Mechanisms involved in the nociception produced by... : PAIN (original) (raw)
1. Introduction
Tissue damage produces an array of chemical mediators that in turn activate or sensitise nociceptors to elicit pain at the site of injury through the modulation of different intracellular signalling pathways (Julius and Basbaum, 2001). It is now well recognised that protein kinase C (PKC) activation is an important step for the nociceptive effects caused by numerous stressful stimuli, including that caused by inflammatory mediators. PKC is known to phosphorylate several cellular components, including enzymes, ion channels and membrane-bound receptors, all that are key regulators in the processes of nociceptor excitation and sensitisation (for review see: Ji and Woolf, 2001). For instance, PKC activation is associated with the excitation and sensitisation of nociceptors in response to bradykinin or histamine in vitro (Dray et al., 1992; Mizumura et al., 1997, 2000). Moreover, PKC inhibitors are able to inhibit the in vivo nociception produced by bradykinin, by epinephrine, or by the inflammatory substance carrageenan, in mice and rats (Aley et al., 2000; Ferreira et al., 2004; Khasar et al., 1999b).
Recent studies have demonstrated the existence of at least 10 different genes codifying 11 distinct isoforms of PKC in mammals (for review see: Newton, 2001; Ohno and Nishizuka, 2002). The up-regulation and activation of PKCγ and PKCβ in the spinal cord appear to exert a key role in the development and maintenance of chronic pain (Ji and Woolf, 2001). Furthermore, reports have appeared on the involvement of peripheral PKCε in nociceptive hypersensitivity produced acutely by epinephrine or TNFα, as well as in chronic models of inflammation or neuropathy (Cesare et al., 1999; Dina et al., 2000, 2001; Khasar et al., 1999a,b; Parada et al., 2003). Thus, the elucidation of targets modulated by PKC could be of particular interest for a better understanding of the mechanisms underlying certain painful disorders.
More direct evidence concerning the role of PKC in nociception may be obtained using the classical exogenous PKC activators, the plant diterpenes phorbol esters (Castagna et al., 1982). Phorbol esters are capable of producing direct excitation of primary sensory neurons in vitro (Leng et al., 1996; Rang and Ritchie, 1988; Schepelmann et al., 1993). Peripheral injection of phorbol esters produces spontaneous nociception, thermal hyperalgesia and mechanical allodynia in mice and rats (Souza et al., 2002; Taniguchi et al., 1997). However, the precise mechanisms involved in the nociceptive effect caused by phorbol esters are relatively unknown. Therefore, the present study aimed to assess some of the mechanisms involved in the spontaneous nociception elicited by peripheral administration of a PKC activator, the plant diterpene phorbol 12-myristate 13-acetate (PMA), in mice.
2. Materials and methods
2.1. Animals
We used non-fasted male Swiss mice (25–35 g) bred in our department, housed at 22±2 °C of temperature, 60–80% of humidity, under a 12:12 light–dark cycle and with access to food and water ad libitum. Experiments were performed during the light phase of the cycle. The animals were acclimatised in the laboratory for at least 2 h before testing and were used only once throughout the experiments. The experiments reported in this study were carried out in accordance with current guidelines for the care of laboratory animals and ethical guidelines for investigation of experimental pain in conscious animals (Zimmermann, 1983). All protocols employed had been approved by the Local Ethics Committee (process numbers 262/CEUA and 23080.035334/2003-16/UFSC). The number of animals used and the intensity of nociceptive stimuli was the minimum necessary to demonstrate the consistent effects of drug treatments.
2.2. PMA-induced nociception
The experiments were carried out as previously described for rats (Taniguchi et al., 1997) with some modifications. Briefly, a volume of 20 μl of phorbol 12-myristate 13-acetate (PMA) solution (16–1600 pmol/paw), diluted in phosphate buffered saline (PBS), was injected intraplantarly (i.pl.) under the plantar surface of the right hind paw. The mice were observed from 0 to 60 min following PMA injection in order to evaluate the temporal characteristics of the nociceptive behaviour. After challenge, the animals were individually placed into glass cylinders, 20 cm in diameter. The time spent licking and biting the injected paw was timed with a chronometer and was considered to be indicative of spontaneous nociception. To confirm the selectivity of PMA, a separate group of animals received a similar i.pl. injection of the PMA inactive analogue α-PMA. To assess if PMA effect could be reproduced by other phorbol ester, a separated group of animals received an i.pl. injection of phorbol 12,13-dibutyrate (PDBu, 500 pmol/paw).
2.3. Study of the peripheral mechanisms involved in PMA-induced nociception
PKC is able to modulate the activity of several cellular processes that are important to pain production, including the release of pro-nociceptive mediators and subsequent actions, in target receptors and/or signalling pathways. To address some of the possible mechanisms through which peripheral injection of PMA causes nociception, distinct groups of animals were treated with different classes of drugs, all locally co-administered with a sub-maximal dose of PMA (50 pmol/paw, i.pl., see below). The choice of the dose of each drug was based on previous data described in the literature (Beirith et al., 2002; Campos et al., 1999; Ferreira et al., 2004; Santos and Calixto, 1997) or on preliminary experiments carried out in our laboratory (data not shown).
To confirm the hypothesis that PMA-induced nociception is mediated by PKC stimulation, animals were treated with the selective PKC inhibitor (GF 109203X; 0.03–1 nmol/paw). Next, the possible contribution of pro-nociceptive substances, peripherally released following PMA stimulation, was investigated. The following drugs were co-administered with PMA: the glutamate NMDA receptor antagonist MK 801 (1 nmol/paw), the tachykinin NK1 receptor antagonist FK888 (0.15 nmol/paw), the β-adrenoceptor antagonist propranolol (50 nmol/paw), the selective β1-adrenoceptor antagonist atenolol (100 nmol/paw), the selective calcitonin gene-related peptide receptor antagonist (CGRP8–37, 1 nmol/paw), the selective kinin B2 receptor antagonist Hoe 140 (3 nmol/paw), the vanilloid receptor 1 (TRPV1) antagonist capsazepine (30 nmol/paw), the selective vanilloid receptor antagonist SB-366791 (1 nmol/paw), the non-selective TRP inhibitor ruthenium red (0.3 nmol/paw), the nitric oxide (NO) synthase inhibitor L-NOARG (1 μmol/paw), or the selective inhibitors of cycloxygenase (COX) type 1 (valeryl salicilate, 10 nmol/paw) or type 2 (rofecoxib, 100 nmol/paw).
To determine any possible participation of some of the cytokines or neurotrophins in the nociception produced by PMA, the animals were co-treated with antibodies against tumor necrosis factor-α (anti-TNFα, 50 ng/paw), nerve growth factor (NGF, 2 μg/paw), brain-derived neurotrophic factor (BDNF, 2 μg/paw) or glial cell line-derived neurotrophic factor (GDNF 2 μg/paw), or with the interleukin-1β-receptor antagonist (IL-RA, 100 μg/paw). Finally, the possible involvement of mitogen-activated protein kinases (MAPK) in PMA-induced nociception was investigated in animals treated with p38 MAPK (SB203580, 10 nmol/paw) or with ERK upstream MAPK-kinase MEK1 (PD98059, 2 nmol/paw) selective inhibitors, both co-administrated with PMA (50 nmol/paw).
2.4. The role of mast cells, capsaicin-sensitive and sympathetic fibres in PMA-induced nociception
To explore further the role of capsaicin-sensitive fibres in the nociceptive effect induced by PMA, neonatal mice (on day 2 of life) received either capsaicin (50 mg/kg, subcutaneously) or the vehicle alone (10% ethanol, 10% Tween-80 and 80% PBS), as described previously (Ferreira et al., 2004). The animals were used 6–7 weeks after this administration of capsaicin or vehicle (used as control).
To analyse the role of sympathetic fibres in the PMA-induced nociception, a chemical sympathectomy was produced by the treatment of mice with guanethidine (30 mg/kg, i.p.) 3 days before PMA challenge, as described previously (Malmberg and Basbaum, 1998).
To assess the participation of mast cells in the PMA-induced nociception, mast cells were degranulated by the treatment with compound 48/80 (120 μg/paw, i.pl.) 12 h before PMA challenge, as described previously (De Campos et al., 1996).
2.5. Preparation of tissues for western blot studies
The right paws of the mice were isolated at different periods of time (1–45 min) after PMA treatment (50 pmol/paw). The skin and connective tissues of the plantar aspect of the hind paws were removed and disrupted using a glass Potter homogeniser in an ice-cold buffer containing protease and phosphatase inhibitors (100 mM Tris–HCl–pH 7.4; 2 mM EDTA; 2 μg aprotinin, 0.1 mM phenylmethanesulfhonyl fluoride, 200 mM NaF and 2 mM of sodium orthovanadate). The homogenate was first centrifuged at 1000×g for 10 min at 4 °C; the pellet was discarded and the supernatant was further centrifuged at 35,000×g for 30 min at 4 °C. The supernatant was collected as a cytoplasm-rich fraction. The resulting pellet was re-suspended and considered as a membrane-rich fraction. The protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA). The samples were aliquotted and stored at −80 °C until western blot analysis.
2.6. Western blot analysis
In order to confirm the activation of PKC or MAP kinases after PMA injection into the mouse paw, western blot analysis was carried out as previously described (André et al., 2004; Leal et al., 2002; Miletic et al., 2000) with minor modifications. Equivalent amounts of proteins (10 and 50 μg for membrane- and cytoplasm-rich fractions, respectively) were mixed in buffer (Tris 200 mM, glycerol 10%, SDS 2%, β-mercaptoethanol 2.75 mM and bromophenol blue 0.04%) and boiled for 5 min. Proteins were resolved in 10% sodium dodecyl sulfate-polyacrilamide gel by electrophoresis (SDS-PAGE) and transferred on to polyvinilidene difluoride menbranes, according to the manufacturer's instructions (Millipore). The membranes were saturated by incubation overnight with 10% non-fat dry milk solution and then incubated with anti-PKCα, anti-PKCε, anti-phospho-ERK (that recognises ERK1 and ERK2) or anti-phospho-p38 antibodies (Santa Cruz Biotechnology, USA). Following washing, the membranes were incubated with adjusted peroxidase-coupled secondary antibodies. The immunocomplexes were visualised using the ECL chemiluminescence detection system (Amersham Biosciences, UK).
2.7. Measurement of neutrophil infiltration to PMA-injected paws
Since PMA is able to induce skin neutrophil infiltration, and considering the fact that inflammatory cells seem to be involved in nociception development (Bennett et al., 1998; De Young et al., 1989), we assessed whether or not neutrophils are involved in PMA-induced licking. The accumulation of neutrophils in the mouse paw was measured by means of tissue mieloperoxydase (MPO) activity, according to a previously described method (De Young et al., 1989). The animals received a 20 μl injection of PMA (50 pmol/paw) into the right paw and were sacrificed 15–360 min after challenge. At the time of sacrifice, the plantar skin of the paw was removed and assayed for MPO. The tissue was weighed and placed in 80 mM sodium phosphate buffer (PBS) pH 5.4 (0.1 ml each 10 mg of tissue) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and then homogenised (45 s at 0 °C) in a motor-driven homogeniser. The sample was centrifuged at 12,000×g for 15 min at 4 °C. Duplicate 30 μl samples of the resulting supernatant were added to 96-well micro titre plates. For the assay, 200 μl of a mixture containing 100 μl of 80 mM PBS pH 5.4, 85 μl of 0.22 M PBS pH 5.4, and 15 μl of 0.017% hydrogen peroxide was added to the wells. The reaction was started by the addition of 20 μl of 18.4 mM tetramethylbenzidine HCl in 8% aqueous dimethylformamide. The plates were incubated for 3 min at 37 °C and then placed on ice where the reaction was halted by the addition of 30 μl of 1.46 M sodium acetate, pH 3.0, to each well. Enzyme activity was determined colourimetrically using a Bio-Tek Ultra Microplate reader (EL 808) set to measure absorbance at 630 nm and expressed as OD per milligram of tissue.
2.8. Sensitivity of PMA-induced nociception to clinically-used analgesics
In these experiments, we sought to determine whether or not drugs used clinically to treat acute or chronic pain were able to alter PMA-induced nociception. To this end, PMA (50 pmol/paw, i.pl.) was injected into mice previously treated with morphine (5 mg/kg, s.c., 30 min prior to PMA), indomethacin (10 mg/kg, p.o., 30 min prior to PMA), lidocaine (30 mg/kg, s.c., 30 min prior to PMA) or dexamethasone (0.5 mg/kg, s.c., 120 min prior to PMA). Control animals received vehicle injection (1% ethanol p.o. or s.c., 30 min prior to PMA).
2.9. Drugs
The following drugs were used: GF 109203X, SB-366791 (Tocris, Baldwin, USA), capsaicin, capsazepine, MK 801 (RBI, Natick, USA), morphine (Merck, Darmstadt, Germany), valeryl salicylate (Cayman Chemical Company, Ann Arbor, USA), guanethidine, lidocaine, propranolol, atenolol, compound 48/80, indomethacin, dexamethasone, PBS tablets, EDTA, EGTA, tetramethylbenzidine, PMA, 4α-PMA, ruthenium red (Sigma Chemical Company, St Louis, USA), IL-RA, anti-TNFα, anti-NGF, anti-BDNF, anti-GDNF (R&D Systems, USA). FK 888, Rofecoxib and Hoe 140 were kindly donated by Fujisawa (Japan), Merck (USA) and Aventis (Germany), respectively. All other reagents used were of a high grade of purity. The stock solutions for most substances were prepared in PBS, with the exception of valeryl salicylate, indomethacin, dexamethasone, GF 109203X, capsazepine, PMA, 4α-PMA, PDBu, SB-366791, FK 888 and rofecoxib which were dissolved in absolute ethanol, kept in siliconised plastic tubes, and maintained at −18 °C. The final concentration of ethanol did not exceed 0.5% and did not cause any effect per se. All drugs were dissolved in phosphate buffered solution (PBS) just before use.
2.10. Statistical analysis
The results are presented as the mean±SE mean of 3–8 animals, except for the ED50 values (i.e. the dose of PMA producing nociceptive responses to the order of 50% of the response relative to the control value) that are reported as geometric means accompanied by their respective 95% confidence limits. The percentages of inhibition are reported as mean±SE mean of inhibitions obtained in each individual experiment at the peak of nociceptive behaviour (15–45 min after injection of PMA). The statistical significance between groups was assessed by means of one-way analysis of variance followed by Student's unpaired, Dunnett's or Student–Newmann–Keuls' tests when appropriate. _P_-values less than 0.05 (P<0.05) or less were considered to be indicative of significance. The ED50 values were determined by linear regression analysis from individual experiments using GraphPad Software 1.0 (GraphPad, USA). The percentages of inhibition were calculated for the maximal developed responses in comparison with vehicle-treated animals.
3. Results
The intraplantar administration of the PKC activator PMA (16–1600 pmol/paw) in mice produced a long-lasting and dose-dependent spontaneous nociception (Fig. 1A and B). This nociceptive effect was developed slowly, being significantly observable as early as 15 min and persisting until at least 45 min after PMA injection (Fig. 1A). For subsequent experiments, we measured the nociception cumulatively between 15 and 45 min after PMA challenge (Fig. 1B). The estimated mean ED50 value for PMA-induced nociception was 91.6 (46.8–136.4) pmol/paw and the maximal nociceptive effect observed was 307.4±33.1 s achieved with the dose of 1600 pmol/paw. The sub-maximal dose of 50 pmol/paw of PMA was then chosen to carry out further experiments in order to avoid supra-maximal stimulation and the consequent unnecessary animal discomfort. On the other hand, the intraplantar injection of 4α-PMA (50 pmol/paw), an inactive PMA analogue to PKC, was not able to produce nociception (licking time of 14.7±6.6, 130.5±16.7 and 13.2±4.9 for vehicle (PBS plus 0.085% of ethanol), PMA (50 pmol/paw) and 4α-PMA (50 pmol/paw), respectively; _P_>0.05). Moreover, the nociceptive effect caused by phorbol esters is not exclusive for PMA, since the i.pl. injection of PDBu (500 pmol/paw) was also capable of causing spontaneous nociception (Fig. 1C). The co-administration of the selective PKC inhibitor GF109203X (0.03–1 nmol/paw) with PMA (50 pmol/paw) dose-dependently blocked the nociception production (Fig. 2).
(A) Time-dependent curves for the spontaneous nociception caused by i.pl. injection of different doses of PMA (16–1600 pmol/paw) in mice. (B) Dose-dependent nociceptive effect produced by PMA observed for 15–45 min after its administration. (C) Time-dependent spontaneous nociception caused by i.pl. injection of PDBu (500 pmol/paw) in mice. The spontaneous nociceptive effect of vehicle (0.085% of ethanol), PMA and PDBu is expressed as licking time (s). Each point or bar on the curve represents the mean of 5–7 animals and vertical lines show the SEM. Asterisks denote the significance levels in comparison with the vehicle-treated group (B: one-way ANOVA followed by Dunnett's test; C: Student's t test). *P<0.05, **P<0.01.
Effect of intraplantar treatment with the selective PKC inhibitor GF109203X on PMA (50 pmol/paw)-induced nociception in mice. Each column represents the mean±SEM of 4–6 animals. The asterisks denote the significance levels. *P<0.05, **P<0.01, compared with PMA-treated mice (one-way ANOVA followed by Dunnett's test).
The participation of PKC was also confirmed in this painful process through western blot analysis. Injection of PMA (50 pmol/paw) into the mouse paw was capable of activating PKCα, and in to a lesser extend PKCε isoforms as charged by their translocation from cytosol- to membrane-rich homogenates achieved in administered tissues (Fig. 3A and B). The activation of PKCα was fast being significantly activated 5 min after PMA injection and lasting for up to 45 min, an effect that coincides with the time-course of the nociceptive action of PMA (Fig. 3A). On the other hand, the activation of PKCε was weaker than PKCα being statistically significant only at 30 min time point after PMA injection (Fig. 3B).
Western blots showing the time course of PKCα (A) and PKCε (B) and MAPKs activation in response to i.pl. injection of PMA (50 pmol/paw) into mice paw. Mice paw tissues were obtained from naive (Basal) or PMA-injected mice at the indicated times and membrane and cytosolic extracts were then prepared. Cytosolic and membrane levels of PKCα or PKCε were determined using specific antibodies. Results were normalised by arbitrarily setting the densitometry of the basal group and are expressed as mean±SEM (_n_=3). *P<0.05 and **P<0.01, as compared with basal values (one-way ANOVA followed by Dunnett's test).
The destruction of thin-diameter capsaicin-sensitive sensory fibres produced by neonatal capsaicin treatment (50 mg/kg, s.c.) almost abolished the PMA-induced nociception (Fig. 4A). Furthermore, the chemical sympathectomy produced by guanethidine (30 mg/kg, i.p., 3 days before) or the previous mast cell degranulation caused by compound 48/80 (120 μg/paw, i.pl., 12 h before) inhibited partially, but significantly, the nociceptive effect produced by PMA (Fig. 4B). However, PMA was unable to produce neutrophil infiltration 15–45 min after its administration, while the nociceptive effect positively did occur, as assessed by the MPO activity (Fig. 4C). In contrast, 1 and 6 h after PMA injection, a significant increase in the neutrophil infiltration was observed.
(A–C) Disruption of the nociceptive effect caused by the intraplantar injection of PMA (50 pmol/paw) produced by treatment of neonate mice with capsaicin (50 mg/kg, s.c., A) or of adult mice with guanethidine (30 mg/kg, i.p., B) or compound 48/80 (120 μg/paw, i.pl., C). (D) Evaluation of the neutrophil infiltration (assessed by the activity of MPO) produced by intraplantar injection of PMA (50 pmol/paw). Each column represents the mean±SEM of 4–6 mice. The asterisks denote the significance levels. (A–C) **P<0.01, compared with the vehicle-injected mice (Student's t test). (D) *P<0.05; **P<0.01, compared with naive mice (one-way ANOVA followed by Dunnett's test).
Subsequently, the possible participation of different signalling pathways in the PMA-induced nociception was investigated. The treatment of animals with the selective NK1, CGRP, NMDA, β-adrenergic or kinin B2 receptor antagonists or with a non-selective TRP channels blocker were all able to partially inhibit the nociception caused by PMA (Fig. 5A and Table 1). The adrenergic receptor involved in PMA-induced nociception seems to be of the β1 subtype, since atenolol (100 nmol/paw) significantly reduced PMA-induced nociception (licking time of 180.0±10.5 and 102.0±17.2 s for PMA alone and PMA plus atenolol, respectively, P<0.05, Student's t test). Moreover, the nociception generated by PMA was reduced by the extracellular chelating of Ca++ or by the selective inhibition of COX-1 or COX-2, but not by the NO synthase inhibitor L-NOARG. Remarkably, treatment with an antagonist of IL-1β receptor or with antibodies against TNFα, NGF or BDNF was also able to partially, but significantly, inhibit PMA (50 pmol/paw)-induced nociception (Fig. 5C). On the other hand, GDNF was not effective in altering the nociception produced by peripheral PMA injection (50 pmol/paw) (Fig. 5A–C). The calculated percentages of inhibition for each drug are shown in Table 1. The selective TRPV1 antagonist SB-366791 (1 nmol/paw) partially reduced PMA-induced nociception (licking time of 186.0±10.5 and 141.7±16.6 s for PMA alone and PMA plus SB-366791, respectively; P<0.05, Student's t test). However, the TRPV1 antagonist capsazepine was not able to alter the nociceptive response caused by PMA (Fig. 5B).
Signalling pathways involved in PMA-induced spontaneous nociception in mice. (A) Effect of the intraplantar co-administration of PMA (50 pmol/paw) with some receptor antagonists MK801 (1 nmol/paw), FK888 (0.15 nmol/paw), CGRP8–37 (1 nmol/paw), propranolol (50 nmol/paw), Hoe 140 (3 nmol/paw), capsazepine (30 nmol/paw) or ruthenium red (0.3 nmol/paw). (B) Effect of the intraplantar co-administration of PMA (50 pmol/paw) with EGTA (2 nmol/paw) or with the following inhibitors: valeryl salicilate (10 nmol/paw), rofecoxib (100 nmol/paw), PD98059 (2 nmol/paw) or SB203580 (10 nmol/paw). (C) Effect of the intraplantar co-administration of PMA (50 pmol/paw) with IL-RA or with antibodies to TFNα (50 ng/paw), NGF (2 μg/paw), BDNF (2 μg/paw) or GDNF (2 μg/paw). (D) Effect of systemic treatment with some clinically used analgesic drugs (morphine, 5 mg/kg, s.c.; indomethacin, 10 mg/kg, p.o.; lidocaine, 30 mg/kg, s.c. or dexamethasone, 0.5 mg/kg, s.c.) on PMA (50 nmol/paw)-induced nociception. Each column represents the mean±SEM of 6–8 mice. The asterisks denote the significance levels. (A–D) *P<0.05; **P<0.01, compared with the PMA plus vehicle-injected mice (black bar, one-way ANOVA followed by Dunnett's test).
Effect of i.pl. co-administration of several classes of drugs in the overt nociception produced by PMA (50 pmol/paw)
The involvement of MAPK in PMA-induced nociception was assessed by both pharmacological and western blot analysis. The co-administration of the selective inhibitors of MEK1 or p38 MAPK was capable of significantly reducing the nociceptive response produced by PMA (Fig. 5B). Likewise, the injection of PMA into the mouse paw resulted in a marked and time-dependent activation of MAPKs. Statistically significant increase in phosphorylation levels of ERK1/2 was observed from 5 to 15 min following PMA administration (Fig. 6A). Moreover, p38 MAPK phosphorylation was significant from 30 to 45 min after PMA injection (Fig. 6B). We did not detect any alteration in β-actin expression after PMA-injection (results not shown).
Western blots showing the time course of ERK1/2 (A) and p38 (B) activation in response to i.pl. injection of PMA (50 pmol/paw) into mice paw. Mice paw tissues were obtained from naive (Basal) or PMA-injected mice at the indicated times. Cytolosic levels of phosphorylated p38 (P-p38) and phosphorylated ERK1/2 (P-ERK1/2) were determined using specific antibodies. Results were normalised by arbitrarily setting the densitometry of the basal group and are expressed as mean±SEM (_n_=3). *P<0.05 and **P<0.01, as compared with basal values (one-way ANOVA followed by Dunnett's test).
Finally, we assessed the sensitivity of the nociception produced by PMA to clinically used analgesic drugs. The systemic administration of morphine (5 mg/kg, s.c.) abolished PMA-induced nociception (Fig. 5D). In the same way, the treatment with indomethacin (10 mg/kg, i.p.) or with lidocaine (30 mg/kg, s.c.), but not with dexamethasone (0.5 mg/kg, s.c.), partially inhibited PMA-induced nociception (Fig. 5D). The calculated percentages of inhibition are shown in Table 1.
4. Discussion
There is a substantial amount of experimental evidence supporting a critical role exerted by PKC in regulating pain sensitivity at the spinal level. As neuronal plasticity occurs not only in spinal dorsal horn neurons, but also at the peripheral nociceptors, we have used different pharmacological tools to investigate further the role played by peripheral PKC activation in producing nociception and also to assess some of the mechanisms underlying this behaviour.
In our study, direct PKC activation by injection of a phorbol ester (PMA) into the mouse paw-induced nociception that was developed slowly (about 15 min after the injection), but lasted for up to 45 min after PMA injection. Similarly, Taniguchi et al. (1997) have found that PMA produces spontaneous nociception in rats from 15 to 45 min after i.pl. administration. This temporal profile forcefully agrees with previous in vitro studies that have demonstrated a slow but sustained development of depolarisation of rat vagus nerve, cat joint and canine visceral nociceptors following phorbol ester application (Leng et al., 1996; Rang and Ritchie, 1988; Schepelmann et al., 1993). Moreover, phorbol esters can produce a delayed inward current in cultured sensory neurons, appearing after a latency of 1 min and taking up to 10 min to reach the peak (Lindsay and Rang, 1988). Diacylglycerol and phorbol esters bind to conventional (α, βI–II, γ) and novel PKCs (δI–III, ε, η, δI–II), but not atypical PKCs(ζ, λ), which in turn stimulate their kinase catalytic activity. Furthermore, PKC activity requires intracellular translocation from cytosol to cytoskeletal and membrane sites of action (for review see: Newton, 2001; Ohno and Nishizuka, 2002). Thus, translocation of PKC from a cytosolic to a membrane-associated location within the cell is a sensitive indicator of activation (Cesare et al., 1999). Notably, all PKC isozymes except PKCγ can be detected in mouse DRG (Khasar et al., 1999a), and PMA can produce translocation of PKCδ and ε to surface membranes in cultured rat DRG neurons (Cesare et al., 1999). We found that i.pl. injection of PMA induces translocation of a classical (α) isoforms, and, to a lesser extend a novel (ε) isoforms, of PKC from cytoplasm to membrane extracts obtained from treated tissues. These results strongly suggest that the spontaneous nociceptive response caused by PMA injection into the mouse paw is probably a consequence of its ability to activate PKC.
PKC phosphorylates several cellular components that are key regulators in the processes of nociceptor excitation and sensitisation. Apparently, an important target for PMA nociceptive action in our model is the vanilloid receptor TRPV1. Phorbol esters, acting on PKC, can directly activate TRPV1 channels, decreasing the temperature threshold to a level which is below normal body temperature (Numazaki et al., 2002; Sugiura et al., 2002). Moreover, PKC increases the capsaicin, anandamide and protons gating of TRPV1 (Premkumar and Ahern, 2000; Vellani et al., 2001). We have found that treatment with the non-selective TRP blocker ruthenium red or with the selective TRPV1 antagonist SB-366791, but not with capsazepine, reduces PMA-mediated nociceptive response. These results are not unexpected, since it has been shown that capsazepine is only able to inhibit capsaicin-evoked calcium influx, but not that produced by pH or PMA, in cells heterologously expressing mouse TRPV1 (Correll et al., 2004). Moreover, TRPV1 sensitisation to pH produced by phorbol ester-activated PKCα is not altered by capsazepine (Olah et al., 2002). On the other hand, SB-366791 is a novel, potent, and selective TRPV1 antagonist that, unlike capsazepine, is effective in inhibiting TRPV1 when activated by different stimuli, such as capsaicin, pH or heat (Gunthorpe et al., 2004). Besides a clear role for TRPV1, PMA is also able to activate tetrodotoxin-resistant sodium currents (Gold et al., 1998), as well as to inhibit potassium currents through a voltage-dependent calcium entry (Zhang et al., 2001), suggesting that other targets could also mediate, at least in part, the nociceptor excitability caused by PMA.
Apart from its direct excitatory effect on sensory neurons, PMA seems to produce nociception indirectly through the release of pro-nociceptive mediators from some of the peripheral structures and resident cells, including capsaicin-sensitive afferent and sympathetic fibres as well as mast cells, but not infiltrating cells, such as neutrophils. Our results, and those recently reported by Tsuchiya et al. (2005), suggest that capsaicin-sensitive fibres (mainly non-myelinated C-fibres) exert a relevant effect on PMA-induced nociception. These fibres are the source of certain mediators involved in the nociceptive effect induced by PMA, including glutamate, substance P and CGRP (Szallasi and Blumberg, 1999). In fact, it has been found that phorbol esters are capable of releasing neuropeptides, namely substance P and CGRP, from cultured DRG neurons and from skin samples (Baber and Vasko, 1996; Kessler et al., 1999). Substance P and glutamate can act on their own receptors found in sensory nerves terminals, amplifying the nociceptive process (Coggeshall and Carlton, 1997). Moreover, substance P and CGRP (acting on NK1 and CGRP1 receptor, respectively) cause plasma extravasation and edema allowing the injected tissue to be accessed by plasma-derived mediators, including kininogens (Campos and Calixto, 2000). In fact, PMA-induced nociception is accompanied by plasma extravasation and oedema formation (J.B. Calixto, unpublished data).
We have also shown in the present study that the previous degranulation of mast cells produced by compound 48/80 treatment efficiently reduces PMA-induced nociception. Beyond their action on vessels, substance P and PMA itself degranulate mast cells, releasing several mediators and enzymes that provide a positive feedback on the nociceptive processes (Julius and Basbaum, 2001). For example, activated mast cells are able to release tryptase that stimulating kallikrein can produce bradykinin from kininogen-derived plasma (Imamura et al., 1996).
The nonapetide bradykinin is a potent pain-producing substance that, acting on B2 receptors found in sensory terminals, induces nociception and neuropeptide release as well acting on sympathetic fibres to release noradrenaline and prostaglandins that amplify its nociceptive process (for review see: Calixto et al., 2000). In the current experiments, we showed that selective inhibitors of COX-1 or COX-2 partially reduce PMA-induced spontaneous nociception. Interestingly, the effects of the non-selective COX inhibitor indomethacin were similar to the sum of all of the selective inhibitory effects, demonstrating that the blockade of both COX isoforms is more efficacious in the production of antinociception. Moreover, sympathetic fibre-released noradrenaline and activation of β1-adrenoceptors are associated with the production of nociception in our model. Similarly, sympathetic fibres and β1-adrenoceptor stimulation have been shown to mediate inflammatory hyperalgesia in rats (Cunha et al., 1991; Safieh-Garabedian et al., 2002).
It is well known that the pro-inflammatory cytokines IL-1β and TNFα, or the neurotrophins NGF and BDNF, produce painful hypersensitivity when injected peripherally in rats (Andreev et al., 1995; Cunha et al., 1992; Safieh-Garabedian et al., 1995; Shu et al., 1999; Woolf et al., 1997). In the present study, we have shown that these mediators are also involved in PMA-induced nociception. It has been reported that substance P, or PMA itself, can peripherally release cytokines and neurotrophin from resident cells, such as mast cells, keratinocytes and fibroblasts (for review see: Schaffer et al., 1998; Theoharides et al., 2004). Furthermore, peripheral injection of TNFα into rat paws induces hyperalgesia via the release of IL-1β and NGF (Safieh-Garabedian et al., 1995; Woolf et al., 1997). Cytokines and NGF can directly activate the nociceptors, since their receptors are found in sensory neurons (Bennett et al., 1996; Copray et al., 2001; Li et al., 2004). However, indirect mechanisms can also occur, as in the case of NGF that induces hyperalgesia dependent on mast cells and sympathetic fibres (Amann et al., 1996; Andreev et al., 1995). The peripheral role of BDNF in nociception is so far not well elucidated. Peripheral injection of BDNF causes hyperalgesia and sensitises C fibres in rats (Shu et al., 1999). However, there is no clear evidence for the existence of the high affinity BDNF receptor trkB in the peripheral terminals of nociceptors. A role of mast cells in BDNF-induced hyperalgesia has been suggested since these cells can express trkB receptors, and behavioural hyperalgesia induced by trkB activation is eliminated by prior mast cell degranulation (Rueff and Mendell, 1996; Shu et al., 1999).
Finally, we assessed whether or not MAPK activation was involved in the nociceptive effect produced by PMA. The MAPK family has three members: the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38 MAPK (for review see: Obata and Noguchi, 2004). Recently published data has shown that the peripheral stimulation of ERK1/2 and p38 kinases is involved in the peripheral nociceptor sensitisation produced by noxious stimuli such as epinephrine, NGF and capsaicin (Aley et al., 2001; Dai et al., 2002; Sweitzer et al., 2004; Zhuang et al., 2004). Our data has shown that activated forms of ERK1/2 and p38 are found in PMA-injected tissues and that the inhibitors of both kinases reduce PMA-induced spontaneous nociception. We suggest that ERK and p38 MAPK activation generates pain hypersensitivity in PMA-induced nociception by way of a non-transcriptional mechanism, given the short time of the nociceptive response (15–45 min).
The PKC signalling pathway appears to exert a critical role in modulating the excitability of sensory neurons. Since PKC activation contributes greatly to the enhancement of the neuronal sensitivity observed in certain inflammatory and neuropathic pain conditions, the development of drugs that selectively antagonise PKC signalling mechanisms could represent an attractive target for the development of new analgesics.
Acknowledgements
This study was supported by CNPq (Conselho Nacional de Desenvolvimento Científico), FINEP (Financiadora de Estudos e Projetos), PRONEX (Programa de Apoio aos Núcleos de Excelência) and FUNCITEC (Fundação de Apoio a Ciência e Tecnologia de Santa Catarina) (Brazil). K.M.T. is an undergraduate and R.M. and J.F. are PhD students in Pharmacology. They thank CNPq (Brazil) for fellowship support.
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Keywords:
Protein kinase C; Phorbol ester; Glutamate; Substance P; MAP kinase; Nociception; Mice
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