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Angiotensin II produces nociceptive behavior through spinal AT1 receptor-mediated p38 mitogen-activated protein kinase activation in mice

Abstract

Background

It has been demonstrated that angiotensin II (Ang II) participates in either the inhibition or the facilitation of nociceptive transmission depending on the brain area. Neuronal Ang II is locally synthesized not only in the brain, but also in the spinal cord. Though the spinal cord is an important area for the modulation of nociception, the role of spinal Ang II in nociceptive transmission remains unclear. Therefore, in order to elucidate the role of Ang II in nociceptive transmission in the spinal cord, we examined the effect of intrathecal (i.t.) administration of Ang II into mice.

Results

I.t. administration of Ang II produced a behavioral response in mice mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by Ang II (3 pmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.1-0.3 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also inhibited dose-dependently by i.t. co-administration of losartan (0.3-3 nmol), an Ang II type 1 (AT1) receptor antagonist, and SB203580 (0.1-1 nmol), a p38 MAPK inhibitor. However, the Ang II type 2 (AT2) receptor antagonist PD123319, the upstream inhibitor of ERK1/2 phosphorylation U0126, and the JNK inhibitor SP600125 had no effect on Ang II-induced nociceptive behavior. Western blot analysis showed that the i.t. injection of Ang II induced phosphorylation of p38 MAPK in the lumbar dorsal spinal cord, which was inhibited by losartan, without affecting ERK1/2 and JNK. Furthermore, we found that AT1 receptor expression was relatively high in the lumbar superficial dorsal horn.

Conclusions

Our data show that i.t. administration of Ang II induces nociceptive behavior accompanied by the activation of p38 MAPK signaling mediated through AT1 receptors. This observation indicates that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.

Background

Angiotensin II (Ang II), a main bioactive component of the renin-angiotensin system (RAS), plays a critical role in sympathetic regulation, cardiovascular control, fluid balance and hormone secretion (for review, see Refs[1, 2]). In the RAS, renin converts angiotensinogen to angiotensin I (Ang I), which in turn is cleaved by angiotensin-converting enzyme (ACE) to Ang II. Ang II mediates its biological effects through Ang II type 1 (AT1) receptors and Ang II type 2 (AT2) receptors, which are seven transmembrane receptors with approximately 30% amino acid sequence similarity. Most species express a single type of AT1 receptors, but two related AT1A and AT1B receptor subtypes are expressed in rodents (for review, see Ref[3]). Ang II is not only generated by circulating ACE, but also produced locally in tissues. The existence of local tissue-based RAS, independent of the classical circulating RAS, has been established in several organs (for review, see Ref[4]). The tissue RAS is characterised by the presence of all RAS components, including angiotensinogen, renin, ACE, Ang I, Ang II and Ang II receptors, and is found in the heart[5], blood vessels[6], kidney[7], pancreas[8], brain[9] and adipose tissue[10]. Evidence indicates that Ang II is involved in the modulation of nociceptive transmission. Namely, Ang II causes hyperalgesia in the caudal ventrolateral medulla (CVLM)[11] and hypoalgesia in the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM)[12–14]. However, the role of spinal Ang II in the modulation of nociceptive transmission remains unclear.

Ang II acts as an activator of mitogen-activated protein kinase (MAPK)[15–17], a family of Ser/Thr kinases that convert extracellular stimuli into a wide range of cellular responses. The MAPKs include extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminus kinase (JNK) and p38 MAPK. These MAPKs have common activation motif (T-X-Y), which are phosphorylated by MAPK kinase. It has been reported that ERK1/2 and JNK are activated in several pain models involving peripheral inflammation, noxious heat and electric stimulation, and that the corresponding nociceptive behaviors are blocked by their respective kinases inhibitor[18–21]. In addition, p38 MAPK, which is activated by cellular stress and proinflammatory cytokines, is considered as a stress-induced kinase and plays a critical role in inflammatory responses. Spinal p38 MAPK is activated by complete Freund's adjuvant (CFA)-induced peripheral inflammation and nociceptive responses accompanying the inflammation are markedly decreased by p38 MAPK inhibitor[22]. Inhibition of p38 MAPK also reduces the mRNA expression of proinflammatory cytokines such as IL-1β, IL-6 and TNFα[22]. These observations indicate that ERK1/2, JNK and p38 MAPK are involved in the facilitation of nociceptive transmission.

We have previously found that intrathecal (i.t.) administration into mice of dynorphin[23, 24], spermine[25], D-cycloserine[26] and serotonin releaser[27] produces nociceptive behavior. In the present study, we found that i.t.-administered Ang II also produced nociceptive behavior. To gain insight into the mechanism of Ang II-induced nociceptive behavior, we determined whether Ang II receptor subtypes and MAPK signaling were involved.

Results

Behavioral response induced by i.t.-administered Ang II

I.t.-administered Ang II (3 pmol) produced a characteristic behavioral response consisting of scratching, biting and licking, which almost disappeared 25 min after the injection (Figure 1a). Two-way repeated-measures ANOVA revealed significant effects of the treatment (F1,18 = 6.89, p < 0.05) and time (F5,90 = 2.41, p < 0.05) but not treatment × time interaction (F5,90 = 0.89, p = 0.17). As seen in Figure 1b, a dose-dependent increase in the total time of scratching, biting and licking for 25 min was observed following i.t. administration of Ang II (1–3 pmol). One-way ANOVA revealed a significant effect of treatment (F3,36 = 3.47, p < 0.05). A post-hoc test demonstrated a significant increase in the behavioral responses induced by injection of Ang II (3 pmol) compared to the Ringer-administered group (p < 0.05). Therefore, the latter dose of Ang II was used in subsequent injections which were followed by a 25 min observation period.

Figure 1
figure 1

Scratching, biting and licking responses induced by i.t.-administered Ang II in mice. (a) Time course of behavioral response induced by Ang II (3 pmol) or Ringer’s solution alone. The ordinate shows the total time of scratching, biting and licking that occurred during each 5 min of measurement. (b) Effects of varying doses of Ang II (1–3 pmol/mouse). The duration of scratching, biting and licking induced by Ang II or Ringer’s solution was determined over a 25 min period starting immediately after i.t. injection. Values represent the means ± S.E.M. for groups of 10 mice. *p < 0.05 compared with Ringer controls.

To determine whether the Ang II-induced behavior is related to nociception, we examined the effect of a pretreatment with morphine. As shown in Figure 2, morphine (0.1-0.3 mg/kg, i.p.) inhibited the Ang II-induced behavior in a dose-dependent manner with an ID50 value of 0.19 (0.14-0.27) mg/kg, suggesting that the behavioral response is related to nociception (one-way ANOVA analysis, F4,45 = 3.34, p < 0.05; post hoc test, p < 0.01 for Ringer versus Ang II, p < 0.05 for Ang II versus Ang II plus 0.3 mg/kg morphine).

Figure 2
figure 2

Effect of morphine on Ang II-induced scratching, biting and licking responses in mice. Morphine was administered i.p. 5 min prior to injection of Ang II (3 pmol). The duration of scratching, biting and licking induced by Ang II was determined over a 25 min period starting immediately after i.t. injection. Values represent the means ± S.E.M. for groups of 10 mice. **p < 0.01 compared with Ringer controls and # p < 0.05 compared with Ang II alone.

Effects of Ang II receptor antagonists on Ang II-induced nociceptive behavior

To determine which type of Ang II receptors is involved in the nociceptive behavior, we compared the effects of losartan, an AT1 receptor antagonist, to PD123319, an AT2 receptor antagonist. Losartan (0.3-3 nmol) co-administered i.t. with Ang II caused a dose-dependent inhibition of Ang II-induced nociceptive behavior with an ID50 value of 0.55 (0.47-0.63) nmol (one-way ANOVA analysis, F4,45 = 3.45, p < 0.05; post hoc test, p < 0.01 for Ringer versus Ang II, p < 0.05 for Ang II versus Ang II plus 1 and 3 nmol losartan, Figure 3a). In contrast, i.t.-administered PD123319 (1 and 3 nmol) did not affect the nociceptive behavior induced by Ang II (one-way ANOVA analysis, F3,36 = 2.74, p < 0.05; post hoc test, p > 0.05 for Ang II versus Ang II plus 1 and 3 nmol PD123319, Figure 3b). These results indicate i.t. Ang II-induced nociceptive behavior is mediated through AT1 receptors but not through AT2 receptors.

Figure 3
figure 3

Effects of Ang II receptor antagonists on i.t. Ang II-induced nociceptive behavior in mice. Losartan (a) or PD123319 (b) was co-administered i.t. with Ang II (3 pmol). The duration of scratching, biting and licking induced by Ang II was determined over a 25 min period starting immediately after i.t. injection. Values represent the means ± S.E.M. for groups of 10 mice. **p < 0.01 compared with Ringer or vehicle (6.8% DMSO) controls and # p < 0.05 compared with Ang II alone. n.s., not significant.

Distribution of AT1 receptors in mouse spinal cord

The distribution of AT1 receptor fluorescence intensity in mouse spinal cord (L5) was determined by microphotometry and categorized into 18 levels (shown as different colors in Figure 4b, with the lowest concentration shown as black and the highest concentration represented by white). Relatively high intensity of AT1 receptor fluorescence was seen in the superficial dorsal horn (laminae I and II).

Figure 4
figure 4

Distribution of the immunohistochemical fluorescence intensity for AT 1 receptors in mouse lumbar spinal cord (L5). (a) Diagram representing segment L5 of the spinal cord. (b) Quantitative immunohistochemical distribution of AT1 receptors in the lumbar spinal cord.

Effects of MEK and MAPK inhibitors on Ang II-induced nociceptive behavior

The role of ERK1/2, JNK and p38 MAPK signaling in Ang II-induced nociceptive behavior was examined using the inhibitors U0126, SP600125, and SB203580, respectively. U0126 (1 and 3 nmol) co-administered i.t. with Ang II did not affect the nociceptive behavior induced by Ang II (one-way ANOVA analysis, F3,36 = 5.11, p < 0.01; post hoc test, p > 0.05 for Ang II versus Ang II plus 1 and 3 nmol U0126, Figure 5a). Similarly, SP600125 (0.3 and 3 nmol) did not affect the nociceptive behavior induced by Ang II (one-way ANOVA analysis, F3,36 = 5.82, p < 0.01; post hoc test, p > 0.05 for Ang II versus Ang II plus 0.3 and 3 nmol SP600125, Figure 5b). On the other hand, i.t.-administered SB203580 (0.1-1 nmol) caused a dose-dependent inhibition of Ang II – induced nociceptive behavior with an ID50 value of 0.34 (0.32-0.37) nmol (one-way ANOVA analysis, F4,45 = 4.72, p < 0.01; post hoc test, p < 0.05 for Ang II versus Ang II plus 1 nmol SB203580, Figure 5c). These results suggest that p38 MAPK, but not ERK1/2 and JNK is critically involved in the nociceptive behavior produced by Ang II.

Figure 5
figure 5

Effects of MEK and MAPK inhibitors on i.t. Ang II-induced nociceptive behavior in mice. U0126 (a), SP600125 (b) and SB203580 (c) were co-administered i.t. with Ang II (3 pmol). The duration of scratching, biting and licking induced by Ang II was determined over a 25 min period starting immediately after i.t. injection. Values represent the means ± S.E.M. for groups of 10 mice. **p < 0.01 compared with vehicle (6.8% DMSO) controls and #p < 0.05 compared with Ang II alone. n.s., not significant.

Phosphorylation of MAPKs in the dorsal spinal cord after i.t. injection of Ang II

To investigate whether spinal MAPKs were activated by i.t. injection of Ang II (3 pmol), we examined the phosphorylation of ERK1/2, JNK and p38 MAPK in the lumber dorsal cord extracted 10 min after i.t. injection by Western blotting. Ang II did not affect the phosphorylation of ERK1/2 (t = 0.47, p > 0.05, Figure 6a) and JNK (t = 0.97, p > 0.05, Figure 6b). As shown in Figure 6c and d, Ang II increased the phosphorylation of p38 MAPK in the lumber dorsal cord. In addition, as seen in Figure 6c, losartan inhibited the p38 MAPK phosphorylation induced by Ang II (one-way ANOVA analysis, F2,9 = 4.50, p < 0.05; post hoc test, p < 0.05 for Ang II versus Ang II plus 3 nmol losartan). In contrast, PD123319 did not affect the p38 MAPK phosphorylation induced by Ang II (one-way ANOVA analysis, F2,9 = 6.99, p < 0.05; post hoc test, p > 0.05 for Ang II versus Ang II plus 3 nmol PD123319, Figure 6d). These results indicate that i.t.-administered Ang II produces p38 MAPK phosphorylation mediated through AT1 receptors but not through AT2 receptors in the lumber dorsal cord.

Figure 6
figure 6

Alterations in spinal MAPKs phosphorylation by Ang II and the effects of losartan and PD123319. Dorsal spinal cord samples were taken 10 min after i.t. injection of Ang II (3 pmol). Phosphorylation of ERK1/2 (a), JNK (b) and p38 MAPK (c, d) were examined by Western blotting. Losartan (3 nmol) or PD123319 (3 nmol) was co-administered i.t. with Ang II. Top: representative Western blot of total- and phospho-MAPKs. Bottom: quantification of phospho-MAPKs to total-MAPKs set as 1.0 in the Ringer- or vehicle (6.8% DMSO)-treated group. Each value represents the means ± S.E.M. of 4 mice in each group. *p < 0.05 compared with Ringer or vehicle controls and #p < 0.05 compared with Ang II alone. n.s., not significant.

Discussion

In the present study, we demonstrated for the first time that i.t.-administered Ang II in mice induced a characteristic behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, indicative of nociceptive responses, accompanied by the activation of p38 MAPK mediated through AT1 receptors.

Ang II was originally discovered as a potent vasoconstrictor, while recent studies have shown that Ang II affects a wide range of central and peripheral components of sensory systems[13, 28, 29]. It has been demonstrated that the administration of Ang II either i.c.v. or directly in key components of the supraspinal pain modulatory system, namely the PAG or RVM (for review, see Ref[30]), induces antinociceptive effects, which are reversed by losartan[12, 13, 31, 32]. On the other hand, Marques-Lopes et al.[11] have recently reported that the microinjection of Ang II into the CVLM induces hyperalgesia through AT1 receptors, and that the effect of Ang II on spinal nociceptive processing is likely indirect, since few AT1 receptor-expressing CVLM neurons were found to project to the spinal cord. These reports lead us to suggest that supraspinal Ang II may participate in both inhibition and facilitation of the nociceptive transmission and its effect is region-dependent. However, the role of Ang II in the modulation of nociceptive transmission in the spinal cord has not been reported until this study. Therefore, it is important to clarify the role of spinal Ang II in the modulation of nociception.

Recently, it has been reported that Ang II is colocalized with calcitonin gene-related peptide (CGRP) and substance P, the neuropeptides established as the key regulators of sensory transmission and nociception, in rat and human dorsal root ganglia (DRG)[33]. Takai et al.[34] have revealed that repeated oral administration of AT1 receptor antagonist and ACE inhibitors show antinociceptive effect in hot-plate test. Furthermore, we have found that i.t.-administered losartan produces antinociceptive effect in a mouse formalin test (data not shown). These findings suggest that Ang II may act as a neurotransmitter and/or neuromodulator in the transmission of nociceptive information in the spinal cord. In the present study, we found that i.t.-administered Ang II (3 pmol) produced a nociceptive behavior consisting of scratching, biting and licking. We also observed that the Ang II-induced nociceptive behavior was inhibited by losartan but not by PD123319, indicationg that receptor type 1 and not type 2 for Ang II is involved. Regarding the distribution of spinal AT1 receptors, Pavel et al.[35] have reported that the receptors are present in high density in the lumbar superficial dorsal horn (laminae I and II) using autoradiography in rat. In this study, we also detected a relatively high intensity of fluorescence for AT1 receptors in the mouse lumbar superficial dorsal horn. Our results obtained with behavioral and immunohistchemical experiments suggest that spinal AT1 receptors are responsible for the nociceptive response.

Ang II induced two peaks of nociceptive behavior, one at 5–10 and the other 20–25 min after injection, although there was no significant difference between time × treatment interaction. The hydrolysis of Ang II by a homogenate of rat ventrolateral PAG forms Ang III, a major hydrolysis product, Ang IV, Ang (1–7) and Ang (1–4)[36]. Moreover, microinjection of Ang III into the ventrolateral PAG produces the antinociceptive effect mediated through AT1 and AT2 receptors[36]. Therefore, we may speculate that in our time course experiment, Ang II is responsible for the first peak while Ang III generated from Ang II is responsible for the second peak.

ERK1/2, JNK and p38 MAPK are phosphorylated in the presence of Ang II in mouse atrial fibroblasts[15] and natural killer cells[16], while only ERK1/2 and p38 MAPK but not JNK are phosphorylated by Ang II in RVM[17]. In addition, Sung et al.[37] have reported that i.t.-administered IL-1β activates only p38 MAPK without affecting ERK1/2 and JNK in the spinal cord. Similarly, in this study, only the spinal p38 MAPK was activated after i.t. administration of Ang II, although the ERK1/2, JNK and p38 MAPK were constitutively expressed in the spinal cord. There are four p38 MAPK isoforms: p38α, p38β, p38γ and p38δ. Whereas p38α and p38β are two of the major isoforms in the mature nervous system, p38α is the most abundant isoform in DRG neuron and spinal cord (for review, see[38]). Therefore, we used SB203580 to inhibit p38 MAPK signaling in the spinal cord since it can inhibit the activity of both p38α and p38β isoforms[39]. In this study, the behavioral observation revealed that Ang II-induced nociceptive response was almost completely inhibited by SB203580. On the other hand, neither U0126 nor SP600125 affected the Ang II-induced nociceptive behavior. Ample evidence suggest that the spinal p38 MAPK is involved in several types of pain. Phosphorylation of spinal p38 MAPK has been observed not only in neuropathic pain models such as chronic constriction injury[40, 41] and spinal nerve ligation[42–44], but also in peripheral inflammation induced by CFA[22], bee-venom[45], formalin[46–48] and capsaicin[48]. Moreover, i.t. administration of N-methyl-D-aspartate (NMDA) produces thermal hyperalgesia through spinal p38 MAPK phosphorylation[49]. Taken together with these previous reports, our present results indicate that the phosphorylation of spinal p38 MAPK, but not of the other MAPKs, is involved in Ang II-induced nociceptive behavior. In addition, since the nociceptive behavior arises rapidly and declines within 25 min to resemble controls, we suggest that the phosphorylation of p38 MAPK leads to the behavior via non-transcriptional mechanisms. Mizushima et al.[50] have reported that intraplantar injection into rats of capsaicin induces phosphorylation of p38 MAPK in DRG neurons and thermal hyperalgesia which peak at 2–5 min after injection. Although the specific target proteins of p38 MAPK are not clearly identified, p38 MAPK signaling pathway leads to Ang II-induced nociceptive behavior through post-transcriptional modifications of kinases, receptors and ion-channels.

Finally, we examined the effects of Ang II receptor antagonists on p38 MAPK phosphorylation in the dorsal spinal cord. Whereas p38 MAPK phosphorylation was inhibited by losartan, it was resistant against PD123319, and these results were consistent with those of the behavioral experiments. It has been reported that Ang II increases the phosphorylation of p38 MAPK in cultured rat neonatal cardiomyocytes, which is attenuated by losartan similarly to SB205380, a p38 MAPK inhibitor, and p38 siRNA[51]. Taken together, the present results suggest that phosphorylation of p38 MAPK mediated through AT1 but not AT2 receptors contributes to i.t. Ang II-induced nociceptive behavior.

Conclusions

In conclusion, our data show that i.t.-administered Ang II induces nociceptive behavior accompanied by p38 MAPK phosphorylation mediated through spinal AT1 receptors. Moreover, it is suggested that Ang II may be a neurotransmitter and/or neuromodulator in the transmission of nociceptive information in the spinal cord.

Methods

Animals

Male ddY strain mice (weighing 22–24 g, Japan SLC, Japan) were used in all experiments. Mice were housed in cages with free access to food and water under conditions of constant temperature (22 ± 2°C) and humidity (55 ± 5%), on a 12 h light–dark cycle (lights on: 08:00 to 20:00). Groups of 10 mice for behavioral experiments and 4 mice for Western blotting and immunohistchemical experiments were used in single experiments. All experiments were performed following the approval from the Ethics Committee of Animal Experiment in Tohoku Pharmaceutical University and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize suffering and to reduce the number of animals used.

Intrathecal injections

The i.t. injections were made in unanaesthetized mice at the L5, L6 intervertebral space as described by Hyden and Wilcox[52]. Briefly, a volume of 5 μl was administered i.t. with a 28-gauge needle connected to a 50-μl Hamilton microsyringe, the animal being lightly restrained to maintain the position of the needle. Puncture of the dura was indicated behaviorally by a slight flick of the tail.

Behavioral observation

Approximately 60 min before the i.t. injection, the mice were habituated to an individual cage (22.0 × 15.0 × 12.5 cm) which was also used as the observation chamber after injection. Immediately after the i.t. injection, the mice were placed in the transparent cage and the accumulated response time of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw and the tail was measured for 25 min with the exception of the 30 min time course experiment in which the response was divided into 5 min intervals.

Drugs and antibodies

The following drugs and chemicals were used: Ang II (Peptide Institute, Japan); morphine hydrochloride (Sankyo, Japan); losartan potassium (LKT Laboratories, USA); 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319), 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), 4-[5-(4-fluorophenyl)-2-[4-(methylsulphonyl) phenyl]-1H-imidazol-4-yl]pyridine hydrochloride (SB203580 hydrochloride) (Tocris Biosciense, USA); anthra (1,9-cd) pyrazol-6(2H)-one, 1,9-pyrazoloanthrone (SP600125) (Alexis, USA); sodium pentobarbital (Dainippon Sumitomo Pharma, Japan); antibodies against ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, p38 MAPK, phospho-p38 MAPK, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology, USA); anti-AT1 receptor antibody (Alpha Diagnostic, USA); enhanced chemiluminescence (ECL) assay kit (GE Healthcare, England). For i.t. injections, Ang II and losartan were dissolved in Ringer’s solution. PD123319, U0126, SB203580 and SP600125 were dissolved in Ringer’s solution containing 6.8% dimethyl sulfoxide (DMSO). When the effects of Ang II receptor antagonists and MAPK-related inhibitors were tested, they were co-injected with Ang II in a volume of 5 μl. Morphine was dissolved in physiological saline and administered intraperitoneally (i.p.) 5 min prior to injection of Ang II.

Immunohistochemical staining

Spinal cords for measurement of AT1 receptors were prepared within 24 h following delivery. Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused through the heart with ice-cold phosphate-buffered saline (PBS, pH 7.2), immediately followed by a fixative containing 4% paraformaldehyde (Sigma–Aldrich, USA) and 0.2% glutaraldehyde (Nacalai Tesque, Japan) in PBS. Spinal cords (lumbar 5; L5) were then postfixed with the same fixative solution at 4°C for 1 h and then placed in a 20% sucrose-buffered solution at 4°C for 12 h. Tissues were frozen on dry ice and cut into 20 μm-thick coronal sections on a cryostat (Micro-edge Instruments Co. Ltd., Germany). The immunohistochemical staining procedure was carried out as previously described[53]. Briefly, a rabbit anti-AT1 receptor antibody (diluted 1:100 with PBS and 5% normal goat serum (NGS); Millipore Co., USA) was applied to spinal cord slices, which were then incubated at 4°C for 12 h. The secondary antibody consisted of FITC-labeled anti-rabbit IgG goat serum (diluted 1:200 with PBS; Millipore Co.), and was allowed to react in the dark at room temperature for 2 h. The stained sections were mounted in Dako Fluorescence Mounting Medium (Dako North America, USA), and kept at 4°C in a dark room until measurements were carried out. The distribution of AT1 receptor immunofluorescence intensities was quantitatively analyzed using a MapAnalyzer (Yamato Scientific Co., Japan). The background value, including non-specific fluorescence originating from glutaraldehyde, was subtracted photometrically from the total fluorescence intensity value at each point measured.

SDS-polyacrylamide gel electrophoresis and immunoblotting

Samples used for immunoblotting were prepared as follows. At 10 min after i.t. injection, mice were decapitated and the whole spinal cord was taken by pressure expulsion with physiological saline. The dorsal part of lumbar spinal cord was dissected quickly on ice-cooled glass dish. The tissue samples were homoginaized in 0.15 ml of CelLytic™ MT Manmalian Tissue Lysis/Extraction Reagent (Sigma Aldrich, USA) and centrifuged the lysis sample at 15,000× g for 15 min at 4°C. Supernatants were dissolved in 4 × Laemmli sample buffer (300 mM Tris–HCl pH 6.8, 8% SDS, 40% glycerol, 12% 2-mercaptoethanol and 0.012% bromophenol blue), and boiled at 95°C for 10 min. Electrophoresis was performed on 10% acrylamide gels. Proteins were transferred electrically from the gel onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Japan) by the semi-dry blotting method. The blots were blocked for 30 min with 5% skim-milk in Tris-buffered saline supplemented with 0.1% Tween-20, and incubated with primary antibodies overnight at 4°C. The blots were washed several times and then incubated at room temperature for 2 h with a secondary antibody (HRP-conjugated anti-rabbit IgG antibody). Blots were developed using an enhanced chemiluminescence assay kit, and visualized by chemiluminescence on Hyper-film ECL. The densities of the bands were analyzed by densitometry (Image-J 1.43u, National Institute of Health).

Statistical methods

Data were expressed as mean ± SEM. The ID50 values with 95% confidence limits were calculated for reduction in Ang II-induced scratching, biting and licking response by a computer-associated curve-fitting program (GraphPad Prism; GraphPad Software, USA). The significant differences were analyzed by a one-way or two-way analysis of variance (ANOVA), followed by Fisher’s PLSD test for multiple-comparisons. Student’s t test was used for comparisons between two groups. In all comparisons, P < 0.05 was considered statistical significance.

Abbreviations

ACE:

angiotensin-converting enzyme

Ang:

Angiotensin

AT1:

Ang II type 1

AT2:

Ang II type 2

CFA:

complete Freund's adjuvant

CGRP:

calcitonin gene-related peptide

CVLM:

caudal ventrolateral medulla

DMSO:

dimethyl sulfoxide

DRG:

dorsal root ganglia

ERK:

extracellular signal-regulated kinase

ID50:

inhibitory dose 50%

i.p.:

intraperitoneal

i.t.:

intrathecal

JNK:

c-Jun N-terminus kinase

MAPK:

mitogen-activated protein kinase

NMDA:

N-methyl-D-aspartate

PAG:

periaqueductal gray

RAS:

renin-angiotensin system

RVM:

rostral ventromedial medulla.

References

  1. Paul M, Poyan Mehr A, Kreutz R: Physiology of local renin-angiotensin systems. Physiol Rev 2006, 86: 747. 10.1152/physrev.00036.2005

    Article  CAS  PubMed  Google Scholar 

  2. de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T: International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 2000, 52: 415–472.

    CAS  PubMed  Google Scholar 

  3. Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, Smith RD: Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 1993, 45: 205–251.

    CAS  PubMed  Google Scholar 

  4. Bader M: Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy. Annu Rev Pharmacol Toxicol 2010, 50: 439–465. 10.1146/annurev.pharmtox.010909.105610

    Article  CAS  PubMed  Google Scholar 

  5. Villarreal FJ, Kim NN, Ungab GD, Printz MP, Dillmann WH: Identification of functional angiotensin II receptors on rat cardiac fibroblasts. Circulation 1993, 88: 2849–2861. 10.1161/01.CIR.88.6.2849

    Article  CAS  PubMed  Google Scholar 

  6. Nguyen Dinh Cat A, Touyz RM: A new look at the renin-angiotensin system--focusing on the vascular system. Peptides 2011, 32: 2141–2150. 10.1016/j.peptides.2011.09.010

    Article  CAS  PubMed  Google Scholar 

  7. Zhuo JL, Li XC: New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II. Peptides 2011, 32: 1551–1565. 10.1016/j.peptides.2011.05.012

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Leung PS: Pancreatic RAS. Adv Exp Med Biol 2010, 690: 89–105. 10.1007/978-90-481-9060-7_6

    Article  PubMed  Google Scholar 

  9. Wright JW, Harding JW: Brain renin-angiotensin–a new look at an old system. Prog Neurobiol 2011, 95: 49–67. 10.1016/j.pneurobio.2011.07.001

    Article  CAS  PubMed  Google Scholar 

  10. Thatcher S, Yiannikouris F, Gupte M, Cassis L: The adipose renin-angiotensin system: role in cardiovascular disease. Mol Cell Endocrinol 2009, 302: 111–117. 10.1016/j.mce.2009.01.019

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Marques-Lopes J, Pinto M, Pinho D, Morato M, Patinha D, Albino-Teixeira A, Tavares I: Microinjection of angiotensin II in the caudal ventrolateral medulla induces hyperalgesia. Neuroscience 2009, 158: 1301–1310. 10.1016/j.neuroscience.2008.11.044

    Article  CAS  PubMed  Google Scholar 

  12. Prado WA, Pelegrini-da-Silva A, Martins AR: Microinjection of renin-angiotensin system peptides in discrete sites within the rat periaqueductal gray matter elicits antinociception. Brain Res 2003, 972: 207–215. 10.1016/S0006-8993(03)02541-1

    Article  CAS  PubMed  Google Scholar 

  13. Pelegrini-da-Silva A, Martins AR, Prado WA: A new role for the renin-angiotensin system in the rat periaqueductal gray matter: angiotensin receptor-mediated modulation of nociception. Neuroscience 2005, 132: 453–463. 10.1016/j.neuroscience.2004.12.046

    Article  CAS  PubMed  Google Scholar 

  14. Yien HW, Chan JY, Tsai HF, Lee TY, Chan SH: Participation of nucleus reticularis gigantocellularis in the antinociceptive effect of angiotensin III in the rat. Neurosci Lett 1993, 159: 9–12. 10.1016/0304-3940(93)90785-J

    Article  CAS  PubMed  Google Scholar 

  15. Gu J, Liu X, Wang QX, Tan HW, Guo M, Jiang WF, Zhou L: Angiotensin II increases CTGF expression via MAPKs/TGF-beta1/TRAF6 pathway in atrial fibroblasts. Exp Cell Res 2012, 318: 2105–2115. 10.1016/j.yexcr.2012.06.015

    Article  CAS  PubMed  Google Scholar 

  16. Yang L, Du C, Chen T, Li S, Nie W, Zhu W, Fan F, Zhu J, Yan H: Distinct MAPK pathways are involved in IL-23 production in dendritic cells cocultured with NK cells in the absence or presence of angiotensin II. Mol Immunol 2012, 51: 51–56. 10.1016/j.molimm.2012.02.004

    Article  CAS  PubMed  Google Scholar 

  17. Chan SH, Hsu KS, Huang CC, Wang LL, Ou CC, Chan JY: NADPH oxidase-derived superoxide anion mediates angiotensin II-induced pressor effect via activation of p38 mitogen-activated protein kinase in the rostral ventrolateral medulla. Circ Res 2005, 97: 772–780. 10.1161/01.RES.0000185804.79157.C0

    Article  CAS  PubMed  Google Scholar 

  18. Ji RR, Baba H, Brenner GJ, Woolf CJ: Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 1999, 2: 1114–1119. 10.1038/16040

    Article  CAS  PubMed  Google Scholar 

  19. Dai Y, Iwata K, Fukuoka T, Kondo E, Tokunaga A, Yamanaka H, Tachibana T, Liu Y, Noguchi K: Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J Neurosci 2002, 22: 7737–7745.

    CAS  PubMed  Google Scholar 

  20. Ma W, Quirion R: Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain 2002, 99: 175–184. 10.1016/S0304-3959(02)00097-0

    Article  CAS  PubMed  Google Scholar 

  21. Galan A, Cervero F, Laird JM: Extracellular signaling-regulated kinase-1 and −2 (ERK 1/2) mediate referred hyperalgesia in a murine model of visceral pain. Brain Res Mol Brain Res 2003, 116: 126–134. 10.1016/S0169-328X(03)00284-5

    Article  CAS  PubMed  Google Scholar 

  22. Boyle DL, Jones TL, Hammaker D, Svensson CI, Rosengren S, Albani S, Sorkin L, Firestein GS: Regulation of peripheral inflammation by spinal p38 MAP kinase in rats. PLoS Med 2006, 3: e338. 10.1371/journal.pmed.0030338

    Article  PubMed Central  PubMed  Google Scholar 

  23. Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K: Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism. Brain Res 2002, 952: 7–14. 10.1016/S0006-8993(02)03180-3

    Article  CAS  PubMed  Google Scholar 

  24. Tan-No K, Takahashi H, Nakagawasai O, Niijima F, Sato T, Satoh S, Sakurada S, Marinova Z, Yakovleva T, Bakalkin G, et al.: Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation. Pain 2005, 113: 301–309. 10.1016/j.pain.2004.11.004

    Article  CAS  PubMed  Google Scholar 

  25. Tan-No K, Taira A, Wako K, Niijima F, Nakagawasai O, Tadano T, Sakurada C, Sakurada T, Kisara K: Intrathecally administered spermine produces the scratching, biting and licking behaviour in mice. Pain 2000, 86: 55–61. 10.1016/S0304-3959(99)00312-7

    Article  CAS  PubMed  Google Scholar 

  26. Tan-No K, Esashi A, Nakagawasai O, Niijima F, Furuta S, Sato T, Satoh S, Yasuhara H, Tadano T: Intrathecally administered D-cycloserine produces nociceptive behavior through the activation of N-methyl-D-aspartate receptor ion-channel complex acting on the glycine recognition site. J Pharmacol Sci 2007, 104: 39–45. 10.1254/jphs.FP0070203

    Article  CAS  PubMed  Google Scholar 

  27. Tan-No K, Takahashi K, Shimoda M, Sugawara M, Nakagawasai O, Niijima F, Sato T, Satoh S, Tadano T: S-(+)-fenfluramine-induced nociceptive behavior in mice: Involvement of interactions between spinal serotonin and substance P systems. Neuropeptides 2007, 41: 33–38. 10.1016/j.npep.2006.10.003

    Article  CAS  PubMed  Google Scholar 

  28. Burkhalter J, Felix D, Imboden H: A new angiotensinergic system in the CNS of the rat. Regul Pept 2001, 99: 93–101. 10.1016/S0167-0115(01)00238-5

    Article  CAS  PubMed  Google Scholar 

  29. Wu W, Zhang Y, Ballew JR, Fink G, Wang DH: Development of hypertension induced by subpressor infusion of angiotensin II: role of sensory nerves. Hypertension 2000, 36: 549–552. 10.1161/01.HYP.36.4.549

    Article  CAS  PubMed  Google Scholar 

  30. Gebhart GF: Descending modulation of pain. Neurosci Biobehav Rev 2004, 27: 729–737. 10.1016/j.neubiorev.2003.11.008

    Article  CAS  PubMed  Google Scholar 

  31. Georgieva D, Georgiev V: The role of angiotensin II and of its receptor subtypes in the acetic acid-induced abdominal constriction test. Pharmacol Biochem Behav 1999, 62: 229–232. 10.1016/S0091-3057(98)00116-6

    Article  CAS  PubMed  Google Scholar 

  32. Raghavendra V, Chopra K, Kulkarni SK: Brain renin angiotensin system (RAS) in stress-induced analgesia and impaired retention. Peptides 1999, 20: 335–342. 10.1016/S0196-9781(99)00040-6

    Article  CAS  PubMed  Google Scholar 

  33. Patil J, Schwab A, Nussberger J, Schaffner T, Saavedra JM, Imboden H: Intraneuronal angiotensinergic system in rat and human dorsal root ganglia. Regul Pept 2010, 162: 90–98. 10.1016/j.regpep.2010.03.004

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Takai S, Song K, Tanaka T, Okunishi H, Miyazaki M: Antinociceptive effects of angiotensin-converting enzyme inhibitors and an angiotensin II receptor antagonist in mice. Life Sci 1996, 59: PL331-PL336. 10.1016/0024-3205(96)00527-9

    Article  CAS  PubMed  Google Scholar 

  35. Pavel J, Tang H, Brimijoin S, Moughamian A, Nishioku T, Benicky J, Saavedra JM: Expression and transport of Angiotensin II AT1 receptors in spinal cord, dorsal root ganglia and sciatic nerve of the rat. Brain Res 2008, 1246: 111–122.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Pelegrini-da-Silva A, Rosa E, Guethe LM, Juliano MA, Prado WA, Martins AR: Angiotensin III modulates the nociceptive control mediated by the periaqueductal gray matter. Neuroscience 2009, 164: 1263–1273. 10.1016/j.neuroscience.2009.09.004

    Article  CAS  PubMed  Google Scholar 

  37. Sung CS, Wen ZH, Chang WK, Chan KH, Ho ST, Tsai SK, Chang YC, Wong CS: Inhibition of p38 mitogen-activated protein kinase attenuates interleukin-1beta-induced thermal hyperalgesia and inducible nitric oxide synthase expression in the spinal cord. J Neurochem 2005, 94: 742–752. 10.1111/j.1471-4159.2005.03226.x

    Article  CAS  PubMed  Google Scholar 

  38. Ji RR, Gereau RW, Malcangio M, Strichartz GR: MAP kinase and pain. Brain Res Rev 2009, 60: 135–148. 10.1016/j.brainresrev.2008.12.011

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Enslen H, Raingeaud J, Davis RJ: Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 1998, 273: 1741–1748. 10.1074/jbc.273.3.1741

    Article  CAS  PubMed  Google Scholar 

  40. Gu YW, Su DS, Tian J, Wang XR: Attenuating phosphorylation of p38 MAPK in the activated microglia: a new mechanism for intrathecal lidocaine reversing tactile allodynia following chronic constriction injury in rats. Neurosci Lett 2008, 431: 129–134. 10.1016/j.neulet.2007.11.065

    Article  CAS  PubMed  Google Scholar 

  41. Xu L, Huang Y, Yu X, Yue J, Yang N, Zuo P: The influence of p38 mitogen-activated protein kinase inhibitor on synthesis of inflammatory cytokine tumor necrosis factor alpha in spinal cord of rats with chronic constriction injury. Anesth Analg 2007, 105: 1838–1844. 10.1213/01.ane.0000287660.29297.7b

    Article  CAS  PubMed  Google Scholar 

  42. Jin SX, Zhuang ZY, Woolf CJ, Ji RR: p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci 2003, 23: 4017–4022.

    CAS  PubMed  Google Scholar 

  43. Terayama R, Omura S, Fujisawa N, Yamaai T, Ichikawa H, Sugimoto T: Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury. Neuroscience 2008, 153: 1245–1255. 10.1016/j.neuroscience.2008.03.041

    Article  CAS  PubMed  Google Scholar 

  44. Tsuda M, Mizokoshi A, Shigemoto-Mogami Y, Koizumi S, Inoue K: Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 2004, 45: 89–95. 10.1002/glia.10308

    Article  PubMed  Google Scholar 

  45. Cui XY, Dai Y, Wang SL, Yamanaka H, Kobayashi K, Obata K, Chen J, Noguchi K: Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia. Mol Pain 2008, 4: 17. 10.1186/1744-8069-4-17

    Article  PubMed Central  PubMed  Google Scholar 

  46. Kim SY, Bae JC, Kim JY, Lee HL, Lee KM, Kim DS, Cho HJ: Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. Neuroreport 2002, 13: 2483–2486. 10.1097/00001756-200212200-00021

    Article  CAS  PubMed  Google Scholar 

  47. Svensson CI, Marsala M, Westerlund A, Calcutt NA, Campana WM, Freshwater JD, Catalano R, Feng Y, Protter AA, Scott B, Yaksh TL: Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing. J Neurochem 2003, 86: 1534–1544. 10.1046/j.1471-4159.2003.01969.x

    Article  CAS  PubMed  Google Scholar 

  48. Sweitzer SM, Peters MC, Ma JY, Kerr I, Mangadu R, Chakravarty S, Dugar S, Medicherla S, Protter AA, Yeomans DC: Peripheral and central p38 MAPK mediates capsaicin-induced hyperalgesia. Pain 2004, 111: 278–285. 10.1016/j.pain.2004.07.007

    Article  CAS  PubMed  Google Scholar 

  49. Svensson CI, Hua XY, Protter AA, Powell HC, Yaksh TL: Spinal p38 MAP kinase is necessary for NMDA-induced spinal PGE(2) release and thermal hyperalgesia. Neuroreport 2003, 14: 1153–1157. 10.1097/00001756-200306110-00010

    Article  CAS  PubMed  Google Scholar 

  50. Mizushima T, Obata K, Yamanaka H, Dai Y, Fukuoka T, Tokunaga A, Mashimo T, Noguchi K: Activation of p38 MAPK in primary afferent neurons by noxious stimulation and its involvement in the development of thermal hyperalgesia. Pain 2005, 113: 51–60. 10.1016/j.pain.2004.09.038

    Article  CAS  PubMed  Google Scholar 

  51. Wang BW, Chang H, Kuan P, Shyu KG: Angiotensin II activates myostatin expression in cultured rat neonatal cardiomyocytes via p38 MAP kinase and myocyte enhance factor 2 pathway. J Endocrinol 2008, 197: 85–93. 10.1677/JOE-07-0596

    Article  CAS  PubMed  Google Scholar 

  52. Hylden JL, Wilcox GL: Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980, 67: 313–316. 10.1016/0014-2999(80)90515-4

    Article  CAS  PubMed  Google Scholar 

  53. Nakagawasai O, Hozumi S, Tan-No K, Niijima F, Arai Y, Yasuhara H, Tadano T: Immunohistochemical fluorescence intensity reduction of brain somatostatin in the impairment of learning and memory-related behaviour induced by olfactory bulbectomy. Behav Brain Res 2003, 142: 63–67. 10.1016/S0166-4328(02)00383-2

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported in part by JSPS KAKENHI Grant Number 21600012 to KT, JSPS KAKENHI Grant Number 22600010 to ON, and Matching Fund Subsidy for Private University from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Koichi Tan-No.

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WN designed, performed the experiments and wrote the manuscript. ON performed immunohistchemical experiment and analyzed the data. FY, SK and SY contributed to design of experimentation. MI and TT supervised the experiments. KT supervised the experiments, and participated in their design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

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Nemoto, W., Nakagawasai, O., Yaoita, F. et al. Angiotensin II produces nociceptive behavior through spinal AT1 receptor-mediated p38 mitogen-activated protein kinase activation in mice. Mol Pain 9, 38 (2013). https://doi.org/10.1186/1744-8069-9-38

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