Kinin Receptors Sensitize TRPV4 Channel and Induce Mechanical Hyperalgesia: Relevance to Paclitaxel-Induced Peripheral Neuropathy in Mice
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Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg9-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg9-[Leu8]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1–2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.
KeywordsPaclitaxel Peripheral neuropathy Neuropathic pain Kinins TRPV4
Peripheral neuropathy caused by paclitaxel (PTX) is a dose-limiting side effect characterized by painful paraesthesia of the hands and feet [1, 2]. Kinins were recently implicated in the physiopathology of PTX-induced peripheral neuropathy in mice . Kinins are algogenic peptides endogenously formed by the cleavage of kininogens by kallikreins. They act on two subtypes of G protein-coupled receptors, called B1 (B1R) and B2 (B2R) receptor. Typically constitutive, B2R receptors are expressed in peripheral tissues and the central nervous system, displaying higher affinity for bradykinin (BK) and kallidin peptides and mediating most of the physiological effects of the kinins. In contrast, B1R shows higher affinity for the kinin metabolites des-Arg9-BK (DABK) and Lys-des-Arg9-BK, and it is usually absent in peripheral tissues under physiological conditions. However, B1R can be upregulated during pathological states [4, 5] and its constitutive expression was previously detected in the central nervous system [6, 7].
The contribution of both kinin receptors to the transduction of pain has been widely demonstrated, particularly in chronic painful states. Of note, B1R and B2R are suggested to be involved in both the mechanical and thermal hypersensitivities induced by PTX in mice, but little is known of the mechanisms underlying these processes . Evidences from in vitro and in vivo studies have shown that the pro-algesic effects of kinins can be mediated by sensitization of transient receptor potential (TRP) channels, including the TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) channels [8, 9, 10, 11, 12, 13, 14]. Recently, BK was shown to sensitize the TRP vanilloid 4 (TRPV4) channel via a protein kinase C (PKC)-dependent mechanism , suggesting this channel might be implicated in the nociceptive actions of kinins.
TRPV4 is a cation channel sensitive to osmolarity changes and mechanical stimuli . Indeed, hypotonicity induces the excitation of dorsal root ganglion (DRG) neurons by activating TRPV4 channels and increasing intracellular Ca2+ levels . Inflammatory mediators have been shown to sensitize rodents to the nociceptive effects of hypotonicity, causing TRPV4-dependent overt nociception and mechanical hyperalgesia [17, 18, 19]. TRPV4 is also involved in PTX-induced peripheral neuropathy, by mediating both mechanical hyperalgesia and increased sensitivity to hypotonicity [20, 21, 22, 23].
Despite the well-characterized involvement of kinin receptors and TRPV4 channels in PTX-induced peripheral neuropathy in rodents, the downstream pathways to kinin receptor activation, as well as the sensitizers of the TRPV4 channel involved in the maintenance of PTX-induced mechanical hypersensitivity, are currently unknown. Herein, we present evidence on the crosstalk between kinins and TRPV4 channels and their contribution to PTX-induced mechanical hyperalgesia in mice.
Male adult Swiss mice (8–10 weeks) were used. Animals were housed in a room with controlled temperature (22 ± 2 °C) and humidity (~60–80%) under a 12:12 h light-dark cycle (lights on at 06:00 a.m.). Food and water were provided ad libitum. Mice were randomly distributed in experimental groups (n = 6–10/group), and behavioural experiments were conducted in a blinded manner in order to reduce experimental bias. The n numbers and the intensity of noxious stimuli used in this study were the minimum necessary to achieve consistent effects. All experimental procedures were carried out in accordance with the National Institutes of Health animal care guidelines (NIH publications No. 8023) and were approved by the Ethics Committee of the Universidade Federal de Santa Catarina (protocol number PP00811). All animal studies are in compliance with the ARRIVE guidelines .
Mechanical Hyperalgesia Induced by Selective Kinin Receptor Agonists
In order to investigate whether TRPV4 contributes to the mechanical hyperalgesia induced by kinins, animals (n = 8/group) received either the selective kinin B2 (BK; 3 nmol/paw; 20 μl) or B1 (DABK; 30 nmol/site; 5 μl) receptor agonist by intraplantar (i.pl.) and intrathecal (i.t.) routes, respectively. Vehicle (saline)-treated animals were used as controls. Mechanical nociceptive thresholds were measured at different time points (20–240 min) following the injection of the stimuli.
Mechanical Hyperalgesia Induced by TRPV4 Activation in Animals Sensitized by Kinin Receptor Agonists
It was investigated whether BK or DABK could sensitize mice to the nociceptive response elicited by the TRPV4 activators, 4α-PDD and hypotonic solution. For this, 10 μl of saline (0.9% NaCl) containing either BK (0.3 nmol/paw) or DABK (20 nmol/paw) were injected intraplantarly into the right mouse hindpaw (n = 8/group). Control groups received an equal volume of saline. After 5 min, the TRPV4 agonist 4α-PDD (1 nmol/paw, 10 μl of 1% DMSO) or hypotonic solution (10 μl of deionized water) was injected into paws previously treated with the algogens. Mechanical thresholds were evaluated 15–120 min after the stimuli. Control groups received 10 μl of vehicle (1% DMSO) or isotonic solution (0.9% NaCl), respectively.
To evaluate the mechanisms implicated in the sensitizing effect of BK to hypotonicity, animals received one of the following drugs: the selective TRPV4 (HC-067047; 3 μg/paw), kinin B1 (des-Arg9-[Leu8]-bradykinin, DALBK; 3 nmol/paw), kinin B2 (HOE 140; 3 nmol/paw) or TRPV1 (SB366791; 1 nmol/paw) receptor antagonists; the non-selective cyclooxygenase (COX) 1/2 (indomethacin; 5 mg/kg, p.o.) or catecholamine release (guanethidine; 30 mg/kg, s.c.) inhibitors, to assess the involvement of prostaglandins and catecholamines, respectively; or the selective phospholipase A2 (PLA2) (PACOCF3; 1 nmol/paw), protein kinase A (PKA) (KT-5720; 3 nmol/paw), phospholipase C (PLC) (U73122; 30 pmol/paw) or PKC (GF109203X; 3 nmol/paw) inhibitors. Indomethacin and guanethidine were given 1 h prior to BK (0.3 nmol/paw). All other drugs were co-injected with BK (5 min prior to hypotonic solution injection). Treatment schedules are represented in Fig. 1(c).
Mechanical Hyperalgesia Induced by Hypotonicity in Animals Sensitized by PKCε Activation
We also evaluated whether PKCε activation sensitizes mice to hypotonicity. Thus, the selective PKCε activator ΨεRACK (0.1 μg/paw) or its vehicle (0.9% NaCl; control group) was intraplantarly injected (in 10 μl of saline) into the right mouse hindpaw (n = 8/group). After 5 min, a hypotonic solution (10 μl of deionized water) was injected into the paws previously treated with the sensitizing agent, and the mechanical thresholds were then evaluated after 15 min. Control groups received 10 μl of isotonic solution (0.9% NaCl).
Mechanical Hyperalgesia Induced by PTX in Mice
Peripheral neuropathy was induced by PTX as previously described  and adapted for mice . Briefly, mice (n = 6–10/group) were injected with PTX (2 mg/kg, i.p.) for five consecutive days (days 0 to 4), using an injection volume of 10 ml/kg. The cumulative dose of PTX was of 10 mg/kg. Control animals received vehicle (0.9% NaCl). The development of peripheral neuropathy was assessed 7 days after the first injection of PTX by testing the mouse sensitivity to mechanical stimuli.
Mechanical Threshold Measurements
Mechanical nociceptive thresholds were evaluated as previously described . Initially, mice were individually placed in clear Plexiglas boxes (9 × 7 × 11 cm) on elevated wire-mesh platforms to allow access to the ventral surface of the right hindpaw. Animals were acclimatized to the boxes for 1 h before the initiation of the behavioural tests. Through the wire-mesh floor of the chamber, a series of eight von Frey hair monofilaments (Stöelting, Wood Dale, IL, USA), calibrated to produce incremental forces of 0.02 to 2 g, were applied to the plantar surface of the right hindpaw for a maximum of 3 s, or until the animal displayed a nocifensive response. Tests were initiated with a 0.6-g filament. In the absence of a nocifensive response, incrementally stronger filaments were used consecutively until a response was elicited. If the 0.6-g filament elicited a response, decrementally weaker filaments were used until one of them failed to cause a nocifensive response. The data collected using this up-down method were then used to calculate paw withdrawal thresholds (in g) . Baseline measurements were taken before the injection of the nociceptive stimuli. Significant decreases in the paw withdrawal thresholds were interpreted as indicatives of mechanical hyperalgesia.
Overt Nociception Induced by Hypotonicity in PTX-Treated Mice
Overt nociception was induced as described previously . Briefly, 7 days after the first PTX treatment, animals (n = 10) were individually placed into inverted glass cylinders of 20 cm in diameter, for at least 30 min, in order to acclimatize them to the experimental environment. Then, 20 μl of hypotonic (deionized water) or isotonic (0.9% NaCl; control) solutions was injected intraplantarly, in both PTX- and vehicle-treated mice, into the right mouse hindpaw, using a 500-μl insulin syringe (30-gauge needle). Mice were then observed for 5 min. The amount of time spent shaking, licking, flinching or elevating the injected paw was measured with a chronometer and considered as an indicative of overt nociception.
In order to assess the contribution of TRPV4, kinin B1 and B2 receptors to the increased sensitivity to hypotonicity in PTX-treated mice, animals received either HC-067047 (3 μg/paw), DALBK (150 nmol/kg, i.p.) or HOE 140 (50 nmol/kg, i.p.), respectively. We also verified the involvement of PKCε in this response by using the selective peptide PKCε inhibitor εV1–2 (9 μg/paw). HC-067047 was co-injected with hypotonic solution 7 days after the first injection of PTX. DALBK, HOE 140 or the εV1–2 peptide was given twice a day (every 12 h) for 6 days, starting at the time of the first PTX treatment. Eight to twelve animals were used per group in each experiment. Treatment schedules are represented in Fig. 2(b).
Membrane and Cytosolic Extract Preparation
To evaluate the contribution of kinin B1R and B2R to the increased levels of PKCε in PTX-treated mice, neuropathic mice received either the selective B1 DALBK (150 nmol/kg, i.p., n = 6–8) or B2 HOE 140 (50 nmol/kg, i.p., n = 6) receptor antagonists, given twice a day (every 12 h) for 6 days, starting at the time of the first PTX treatment. Vehicle-treated mice were used as controls (n = 6). Seven days after the first PTX treatment, animals (under isoflurane anaesthesia) were killed by decapitation, and the hindpaw skins and L1 to L6 DRGs were collected. PKCε levels were assessed as previously described , with minor modifications. Hindpaw skin samples were homogenized in 500 μl of ice-cold buffer A (10 mM HEPES; pH 7.4, 2 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 50 mm sodium fluoride, 10 mm β-glycerophosphate, 1 mM dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 1 mM sodium orthovanadate). Homogenates were vigorously shaken and chilled on ice for 15 min and then centrifuged at 14,000 rpm for 45 min, at 4 °C. Supernatants (cytosolic fraction) were stored at −70 °C until further use. For the obtention of membrane extracts, the resulting pellets were washed twice in ice-cold PBS and suspended in 200 μl of buffer A containing Triton X-100 (1%). Samples were sonicated for 5 min, centrifuged at 14,000 rpm, at 4 °C, for 45 min, and the supernatant was collected and stored at −70 °C until further use. Dorsal root ganglion tissue samples from L1 to L6 segments were homogenized in 50 μl of RIPA buffer containing protease inhibitors. The lysates were centrifuged twice at 14,000g for 10 min, at 4 °C, and the supernatant was collected and stored at −70 °C until further use.
Protein concentrations were determined by spectrophotometry (NanoDrop Spectrophotometer ND-1000; Thermo Scientific, Rockford, IL, EUA). Samples were then diluted in 5× Laemmli buffer (25% of 62.5 mM Tris-HCl (pH 6.8), 2% glycerol, 0.01% SDS and bromophenol blue) containing 5% β-mercaptoethanol, adjusted to the same amount of protein and boiled at 100 °C for 5 min.
Western Blotting Assay
Fifteen micrograms of protein were loaded per lane and electrophoretically separated using 12% denaturing SDS polyacrylamide gel electrophoresis (PAGE). Proteins were then transferred to nitrocellulose membranes using a Mini Trans-Blot Cell System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer’s protocol. Membranes were blocked using 10% BSA in 0.05% TBST solution for 1 h, at 4 °C, and probed with the following antibodies: mouse anti-β-actin (sc-81178, 1:1000) and rabbit anti-PKCε (sc-214, 1:1000), both from Santa Cruz Technology Biotechnology, Inc. (Dallas, TX, USA), diluted in blocking buffer and incubated at 4 °C, overnight. Following incubation, membranes were washed and incubated with specific secondary antibodies conjugated to horseradish peroxidase (1:25.000; Cell Signaling Technology, Danvers, MA, USA). Immunocomplexes were visualized using SuperSignal West Femto Chemiluminescent Substrate Detection System (Thermo Fischer Scientific, Rockford, IL, USA). Protein levels were quantified by optical density using ImageJ Software®, and densitometric values were normalized as the ratio to β-actin in arbitrary units (AU). Results are expressed as a percentage of PKCε expression in relation to the control group.
The following drugs were used: paclitaxel (PTX; Dosa S.A., Buenos Aires, Argentina); 4α-phorbol 12,13-didecanoate (4α-PDD), bradykinin (BK), des-Arg9-bradykinin (DABK), des-Arg9-[Leu8]-bradykinin (DALBK), guanethidine, HC-067047, indomethacin and SB366791 (Sigma Chemical, Saint Louis, MO, USA); GF109203X, KT-5720, PACOCF3 and U73122 (Tocris Bioscience, Bristol, UK); and εV1–2 (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) and ΨεRACK (His-Asp-Ala-Pro-IIe-Gly-Tyr-Asp) (GenScript USA, Inc., Piscataway, NJ, USA). PTX stock solution (6 mg/ml in Cremophor EL®) was diluted in saline (0.9% NaCl) to a concentration of 0.2 mg/ml (injection solution). 4α-PDD and HC-067047 were diluted in 1% dimethyl sulfoxide (DMSO) in saline. Indomethacin was diluted in 5% Na2CO3. SB366791, GF109203X, KT-5720, PACOCF3 and U73122 stock solutions (10−3 M) were prepared in ethanol. For in vivo experiments, the final concentration of ethanol did not exceed 1%. All other drugs were dissolved in saline. The doses of TRPV4 and kinin B1 and B2 receptor antagonists were selected based on previous studies [3, 30].
Results are presented as the mean ± SEM of 6–10 animals for each experimental group. Statistical comparisons were performed by two-way ANOVA followed by the Bonferroni post test, one-way ANOVA followed by the Newman–Keuls post test or Student’s t test, in GraphPad Prism software, version 5.01. P values <0.05 were considered significant. Power analysis was performed in order to calculate the size of the experimental groups.
TRPV4 Mediates the Mechanical Hyperalgesia Induced by Kinin Receptor Agonists
BK Sensitizes Mice to TRPV4 Activators Causing Mechanical Hyperalgesia
Effect of different classes of drugs on the sensitizing effect of bradykinin to hypotonicity
Drugs and doses
Mechanical threshold (g)
Hypotonicity (10 μl of deionized water)
BK (0.3 nmol/paw)
HC-067047 (3 μg/paw)
0.88 ± 0.12
0.66 ± 0.10
0.12 ± 0.05*
0.54 ± 0.16#
78 ± 30%
HOE 140 (3 nmol/paw)
0.87 ± 0.11
0.66 ± 0.11
0.15 ± 0.04*
0.56 ± 0.09#
80 ± 18%
DALBK (3 nmol/paw)
0.87 ± 0.11
0.66 ± 0.11
0.15 ± 0.04*
0.14 ± 0.07*
Indomethacin (5 mg/kg, p.o.)
0.87 ± 0.11
0.62 ± 0.05
0.13 ± 0.05*
0.18 ± 0.05*
Guanethidine (30 mg/kg, s.c.)
Sympathetic amine release inhibitor
0.89 ± 0.15
0.76 ± 0.13
0.15 ± 0.04*
0.21 ± 0.09*
PACOCF3 (1 nmol/paw)
1.02 ± 0.10
0.75 ± 0.05
0.24 ± 0.08*
0.21 ± 0.07*
KT-5720 (3 nmol/paw)
0.98 ± 0.05
0.76 ± 0.13
0.22 ± 0.09*
0.27 ± 0.08*
SB366791 (1 nmol/paw)
0.84 ± 0.13
0.62 ± 0.08
0.19 ± 0.10*
0.26 ± 0.11*
U73122 (30 pmol/paw)
1.02 ± 0.13
0.77 ± 0.06
0.24 ± 0.08*
0.65 ± 0.12#
77 ± 22%
GF109203X (3 nmol/paw)
0.95 ± 0.10
0.70 ± 0.06
0.20 ± 0.05*
0.48 ± 0.10#
56 ± 20%
The Sensitizing Effect of BK to Hypotonicity Is Dependent on PLC and PKC Activation
As depicted in Table 1, treatment with the selective PLC (U73122; 30 pmol/paw) or PKC (GF00109203X; 3 nmol/paw) inhibitors significantly attenuated the mechanical hyperalgesia induced by hypotonicity in animals pre-injected with BK (0.3 nmol/paw). However, neither the treatment with the selective PLA2 (PACOCF3; 1 nmol/paw) nor the PKA (KT-5720; 3 nmol/paw) inhibitor prevented this response (Table 1). Similarly, neither the i.pl injection with the selective TRPV1 antagonist SB366791 (1 nmol/paw) nor the systemic treatment with the non-selective COX 1/2 (indomethacin; 5 mg/kg, p.o., 1 h) or the catecholamine release (guanethidine; 30 mg/kg, s.c., 1 h) inhibitor was able to interfere with the BK-induced sensitization to hypotonicity (Table 1).
PTX-Induced Mechanical Hyperalgesia Is Similarly Reduced by Both Kinin Receptors and TRPV4 Antagonism
Kinin Receptors Mediate Hypotonicity-Induced Overt Nociception in PTX-Treated Mice
PTX-treated mice exhibited increased overt nociception following the i.pl. injection of hypotonic solution (10 μl of deionized water) when compared with vehicle-treated animals (Fig. 5b). This response was prevented by the co-treatment with the selective TRPV4 antagonist HC-067047 (3 μg/paw) (data not shown). Similarly, mice systemically treated with either the selective kinin B1R (DALBK; 100 nmol/kg) or B2R (HOE 140; 50 nmol/kg) antagonists (Fig. 5c) did not exhibit significant overt nociception.
Kinin Receptor Antagonism Inhibited the Increased Levels of PKCε in PTX-Treated Mice
PKCε Mediates Hypotonicity-Induced Overt Nociception in PTX-Treated Mice
Paclitaxel is one of the most effective and commonly used anti-neoplastic drug. However, its major dose-limiting side effect is the development of peripheral sensory neuropathy, which remains without satisfactory treatment and compromises patient’s quality of life [2, 31]. Hence, understanding the mechanisms underlying this syndrome is critical to the discovery of new molecular targets and to the development of more effective analgesic drugs. We have recently reported that in the absence of kinin receptor (B1R and B2R) activation, there is a decrease in PTX-induced mechanical hyperalgesia . Similarly, the mechanical hyperalgesia induced by PTX was shown to be reduced in TRPV4-deficient mice  and in animals treated with TRPV4 antisense oligodeoxynucleotides  or selective TRPV4 antagonists [22, 23]. Here, we show that TRPV4 channels mediate the mechanical hyperalgesia induced by the activation of kinin receptors and that this phenomenon may contribute to PTX-induced peripheral neuropathy in mice.
Although it is well known that BK causes hypersensitivity to mechanical stimuli, the mechanisms underlying this response are not completely understood . Importantly, TRPV4 is a mechanotransducer channel that contributes to the mechanical hyperalgesia induced by pro-inflammatory mediators such as prostaglandins and proteases [18, 19]. Here, we investigated whether TRPV4 mediates the mechanical hyperalgesia induced by BK (a B2R agonist) and DABK (a B1R agonist) in mice. We showed that the treatment with the selective TRPV4 antagonist HC-067047 significantly inhibits the nociceptive responses elicited by both BK and DABK, suggesting that TRPV4 sensitization occurs downstream to B2R and B1R activation in order to induce mechanical hypersensitivity in mice. Since the i.pl. injection of DABK is not capable of producing any detectable overt nociceptive response  or mechanical hyperalgesia in mice (data not shown), DABK was given i.t. Indeed, this approach is known to produce increased sensitivity to both thermal and mechanical stimuli in mice [33, 34]. In fact, B1 receptors are reported to be constitutively and functionally expressed in the central nervous system [6, 33, 34, 35]. To our knowledge, we present here the first evidences on that kinin receptors and TRPV4 interact in vivo.
Corroborating the above data, a sub-effective dose of BK sensitized mice to the TRPV4 activator 4α-PDD or hypotonic solution and caused mechanical hyperalgesia. Nonetheless, the pre-injection of DABK (20 nmol/paw) was not able to sensitize mice to hypotonicity (data not shown). Antagonism of B2R or TRPV4, but not B1R, significantly attenuated the mechanical hyperalgesia induced by the combination of BK and hypotonicity, providing, therefore, additional evidence on the involvement of these receptors in this response. Conversely, the antagonism of B1R was not effective, possibly because BK acts preferentially on B2R and needs to be degraded in DABK in order to activate B1R . Additionally, B1R is suggested to be non-functional in peripheral tissues under normal conditions and needs to be stimulated in order to mediate nociception [11, 36].
Our findings also indicate that the sensitizing effect of BK to hypotonicity is dependent on PLC and PKC activation as inhibition of their activity significantly attenuated the mechanical hyperalgesia induced by hypotonicity in BK-injected animals. The PLC/PKC signalling pathway was previously suggested to be involved in the sensitization of TRPV4 by inflammatory mediators in HEK293 cells . Nonetheless, BK can also sensitize nociceptors by mechanisms other than the PLC/PKC pathway, including indirect sensitization by releasing of prostaglandins or sympathomimetic amines , increases in the intracellular Ca2+ concentration by opening of TRPV1 channels [12, 13] or activation of PLA2 or PKA enzymes [14, 38]. However, the sensitizing effect of BK to hypotonicity seems to be independent of these mechanisms as the blockade of such pathways did not interfere with the mechanical hyperalgesia induced by hypotonicity in BK-injected mice. Therefore, we can conclude that the sensitizing effect of BK to hypotonicity is mainly dependent on PLC and PKC activation.
It was previously demonstrated that TRPV4 and kinin receptors play a crucial role in the peripheral neuropathy induced by PTX in rodents [3, 20]. Indeed, the deletion of TRPV4 or the treatment with TRPV4 antisense oligodeoxynucleotides  or selective antagonists for this channel [22, 23] inhibits PTX-induced mechanical hyperalgesia in rodents. Similar results were found in mice lacking kinin receptors and also in those treated with selective kinin B1R or B2R antagonists . However, to date, the downstream pathways activated by kinin receptors to maintain PTX-induced mechanical hyperalgesia are unknown. Herein, the treatment with the selective kinin receptor antagonists, DALBK and HOE 140, presented similar efficacy to that observed for the selective TRPV4 antagonist (HC-067047) on the PTX-induced mechanical hyperalgesia in mice. These results suggest that kinin receptors modulate TRPV4 channel activity. Additionally, it is possible that these receptors may act through common anti-nociceptive pathways, as both the TRPV4 and the kinin receptor antagonists exhibited similar inhibition profiles in our model.
To investigate this hypothesis, we evaluated the effect of kinin receptor antagonists on the overt nociception induced by hypotonicity in PTX-treated mice. We found that in addition to the mechanical hyperalgesia, peripheral neuropathy induced by PTX is accompanied by increased sensitivity to hypotonicity, characterized by TRPV4-dependent overt nociception [17, 20]. We show that the treatment with either DALBK (B1R antagonist) or HOE 140 (B2R antagonist) reduces hypotonicity-induced overt nociception in PTX-treated animals, suggesting that kinins act on their receptors and sensitize TRPV4 channels, thus contributing to the peripheral neuropathy induced by PTX in mice.
PKCε activation was previously suggested to contribute to the mechanical hyperalgesia caused by PTX  and to the sensitization of TRP channels by inflammatory mediators [9, 40, 41, 42]. Thus, we investigated whether PKCε activation acts downstream to the B1R and B2R activation in order to sensitize TRPV4 channels in our model. PTX treatment increased the levels of PKCε but did not change the ratio between membrane and cytoplasm protein expression (data not shown), suggesting a change in PKCε expression rather than in its trafficking to the cell membrane. Importantly, treatment with the selective kinin receptor antagonists reduced the levels of PKCε in both the membrane and cytosolic extracts from the plantar skin of neuropathic mice, indicating that kinins may regulate its expression. Corroborating these findings, kinin receptor antagonism reduced the levels of PKCε in DRG total extracts from PTX-treated animals. Indeed, it was recently shown that PKCε mediates the pro-nociceptive response to kinins in a mouse model of muscle pain induced by formalin .
We also reported here that the selective PKCε activator (ψεRACK) is able to sensitize mice to hypotonicity and, most importantly, that the treatment with the selective PKCε inhibitor εV1–2 reduces hypotonicity-induced overt nociception in PTX-treated mice in a similar manner to that observed for the kinin receptor antagonists. Collectively, our findings suggest that PKCε acts downstream to B1R and B2R activation to sensitize TRPV4 channels in PTX-treated mice. Indeed, PKCε activation was implicated in the sensitization of TRPV4 by inflammatory mediators, contributing to mechanical hyperalgesia . Additionally, Chen and collaborators  proposed that PKCε modulates TRPV4 activity in PTX-induced peripheral neuropathy in mice, based on the similar efficacy observed for a PKCε inhibitor and a TRPV4 antagonist in mechanical hyperalgesia.
Overall, we demonstrate that kinins sensitize TRPV4 to cause mechanical hyperalgesia and that this phenomenon may contribute to PTX-induced peripheral neuropathy.
In summary, this study suggests that kinins sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. Our results also demonstrate that this mechanism of interaction seems to contribute to the maintenance of mechanical hyperalgesia induced by PTX through the activation of PKCε. These evidences are novel and support the notion that these receptors are potential pharmacological targets for the development of new and effective analgesics to treat chronic pain.
The study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) e Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA). M.A.B and M.N.M. are PhD students funded by the CNPq. G.C.S. and F.C.D are master’s students funded by the CAPES.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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