Home Medicine PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system
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PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system

  • Flavia Karine Rigo , Mateus Fortes Rossato , Gabriela Trevisan , Samira Dal-Toé De Prá , Rafael Porto Ineu , Mariane Bernardo Duarte , Célio José de Castro Junior , Juliano Ferreira and Marcus Vinicius Gomez EMAIL logo
Published/Copyright: October 1, 2017
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Scandinavian Journal of Pain
From the journal Scandinavian Journal of Pain Volume 17 Issue 1

Abstract

Background and aims

Cholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice.

Methods

Male albino Swiss mice (25–35 g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider’s venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001 mg/kg, i.p.) and the PhKv toxin (30 pmol/site i.t.) or neostigmine (100 pmol/site) were applied 15 min before the intraplantar capsaicin (1 nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1 mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37 °C for 10 min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405 nm.

Results

PhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4 h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of capsaicin (1 nmol/paw) caused a maximal antinociceptive effect of 69.5 ± 4.9% and 85 ± 2.5%, respectively. A pretreatment in mice with atropine; 5 mg/kg, i.p. or mecamylamine 0.001 mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED50 of 7.6 (4.6–12.6 pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3 nM), shifting the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE activity by PhKv.

Conclusions

Our findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response.

Implications

Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.

Keywords: PhKv; Pain; Antinociception; Muscarinic receptors; Nicotinic receptors; Acetylcholinesterase

1 Introduction

The agonists of nicotinic [1] and muscarinic [2] acetylcholine receptors (AChRs) are being evaluated as candidate analgesics for the treatment of pain. The antinoceptive effects of (–)- nicotine hydrogen tartrate (nicotine), a relatively non-selective nAChR agonist, have been demonstrated in preclinical and clinical studies [3,4]. In wild-type mice, a systemic intrathecal or an intracerebroventricular administration of centrally active muscarinic agonists resulted in robust analgesic effects, indicating that muscarinic analgesia mediates both the spinal and the supraspinal mechanisms. Centrally active muscarinic agonists display pronounced analgesic effects that are not observed in M2/M4 double-KO mice, indicating that both M2 and M4 mAChRs are involved in mediating a muscarinic analgesia [2].

The hydrolysis of ACh by acetylcholinesterase (AChE) has a key role in limiting the activation of both nicotinic and muscarinic receptors and cholinesterase inhibitors have shown an activity in clinical trials for pain [5]. A neostigmine treatment reversed a non-noxious stimulus of neuropathic pain in the streptozo- tocin neuropathic pain of rats, suggesting a potential therapeutic role for diabetic neuropathy [6]. A major site of action for choli- nomimetics in analgesia is the spinal cord. Intrathecal cholinergic agents cause antinociception by mimicking the release of ACh from the spinal cholinergic nerves. Drugs that modulate the cholinergic system (muscarinic/nicotinic agonists, acetylcholinesterase inhibitors) induce antinociception. Furthermore, the release of ACh acts as a modulator of the nociceptive signals in the spinal cord [7].

Various studies showing that spider venoms and some of its toxins are both nociceptive and antinociceptive [8]. Toxins isolated from spider venoms either inhibit or activate a vast number of targets, such as ion channels, with a high selectivity and an affinity [9]. For this selectivity, these animals have received the help of several million years. Thus, nature has evolved venoms into a huge pharmacological library of active pharmaceuticals with high selectivities and affinities, which can be explored as being therapeutics or to serve as templates for drug design. The spider toxins Pha1P and PhTx3–3 are calcium channel blockers that are effective in different rodent models of pain and in the control of chronic pathological pain, especially neuropathic [10,11] and cancer-related pain [12]. One component of Phoneutria nigriventer venom originally named PnTx3–1 [13] blocks voltage activated A-type potassium currents in the GH3 neuroendocrinal cell line [14]. In light of its potassium channel blocking activity, this toxin was renamed PhKv. This toxin reduced the ventricular arrhythmias that were induced by an occlusion of the left anterior descending coronary artery following a reperfusion [15]. This inhibitory effect of PhKv on cardiac arrhythmias was caused by a toxin-induced increase in the ACh release and it was blocked by atropine, a muscarinic receptor antagonist. Since the agonists of muscarinic and nicotinic ACh receptors display pronounced analgesic effects, it was decided to investigate the possible antinociceptive effects of PhKv in the cholinergic system.

2 Material and methods

2.1 Animals

Three-month-old male albino Swiss mice (25–35 g) that were bred in our animal house were used in this study. The animals were housed in a controlled temperature (22 ±2°C) with a 12 h light/dark circle, with the lights on at 6 a.m.

They were provided with standard rodent chow and tap water ad libitum. The animals were habituated in the experimental room for at least 1 h before the experiments. Each animal was used for only one experiment. The Ethics Committee of the Federal University of Minas Gerais authorised the studies, Protocol 347/2012. The experiments were performed in accordance with the current ethical guidelines for the investigation of experimental pain in conscious animals [16].

2.2 Drugs and chemicals

The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider’s venom according to Cordeiro, 1993 [13]. The PhKv toxin, previously named PnTx3–1, contained 40 amino acids (AECAAVYERCGKGYKRCCEERPCKCN IVNDNCTCKKFISE) with a molecular weight of 4575.4 dalton (Da) [17]. Capsaicin, atropine, mecamylamine, neostigmine, acetylthiocholine iodide, and 5,5’- dithiobis-2-nitrobenzoicacid (DTNB), were all purchased from Sigma (St Louis , MO, USA). The other reagents were of an analytical grade.

2.3 Intrathecal administration

Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system (CNS) by an intrathecal (i.t.) route as described elsewhere [18]. The injections were made with a 28-gauge needle that was connected to a Hamilton micro syringe with a volume of 2.5 μl/site. Before injecting the toxin and in order to validate the method, the experimenters had to perform a prior training with the i.t. administration of an anaesthetic (lidocaine 1%) followed by an observation for the development of a spinal blockade, indicated by a paralysis of both hind limbs. The experimenters were only accepted if they had previously performed i.t. injections of toxins and had achieved more than 90% accuracy in all of their injections. The correct puncture of the dura was indicated by a slight flick of the tail.

2.4 Thermal sensitivity

In order to evaluate whether PhKv could change the normal thermal sensitivity, we used a plantar test apparatus (Ugo Basile, Varese, Italy), according to Hargreaves 1988 [19]. Briefly, the animals were habituated in a place of observation that consisted of a Plexiglas chamber, for at least 60 min before the experiments. A radiant light beam that was generated by a 60 W light bulb was directed at the right hind paw in order to determine the basal withdrawal latency. The time that elapsed between the onset of the stimulus and the manifestation of the paw withdrawal response was measured automatically. This was taken as an index of the thermal nociceptive threshold. Any significant reduction in paw withdrawal latency were interpreted as being indicative of heat- induced hyperalgesia.

2.5 Mechanical sensitivity

The mechanical sensitivity was evaluated by using the up-and- down method, as described by Dixon (1980) [20], when using the von Frey filaments. Briefly, the mice were placed in cages with a wire mesh bottom which allowed for full access to the paws. The paw was touched with one of a series of seven von Frey hairs with logarithmic increments (0.02; 0.07; 0.16; 0.4; 1.0; 4.0; 8.0).The von Frey hairs were applied perpendicular to the plantar surface with sufficiently enough force in order to cause a slight buckling against the paw and they were held there for approximately 2–4 s. The stimuli were presented at intervals of several seconds, allowing for an apparent resolution of any behavioural responses to the previous stimuli [21].

2.5.1 Chronic constriction injury (CCI)

In order to induce neuropathic pain, the mice were anaesthetized with a mixture of ketamine (90 mg/kg) and xylazine (30 mg/kg) by an intraperitoneal (i.p.) injection. The chronic mononeuropathy damage was induced by a chronic constriction injury (CCI) of the sciatic nerve by using a similar procedure as previously described for rats [22] and it was adapted for mice [23]. The nerves were exposed in the midline of the thigh and close to its trifurcation and they were held by three loose ligatures with a distance from each other of ±1 mm, when using nylon thread (8.0). The incisions were sutured by using a nylon suture (6.0). In the sham surgery, the animals were anaesthetised and the sciatic nerves were exposed without performing a constriction. The sham-operated animals were used as neuropathic controls. The nociceptive tests (thermal and mechanical sensitivities) with PhKv (3–30 pmol/site i.t.) were performed 14 days after the procedures, since the mice did not present pain for seven days after surgery.

2.6 Capsaicin test

The peripheral capsaicin tests were carried out as has been previously described [24,25]. The animals were habituated in an observation location that consisted of a glass chamber for at least 60 min before the experiments. Following the habituation, PhKV was administrated 15 min before 1 nmol of capsaicin injected into the right hind paw of the mice and the total time that was spent licking the injected paws over 5 min was measured as an indicator of nociception. A vehicle solution (0.15% ethanol in saline) was prepared and it was used as a control treatment. The vehicle administrations did not evoke nociceptive behaviour themselves [26].The PhKv (3–100 pmol i.t.) or neostigmine (100pmol) pre-treatments were performed 15 min before the intraplantar capsaicin injections (1 nmol/paw).

2.6.1 Participation of the cholinergic system on the PhKv induced antinociception

In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5 mg/kg, i.p.) or mecamylamine (0.001 mg/kg, i.p.) and the PhKv toxin (30 pmol/site i.t.) or neostigmine (100 pmol/site) were applied 15 min before the intraplantar capsaicin (1 nmol/paw) administrations.

2.7 Acetylcholinesterase (AChE), in vitro assay

To investigate the effects of PhKv on the AChE activities, we performed an in vitro and an ex vivo assay that was based on the method as described by Pereira [27] and Walker [28]. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1 mg/ml) were used as source of AChE activity. The supernatants were incubated with the substrate acetylthiocholine iodide (10-7-10-1 M), DTNB (15mM), PhKv (100nM), boiled PhKv (100 nM), as negative control and PBS for the competitive assay. The reaction was monitored at 37 °C for 10 min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405 nm. The results were expressed as nM of ATC hydrolysed/min/mg protein of homogenates.

2.8 Acetylcholinesterase (AChE), ex vivo assay

For the ex vivo experiment, PhKv (3–100 pmol/site), boiled PhKv (100 pmol/site), neostigmine (100 pmol/site) or PBS were injected, intrathecally, in the mice as previously described. Fifteen minutes after the i.t. treatments, the mice were sacrificed and the lumbar portion of the spinal cord was collected, homogenised in PBS, centrifuged at 5000 × g (4°C) for 5 min and the supernatant was diluted to 1 mg of protein/ml, Bradford method. The supernatant was used as source of AChE activity of the central nervous system. The reaction was started by mixing 25 μl of supernatant with 25 μl DTNB (15 mM), 25 μl of acetylthiocholine (100 mM) and 25 μl of PBS. The reaction was monitored for 10 min (37 °C) in a FlexStation 3 MultiMode Microplate Reader (Molecular Devices) at 405 nm. The results were expressed as AChE activity (μmol/min/mg protein).

2.9 Experimental design

First set: Normal sensation - To investigate the effect of PhKv in the mechano/thermal sensations, mice were divided into three groups and treated with PBS, PhKv 30 pmol/site or 100 pmol/site. In different time points after, mechano/thermal sensation PhKv was evaluated as described before (3 groups, 5 mice each, 15 mice total).

Second set: Antinociceptive effect - To investigate the antinociceptive effect of PhKv CCI clinically relevant pain model, mice were initially divided into 4 groups CCI-PBS, CCI-PhKv, SHAM-PBS and SHAM-PhKv, (4 groups, 5 mice each, 20 mice total), to determine the time-course of its analgesic effect. Once the best time-point was selected, a new group of mice was divided into 8 groups, been treated with different doses of PhKv (0–30 pmol/site) to determine the best dose, Emax and ED50 values (8 groups, 5 mice each, 40 mice total).

Third set: Mechanism of action - To investigate the antinociceptive effect of PhKv in the capsaicin nociception test and investigate whether it could be related with AChE inhibition, we divided mice into 8 groups, treated with PBS, neostigmine, boiled PhKv, native PhKv (1–100 pmol/site) and subjected mice to capsaicin test. Right after the end of the capsaicin test (5 min), mice were sacrificed and the lumbar portion of spinal cord was collected for AChE ex vivo assay (8 groups, 5 mice each, 40 mice). To investigate the participation of muscarinic and nicotinic receptors in the PhKv and neostigmine antinociceptive effect, a new group of mice were divided into 6 groups: Vehicle-Vehicle, Vehicle-Neostigmine, Vehicle-PhKv, Atropine-Vehicle, Atropine-Neostigmine, Atropine- PhKv, Mecamylamine-Vehicle, Mechamylamine-Neostigmine and Mecamylamine-PhKv, and subjected to the capsaicin test (6 groups, 5 mice each, 30 mice total).

Fourth set: In vitro assay - To investigate the in vitro AChE inhibitory effect of PhKv, six mice were sacrificed and the spinal cord tissue was collected as biological source of AChE. (total 6 mice).

Summary - A total of 131 mice were used in the project, all subjected to the lowest dose enough to elicit the biological effect desired. During all the experiments, mice were observed for the development of signs of stress/distress or toxicity, also described in Table 1.

Table 1

Adverse effects in mice after spider peptide PhKv intrathecal injection (10, 30, 100 pmol/site).

Drug Dose (pmol/site, i.t.) Adverse effects
Body shake Serpentine tail Dynamic allodynia
PBS NA 0/7 0/7 0/7
PhKv 10 0/8 0/8 0/8
30 0/8 0/8 0/8
100[a] 3/3[**] 3/3[**] 3/3[**]

  1. Data expressed as the number of animals showing behaviours/total injected animals and were analyzed by χ2 test (n = 6–8)

2.10 Statistical analyses

The results were expressed as mean ±S.E.M. and the statistical analyses were performed by using the Student’s t-test, plus one-way or two-way analysis of Variance (ANOVA), followed by Dunnett’s post-test or Bonferroni’s post-test. The effective dose/concentrations that were capable of reducing the described effects by 50% (ED50 or EC50) were calculated by a non-linear regression of a dose-response curve, when using Graph Pad Prism 5.0 software and they were expressed as mean and the confidence intervals. The level of significance was set at p <0.05.

3 Results

3.1 Thermal sensitivity and mechanical threshold

PhKv (30 or 100 pmol/site i.t.) did not alter the thermal sensitivity (Fig. 1A), or the mechanical nociceptive thresholds, that were detected by using the von Frey filaments on the mice (Fig. 1B).

Fig. 1 Thermal and mechanical sensitivity of the mice that were treated intrathecally with PhKv. Normal (A) thermal and (B) mechanical sensitivity of the mice treated with PhKv (30 and 100 pmol/site) in mice without any nociceptive stimulation (naive). Statistical analyses were performed by using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test (n = 4).
Fig. 1

Thermal and mechanical sensitivity of the mice that were treated intrathecally with PhKv. Normal (A) thermal and (B) mechanical sensitivity of the mice treated with PhKv (30 and 100 pmol/site) in mice without any nociceptive stimulation (naive). Statistical analyses were performed by using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test (n = 4).

3.2 Antinociceptive effects of PhKv

In order to evaluate the mechanisms that were involved in neuropathic pain and in order to develop new therapeutic approaches, different experimental models of neuropathic pain have been developed, such as the CCI procedures on the sciatic nerves in rodents [22]. We used this experimental approach in order to examine the antinociceptive effects of PhKv. This peptide reduced the mechanical hyperalgesia that was induced by the CCI procedure and the antinociceptive effects were unrelated to any side effects.

PhKv toxin (3–30 pmol/site i.t.) generally reduced the neuropathic nociception that was caused by the CCI procedures and this was not observed in the sham operated animals (data not showed). The PhKv-mediated inhibition of nociception that was induced by the CCI procedures was concentration dependent, showing a maximum inhibitory effect at 10 pmol/site i.t. (Fig. 2A) and ED50 2.8 (1.29–3.43) pmol/site i.t. The antinociceptive effects of PhKv on the hyperalgesia that was induced by the CCI procedures lasted for 4 h, Fig. 2B. Boiled PhKv abolished the peptide antinociceptive effect (data not shown).

Fig. 2 Effects produced by a PhKv intrathecal administration in mice with chronic constriction injury. (A) Dose response curve of the antinociceptive effects of PhKv (3; 10; 30 pmol/site, i.t.) that were observed one hour after injection and (B) time- course of PhKv (10pmol/site, i.t.) over the mechanical allodynia (von Frey test) induced by CCI. Each point and vertical line represents the mean and the SEM of the values that were obtained in 7–8 animals. The effects are expressed as a paw withdrawal threshold (PWT) ing. Statistically significant differences between PhKv- and the saline-treated groups on the same day after treatment: *p < .0.05;**p < .0.01; ***p <0.001 (1- and 2-way repeated measures of ANOVA followed by Dunnett’s test and Bonferroni’s test, respectively) and #p<.0.001 denotes significant difference when compared with the baseline group (B Group).
Fig. 2

Effects produced by a PhKv intrathecal administration in mice with chronic constriction injury. (A) Dose response curve of the antinociceptive effects of PhKv (3; 10; 30 pmol/site, i.t.) that were observed one hour after injection and (B) time- course of PhKv (10pmol/site, i.t.) over the mechanical allodynia (von Frey test) induced by CCI. Each point and vertical line represents the mean and the SEM of the values that were obtained in 7–8 animals. The effects are expressed as a paw withdrawal threshold (PWT) ing. Statistically significant differences between PhKv- and the saline-treated groups on the same day after treatment: *p < .0.05;**p < .0.01; ***p <0.001 (1- and 2-way repeated measures of ANOVA followed by Dunnett’s test and Bonferroni’s test, respectively) and #p<.0.001 denotes significant difference when compared with the baseline group (B Group).

Next, we assessed the effects of PhKv and neostigmine on the nociception that was triggered by the capsaicin agonist of the TRPV1 (transient receptor potential vanilloid 1). TRPV1 is a cation channel that serves as a polymodal detector of painful stimuli (such as capsaicin, acid and heat) and it is expressed as a subtype of the C-type afferent fibres [29]. Neostigmine (100pmol) and PhKv (10, 30 and 100pmol) pre-treatments reduced the nociceptive response that was induced by capsaicin (Fig. 3A). The maximal inhibitory effect of PhKv was 69 ±4.9% (30 nmol/site i.t.) and an ED50 of 19.6 (7.2 to 53.5) nmol/site i.t. Boiled PhKv had no effect on the capsaicin induced nociception (Fig. 3A). The inhibitory effect of 100pmol neostigmine on capsaicin induced nociception was 85 ± 2.1% (Fig. 3A). The effect of the neostigmine, inhibitor of AChE activity, inhibiting capsaicin induced nociception, lead us to test the effect of PhKv on the hydrolysis of ACh. The effects of neostigmine and PhKv boiled/not boiled on AChE activity were performed in the ex vivo experiments, as previously described and is shown in Fig. 3B. Mice treatments with neostigmine (100 pmol, i.t.) and PhKv (10–100 pmol, i.t.) reduced the AChE activity of the spinal cord homogenates by 89 ± 2% and 83 ±1.9%, respectively (Fig. 3B). Boiled PhKv (100pmol, i.t.) had no effect on the ex vivo, AChE activity of the spinal cord homogenates, Fig. 3B.

Fig. 3 (A) Antinociceptive efects produced by a PhKv, boiled/not boiled (1.0, 3.0, 10, 30 and 100pmol/site) and neostigmine (neo, 100pmol/site) in mice subjected to the intraplantar capsaicin (1 nmol/paw), the effects are expressed as time spent licking paw (s). (B) Ex vivo activity of AChE in the presence of PhKv boiled/not boiled (3–100pmol/site) or neostigmine, neo (100pmol/site). The AChE activity is expressed as μmol/min/mg protein. For details see the Methods section Acetylcholinesterase (AChE), ex vivo Assay. *p <0.05, **p < 0.01 when compared with PBS group. ***p <0.001 when compared with vehicle group according one-way analysis of variance (ANOVA) followed by Dunnett’s post test (n = 4).
Fig. 3

(A) Antinociceptive efects produced by a PhKv, boiled/not boiled (1.0, 3.0, 10, 30 and 100pmol/site) and neostigmine (neo, 100pmol/site) in mice subjected to the intraplantar capsaicin (1 nmol/paw), the effects are expressed as time spent licking paw (s). (B) Ex vivo activity of AChE in the presence of PhKv boiled/not boiled (3–100pmol/site) or neostigmine, neo (100pmol/site). The AChE activity is expressed as μmol/min/mg protein. For details see the Methods section Acetylcholinesterase (AChE), ex vivo Assay. *p <0.05, **p < 0.01 when compared with PBS group. ***p <0.001 when compared with vehicle group according one-way analysis of variance (ANOVA) followed by Dunnett’s post test (n = 4).

3.3 Participation ofthe cholinergic system on the PhKv effects

To test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (muscarinic receptor antagonist, 5 mg/kg, i.p.) or mecamylamine (nicotinic antagonist 0.001 mg/kg, i.p.). In these pre-treated mice, PhKv (30 pmol/site i.t.) or neostigmine (100 pmol/site, i.t.) were applied 15 min before the intraplantar capsaicin injections (1 nmol/paw).

The pre-treatments with atropine or mecamylamine reversed neostigmine antinociceptive effect and attenuated the effect of PhKv by 59% (Fig. 4A) and 72% (Fig. 4B), respectively. Meanwhile, mice no pre-treated with atropine or mecamylamine showed that neostigmine (100 pmol/site, i.t.) or PhKv (30 pmol/site i.t.) inhibited the capsaicin induced nociception by 85 ±2.1 and 69 ±4.1%, respectively (Fig. 4 A and B).

Fig. 4 Participation ofthe cholinergic system in the antinociceptive effect of PhKv and neostigmine. The effects produced by a pre-treatment of (A) muscarinic receptor antagonist atropine (5 mg/kg, i.p.) and (B) nicotinic receptor antagonist mecamylamine (0.001 mg/kg, i.p.) in mice subjected to the intraplantar capsaicin (1 nmol/paw). Atropine or mecamylamine. were injected 30 min before PhKv (30 pmol/site i.t.) and neostigmine (neo,100 pmol/site) in mice subjected to the intraplantar capsaicin (1 nmol/paw). Statistical analyses were performed by using a one-way analysis of variance (ANOVA) followed by Dunnett’s post-test. ***p < 0.001 denotes the difference when compared PhKv or neostigmine with PBS and #p >0.05 denotes the difference when compared the PhKv or neostigmine with PBS group (n = 6).
Fig. 4

Participation ofthe cholinergic system in the antinociceptive effect of PhKv and neostigmine. The effects produced by a pre-treatment of (A) muscarinic receptor antagonist atropine (5 mg/kg, i.p.) and (B) nicotinic receptor antagonist mecamylamine (0.001 mg/kg, i.p.) in mice subjected to the intraplantar capsaicin (1 nmol/paw). Atropine or mecamylamine. were injected 30 min before PhKv (30 pmol/site i.t.) and neostigmine (neo,100 pmol/site) in mice subjected to the intraplantar capsaicin (1 nmol/paw). Statistical analyses were performed by using a one-way analysis of variance (ANOVA) followed by Dunnett’s post-test. ***p < 0.001 denotes the difference when compared PhKv or neostigmine with PBS and #p >0.05 denotes the difference when compared the PhKv or neostigmine with PBS group (n = 6).

3.4 Acetylcholinesterase assay

The hydrolysis of ACh by acetylcholinesterase (AChE) limits the activation of the nicotinic and muscarinic receptors [5]. An increase in the acetylcholine concentrations that were induced by cholinesterase inhibition caused antinociception effects by the activation of the nACh and mACh receptors [30,31]. In this context, we tested the in vitro effect of PhKv on the AChE activity of spinal cord homogenates of untreated mice, as source of the cholinesterase enzyme. PhKv inhibited AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3 nM), Fig. 5A. PhKv (100 nM) increased the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE (Fig. 5B).

Fig. 5 (A) In vitro concentration curve for the inhibitory effect of PhKv (10-13–10-6 M) on AChE activity of spinal cord homogenates. (B) Competitive assay of AChE activity in the presence of PhKv (100 nM) and substrate acetylthiocholine (10-7-1.0M). The AChE activity is expressed as nmol/min/mg of homogenates protein of 4 experiments. For other details see the Methods section Acetylcholinesterase (AChE), ex vivo Assay.
Fig. 5

(A) In vitro concentration curve for the inhibitory effect of PhKv (10-13–10-6 M) on AChE activity of spinal cord homogenates. (B) Competitive assay of AChE activity in the presence of PhKv (100 nM) and substrate acetylthiocholine (10-7-1.0M). The AChE activity is expressed as nmol/min/mg of homogenates protein of 4 experiments. For other details see the Methods section Acetylcholinesterase (AChE), ex vivo Assay.

3.4.1 Side effects

Behavioural adverse effects, such as serpentine-like tail movements, body shaking and dynamic allodynia, were all scored on day 14 after the induction of neuropathic pain by CCI. Table 1 shows that only a high dose of PhKv (100 pmol/site i.t.) caused side effects. Although adverse effects were only seen at a high dose of 100 pmol/site, i.t. this dose was nevertheless regularly used in other protocols, e.g., Figs. 1,3 and 5, so it can hardly be considered to be ‘far from the maximal therapeutic dosage’. At high dose (100 pmol/site i.t.), PhKv may have an action activating A-type potassium currents [14].

4 Discussion

Previously, we found that ventricular arrhythmias that were induced by an occlusion of the left anterior descending coronary artery were partially abolished by PhKv. The PhKv effects were blocked by atropine and they were potentiated by pyridostigmine, indicating a modulation of the cholinergic system [15]. PhKv induced an acetylcholine release in the neuromuscular junctions [15] suggesting, at least in part, that the antiarrhythmogenic effects that were evoked by PhKv were related to the release of acetylcholine. For several painful processes, the activation of the cholinergic system is well known [32,33]. In this study, we have investigated the antinociceptive effects of PhKv i.t. in neuropathic pain models with sciatic nerve injuries (CCI) and intraplantar capsaicin injections.

In the CCI models, PhKv caused a robust reduction on the mechanical withdrawals in the mice with maximum effects at 10 pmol/site i.t. and at ED50 2.8 (1.29–3.43) pmol/site i.t. We have used nerve injuries (CCI) model since it has a pathological similarity in relation to the painful diseases in humans. Neuropathic pain management is currently unsatisfactory and it remains a challenge in clinical practices. Neuropathic pain is considered as one of the most difficult types of pain to manage with conventional analgesics. Even with the numerous analgesic drugs available, neuropathic pain is still difficult to treat. The current pharmacological treatments for neuropathic pain are tricyclic antidepressants (amitriptyline) and certain anticonvulsants (gabapentin and pre- gabalin), [34,35]. However, these drugs are only able to clinically produce a 50% relief of neuropathic pain (in 40–60% of patients) and they are also associated with several side effects.

An intradermal injection of capsaicin causes pain, which was indicated by the behavioural manifestations, such as in the animal’s primary hyperalgesia and hypersensitivity [36]. PhKv (3–100 pmol/site i.t.) administrated before the intraplan- tar injections of capsaicin induced antinociception in a dose dependent manner with maximal inhibitory effects of 69.5 ± 4.9% (30 nmol/site i.t.) and an ED50 of 19.6 (7.2–53.5) nmol/site. Neostigmine also inhibited by 85 ± 2.1% the capsaicin induced nociception.

The study explored whether cholinergic receptors were involved in the PhKv antinociceptive actions. Atropine, an antagonist of the muscarinic receptor, and mecamylamine, an antagonist of the nicotinic receptor, caused an inhibition of the PhKv antinociceptive effects. Similarly, the antihypersensitivity effects of donepezil, a cholinesterase inhibitor, in the spinal nerves of rats were reduced by an intrathecal pre-treatment with atropine and mecamylamine [37].

In vitro experiments, using spinal cord homogenates of untreated mice, as source of the enzyme activity of AChE of central nervous system, showed that PhKv inhibited AChE with an EC50 of 20.8 (11.6–37.3 nM), shifting the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE activity by PhKv 100 nM, The inhibition of PhKv on AChE activity was also observed in ex vivo experiments. Mice treatments with intrathechal injection of neostigmine (100pmol, i.t.) or PhKv (10–100 pmol,) had reduced the AChE activity of the spinal cord homogenates by 89 ± 2% and 83 ±1.9%, respectively. Boiled PhKv (100 pmol, i.t.) had no effect on the AChE activity of the mice spinal cord homogenates. Clinical trials have shown that cholinesterase inhibition results in antinociception. Our results have agreed with studies suggesting that neostigmine inhibits the capsaicin noxious stimuli [30,31] and that cholinesterase inhibitors produce analgesia by the spinal mechanisms [38].

The inhibition of spinal AChE by PhKv presumably increase the availability of ACh in central cholinergic synapses, resulting in the activation of the muscarinic and nicotinic receptors and the consequent antinociception. The hydrolysis of ACh by AChE limits the activation of both nicotinic and muscarinic receptor stimulation [5]. PhKv may also stimulate the release of ACh from the nerve terminals, as has been previously observed [15] and this could enhance the antinociception. The inhibition of AChE by intrathecal PhKv would be facilitated by the abundance of this enzyme in the cholinergic synapses of spinal cords of rodents. Our findings have agreed with those studies that have shown that the major site of action for the cholinomimetics in analgesia is the spinal cord [5] and that painful stimuli are known to increase acetylcholine in the spinal cord [39]. There are other non-Phoneutria venom peptides that also inhibit AChE. A classic example in this case are fasciculins in the venom of the Easter green mamba snake, Dentroaspis angusticeps [40,41]. Fasciculins produce marked skeletal muscle fasciculation as a consequence of the AChE inhibition [42], but the potential analgesic effects of such an inhibition have not yet been investigated.

Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities, not only for health organisations, but also for pharmaceutical companies. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.

5 Conclusion

PhKv induces antinociception in chronic constriction injury and an intraplantar injection of capsaicin in mice. The antinociceptive effect of PhKv and neostigmine on nociceptive response induced by capsaicin is inhibited by agonists of muscarinic or nicotinic receptors, suggesting a cholinergic mechanism. Ex vivo data showed that PhKv and neostigmine reducing the capsaicin nociceptive process is simultaneous with the inhibition of AChE activity of the spinal cord homogenates. In vitro, PhKv (100 nM) inhibited AChE of central nervous system increasing the Km without affecting the Vm, characteristic of a competitive inhibition.

Highlights

  • PhKv, a peptide from P. nigriventer spider venom, does not affect thermal or mechanical sensitivity in mice.

  • PhKv caused antinociception in the chronic constriction injury model and after intraplantar injection of capsaicin.

  • Pretreatment of mice with atropine or mecamylamine inhibited neostigmine and PhKv-induced antinociception.

  • The antinociceptive activity of PhKv may be mediated by inhibition of AChE.

  • The inhibition of AChE activity by PhkV is competitive.

DOI of refers to article: http://dx.doi.org/10.1016/j.sjpain.2017.09.022.

Instituto de Ensino e Pesquisa Santa Casa Belo Horizonte, Rua Domingos Vieira, 590, 30150-240 Belo Horizonte, MG, Brazil

1These authors contributed equally to the development of this project.

  1. Ethical issues: The experiments were performed in accordance with the current ethical guidelines for the investigation of experimental pain in conscious animals and complying with ARRIVE-guidelines (16)(16).

  2. Conflict of interest: We wish to confirm that all the authors of this manuscript have no conflicts of interest and the manuscript was read and approved by all the authors. We also confirmed that the order of authors listed in the manuscript has been approved by all the authors. The corresponding author is the sole contact for the Editorial process.

  3. Funding This work was supported by REDE 00006–14 FAPEMIG (Minas Gerais State Agency for Research and Development), CNPq (471070/2012–2), CAPES Toxinology (AUX-PE 1444/2011) and FAPEMIG (PPM-00482–15). F.K. Rigo, M.F. Rossato and R.P. Ineu are Postdoctoral Fellows.

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Received: 2017-08-11
Revised: 2017-09-20
Accepted: 2017-09-21
Published Online: 2017-10-01
Published in Print: 2017-10-01

© 2017 Scandinavian Association for the Study of Pain

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https://doi.org/10.1016/j.sjpain.2017.09.019
Keywords for this article
PhKv; Pain; Antinociception; Muscarinic receptors; Nicotinic receptors; Acetylcholinesterase

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  1. Observational study
  2. Perceived sleep deficit is a strong predictor of RLS in multisite pain – A population based study in middle aged females
  3. Clinical pain research
  4. Prospective, double blind, randomized, controlled trial comparing vapocoolant spray versus placebo spray in adults undergoing intravenous cannulation
  5. Clinical pain research
  6. The Functional Barometer — An analysis of a self-assessment questionnaire with ICF-coding regarding functional/activity limitations and quality of life due to pain — Differences in age gender and origin of pain
  7. Clinical pain research
  8. Clinical outcome following anterior arthrodesis in patients with presumed sacroiliac joint pain
  9. Observational study
  10. Chronic disruptive pain in emerging adults with and without chronic health conditions and the moderating role of psychiatric disorders: Evidence from a population-based cross-sectional survey in Canada
  11. Educational case report
  12. Management of patients with pain and severe side effects while on intrathecal morphine therapy: A case study
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  14. Behavioral inhibition, maladaptive pain cognitions, and function in patients with chronic pain
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  17. Original experimental
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  19. Topical review
  20. New evidence for a pain personality? A critical review of the last 120 years of pain and personality
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  22. A multi-facet pain survey of psychosocial complaints among patients with long-standing non-malignant pain
  23. Clinical pain research
  24. Pain patients’ experiences of validation and invalidation from physicians before and after multimodal pain rehabilitation: Associations with pain, negative affectivity, and treatment outcome
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  28. COMBAT study – Computer based assessment and treatment – A clinical trial evaluating impact of a computerized clinical decision support tool on pain in cancer patients
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  34. Effects of validating communication on recall during a pain-task in healthy participants
  35. Original experimental
  36. Comparison of spatial summation properties at different body sites
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  48. Opioids in chronic pain – Primum non nocere
  49. Editorial comment
  50. Finally a promising analgesic signal in a long-awaited new class of drugs: TRPV1 antagonist mavatrep in patients with osteoarthritis (OA)
  51. Observational study
  52. The relationship between chronic musculoskeletal pain, anxiety and mindfulness: Adjustments to the Fear-Avoidance Model of Chronic Pain
  53. Clinical pain research
  54. Opioid tapering in patients with prescription opioid use disorder: A retrospective study
  55. Editorial comment
  56. Sleep, widespread pain and restless legs — What is the connection?
  57. Editorial comment
  58. Broadening the fear-avoidance model of chronic pain?
  59. Observational study
  60. Identifying characteristics of the most severely impaired chronic pain patients treated at a specialized inpatient pain clinic
  61. Editorial comment
  62. The burden of central anticholinergic drugs increases pain and cognitive dysfunction. More knowledge about drug-interactions needed
  63. Editorial comment
  64. A case-history illustrates importance of knowledge of drug-interactions when pain-patients are prescribed non-pain drugs for co-morbidities
  65. Editorial comment
  66. Why can multimodal, multidisciplinary pain clinics not help all chronic pain patients?
  67. Topical review
  68. Individual variability in clinical effect and tolerability of opioid analgesics – Importance of drug interactions and pharmacogenetics
  69. Editorial comment
  70. A new treatable chronic pain diagnosis? Flank pain caused by entrapment of posterior cutaneous branch of intercostal nerves, lateral ACNES coined LACNES
  71. Clinical pain research
  72. PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system
  73. Clinical pain research
  74. Lateral Cutaneous Nerve Entrapment Syndrome (LACNES): A previously unrecognized cause of intractable flank pain
  75. Editorial comment
  76. Towards a structured examination of contextual flexibility in persistent pain
  77. Clinical pain research
  78. Context sensitive regulation of pain and emotion: Development and initial validation of a scale for context insensitive avoidance
  79. Editorial comment
  80. Is the search for a “pain personality” of added value to the Fear-Avoidance-Model (FAM) of chronic pain?
  81. Editorial comment
  82. Importance for patients of feeling accepted and understood by physicians before and after multimodal pain rehabilitation
  83. Editorial comment
  84. A glimpse into a neglected population – Emerging adults
  85. Observational study
  86. Assessment and treatment at a pain clinic: A one-year follow-up of patients with chronic pain
  87. Clinical pain research
  88. Randomized, double-blind, placebo-controlled, dose-escalation study: Investigation of the safety, pharmacokinetics, and antihyperalgesic activity of L-4-chlorokynurenine in healthy volunteers
  89. Clinical pain research
  90. Prevalence and characteristics of chronic pain: Experience of Niger
  91. Observational study
  92. The use of rapid onset fentanyl in children and young people for breakthrough cancer pain
  93. Original experimental
  94. Acid-induced experimental muscle pain and hyperalgesia with single and repeated infusion in human forearm
  95. Original experimental
  96. Swearing as a response to pain: A cross-cultural comparison of British and Japanese participants
  97. Clinical pain research
  98. The cognitive impact of chronic low back pain: Positive effect of multidisciplinary pain therapy
  99. Clinical pain research
  100. Central sensitization associated with low fetal hemoglobin levels in adults with sickle cell anemia
  101. Topical review
  102. Targeting cytokines for treatment of neuropathic pain
  103. Original experimental
  104. What constitutes back pain flare? A cross sectional survey of individuals with low back pain
  105. Original experimental
  106. Coping with pain in intimate situations: Applying the avoidance-endurance model to women with vulvovaginal pain
  107. Clinical pain research
  108. Chronic low back pain and the transdiagnostic process: How do cognitive and emotional dysregulations contribute to the intensity of risk factors and pain?
  109. Original experimental
  110. The impact of the Standard American Diet in rats: Effects on behavior, physiology and recovery from inflammatory injury
  111. Educational case report
  112. Erector spinae plane (ESP) block in the management of post thoracotomy pain syndrome: A case series
  113. Original experimental
  114. Hyperbaric oxygenation alleviates chronic constriction injury (CCI)-induced neuropathic pain and inhibits GABAergic neuron apoptosis in the spinal cord
  115. Observational study
  116. Predictors of chronic neuropathic pain after scoliosis surgery in children
  117. Clinical pain research
  118. Hospitalization due to acute exacerbation of chronic pain: An intervention study in a university hospital
  119. Clinical pain research
  120. A novel miniature, wireless neurostimulator in the management of chronic craniofacial pain: Preliminary results from a prospective pilot study
  121. Clinical pain research
  122. Implicit evaluations and physiological threat responses in people with persistent low back pain and fear of bending
  123. Original experimental
  124. Unpredictable pain timings lead to greater pain when people are highly intolerant of uncertainty
  125. Original experimental
  126. Initial validation of the exercise chronic pain acceptance questionnaire
  127. Clinical pain research
  128. Exploring patient experiences of a pain management centre: A qualitative study
  129. Clinical pain research
  130. Narratives of life with long-term low back pain: A follow up interview study
  131. Observational study
  132. Pain catastrophizing, perceived injustice, and pain intensity impair life satisfaction through differential patterns of physical and psychological disruption
  133. Clinical pain research
  134. Chronic pain disrupts ability to work by interfering with social function: A cross-sectional study
  135. Original experimental
  136. Evaluation of external vibratory stimulation as a treatment for chronic scrotal pain in adult men: A single center open label pilot study
  137. Observational study
  138. Impact of analgesics on executive function and memory in the Alzheimer’s Disease Neuroimaging Initiative Database
  139. Clinical pain research
  140. Visualization of painful inflammation in patients with pain after traumatic ankle sprain using [11C]-D-deprenyl PET/CT
  141. Original experimental
  142. Developing a model for measuring fear of pain in Norwegian samples: The Fear of Pain Questionnaire Norway
  143. Topical review
  144. Psychoneuroimmunological approach to gastrointestinal related pain
  145. Letter to the Editor
  146. Do we need an updated definition of pain?
  147. Narrative review
  148. Is acetaminophen safe in pregnancy?
  149. Book Review
  150. Physical Diagnosis of Pain
  151. Book Review
  152. Advances in Anesthesia
  153. Book Review
  154. Atlas of Pain Management Injection Techniques
  155. Book Review
  156. Sedation: A Guide to Patient Management
  157. Book Review
  158. Basics of Anesthesia
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