.H., Larson, A.A., 1990. Differential effects of C- and Nterminal substance P metabolites on the release of amino acid neurotransmitters from the spinal cord: potential role of nociception. J. Neurosci 10, 1309e1318. Thomas, K.L., Hunt, S.P., 1993. The regional distribution of extracellularly regulated kinase-1 and -2 messenger RNA in the adult rat central nervous system. Neuroscience 56, 741e757. Tsuda, M., Ueno, H., Kataoka, A., Tozaki-Saitoh, H., Inoue, K., 2008. Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling. Glia 56, 378e386. Wang, H., Dai, Y., Fukuoka, T., Yamanaka, H., Obata, K., Tokunaga, A., Noguchi, K., 2004. Enhancement of stimulation-induced ERK activation in the spinal dorsal horn and gracile nucleus in rats with peripheral nerve injury. Eur. J. Neurosci. 19, 884e890. Widmann, C., Gibson, S., Jarpe, M.B., Johnson, G.L., 1999. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143e180. Zhao, P., Waxman, S.G., Hains, B.C., 2007. Extracellular signal-regulated microglianeuron signaling by prostaglandin E2 contributes to pain after spinal cord injury. J. Neurosci. 27, 2357e2368. Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is essentially activated in neurons, microglia, and astrocytes by spinal ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114, 149e159. Zimmermann, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109e110.
Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Inhibition of ERK phosphorylation by substance P N-terminal fragment decreases capsaicin-induced nociceptive response Takaaki Komatsu a, Hirokazu Mizoguchi b, Mika Sasaki a, Chikai Sakurada c, Minoru Tsuzuki c, Shinobu Sakurada b, **, Tsukasa Sakurada a, * a b c
First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Department of Biochemistry, Nihon Pharmaceutical University, 10281 Komuro Ina-Machi Kitaadachi-gun, Saitama 362-0806, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 June 2010 Received in revised form 9 April 2011 Accepted 20 April 2011
Previous research has demonstrated that substance P N-terminal fragments produced by the action of several different enzymes in the spinal cord could reduce nociception when injected intrathecally (i.t.) into mice. The present study examined the possible involvement of spinal extracellular signal-regulated protein kinase (ERK), a mitogen-activated protein kinase (MAPK), in i.t. substance P (1e7)-induced antinociception as assayed by the capsaicin test. The i.t. injection of substance P (1e7) (20e80 nmol) into mice resulted in a dose-dependent attenuation of paw-licking/biting behavior induced by intraplantar injection of capsaicin, which was reversed by co-injection of [D-Pro2, D-Phe7]substance P (1e7), a D-isomer and antagonist of substance P (1e7). In Western blot analysis, intraplantar injection of capsaicin (400 and 1600 ng/paw) produced an increase of ERK phosphorylation in the dorsal spinal cord, whereas expression of p38 and c-Jun N-terminal kinase (JNK) phosphorylation was unchanged by capsaicin treatment. In parallel to the behavioral results, i.t. substance P (1e7) inhibited capsaicin-induced EKR phosphorylation, which was reversed by [D-Pro2, D-Phe7]substance P (1e7), a substance P (1e7) antagonist. Both nociceptive behavioral response and spinal ERK activation induced by intraplantar capsaicin were reduced by U0126, an upstream inhibitor of ERK phosphorylation. Taken together, these findings suggest that the activation of ERK, but not p38 and JNK MAPKs in the spinal cord, contributes to intraplantar capsaicininduced nociception, and that blocking ERK activation via substance P (1e7) binding sites may provide significant antinociception at the spinal cord level. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: N-Terminal fragments of substance P Capsaicin-induced nociceptive response Spinal antinociception Intrathecal injection Extracellular signal-regulated protein kinase (ERK)
1. Introduction Numerous studies have documented the presence of substance P in sensory afferent fibers in the dorsal root ganglia and the dorsal horn of the spinal cord (Pernow, 1983). Substance P is released from the spinal cord following various nociceptive stimuli (Duggan et al., 1987; Kantner et al., 1985; Schaible et al., 1990) and administrations of capsaicin through peripheral and intrathecal (i.t.) routes (Gamse et al., 1979; Go and Yaksh, 1987). Following release and binding to neurokinin1 (NK1) receptor, the biological action of substance P is terminated by enzymatic degradation (Lee et al., 1981; Matsas et al., 1984; Nyberg and Terenius, 1991). Substance P can be cleaved by several degrading enzymes such as neutral endopeptidase-24.11 * Corresponding author. Tel.: þ81 925410161; fax: þ81 925535698. ** Corresponding author. E-mail addresses: [email protected] (S. Sakurada), [email protected] (T. Sakurada). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.04.035
(EC 3.4. 24.11.: enkephalinase). Some of the substance P fragments generated by enzymatic hydrolysis are present in significant concentrations in the central nervous system (CNS), particularly in the dorsal part of spinal cord (Sakurada et al., 1985, 1991). Of several substance P fragments, substance P (1e7) was chosen for the present study, because the N-terminal peptide is the major fragment in the CNS. Substance P (1e7) is able to produce behavioral effects which are different from those produced by the C-terminal fragments and the parent compounds (Hall and Stewart, 1983). In several nociceptive assays, substance P (1e7) has been found to be antinociceptive in mice including the formalin (Goettl and Larson, 1996) and capsaicin (Komatsu et al., 2009) tests. We have recently reported that the elevated concentrations of glutamate and nitric oxide metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine were inhibited by i.t. pretreatment with substance P (1e7) (Sakurada et al., 2007). Specific binding sites for substance P (1e7) have been identified in the mouse brain and spinal cord, which are distinct from neurokinin-1 receptors (Igwe et al., 1990).
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
In the current studies there are interesting data, suggesting that substance P (1e7) binding sites possess high affinity for endomorphin-2, which are distinct from the m-opioid receptor in the spinal cord and the ventral tegmental area of rats (Botros et al., 2006, 2008). The mitogen-activated protein kinase (MAP) family including extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 in the spinal transmission of pain tranduces extracellular stimulation into intracellular post-translational and transcriptional responses (Lewis et al., 1998). It is now established that ERK signaling pathway contributes to nociceptive information and central sensitization produced by noxious stimuli (Ciruela et al., 2003; Galan et al., 2003; Ji et al., 1999, 2002 Wang et al., 2004; Zhuang et al., 2005). The ERKs are expressed both in primary afferents as well as in glial cells within the dorsal spinal cord (Zhuang et al., 2005; Katsura et al., 2006; Zhao et al., 2007; Tsuda et al., 2008). Phosphorylation of spinal ERK has been reported by acute noxious stimuli of formalin and capsacin (Thomas and Hunt, 1993; Ji et al., 1999; Karim et al., 2001; Galan et al., 2002). Capsaicin activates C-fibers via the transient receptor potential vanilloid 1 (TRPV1) ion channel (formerly the VR1 receptor) (Caterina and Julius, 2001), and induces phosphorylated ERK expression (Ji et al., 1999; Karim et al., 2001) to produce primary and secondary hyperalgesia in animals (Reeh et al., 1986; Simone et al., 1991) and human (Koltzenburg et al., 1992; LaMotte et al., 1992). Spinal ERK is activated by not only shortlived pain, but also persistent inflammatory hyperalgesia induced by carrageenan and Freund’s adjuvant (Galan et al., 2002; Ji et al., 2002). In addition, inhibition of ERK phosphorylation has been found to be prevented or reduced in numerous pain models (Ji et al., 1999; Dai et al., 2002; Galan et al., 2003). Recently, we reported that phosphorylation of ERK in the dorsal spinal cord occurred in response to nociceptive behavioral responses following i.t. injection of high-dose morphine or morphine-3-glucuronide, a major metabolite of morphine (Komatsu et al., 2007, 2009). However, except for the specific inhibitor of ERK phosphorylation, the contribution of the ERK cascade to the antinociceptive changes produced by compounds remains to be elucidated. The present study was performed in order to examine the possible involvement of the MAP family including p38 and JNK as well as ERK in the spinal cord when capsaicin was injected into the hindpaw. Our findings support the hypothesis that spinal ERK signaling contributes to the pain-related behavioral response induced by intraplantar capsaicin injection. In addition, we have shown that substance P (1e7)-induced antinociception could be attributed to the inhibition of ERK phosphorylation in the spinal cord. 2. Materials and methods 2.1. Animals Male ddY mice (Shizuoka Laboratory Center, Japan) weighing 22e26 g were used in this study. All protocols were approved by the Committee of Animal Experiments in Daiichi College of Pharmaceutical Sciences and Tohoku Pharmaceutical University. These experiments conformed to the ethical guidelines of the International Association for the Study of Pain in conscious animals published (Zimmermann, 1983). The animals were housed under conditions of a 12 h lighte dark cycle, a constant temperature of 22e24 C and 55e60% relative humidity. Animals had free access to food (Clea Japan, Osaka, Japan) and drinking before the experiments. 2.2. Intrathecal injection The i.t. injection procedure was adapted from the method of Hylden and Wilcoxon (1980). Briefly, the lumbar puncture was performed using a 28 gauge needle attached to a 50-ml Hamilton microsyringe. The needle was inserted between L5 and L6, and drugs were delivered in a volume of 5 ml in conscious mice. The mice were not anesthetized during these procedures. Puncture of the dura was behaviorally indicated by a slight flick of the tail.
609
2.3. Assessment of behavioral response The capsaicin test was performed as described in detail previously (Sakurada et al., 1992). To reduce variability each mouse was acclimatized to an acrylic observation chamber (22.0 15.0 12.5 cm) for approximately 1 h before the injection of capsaicin. The mouse was injected 20 ml of a solution of capsaicin (400 ng and 1600 ng/paw) beneath the skin the plantar surface of the right hindpaw using a microsyringe with a 26-gauge needle. Licking/biting behavior induced by intraplantar injection of capsaicin was observed as an indicator of nociceptive response. The accumulated response time (in second) spent in licking/biting the capsaicin-injected paw was measured for a period of 5 min immediately after subcutaneous (s.c.) injection of capsaicin. In the paw-withdrawal test, the latencies were recorded as the time from the start of the radiant heat stimulus to withdrawal of the hindpaw. A radiant heat source was placed under one of the hindpaws, and the time to withdrawal of the stimulated paw was measured. The intensity was set so that the reaction time to paw-withdrawal under control conditions was 10e12 s. Control reaction time was determined by a total of two consecutive measurements each separated by 10 min. 2.4. Western blotting analysis To examine the spinal expression of ERK, p38 and JNK, the mice were decapitated and the whole spinal cord was taken by pressure expulsion with physiological saline. The dorsal and ventral parts of lumbar spinal cord were dissected on an icecooled glass dish. Tissue samples were homogenized in 0.1 ml of lysis buffer reagent (150 mM NaCl, 1.0% NP-40, 50 mM TriseHCl pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mM sodium vanadate and 1 mM EDTA pH 8.0) and centrifuged at 16,000 g for 30 min at 4 C. Supernatants were collected and total protein amounts were measured using the Protein Assay (BIO-RAD, Hercules, CA). An equal volume of 2 sample buffer (100 mM TriseHCl pH 6.8, 2.5% SDS, 20% glycerol, 0.006% bromophenol blue and 10% b-mercaptoethanol) was added to 30 mg of totaprotein. The samples were boiled, and then electrophoresed in a 10e15% SDS-polyacrylamide gel (BIO-RAD, Hercules, CA) and transferred to a Hybond-P membrane (Amersham Bioscience). The blotted membrane was then incubated overnight with 5% skim milk (Wako Pure Chemical Industries Ltd, Osaka, Japan) in T-PBS (PBS containing 0.1% v/v Tween 20). All antibody applications were done in T-PBS. After the membranes were washed with T-PBS, the membranes were incubated with phospho-p44/42 MAP kinase antibody (1:1000), p44/42 MAP kinase antibody (1: 1000), phospho-p38 MAP kinase antibody (1:1000) and phospho-SNAP/JNK antibody (1:1000) (Cell Signaling) for 2 h at room temperature. The membranes were extensively washed with T-PBS and incubated for 2 h with the secondary antibody (anti-rabbit IgG peroxidase-conjugated antibody 1:5000) (Amersham Bioscience). After washing, the proteins were detected using the ECL-Plus Western blotting detection system (Amersham Biosciences) and visualized with the Dolphine-Chemi Image System (Wealtec). MagicMark western protein standard (Invitrogen) was simultaneously resolved on the gel, and the molecular weight of protein was estimated. 2.5. Drugs Substance P (1e7) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). [D-Pro2, D-Phe7]substance P (1e7) was synthetized by conventional solid phase methods in our laboratory (Sakurada et al., 1991). For i.t. injections, substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7) were dissolved in sterile artificial cerebrospinal fluid (CSF) containing 126.6 mM NaCl, 10.0 mM NaHCO3, 2.5 mM KCl, 2.0 mM MgCl2, 1.3 mM CaCl2 and 1.0 mM glucose. Capsaicin (Sigma, St. Louis, MO) was initially dissolved in 100% dimethylsulfoxide (DMSO) at the concentration of 12.8 mg/20 ml as stock solution, further diluted to 400 ng/20 ml and 1600 ng/20 ml by physiological saline (0.9% wt/vol). The final concentration of DMSO for 400 ng/20 ml and 1600 ng/20 ml of capsaicin was 3.125% and 12.5%, respectively. These concentrations of DMSO were used as vehicle controls for capsaicin-injected groups. Injections of capsaicin into the hindpaw were made with a 26-gauge needle attached to a 50 ml Hamilton microsyringe. Substance P (1e7) was injected i.t. 3 min prior to intraplantar capsaicin. In the experiment with i.t. combined administration, capsaicin was injected in a volume of 5 ml into the hindpaw 3 min after i.t. co-administration of substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7). The MEK inhibitor, 1,4-diamino-2,3-dicyano-1,4bis(2-amino-phenylthio)butadiene (U0126) was obtained from Calbiochem, Darmstadt, Germany. U0126 was initially dissolved in 100% DMSO at the concentration of 26.28 mM as stock solution, further diluted to 0.5, 1.25 or 2.5 nmol/5 ml by artificial CSF, and adjusted to 6.71% DMSO as the final concentration. To study the importance of ERK phosphorylation in capsaicin-induced nociception, U0126, an ERK upstream kinase, was injected i.t. 5 min before intraplantar injection of capsaicin. The 5 ml of 6.71% DMSO used as vehicle control for i.t. injection gave no significant effect on capsaicin-induced licking/biting response when compared to artificial CSF controls. 2.6. Analyses of data All values are expressed as means S.E.M. Statistical differences between groups were established using Dunnett’s test for multiple comparisons after analysis
610
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
of variance (ANOVA). The 5% level of statistical significance was set in all experiments.
3. Results 3.1. Intraplantar capsaicin evokes licking/biting behavioral response and hyperalgesia An injection of capsaicin (400 and 1600 ng/paw) into the plantar surface of the hindpaw evoked a spontaneous and severe licking/ biting response toward the injected paw in mice (Fig. 1A). The nociceptive response started immediately after intraplantar injection of capsaicin, reached a peak and gradually subsided to baseline within approximately 5 min. The behavioral characterization of intraplantar capsaicin confirms previously reported data (Sakurada et al., 2003). The 6.25 ng dose capsaicin produced a decrease in reaction time to paw-withdrawal from the radiant heat stimulus 15 min after intraplantar injection (Fig. 1B). A similar decrease in reaction time with response to the magnitude of the effect of capsaicin (6.25 ng) was observed 5 min post-injection, and the reaction time returned to pre-injection values at 20 and 30 min (data not shown). A higher dose (12.5 ng) of capsaicin produced a slight but significant change in withdrawal latency, whereas 25 and 100 ng capsaicin did not alter withdrawal latency. 3.2. Phosphorylation of ERK, p38 and JNK in the spinal cord by intraplantar capsaicin To investigate whether spinal ERK1 and/or ERK2, p38 and JNK are activated by intraplantar capsaicin (400 and 1600 ng/paw), we
compared the effect of capsaicin with that of vehicle control in the lumbar dorsal spinal cord extracted 5 min after intraplantar capsaicin. Western blot analysis revealed an activation of ERK in the dorsal spinal cord after intraplantar capsaicin (Fig. 1B). Both phosphorylated ERK1 (P44 MAPK) and phosphorylated ERK2 (P42 MAPK) levels were significantly increased by 1600 ng capsaicin as compared to vehicle-treated control. A lower dose of capsaicin (400 ng) induced a significant activation of ERK2 without affecting ERK1 level. In contrast to the results of ERK1 and ERK2, intraplantar capsaicin (400 and 1600 ng) did not increase phosphorylation of p38 and JNK MAPKs in the lumbar spinal cord 5 min post-injection (Fig. 2). To examine the specificity of ERK1 and ERK2 activation by intraplantar capsaicin (1600 ng), the upstream inhibitor of ERK phosphorylation, U0126 was used. In the behavioral test, i.t. U0126 (1.25e5.0 nmol, 5 min pre-capsaicin) significantly inhibited nociceptive behavioral response after intraplantar capsaicin (Fig. 3A). The results of western blot study showed that i.t. injection of U0126 at a dose of 2.5 nmol suppressed the increase of phosphorylated ERK in the dorsal spinal cord detectable at 5 min after capsaicin injection (Fig. 3B). These results further confirm that activation of the ERK signaling pathway mediates capsaicin-induced nociceptive response. 3.3. Substance P (1e7) inhibited nociceptive response and spinal ERK activation after intraplantar capsaicin Substance P (1e7) (20e80 pmol), injected i.t. 3 min prior to capsaicin, produced a dose-dependent inhibition of capsaicininduced paw-licking/biting (Fig. 4A). The inhibitory effect of substance P (1e7) (80 pmol) was reversed significantly by i.t. co-injection of [D-Pro2, D-Phe7]substance P (1e7) (8.0 nmol), a substance P (1e7) antagonist. In accordance with the behavioral results, western blot studies showed that i.t. substance P (1e7) at a dose of 80 pmol significantly suppressed the increase of phosphorylated ERKs when compared with the artificial CSF-treated controls (Fig. 4B). The inhibition of phosphorylated ERKs by substance P (1e7) was revsered significantly by i.t. [D-Pro2, D-Phe7] substance P (1e7) (8.0 nmol). Thus our results indicate that spinal ERK activation induced by intraplantar capsaicin could be mediated through substance P (1e7) binding sites in the synapse of the dorsal spinal cord. 4. Discussion
Fig. 1. Behavioral effect after intraplantar injection of capsaicin in mice. (A) The duration of licking/biting response was determined using the 5-min period starting immediately after injection of capsaicin (400 and 1600 ng/paw) into the right hindpaw. The data are given as the mean SEM for groups of 10 mice. **P < 0.01 when compared to corresponding controls (3.1% and 12.5% DMSO). (B) Effect of intraplantar capsaicin on reaction time in the paw-withdrawal test. Paw-withdrawal latencies were measured 15 min after injection of capsaicin (4.68e100 ng). The data are given as the mean SEM for groups of 10 mice. **P < 0.01 when compared to DMSO control.
In the present study, we have shown that i.t. injection of substance P (1e7) attenuated phosphorylation of spinal ERK following intraplantar injection of capsaicin. At the behavioral level, spontaneous pain-related response induced by intraplantar injection of capsaicin was reduced dose-dependently by i.t. substance P (1e7). Our data further showed that the substance P (1e7) antagonist [D-Pro2, D-Phe7]substance P (1e7), reversed the reduced actions of substance P (1e7) on nociceptive behavioral response and spinal ERK activation induced by intraplantar capsaicin. Functionally, i.t. U0126, an inhibitor of ERK1/2 phosphorylation, attenuated both spinal ERK activation and nociceptive response evoked by intraplantar capsaicin. These results for the first time suggest that spinally administered substance P (1e7) may inhibit capsaicin-induced nociceptive behavior through the ERK signaling pathway in the primary afferents. Consistent with previous studies in animals (Sakurada et al., 1992; Reeh et al., 1986; Simone et al., 1991), we observed that capsaicin could induce spontaneous pain-related behavior and hyperalgesia when locally injected the hindpaw. There were differences between licking/biting response and hyperalgesia in their time course and doses. The licking/biting behavior induced by
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
611
Fig. 3. Effect of intrathecal U0126, a MEK inhibitor, on spinal ERK activation and behavioral response induced by intraplantar injection of capsaicin. (A) Inhibition of capsaicin-induced licking/biting response by U0126 (1.25e5.0 nmol). U0126 was given intrathecally 5 min prior to intraplantar capsaicin (1600 ng/paw). The duration of licking/ biting response induced by capsaicin was determined over a 5 min period starting immediately after intraplantar injection. The data are given as the mean SEM for groups of 10 mice. **P < 0.01, *P < 0.05, when compared to vehicle controls. (B) Western blots of the dorsal spinal homogenates for phospho-ERK1/2 and total ERK1/2. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (1600 ng/paw). Total ERK1/2 was used as a loading control. U0126 (2.5 nmol) was given intrathecally 5 min prior to intraplantar capsaicin. This western blot is representative of four independent experiments.
Fig. 2. Expression of phospho-ERK, phospho-p38 and phospho-SAPK/JNK in the dorsal spinal cord following intraplantar injection of capsaicin. Representative western blots (top) and quantitative data (bottom) for the expression of phospho-ERK (A), phosphop38 (B) and phospho-SAPK/JNK (C) in spinal cord membranes. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (400 and 1600 ng/ paw). Total ERK, p38 and total SAPK/JNK were used as a loading control, respectively. The fold changes for the density of phospho-ERK, phospho-p38 and phospho-SAPK/JNK bands are calculated after normalization with vehicle controls. Phospho-ERK, phosphop38 and phospho-SAPK/JNK levels in vehicle control group were set at 1 for quantification. Data are presented as mean SEM of four mice in each group.
capsaicin was of shorter duration than hyperalgesia to heat, and hyperalgesic doses of capsaicin were not sufficient to induce he licking/biting behavior. Hence, it seems apparent that the mechanisms underlying pain-related behavior and hyperalgesia induced by capsaicin differ. It is of importance to note that capsaicin (30 and
100 mg/100 ml) injected subcutaneously into the rostral part of the back failed to induce significant scratching, suggestive of itch-associated response (Kuraishi et al., 1995). Moreover, licking/ biting behavior induced by intraplantar capsaicin at the dose we used (1600 ng/20 mg) was found to be inhibited dose-dependently by i.p. injection of morphine (Sakurada et al., 1994), suggesting that capsaicin-induced licking/biting may be a pronociceptive behavioral response, but not due to itch. Capsaicin stimulates C-fiber nociceptors TRPV1 receptors (Caterina and Julius, 2001), leading to nociceptive behavioral response, neurogenic oedema or thermal hyperalgesia (Reeh et al., 1986; LaMotte et al., 1992). Several lines of evidence have indicated that intense C-fiber stimuli induce phosphorylated ERK expression in dorsal horn neurons, and inhibitors of ERK phosphorylation are able to inhibit thermal hyperalgesia (Ji et al., 1999; Dai et al., 2002; Kawasaki et al., 2006). It seems therefore that phosphorylation of ERK may be a good marker of activity in the spinal neurons mediating thermal hyperalgesia. In this study, intraplantar injection of capsaicin increased the spinal phosphorylated ERK expression. The i.t. injection of U0126 significantly inhibited phosphorylated ERK expression in the dorsal spinal cord. In the behavioral experiment, the MEK inhibitor markedly prevented capsaicin-induced paw-licking/biting response. These results suggest that activation of spinal ERK may contribute to elicitation of pain-related licking/biting behavior. Recent studies indicate that the signaling pathway of p38 MAPK and JNK in dorsal root ganglia and spinal cord is involved in pain models such as inflammatory pain and neuropathic pain. However, the present study shows that activation of TRPV1 by capsaicin could not lead to activation of spinal p38 MAPK and JNK. It seems thus that capsaicin-induced nociceptive response may be related to spinal ERK activation rather than p38 MAPK and JNK, which are stress-induced protein kinases and participate injury responses and cell death (Widmann et al., 1999; Ji and Woolf, 2001).
612
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613
substance P (1e7) may be readily cleaved into inactive shorter fragments by spinal enzymatic process. The central sensitization produced by noxious stimuli is in part mediated by activation of NMDA receptors in the dorsal horn neurons (Coderre, 1993; Dubner and Ruda, 1992; Meller and Gebhart, 1993). Previously, pharmacological i.t. treatments of NMDA receptor antagonists have been found to attenuate capsaicininduced nociception (Sakurada et al., 1998). In vitro studies show that glutamate signaling via NMDA and mGluR1 receptors is the essential component of C-fiber transmission required to initiate ERK phosphorylation of mouse dorsal horn neurons (Adwanikar et al., 2004; Karim et al., 2001; Lever et al., 2003). In the behavioral experiments, i.t. NMDA-induced nociceptive behavior could be reduced by i.t. co-administration of substance P (1e7) (Hornfeldt et al., 1994), suggesting the possible synaptic inhibition of substance P (1e7). Collectively, it is inferred that i.t. SP (1e7) may induce antinociception through the synaptic transmission in the dorsal spinal cord, reducing nociceptive transmitter release presynaptically and acting at the postsynaptic substance P (1e7) binding sites. Indeed, it has been shown that substance (1e7) decreases the basal release of glutamate from the rat spinal cord (Skilling et al.,1990) and the elevated release of glutamate evoked by i.t. injection of highdose morphine (Sakurada et al., 2007). Therefore, we propose that spinal ERK activation induced by intraplantar capsaicin may be inhibited by substance P (1e7) through the decreased release of glutamate and postsynaptic action on substance P (1e7) binding sites in the spinal cord. In conclusion, our data show that activation of the ERK signaling pathway in the dorsal spinal cord contributes to the spontaneous nociceptive behavior induced by intraplantar capsaicin. Inhibition of ERK with i.t. substance P (1e7) attenuated capsaicin-induced nociceptive response, which was reversed by [D-Pro2, D-Phe7] substance P (1e7). This implies that inhibition of the ERK pathway has potential for therapeutics to reduce pain hypersensitivity. Fig. 4. Effects of intrathecal substance P (1e7) and [D-Pro2, D-Phe7]substance P (1e7) on licking/biting response and ERK activation induced by intraplantar capsaicin. (A) Substance P (1e7) was injected intrathecally 3 min prior to intraplantar capsaicin (1600 ng/paw). [D-Pro2, D-Phe7]substance P (1e7) was co-injected intrathecally with substance P (1e7) in a total volume of 5 ml. The duration of licking/biting response induced by capsaicin (1600 ng/paw) was determined over a 5 min period starting immediately after intraplantar injection. The data are given as the mean SEM for groups of 10 mice. **P < 0.01, *P < 0.05, when compared to artificial CSF (A-CSF) controls. #P < 0.05, when compared to A-CSF plus SP(1e7) (80 pmol). (B) Representative western blots (top) and quantitative data (bottom) for the expression of phospho-ERK1/2 and total ERK1/2. Dorsal spinal cord samples were taken 5 min after intraplantar injection of capsaicin (1600 ng/paw). Total ERK1/2 was used as a loading control. The fold change for the density of phospho-ERK bands is calculated after normalization with controls. Phospho-ERK1/2 levels in vehicle control group were set at 1 for quantification. Data are presented as mean SEM of four mice in each group. Statistically significant difference compared with artificial CSF (A-CSF) controls is indicated by **P < 0.01, *P < 0.05.
Substance P (1e7) has been found to inhibit pain-related behavior induced by i.t. injection of substance P (Sakurada et al., 1988) and nociceptive behavioral responses as assayed by the tail-flick and formalin tests (Goettl and Larson, 1996; Sakurada et al., 2002). Recently, we reported that spontaneous pain-related behavior evoked by i.t. high-dose morphine was attenuated by i.t. pretreatment with substance P (1e7) in rats (Sakurada et al., 2007). In agreement with these previous reports, the present study has revealed that substance P (1e7), injected i.t. 3 min prior to capsaicin, produced a dose-dependent reduction on the nociceptive behavior evoked by intraplantar capsaicin. Antinociception observed 3 min after i.t. substance P (1e7) (40 pmol) in the present study was more effective than that observed 5 min after i.t. substance P (1e7) at the same dose (Komatsu et al., 2007). These results indicate that
Acknowledgments This work was supported by The Science Research Promotion Fund from The Promotion and Mutual Aid Corporation for Private Schools of Japan, a Grant-in-Aid for Scientific Research (C) (KAKENHI 16590058 and 17590065) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for High Technology Research Program from the Ministry of Education, Culture, Sports, Science and Technology Japan. References Adwanikar, H., Karim, F., Gereau, R.W., 2004. Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation. Pain 111, 125e135. Botros, M., Hallberg, M., Johansson, T., Zhou, Q., Lindeberg, G., Frandberg, P.-A., Tomboly, C., Toth, G., Le Greves, P., Nyberg, F., 2006. Endomorphin-1 and endomorphin-2 differently interact with specific binding sites for substance P (SP) aminoterimnal SP (1-7) in the rat spinal cord. Peptides 27, 753e759. Botros, M., Johansson, T., Zhou, Q., Lindeberg, G., Tomboly, C., Toth, G., Le Greves, P., Nyberg, F., Hallberg, M., 2008. Endomorphins interact with the substance P (SP) aminoterminal SP (1-7) binding in the ventral tegmenrtal area of the rat brain. Peptides 29, 1820e1824. Caterina, M.J., Julius, D., 2001. The vanilloid receptor: a molecular gateway to the pain pathway. Annu. Rev. Neurosci. 24, 487e517. Ciruela, A., Dixon, A.K., Bramwell, S., Gonzalez, M.I., Pinnock, R.D., Lee, K., 2003. Identification of MEK1 as a novel target for the treatment of neuropathic pain. Br. J. Pharmacol. 138, 751e756. Coderre, T.J., 1993. The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats. Mol. Neurobiol. 7, 229e246. Dai, Y., Iwata, K., Fukuoka, T., Kondo, E., Tokunaga, A., Yamanaka, H., Tachibana, T., Liu, Y., Noguchi, K., 2002. Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J. Neurosci. 22, 7737e7745.
T. Komatsu et al. / Neuropharmacology 61 (2011) 608e613 Dubner, R., Ruda, M.A., 1992. Activity-dependent neuronal plasticity following tissue injury and inflammation. Trends Neurosci. 15, 96e103. Duggan, A.W., Morton, C.R., Zhao, Z.Q., Hendry, I.A., 1987. Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes. Brain Res. 403, 345e349. Galan, A., Lopez-Garcia, J.A., Cervero, F., Laird, J.M.A., 2002. Activation of spinal extracellular signaling-regulated kinase-1 and -2 by intraplantar carrageenan in rodents. Neurosci. Lett. 322, 37e40. Galan, A., Cervero, F., Laird, J.M.A., 2003. Extracellular signaling-regulated kinase-1 and -2 (ERK1/2) mediate referred hyperalgesia in a murine model of visceral pain. Neuropharmacology 116, 126e134. Gamse, R., Molnar, A., Lembeck, F., 1979. Substance P release from spinal cord slices by capsaicin. Life Sci. 25, 629e636. Go, V.L.W., Yaksh, T.L., 1987. Release of substance P form the cat spinal cord. J. Physiol. 391, 141e167. Goettl, V.M., Larson, A.A., 1996. Nitric oxide mediate long-term hyperalgesic and antinociceptive effects of the N-terminus of substance P in the formalin assay in mice. Pain 67, 435e441. Hall, M.E., Stewart, J.M., 1983. Substance P and behavior: opposite effects of N-terminal and C-terminal fragments. Peptides 4, 763e768. Hornfeldt, C., Sun, X., Larson, A.A., 1994. The NH2-terminus of substance P modulates NMDA-induced activity in the mouse spinal cord. J. Neurosci. 14, 3364e3369. Hylden, J.L.K., Wilcoxon, G.L., 1980. Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 17, 313e316. Igwe, O.J., Kim, D.C., Seybold, V.S., Larson, A.A., 1990. Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes. J. Neurosci. 10, 3653e3663. Ji, R.R., Woolf, C.J., 2001. Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol. Dis. 8, 1e10. Ji, R.R., Baba, H., Brenner, G.J., Woolf, C.J., 1999. Nociceptive specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat. Neurosci. 2, 1114e1119. Ji, R.R., Befort, K., Brenner, G.J., Woolf, C.J., 2002. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J. Neurosci. 22, 478e485. Kantner, R.M., Kirby, M.L., Goldstein, B.D., 1985. Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus. Brain Res. 338, 196e199. Karim, F., Wang, C.C., Gereau, R.W., 2001. Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice. J. Neurosci. 21, 3771e3779. Katsura, H., Obata, K., Mizushima, T., Sakurai, J., Kobayashi, K., Yamanaka, H., Dai, Y., Fukuoka, T., Sakagami, M., Noguchi, K., 2006. Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury. J. Neurosci. 26, 8680e8690. Kawasaki, Y., Kohno, T., Ji, R.R., 2006. Different effects of opioid and cannabinoid receptor agonists on C-fiber-induced extracellular signal-regulated kinase activation in dorsal horn neurons in normal and spinal nerve-ligated rats. J. Pharmacol. Exp. Ther. 316, 601e607. Koltzenburg, M., Lundberg, L.E., Torebjork, H.E., 1992. Dynamic and static components of mechanical hyperalgesia in human hairy skin. Pain 51, 207e219. Komatsu, T., Sakurada, C., Sasaki, M., Sanai, K., Tsuzuki, M., Bagetta, G., Sakurada, S., Sakurada, T., 2007. Extracellular signal-regulated kinase (ERK) and nitric oxide synthase mediate intrathecal morphine- induced nociceptive behavior. Neuropharmacology 52, 1237e1243. Komatsu, T., Sasaki, M., Sanai, K., Kuwahata, H., Sakurada, C., Tsuzuki, M., Iwata, Y., Sakurada, S., Sakurada, T., 2009. Intrathecal substance P auguments morphineinduced antinociception: possible relevance in the production of substance P N-terminal fragments. Peptides 30, 1689e1696. Kuraishi, Y., Nagasawa, T., Hayashi, K., Satoh, M., 1995. Scratching behavior induced by pruritogenic but not algesiogenic agents in mice. Eur. J. Pharmacol. 275, 229e233. LaMotte, R.H., Lundberg, L.E., Torebjork, H.E., 1992. Pain, hyperalgesia and activity in nociceptive C units in humans after intradermal injection of capsaicin. J. Physiol. 448, 749e764. Lee, C.M., Sandberg, B.E.B., Hanley, M.R., Iversen, L.L., 1981. Purification and characterization of a membrane-bound substance P degrading enzyme from human brain. Eur. J. Biochem. 114, 315e327. Lever, I.J., Pezet, S., McMahon, S.B., Malcangio, M., 2003. The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn. Mol. Cell Neurosci. 24, 259e270.
613
Lewis, T.S., Shapiro, P.S., Ahn, N.G., 1998. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74, 49e139. Matsas, R., Kenny, A.J., Turner, A.J., 1984. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues by endopeptidase-24.11. J. Biochem. 223, 433e440. Meller, S.T., Gebhart, G.F., 1993. Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 52, 127e136. Nyberg, F., Terenius, L., 1991. Enzymatic inactivation of neuropeptides. In: Henriksen, J.H. (Ed.), Degradation of Bioactive Substances, Physiology and Pathophysiology. CRC Press Inc., New York, pp. 189e200. Pernow, B., 1983. Substance P. Pharmacol. Rev. 35, 85e141. Reeh, P.W., Kocher, L., Jung, S., 1986. Does neurogenic inflammation alter the sensitivity of unmyelinated nociceptors in the rats? Brain Res. 384, 42e50. Sakurada, T., Le Greves, P., Terenius, L., 1985. Measurement of substance P metabolites in rat CNS. J. Neurochem. 44, 718e722. Sakurada, T., Kuwahara, H., Takahashi, K., Sakurada, S., Kisara, K., Terenius, L., 1988. Substance P (1-7) antagonizes substance P-induced aversive behaviour in mice. Neurosci. Lett. 95, 281e285. Sakurada, T., Yamada, T., Tan-No, K., Manome, Y., Sakurada, S., Kisara, K., Ohba, M., 1991. Differential effects of substance P analogs on neurokinin 1 receptor agonists in the mouse spinal cord. J. Pharmacol. Exp. Ther. 259, 205e210. Sakurada, T., Katsumata, K., Tan-No, K., Sakurada, S., Kisara, K., 1992. The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord. Neuropharmacology 12, 1279e1285. Sakurada, T., Yogo, H., Katsumata, K., Tan-No, K., Sakurada, S., Kisara, K., Ohba, M., 1994. Differential antinociceptive effects of senside, a NK-receptor antagonist, and morphine in the capsaicin test. Brain Res. 649, 319e322. Sakurada, T., Wako, K., Sugiyama, A., Sakurada, C., Tan-No, K., Kisara, K., 1998. Involvement of spinal NMDA receptors in capsaicin-induced nociception. Pharmacol. Biochem. Behav. 59, 339e345. Sakurada, C., Watanabe, C., Sakurada, T., 2002. Occurrence of substance P (1-7) in the metabolism of substance P and its antinociceptive activity at the mouse spinal cord level. Methods Find. Exp. Clin. Pharmacol. 24, 39e44. Sakurada, T., Matsumura, T., Moriyama, T., Sakurada, C., Ueno, S., Sakurada, S., 2003. Differential effects of intraplantar capsazepine and ruthenium red on capsaicininduced desensitization in mice. Pharmacol. Biochem. Behav. 75, 115e121. Sakurada, T., Komatsu, T., Kuwahata, H., Watanabe, C., Orito, T., Sakurada, C., Tsuzuki, M., Sakurada, S., 2007. Intrathecal substance P (1-7) prevents morphine-evoked spontaneous pain behavior via spinal NMDA-NO cascade. Biochem. Pharmacol. 74, 758e767. Schaible, H.G., Jarrott, B., Hope, P.J., Duggan, A.W., 1990. Release of immunoreactive substance P in the spinal cord during development of acute arthritis in the knee joint of the cat: a study with antibody microprobes. Brain Res. 529, 214e223. Simone, D.A., Sorkin, L.S., Oh, U., Chung, J.M., Owens, C., LaMotte, R.H., Willis, W.D., 1991. Neurogenic hyperalgesia: central neural correlates in responses of spinothalamic tract neurons. J. Neurophysiol. 66, 228e246. Skilling, S.R., Smullin, D.H., Larson, A.A., 1990. Differential effects of C- and Nterminal substance P metabolites on the release of amino acid neurotransmitters from the spinal cord: potential role of nociception. J. Neurosci 10, 1309e1318. Thomas, K.L., Hunt, S.P., 1993. The regional distribution of extracellularly regulated kinase-1 and -2 messenger RNA in the adult rat central nervous system. Neuroscience 56, 741e757. Tsuda, M., Ueno, H., Kataoka, A., Tozaki-Saitoh, H., Inoue, K., 2008. Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signaling. Glia 56, 378e386. Wang, H., Dai, Y., Fukuoka, T., Yamanaka, H., Obata, K., Tokunaga, A., Noguchi, K., 2004. Enhancement of stimulation-induced ERK activation in the spinal dorsal horn and gracile nucleus in rats with peripheral nerve injury. Eur. J. Neurosci. 19, 884e890. Widmann, C., Gibson, S., Jarpe, M.B., Johnson, G.L., 1999. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79, 143e180. Zhao, P., Waxman, S.G., Hains, B.C., 2007. Extracellular signal-regulated microglianeuron signaling by prostaglandin E2 contributes to pain after spinal cord injury. J. Neurosci. 27, 2357e2368. Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is essentially activated in neurons, microglia, and astrocytes by spinal ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114, 149e159. Zimmermann, M., 1983. Ethical guidelines for investigation of experimental pain in conscious animals. Pain 16, 109e110.