Activation of TRPV1 by capsaicin induces functional kinin B(1) receptor in rat spinal cord microglia

Abstract

Background: The kinin B(1) receptor (B(1)R) is upregulated by pro-inflammatory cytokines and oxydative stress, which are enhanced by transient receptor potential vanilloid subtype 1 (TRPV1) activation. To examine the link between TRPV1 and B(1)R in inflammatory pain, this study aimed to determine the ability of TRPV1 to regulate microglial B(1)R expression in the spinal cord dorsal horn, and the underlying mechanism.

Methods: B(1)R expression (mRNA, protein and binding sites) was measured in cervical, thoracic and lumbar spinal cord in response to TRPV1 activation by systemic capsaicin (1-50 mg/kg, s.c) in rats pre-treated with TRPV1 antagonists (capsazepine or SB-366791), the antioxidant N-acetyl-L-cysteine (NAC), or vehicle. B(1)R function was assessed using a tail-flick test after intrathecal (i.t.) injection of a selective B(1)R agonist (des-Arg(9)-BK), and its microglial localization was investigated by confocal microscopy with the selective fluorescent B(1)R agonist, [Nα-bodipy]-des-Arg(9)-BK. The effect of i.t. capsaicin (1 μg/site) was also investigated.

Results: Capsaicin (10 to 50 mg/kg, s.c.) enhanced time-dependently (0-24h) B(1)R mRNA levels in the lumbar spinal cord; this effect was prevented by capsazepine (10 mg/kg, i.p.; 10 μg/site, i.t.) and SB-366791 (1 mg/kg, i.p.; 30 μg/site, i.t.). Increases of B(1)R mRNA were correlated with IL-1β mRNA levels, and they were significantly less in cervical and thoracic spinal cord. Intrathecal capsaicin (1 μg/site) also enhanced B(1)R mRNA in lumbar spinal cord. NAC (1 g/kg/d × 7 days) prevented B(1)R up-regulation, superoxide anion production and NF-kB activation induced by capsaicin (15 mg/kg). Des-Arg(9)-BK (9.6 nmol/site, i.t.) decreased by 25-30% the nociceptive threshold at 1 min post-injection in capsaicin-treated rats (10-50 mg/kg) while it was without effect in control rats. Des-Arg(9)-BK-induced thermal hyperalgesia was blocked by capsazepine, SB-366791 and by antagonists/inhibitors of B(1)R (SSR240612, 10 mg/kg, p.o.), glutamate NMDA receptor (DL-AP5, 10 μg/site, i.t.), substance P NK-1 receptor (RP-67580, 10 μg/site, i.t.) and nitric oxide synthase (L-NNA, 10 μg/site, i.t.). The B(1)R fluorescent agonist was co-localized with an immunomarker of microglia (Iba-1) in spinal cord dorsal horn of capsaicin-treated rats.

Conclusion: This study highlights a new mechanism for B(1)R induction via TRPV1 activation and establishes a link between these two pro-nociceptive receptors in inflammatory pain.

Figure 1

(A) Changes of B₁R mRNA levels in the lumbar spinal cord of capsaicin-treated rats (0-50 mg/kg, s.c.). Data represent the means ± S.E.M. of 4 to 7 rats per group (a single dose of capsaicin per group, 24 h earlier). Comparison with the 0 mg/kg group is indicated by * P < 0.05; ** P < 0.01. (B) Time-course effect (0 - 48 h) of 15 mg/kg (•) and 50 mg/kg (□) capsaicin on lumbar spinal cord B₁R mRNA expression levels. Data represent the means ± S.E.M. of 4 rats per group. Comparison with the 0 h group (*) is indicated by ** P < 0.01; *** P < 0.001. Shown are the responses in rats housed under constant light (A) or a 12 h-12 h light-dark cycle (B).

Figure 2

Effect of 24 h capsaicin (15 mg/kg, s.c.) or its vehicle on B₁R mRNA expression levels in cervical (C2), thoracic (T4) and lumbar (L5) segments of rat spinal cord. Data represent the means ± S.E.M. of 4 rats per group. Comparison with vehicle (*) or lumbar segment (+) is indicated by +,* P < 0.05; ** P < 0.01.

Figure 3

Effect of intrathecally injected capsaicin (1 μg/site, 24 h) or its vehicle on B₁R mRNA expression level in lumbar spinal cord. Data represent the means ± S.E.M. of 4 rats per group. Comparison with vehicle (*) is indicated by * P < 0.05.

Figure 4

Effect of 1 h pre-treatment with TRPV1 antagonists (capsazepine (10 mg/kg, i.p. or 10 μg/site, i.t.) and SB-366791(1 mg/kg, i.p. or 30 μg/site, i.t.)) or vehicle (filled dotted bars) on the increased lumbar spinal cord B₁R mRNA level induced by capsaicin (15 mg/kg, s.c.). Data represent the means ± S.E.M. of 4 rats per group. Comparison with capsaicin pre-treated with the vehicle is indicated by + P < 0.01.

Figure 5

Autoradiograms (A) and quantitative densitometric analysis (B) of specific B₁R binding sites in the grey matter of lumbar spinal cord of rats treated with capsaicin (15 and 50 mg/kg, s.c.) or vehicle, 24 h earlier. Data represent the means ± S.E.M. of n rats per group. Comparison with vehicle (*) is indicated by ** P < 0.01.

Figure 6

(A) Effect of des-Arg⁹-BK-induced thermal hyperalgesia in rats treated with capsaicin (1-50 mg/kg, s.c.) or vehicle, 24 h earlier. (B) Effect of the selective B₁R antagonist (SSR240612, 10 mg/kg, p.o.) administered 3 h prior to des-Arg⁹-BK in capsaicin-treated rats (50 mg/kg, s.c.). (C) Effect of two TRPV1 antagonists (capsazepine 10 mg/kg, i.p. or 10 μg/site, i.t. and SB-366791 1 mg/kg, i.p. or 30 μg/site, i.t.) given 1h prior to capsaicin on thermal hyperalgesia induced by des-Arg⁹-BK and SP in capsaicin-treated rats (15 mg/kg, s.c.). Shown are the maximal hyperalgesic responses measured at 1 min post-injection of the agonist. Data represent the means ± S.E.M. of 5-7 rats per group. Comparison with the vehicle (*) is indicated by: * P < 0.05; ** P < 0.01; ***P < 0.001.

Figure 7

Time-course effect of des-Arg⁹-BK (9.6 nmol/site, i.t.) on thermal hyperalgesia in the tail-flick test from rats treated 24 h earlier with capsaicin (15 mg/kg, s.c.). Shown are the responses in rats housed under constant light (A) or a 12 h-12 h light-dark cycle (B). Data represent the means ± S.E.M. of 4 rats per group. Comparison with the 0 min group (*) is indicated by ** P < 0.01. No statistical significance was seen between the two groups.

Figure 8

Tail-flick reaction time of control rats injected with (A) vehicle or capsazepine (10 mg/kg, i.p. or 10 μg/site, i.t.) or (B) vehicle or SB366791 (1 mg/kg, i.p. or 30 μg/site, i.t.). Tail-flick reaction time was assessed using either a tungsten lamp (A) or a hot water bath (50°C) as a heat source (B). Data represent the means ± S.E.M. of 4 rats per group.

Figure 9

Effect of several pharmacological treatments on des-Arg⁹-BK- (9.6 nmol/site, i.t.) and SP- (6.6 nmol/site, i.t.) induced thermal hyperalgesia in capsaicin-treated rats (15 mg/kg, s.c., 24 h earlier). Shown are the maximal hyperalgesic responses measured 1 min post-injection of the agonist. Rats received intrathecally (10 μg/site, 15 min earlier) either L-NNA, RP-67580, DL-AP5 or vehicle. Control rats were naïve. Data represent the means ± S.E.M. of 5-7 rats in each group. Comparison to aCSF (*), des-Arg⁹-BK in control rats (a) or substance P in control rats (b) is indicated by *P < 0.05, **P < 0.01, *** ^{aaa bbb} P < 0.001.

Figure 10

Confocal microscopy pictures of coronal sections of lumbar spinal cord dorsal horn isolated from 15 mg/kg capsaicin-treated rats. Microglia were labelled with anti-Iba-1 and are shown in panel A. B₁R was labelled with N^α-bodipy-des-Arg⁹-BK and is shown in panel B. Co-localization is shown in yellow in panel C. Images are representative of at least 4 sections/rat from 4 rats/group. Scale bar = 8.58 μmeter. No B₁R labeling was found in spinal cord of control spinal cord (not shown).

Figure 11

Confocal microscopy pictures of DHE labelling in spinal cord dorsal horn of 15 mg/kg capsaicin-treated (C, D) and control (A, B) rats, pre-treated for 7 days with the potent antioxidant N-acetyl-L-cysteine (1 g/kg/day) (B, D) or its vehicle (A, C). Nuclei were labelled with TO-PRO-3 (blue) and superoxide anion with DHE (red). Scale = 31.75 μm. Data represent the means ± S.E.M. of at least 4 sections/rat from 4 rats/group. Comparison with control + vehicle (*) or capsaicin + vehicle (+) is indicated by: * ⁺ P < 0.05.

Figure 12

Control and capsaicin (CAPS, 15 mg/kg, s.c.)-injected rats were pre-treated for 7 days with N-acetyl-L-cysteine (NAC, 1 g/kg/d) or its vehicle. Protein expression for B₁R (37 kDa), p65NF-κB (65 kDa) and dynein (70 kDa) was measured on lumbar spinal cord by western blot. Representative immunoblots are shown in the upper panels while densitometric quantifications of protein expression are shown in lower panels. Data represent the means ± S.E.M. of 3-4 rats in each group. Comparison with control + vehicle (*) or capsaicin + vehicle (+) is indicated by ** ++ P < 0.01.

Figure 13

Changes in IL-1β and TNF-α mRNA levels in lumbar spinal cord of capsaicin-treated rats (0-50 mg/kg, s.c. 24 h earlier). Also shown is the effect of the TRPV1 antagonist capsazepine (10 mg/kg, i.p.) administered 1 h prior to capsaicin. Data represent the means ± S.E.M. of 5-7 rats in each group. Comparison with vehicle (0 mg/kg) (*) is indicated by * P < 0.05.