Inflammatory muscle pain is dependent on the activation of kinin B₁ and B₂ receptors and intracellular kinase pathways

Abstract

Background and purpose: B(1) and B(2) kinin receptors are involved in pain transmission but they may have different roles in the muscle pain induced by intense exercise or inflammation. We investigated the contribution of each of these receptors, and the intracellular pathways involved, in the initial development and maintenance of the muscle pain associated with inflammation-induced tissue damage.

Experimental approach: Mechanical hyperalgesia was measured using the Randall-Selitto apparatus after injecting 5% formalin solution into the gastrocnemius muscle in mice treated with selective antagonists for B(1) or B(2) receptors. The expression of kinin receptors and cytokines and the activation of intracellular kinases were monitored by real-time PCR and immunohistochemistry.

Key results: The i.m. injection of formalin induced an overexpression of B(1) and B(2) receptors. This overexpression was associated with the mechanical hyperalgesia induced by formalin because treatment with B(1) receptor antagonists (des-Arg(9) [Leu(8)]-BK, DALBK, and SSR240612) or B(2) receptor antagonists (HOE 140 and FR173657) prevented the hyperalgesia. Formalin increased myeloperoxidase activity, and up-regulated TNF-α, IL-1β and IL-6 in gastrocnemius. Myeloperoxidase activity and TNF-α mRNA expression were inhibited by either DALBK or HOE 140, whereas IL-6 was inhibited only by HOE 140. The hyperalgesia induced by i.m. formalin was dependent on the activation of intracellular MAPKs p38, JNK and PKC.

Conclusions and implications: Inflammatory muscle pain involves a cascade of events that is dependent on the activation of PKC, p38 and JNK, and the synthesis of IL-1β, TNF-α and IL-6 associated with the up-regulation of both B(1) and B(2) kinin receptors.

Figure 1

Expression of mRNA of B₁ and B₂ receptors in formalin-induced inflammatory muscle pain. The quantification of mRNA for (A) B₁ and (B) B₂ was performed by RT-PCR. Data were normalized to mRNA levels for GAPDH. ‘N’ represents the naive group and ‘S’ represents non-inflamed mice that received an i.m. injection of 0.9% saline solution (50 µL per site). Data represent the mean ± SEM (n= 5). The symbols denote a significant difference: *P < 0.05 compared to the saline group. Statistical analyses were performed using one-way anova followed by Bonferroni's test.

Figure 2

Effect of pre- and post-treatment with the kinin B₁ receptor antagonists on the mechanical hyperalgesia induced by formalin in gastrocnemius muscle. (A) DALBK or (B) SSR240612 was administered i.p. 30 min before formalin injection and hyperalgesia was assessed 3, 6 and 24 h after the formalin injection. (C) DALBK was administered i.p. 24 h after the formalin injection (post-treatment). The hyperalgesia was assessed 0.5, 2 and 4 h after the DALBK injection. B, represents the baseline measurement (immediately before formalin injection). Data represent the mean ± SEM (n= 5–7). The symbols denote a significant difference: *P < 0.05 compared to the naive group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed using one-way anova (repeated measures) followed by Bonferroni's test.

Figure 3

Effect of pre- and post-treatment with the kinin B₂ receptor antagonists on the mechanical hyperalgesia induced by formalin in gastrocnemius muscle. (A) HOE 140 or (B) FR173657 was administered i.p. 30 min before the formalin injection and hyperalgesia was assessed 3, 6 and 24 h after the formalin injection. (C) HOE 140 was administered i.p. 24 h after the formalin injection (post-treatment). The hyperalgesia was assessed 0.5, 2 and 4 h after DALBK injection. B, represents the baseline measurement (immediately before formalin injection). Data represent the mean ± SEM (n= 5–7). The symbols denote a significant difference: *P < 0.05 compared to the naive group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed using one-way anova (repeated measures) followed by Bonferroni's test.

Figure 4

Effect of the co-treatment with the kinin B₁ and B₂ receptor antagonists on the mechanical hyperalgesia induced by formalin in gastrocnemius muscle. DALBK (0.3 mg·kg⁻¹ i.p.) or HOE 140 (0.017 mg·kg⁻¹) or both were administered 30 min before formalin in (A) or 24 h after formalin in (B). The assessment of hyperalgesia was performed as described in Figures 2 and 3. B, the baseline measurement (immediately before formalin injection). Data represent the mean ± SEM (n= 5–7). The symbols denote a significant difference: *P < 0.05 compared to the naive group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed using one-way anova (repeated measures) followed by Bonferroni's test.

Figure 5

Myeloperoxidase (MPO) activity and expression of mRNA for TNF-α, IL-1β and IL-6 in formalin-induced inflammatory muscle pain. (A) Myeloperoxidase activity and mRNA quantification for (B) TNF-α, (C) IL-1β and (D) IL-6 in the muscle of formalin-injected mice. Mice were pretreated, i.p., with saline, DALBK or HOE 140. Data were normalized to mRNA levels for GAPDH. Control mice received an i.m. injection of 0.9% saline solution (50 µL per site). Data represent the mean ± SEM (n= 3–5). The symbols denote a significant difference: *P < 0.05 compared to the control (saline i.m.) group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed by one-way anova followed by Bonferroni's test.

Figure 6

Activation of MAPKs in formalin-induced inflammatory muscle pain. Mice received 5% formalin via the i.m. route. The tissues were obtained at different time points after formalin injection for immunohistochemical analyses of (A) p-p38 or (B) p-JNK. Two other groups of mice were pretreated with DALBK (1 mg·kg⁻¹. i.p.) or HOE 140 (0.057 mg·kg⁻¹, i.p.) 30 min before formalin. One hour after formalin injection, the tissues were obtained for immunohistochemical analyses for (C) p-p38 and p-JNK. (D) Representative photomicrography of (C). Control mice received an i.m. injection of 0.9% saline solution (50 µL per site) (S). Data represent the mean ± SEM (n= 5). The symbols denote a significant difference: *P < 0.05 compared to the control group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed using one-way anova followed by Bonferroni's test.

Figure 7

Involvement of MAPK kinases and PKC in the mechanical hyperalgesia induced by formalin in gastrocnemius muscle. Mice were treated with the protein kinase inhibitors (A) p38 (SB203580 11 µg per site, i.m.), (B) JNK (SP60015 7 µg per site, i.m.) or (C) PKC (GF109203X 0.4 µg per site, i.m.) 5 min before the i.m. injection of formalin. The mechanical hyperalgesia was evaluated from 1 h after formalin injection. B, baseline measurement (before formalin injection). Data represent the mean ± SEM (n= 5). The symbols denote a significant difference: *P < 0.05 compared to the naive group and ^#P < 0.05 compared to the formalin i.m. group. Statistical analyses were performed using one-way anova (repeated measures) followed by Bonferroni's test.

Figure 8

Effect of the B₁ and B₂ antagonists on the mechanical hyperalgesia induced by the PKCε activator octapeptide ψεRACK. Mice received DALBK or HOE 140 i.p. 30 min before an i.m. injection of the octapeptide ψεRACK. The mechanical hyperalgesia was evaluated from 15 min after the injection of octapeptide ψεRACK. Data represent the mean ± SEM (n= 5). The symbols denote a significant difference: *P < 0.05 compared to the naive group and ^#P < 0.05 compared to the octapeptide ψεRACK i.m. group. Statistical analyses were performed using one-way anova (repeated measures) followed by Bonferroni's test.