Chronic neuropathic pain is the result of disease or damage to the peripheral or the central nervous system and is characterized by hyperalgesia, allodynia, and spontaneous pain (Dworkin et al., 2003; Jarvis and Boyce-Rustay, 2009). Different types of drugs, including opioids, are used to relieve neuropathic pain, but many patients suffering from this type of pain are refractory to the existing treatments (Caviedes and Herranz, 2002; Dworkin et al., 2003, 2010; Furlan et al., 2006). In terms of the opioids, reduced sensitivity to systemic opioids is observed in neuropathic pain, and an increase in their dose may be necessary for adequate pain relief (Portenoy, 1990; Portenoy and Hagen, 1990; Przewlocki and Przewlocka, 2001; Eisenberg et al., 2005). In addition, side-effects secondary to the central nervous system actions of opioids present barriers to their clinical use in managing neuropathic pain. Therefore, the lack of effective treatments and the complex mechanisms involved in its genesis and maintenance make neuropathic pain an area of unmet therapeutic need.
We have recently demonstrated that crotalphine, a structural analogue to a novel analgesic peptide first identified and isolated from the crude venom of the South American rattlesnake Crotalus durissus terrificus (Konno et al., 2008), induces potent antinociceptive effects in a rat model of neuropathic pain produced by chronic constriction injury (CCI) of the sciatic nerve (Gutierrez et al., 2008). The peptide, administered in low doses (0.2–5 µg/kg) by the oral route, induces a long-lasting (3 days) antinociceptive effect in the CCI model, mediated by activation of peripheral opioid receptors (Gutierrez et al., 2008).
It is important to stress that despite having been first characterized in snake crotalid venom, crotalphine could not be considered a toxin, as its amino acid sequence is identical to the γ-chain of crotapotin (Konno et al., 2008), a nontoxic and nonenzymatic component of this venom. Our previous experimental data have also indicated that the antinociceptive effect of crotalphine is not attributable to alterations in general activity, as the frequencies of locomotion and rearing, determined in the open field test, were not altered by the peptide (Gutierrez et al., 2008).
Despite the demonstration of the involvement of peripheral opioid receptors in the antinociceptive effect of crotalphine in the CCI model (Gutierrez et al., 2008), the type of opioid receptor and the subsequent molecular mechanisms responsible for this effect have not yet been characterized. Konno et al. (2008) have shown that the antinociceptive effect of crotalphine in the prostaglandin E2-induced and carrageenin-induced mechanical hyperalgesia models in rats is mediated by the activation of peripheral κ-opioid receptors. However, as pointed out by these authors, crotalphine does not directly activate opioid receptors, indicating that the opioid-mediated effects of the peptide lie downstream of its direct actions (Konno et al., 2008).
Several lines of evidence have indicated that the molecular mechanisms involved in the action of μ-opioid, δ-opioid, and κ-opioid agonists include, at the peripheral level, activation of the L-arginine–nitric oxide (NO)–cyclic GMP (cGMP) pathway and opening of ATP-sensitive K+ channels (Cunha et al., 1991; Nozaki-Taguchi and Yamamoto, 1998; Rodrigues and Duarte, 2000; Sachs et al., 2004). In addition, we have previously demonstrated that the antinociceptive activity of C. d. terrificus crude venom, against carrageenin-induced and prostaglandin E2-induced hyperalgesia, is mediated by the activation of κ-opioid or δ-opioid receptors, with the subsequent activation of the L-arginine–NO–cGMP pathway and opening of the ATP-sensitive K+ channels (Picolo et al., 2000; Chacur et al., 2003; Picolo and Cury, 2004).
The role of the L-arginine–NO–cGMP pathway in neuropathic pain is still controversial, as there are several studies showing that NO, the NO–cGMP pathway, or cGMP–protein kinase G (PKG) have pronociceptive effects (Yoon et al., 1998; Song et al., 2006). However, Wang et al. (2008) have recently demonstrated that the spinal NO–cGMP–protein kinase G–K+ channel pathway mediates the antihyperalgesic effects of bovine lactoferrin in the CCI model of neuropathic pain in rats. In addition, the local application of NO-releasing drugs exerted an antiallodynic effect in human diabetic neuropathies (Yuen et al., 2002), indicating an analgesic role for peripheral NO in neuropathic pain conditions.
On the basis of our previous work, crotalphine induces a potent and long-lasting antinociceptive effect in a rat model of neuropathic pain produced by CCI of the sciatic nerve, which is mediated by the activation of peripheral opioid receptors; the present work was carried out to characterize the type of opioid receptor involved in this effect and to verify the possible contribution of the peripheral L-arginine/NO/cGMP pathway and opening of K+ channels. In this study, crotalphine was administered by the oral route, at doses of 0.2 and 5 µg/kg. These doses were chosen because Gutierrez et al. (2008) observed that 0.2 μg/kg of the peptide partially inhibited the nociceptive phenomena induced by CCI, whereas the dose of 5 μg/kg blocked these phenomena and also increased the nociceptive threshold, as compared with basal values. Furthermore, the peptide was also administered by the intraplantar route (0.0006 μg/paw), in order to confirm its peripheral (local) antinociceptive effect.
Male Wistar rats (170–190 g) were used throughout this study. Animals were housed in a temperature-controlled (21±2°C) and light-controlled (12/12 h light/dark cycle) room. All behavioral tests were performed between 09:00 and 16:00 h. Standard food and water were freely available until 2 h before crotalphine oral administration. After this period, only water was available. The animals were deprived of food for no longer than 5 h. All procedures were performed in accordance with the guidelines for the ethical use of conscious animals in pain research published by the International Association for the Study of Pain (Zimmermann, 1983) and were approved by the Institutional Animal Care Committee of the Butantan Institute (CEUAIB, protocol number 098/2002).
Chronic constriction injury
For induction of neuropathic pain, chronic constriction of the sciatic nerve was performed as described by Bennett and Xie (1988). Rats were anesthetized with halothane. The common sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the trifurcation of the sciatic nerve, about 7 mm of the nerve was freed of adhering tissue and four ligatures (4.0 chromic gut) were tied loosely around it with about 1 mm spacing. Great care was taken to tie the ligatures so that the diameter of the nerve was seen to be just barely constricted. The incision was closed in layers. For characterization of neuropathic pain, the presence of hyperalgesia and allodynia was assessed on day 14 after nerve constriction (Gutierrez et al., 2008). As previously observed by Gutierrez et al. (2008), sham-operated rats, in which the sciatic nerve was exposed but left unaffected, did not present alterations in threshold measurements as compared with the basal values.
Evaluation of mechanical hyperalgesia
The rat paw pressure test (Randall and Selitto, 1957) was used for the determination of hyperalgesia. A Ugo–Basile pressure apparatus was used to assess paw pressure thresholds before nerve ligation and again at day 14 after surgery. Testing was blind with respect to group designation. Briefly, increasing force (in g, 16 g/s) was applied to the hind paw. The force needed to induce paw withdrawal was recorded as the nociceptive threshold. To reduce stress, the rats were habituated to the testing procedure the day before the experiment.
Evaluation of low-threshold mechanical allodynia
The von Frey test (Chaplan et al., 1994) was used to assess low-threshold mechanical paw thresholds before nerve ligation and again at day 14 after surgery. Testing was blind with respect to group designation. This test was performed as previously described in detail, using the modified up-down method (Milligan et al., 2000). Briefly, a logarithmic series of 10 calibrated Semmes–Weinstein monofilaments (von Frey hairs; Stoelting, Wood Dale, Illinois, USA) was applied to the right hind paw to determine the stimulus intensity threshold stiffness required to elicit a paw withdrawal response. Log stiffness of the hairs is determined by log 10 (milligrams×10) and ranged from 3.61 (407 mg) to 5.18 (15 136 mg). Basal line assessment was initiated with the 2041 mg hair. In the event of a paw withdrawal, the same hair was again presented 30–60 s later. If the response was again elicited, the 407 mg monofilament was presented. In the absence of a paw withdrawal response to the 407 mg stimulus, the next stronger monofilament was presented (692 mg). The monofilament that elicited a clear response was recorded, and was presented once again 30–60 s later. If the animal withdrew its paw in two consecutive trials with the same stiffness value, no further von Frey hairs were tested. However, in the absence of a response to the initial 2041 mg monofilament, presentation of monofilaments continued in ascending order until two consecutive responses were elicited from the same monofilament. All single responses were recorded, but assessment was complete only after two consecutive responses were elicited from the same monofilament. In instances when rats failed to respond to the strongest stimulus (15 136 mg), it was considered the cut-off value. Responses that occurred to the weakest stimulus (407 mg) were assigned the lower cut-off value for that time point. To reduce stress, rats were habituated to the experimental environment on each of four days before experiments. Behavioral responses were used to calculate the 50% paw withdrawal threshold (absolute threshold), by fitting a Gaussian integral psychometric function using a maximum-likelihood fitting method, which allows parametric analyses (Harvey, 1986; Treutwein and Strasburger, 1999; Milligan et al., 2000, 2001, 2003).
The doses of the antagonists and inhibitors used were on the basis of our previous studies (Picolo et al., 2000; Chacur et al., 2003; Picolo and Cury, 2004; Guan et al., 2008). Drugs administered by the intraplantar route were diluted in a volume of 100 μl and drugs administered orally were diluted in a volume of 2 ml.
Opioid receptor antagonists
Gutierrez et al. (2008) have demonstrated, using naloxone (a nonselective opioid receptor antagonist, 1 μg/paw), that peripheral opioid receptors are involved in the antinociceptive effect of crotalphine or crude crotalid venom in the CCI model. In order to characterize the type of opioid receptor involved in this effect, D-Phe–Cys–Tyr–D-Trp–Orn–Thr–Pen–Thr amide (CTOP; Sigma-Aldrich, Saint Louis, Missouri, USA; 20 or 60 μg/paw), norbinaltorphimine dihydrochloride (nor-BNI; Sigma-Aldrich; 50, 100, or 200 μg/paw) or naltrindole hydrochloride (Sigma-Aldrich; 45 μg/paw), μ-opioid, κ-opioid, and δ-opioid receptor antagonists, respectively, were injected by the intraplantar route immediately after oral administration of crotalphine or venom.
Inhibitors of the L-arginine–nitric oxide–cyclic GMP pathway
To evaluate the role of NO effect on CCI-induced hyperalgesia, NG-methyl-L-arginine (L-NMMA; Sigma-Aldrich; 50 μg/paw), a nonspecific inhibitor of nitric oxide synthase (NOS) was injected, intraplantarally, 60 min before crotalphine or venom.
In order to characterize the isoform of NOS responsible for NO generation, L-N6-(1-iminoethyl) lysine (L-NIL; Sigma-Aldrich; 50 µg/paw) or 7-nitroindazole (7-NI; Sigma-Aldrich; 50 µg/paw), inhibitors of inducible and neuronal NOS, respectively, was injected, intraplantarlly, 60 min before crotalphine or venom. To evaluate the role of cGMP on crotalphine and venom effects, 1H-(1,2,4) oxadiazolo[4,3-a]quinoxaline-1-one (ODQ; Sigma-Aldrich; 8 µg/paw), a selective guanylate cyclase inhibitor or Rp-cGMP triethylamine (Sigma-Aldrich; 1.5 µg/paw), a cGMP-dependent protein kinase inhibitor, was injected 30 min before crotalphine or venom.
Participation of K+ channels
In order to evaluate the role of K+ channels in the antinociceptive effect of crotalphine and venom, glibenclamide (Sigma-Aldrich; 80 μg/paw), an ATP-sensitive K+ channel blocker, charybdotoxin (Sigma-Aldrich; 2 μg/paw), a selective blocker of large-conductance Ca2+-activated K+ channels, and 4-aminopyridine (Sigma-Aldrich; 100 μg/paw) or tetraethylammonium (Sigma-Aldrich; 640 μg/paw), voltage-dependent K+ channel blockers, were injected immediately after the administration of crotalphine or venom.
The peptide crotalphine (<E–F–S–P–E–N–C–Q–G–E–S–Q–P–C, where <E is a pyroglutamic acid and there is a disulfide bond between 7C–14C) was synthesized by the American Peptide Co. (Sunnyvale, California, USA; product number 331065, lot number U07122A1, 98% purity, molecular weight 1534,6 Da), with Fmoc chemistry in the solid phase, as described by Konno et al. (2008). The synthesized crotalphine was stored at –20°C until use. The peptide was diluted in sterile saline and administered by the oral (gastral cannula, 2 ml) route on day 14 after surgery. The antinociceptive activity of the peptide was evaluated 1 h after treatment. Sterile saline was used as a control. The doses of crotalphine (0.2 or 5 μg/kg) were on the basis of our previous work (Gutierrez et al., 2008).
Results are presented as mean±SEM. Statistical evaluation of data was carried out by two-way analysis of variance. The factors analyzed were treatments, time, and time by treatment interaction. When there was a significant time by treatment interaction, a one-way analysis of variance, followed by a Tukey contrast analysis at P<0.05 (Gad, 1989) were performed at each time point, to compare the groups.
CCI of the sciatic nerve caused a significant decrease in the nociceptive threshold (measured by the Randall and Selitto test), and lowered mechanical withdrawal thresholds (measured by the von Frey test), as compared with the baseline values obtained before surgery (represented by the dashed lines in Figs 1–6). Oral and intraplantar administration of crotalphine inhibited the nociceptive phenomena induced by CCI over time [Fig. 1a: F(4,25)=321.28, P<0.001; Fig. 1b: F(4,25)=4438.34, P<0.001].
δ-opioid and κ-opioid receptors are involved in the antinociceptive effect of crotalphine on neuropathic pain
As previously observed by Gutierrez et al. (2008), oral administration of 0.2 μg/kg of crotalphine partially inhibited mechanical hyperalgesia (Fig. 1a) and low-threshold mechanical allodynia (Fig. 1b), whereas a dose of 5 µg/kg blocked these phenomena (Fig. 1). Crotalphine was also administered by the intraplantar route, in order to confirm the peripheral antinociceptive action of the peptide. Previous work has demonstrated that administration of 0.0006 μg/paw of crotalphine induces an antinociceptive effect only in the injected paw, and not in the contralateral one, indicating that under these experimental conditions, the peptide does not display systemic effects (Konno et al., 2008). Crotalphine, at this dose, inhibited mechanical hyperalgesia (Fig. 1a) and low-threshold mechanical allodynia (Fig. 1b) induced by nerve constriction. Crotalphine administered by the oral route, at the dose of 5 µk/kg, and by the intraplantar route (0.0006 μg/paw) also increased the mechanical threshold in the Randall and Selitto test, to values greater than those recorded at baseline (i.e. antinociception) (Fig. 1a).
We have previously demonstrated that the antinociceptive effect of crotalphine in this model of neuropathic pain is mediated by the activation of peripheral (local) opioid receptors. (Gutierrez et al., 2008) To characterize the type of opioid receptor involved in this effect, rats were injected, by an intraplantar route, with selective antagonists of opioid receptors. Analysis revealed significant treatment×time interactions [Fig. 2a: F(8,45)=416.33, P<0.001; Fig. 2b: F(8,45)=65.44, P<0.001; Fig. 2c: F(14,32)=146.82, P<0.001; Fig. 2d: F(14,32)=134 678, P<0.001; Fig. 2e: F(10,55)=111.502, P<0.001; Fig. 2f: F(10,55)=42.8; Fig. 3a: F(7,40)=237.96, P<0.001; Fig. 3b: F(7,40)=53.73, P<0.001]. Oral (Fig. 2) or intraplantar (Fig. 3) administration of crotalphine, following nerve constriction, inhibited mechanical hyperalgesia (measured by the Randall and Selitto test, Fig. 2a, c, e and Fig. 3a) and low-threshold mechanical allodynia (measured by the von Frey test, Fig. 2b, d, f and Fig. 3b). The antinociceptive activity of crotalphine (Figs 2 and 3) was abolished in the paw injected with naltrindole (45 μg/paw), an antagonist of δ-opioid receptors, whereas nor-BNI (50 μg/paw), an antagonist of κ-opioid receptors, partially reversed this effect. However, CTOP (20 μg/paw), a μ-opioid receptor antagonist, did not alter the action of crotalphine (Figs 2 and 3, P=1.00). In another set of experiments, the effect of different doses of the antagonists on the antinociceptive action of crotalphine (orally, 5 μg/kg) was determined. The results demonstrated that CTOP, also at a dose of 60 μg/paw, did not interfere with the effect (Fig. 2a and b). The effectiveness of the doses of CTOP was confirmed in experiments using morphine (morphine sulfate; União Quimica, São Paulo, Brazil; 5 mg/kg, subcutaneously) as a positive control (Fig. 2a and b). CTOP (20 and 60 μg/paw), injected into the ipsilateral paw, inhibited the antinociceptive effect of morphine. However, it was ineffective when injected into the contralateral paw (Fig. 2a and b), indicating a peripheral site of action. Nor-BNI, at doses of 100 and 200 μg/paw, only partially inhibited the antinociceptive effect of crotalphine, confirming the results obtained with a dose of 50 μg/paw of the antagonist (Fig. 2c and d). The results also demonstrated that the dose of 200 μg/paw of nor-BNI induces a systemic effect, as it was also effective in inhibiting the action of crotalphine when injected into the contralateral paw. Regarding naltrindole, the results showed that the dose of 45 μg/paw induced only a local effect, as it did not interfere with the antinociceptive effect of crotalphine when administered in the contralateral paw (Fig. 2e and f). The antagonists, per se, did not interfere with hyperalgesia and allodynia induced by CCI (Figs 2 and 3). Taken together, these results suggest that crotalphine displays peripheral antinociception that is mediated by the activation of δ-opioid and κ-opioid receptors.
Nitric oxide and cyclic GMP are involved in the antinociceptive action of crotalphine on neuropathic pain
Previous data have shown that peripheral activation of the L-arginine–NO–cGMP pathway mediates the antinociceptive effect of drugs with opioid activity. (Cunha et al., 1991; Nozaki-Taguchi and Yamamoto, 1998; Picolo and Cury, 2004; Sachs et al., 2004)
To analyze the involvement of NO in the effect of crotalphine, L-NMMA, a nonspecific inhibitor of NOS, was administered to the rats by the intraplantar route. The results showed that the L-NMMA abolished the antihyperalgesic and antiallodynic effects of the peptide and indicate that NO is involved in the antinociceptive effect of the peptide in neuropathic pain. (e.g. the paw withdrawal threshold was 103±1.2 g in crotalphine-treated animals and 32±1.0 g in crotalphine plus L-NMMA animals: data not shown). In order to characterize the type of NOS involved in this effect, L-NIL or 7-NI, inhibitors of the inducible and neuronal form of NOS, respectively, were injected by the intraplantar route. Analysis of Fig. 4a and b revealed significant treatment×time interactions [Fig. 4a: F(5,30)=327.00, P<0.001; Fig. 4b: F(5,30)=562.32, P<0.001]. Oral administration of crotalphine inhibited mechanical hyperalgesia (measured using the Randall and Selitto test, Fig. 4a) and low-threshold mechanical allodynia (measured using the von Frey test, Fig. 4b) induced by nerve constriction. The administration of 7-NI partially inhibited the antinociceptive effect of crotalphine, whereas L-NIL did not alter this effect (Fig. 4). The inhibitors per se did not interfere with hyperalgesia and allodynia induced by CCI (Fig. 4).
To determine the involvement of cGMP in the effect of crotalphine, ODQ, a selective guanylate cyclase inhibitor, was administered to the rats by the intraplantar route. Analysis of Figure 5a and b revealed significant treatment×time interactions [Fig. 5a: F(6,36)=31.73, P<0.001; Fig. 5b: F(6,36)=231.97, P<0.001]. Oral administration of crotalphine inhibited mechanical hyperalgesia (measured using the Randall and Selitto test, Fig. 5a) and low-threshold mechanical allodynia (measured using the von Frey test, Fig. 5b) induced by nerve constriction. Pretreatment with ODQ also inhibited antinociception induced by crotalphine (Fig. 5), indicating the involvement of cGMP in this effect. To further characterize the molecular mechanisms involved in crotalphine and venom antinociception, Rp-cGMPS, an inhibitor of cGMPc-dependent protein kinase, was injected by the intraplantar route. This inhibitor, at a dose of μg/paw, partially reduced the antihyperalgesic (Fig. 5a) and antiallodynic (Fig. 5b) effects of the peptide. The inhibitors per se did not interfere with hyperalgesia and allodynia induced by CCI (Fig. 5).
ATP-sensitive K+ channels are involved in the antinociceptive effect of crotalphine on neuropathic pain
Experimental data have indicated a link between the activation of the NO–cGMP pathway and the opening of ATP-sensitive K+ channels (Rodrigues and Duarte, 2000; Chacur et al., 2003). This link was also demonstrated for the antinociceptive effect of the crude crotalid venom on prostaglandin E2-induced hyperalgesia in rats (Chacur et al., 2003). In order to investigate the involvement of K+ channels in the antinociceptive effect of crotalphine, and also of the crude venom, in the CCI model, distinct K+ channels blockers were administered by the intraplantar route.
Analysis of Figure 6a and b revealed significant treatment×time interactions [Fig. 6a: F(8,45)=253.53, P<0.001; Fig. 6b: F(8,45)=41.65, P<0.001]. Oral administration of crotalphine inhibited mechanical hyperalgesia (measured using the Randall and Selitto test, Fig. 6a) and low-threshold mechanical allodynia (measured using the von Frey test, Fig. 6b) induced by nerve constriction. Local pretreatment with charybdotoxin as well as with either 4-aminopyridine or tetraethylammonium did not modify the antihyperalgesic or the antiallodynic effect of crotalphine (Fig. 6a and b, respectively). However, intraplantar administration of glibenclamide, a blocker of ATP-sensitive K+ channel, abolished the antinociceptive effect of the peptide (Fig. 6). None of the drugs per se interfered with hyperalgesia and allodynia induced by CCI (Fig. 6).
We have recently shown that crotalphine, a 14 amino acid peptide obtained from the venom of C. d. terrificus, induces a long-lasting and opioid receptor-mediated antinociceptive effect in a rat model of neuropathic pain induced by constriction of the sciatic nerve (Gutierrez et al., 2008). In the present study, we further characterize the mechanisms involved in this effect, determining the type of opioid receptor involved, and also investigating whether transduction mechanisms related to the release of NO could be responsible for the action of crotalphine.
In this study, crotalphine was administered orally (0.2 or 5 μg/kg) on day 14 after nerve constriction. This protocol, on the basis of previous work (Gutierrez et al., 2008), shows that (a) on that day, the nociceptive phenomena caused by nerve injury were not due to the presence of inflammatory mediators, such as prostanoids, as nonsteroidal anti-inflammatory drugs, administered on that day, did not modify hyperalgesia or allodynia induced by nerve constriction, and (b) oral administration of 0.2 μg/kg of crotalphine partially inhibited the nociceptive phenomena induced by CCI, whereas a dose of 5 μg/kg blocked these phenomena and also increased the nociceptive threshold, as compared with the basal values. It is important to stress that the antinociceptive effect of crotalphine is not due to alterations in the general activity of the animals, as Gutierrez et al. (2008) demonstrated that oral administration of the peptide (5 μg/kg) had no effect on the frequency of locomotion and rearing of the animals, evaluated in the open field test.
The present data indicate that, for both oral doses of crotalphine, peripheral δ-opioid and κ-opioid receptors, but not μ-opioid receptors, are involved in the antinociceptive effect. The results also show that δ-opioid receptors are more prominent for the effect of crotalphine, as the antagonist of δ-opioid receptors blocked the antinociceptive action of the peptide, whereas the κ-opioid receptor antagonist only partially inhibited this effect. These data also confirm our previous observation, using the carrageenin-induced and prostaglandin E2 (PGE2)-induced hyperalgesia models (Picolo and Cury, 2004; Gutierrez et al., 2008), that μ-opioid receptors are not involved in the effect of crotalphine.
It is important to stress that the effectiveness of the doses of the antagonists used in this study was demonstrated in experiments using dose–response curves and in assays using morphine as a positive control. Furthermore, the involvement of peripheral (local) opioid receptors in the action of crotalphine was confirmed in experiments showing that injection of the effective dose of the antagonist into the contralateral paw did not alter the antinociceptive effect. Also, the peripheral opioidergic activity of crotalphine was further confirmed using the intraplantar route, at a dose that, as previously demonstrated (Gutierrez et al., 2008; Konno et al., 2008), does not display systemic effects.
These results indicate that peripheral δ-opioid receptors, as also observed by others (Kabli and Cahill, 2007; Obara et al., 2009), are an important target for the control of neuropathic pain. We have previously reported that in the carrageenin-induced and PGE2-induced hyperalgesia models, the effect of crotalphine is only mediated by peripheral κ-opioid receptors (Gutierrez et al., 2008). Taken together, these results indicate that the opioid mechanisms involved in the effect of crotalphine in models of inflammatory hyperalgesia differ partially from those observed for chronic neuropathic pain. Several lines of evidence have suggested that inflammation increases the expression, axonal transport, and density of neuronal opioid receptors (Hassan et al., 1993; Cahill et al., 2003) and, depending on the nature and the stage of the inflammatory reaction, different types of opioid receptors become activated (Obara et al., 2009). Recent results obtained by our group, using immunoblotting assays, demonstrate that PGE2-induced hyperalgesia and CCI increase the expression of opioid receptors in paw nerve and dorsal root ganglia on the side of the tissue/nerve injury. However, opioid receptor expression is distinctly regulated by the presence of acute or chronic injury, as PGE2 increases the expression of κ-opioid receptors and decreases the expression of δ-opioid receptors, whereas CCI upregulates δ-opioid receptors (V.O. Zambelli and Y. Cury, unpublished data). Therefore, it is reasonable to suggest that the different patterns of κ-opioid and δ-opioid receptors expression caused by acute or chronic injury could contribute to the distinct types of opioid receptors involved in the effect of crotalphine.
The pharmacological profile of crotalphine (antagonized fully by the δ-antagonist and partially by the κ-antagonist) could also be related to an action of the peptide on heterodimers. Dimerization of κ-opioid and δ-opioid receptors has been demonstrated (Jordan and Devi, 1999; Wessendorf and Dooyema, 2001). Furthermore, data have indicated that this heterodimer displays a strong affinity for partially selective ligands (Jordan and Devi, 1999) and that some opioid agonists selectively activate opioid receptor heterodimers, including the κ-heterodimer and δ-heterodimer (Waldhoer et al., 2005). However, it is important to stress that a direct action of crotalphine on opioid receptors was not detected (Konno et al., 2008), suggesting that (a) the opioid-mediated effects of crotalphine lie downstream of its direct actions and (b) the peripheral opioid activity of crotalphine could result from the release of endogenous opioids or from functional interactions between the opioidergic system and other types of receptors, such as cannabinoid receptors (Cichewicz, 2004; Bushlin et al., 2010). The preliminary results obtained by our group indicate that cannabinoid receptors are involved in the antinociceptive effect of crotalphine. These results suggest a possible contribution of the cannabinoid system to the opioidergic activity of the peptide. In addition, recent results demonstrated that the antinociceptive effect of crotalphine in the PGE2-induced hyperalgesia model was reversed by an intraplantar injection of dynorphin A antiserum, indicating the involvement of endogenous opioids in this effect (G. Picolo and F. Machado, unpublished data). Studies using dorsal root ganglia cell culture are now in progress in order to evaluate the possible crotalphine-induced opioid release from these cells.
As pointed out in our previous work (Konno et al., 2008), it is intriguing that crotalphine, a 14 amino acid peptide, displays antinociceptive effects after oral administration. Several lines of evidence have indicated that various peptides do display bioactivity, including antinociception, when administered orally (Karaki et al., 1993; Oluyomi et al., 1994; Seppo et al., 2003; Heimann et al., 2007). The results obtained with crotalphine, administered by the intraplantar route, at a dose devoid of systemic effects, indicate that its antinociceptive effect involves peripheral opioid mechanisms, regardless of the peptide’s pharmacokinetics.
It has been shown that, at a peripheral level, activation of the NO–cGMP pathway and opening of ATP-sensitive K+ channels are relevant for the antinociceptive activity of opioid drugs (Cunha et al., 1991; Nozaki-Taguchi and Yamamoto, 1998; Rodrigues and Duarte, 2000). In the present study, the involvement of the NO–cGMP pathway in the effect of crotalphine was confirmed by the observation that an intraplantar injection of NO synthase inhibitors and also of a selective guanylate cyclase inhibitor blocked the effect of the peptide. Furthermore, the activity of neuronal NO synthase is, at least in part, important for the synthesis of the NO involved in the action of the peptide, as the neuronal NOS inhibitor partially inhibited the antinociceptive effect of crotalphine, whereas the inhibitor of the inducible NOS did not alter this effect. The results also indicate that the effects of crotalphine and crude venom in this model of neuropathic pain are partially dependent on the activation of PKG, as Rp-cGMP triethylamine partially inhibited the antihyperalgesic and antiallodynic action. It is well-known that cGMP regulates ion channel activity either directly or indirectly, mainly through the activation of PKG (Ownby et al., 1999). PKG is a protein kinase that is stimulated selectively, but not exclusively, by cGMP. Once stimulated, PKG induces the inhibition of phospholipase C activity, stimulation of Ca2+-ATPase activity, inhibition of inositol 1,4,5-triphosphate, inhibition of Ca2+ channels, or stimulation of K+ channel activity (Wang et al., 1997; Lucas et al., 2000).
The involvement of peripheral K+ channels in the effect of crotalphine in the CCI model was suggested by the experiments showing that glibenclamide, a blocker of ATP-sensitive K+ channels, administered by an intraplantar route, inhibited the antihyperalgesic and antiallodynic effects of the peptide. Thus, crotalphine seems to act by restoring the normal high threshold of nociceptors through the increase in the permeability to K+. Interestingly, blockers of other types of K+ channels, such as voltage-dependent and Ca2+-activated K+ channels, at doses reported in the literature to be effective in various models (Yonehara and Takiuchi, 1997; Ortiz et al., 2003), did not modify antinociception induced by crotalphine. These results are in agreement with data showing the involvement of peripheral ATP-sensitive K+ channels, but not of the other types of K+ channels, in the antinociceptive effect of opioid drugs (Rodrigues and Duarte, 2000; Sachs et al., 2004). Furthermore, several lines of evidence have indicated that drugs capable of activating the NO–cGMP pathway, including opioids, cause direct blockade of acute and persistent hypernociception by opening of ATP-sensitive K+ channels, through stimulation of PKG (Sachs et al., 2004; Wang et al., 2008). Therefore, our data strongly suggest that in the CCI model, the effect of crotalphine involves local activation of δ-opioid and κ-opioid receptors, followed by stimulation of neuronal NO and cGMP, with subsequent opening of ATP-sensitive K+ channels. However, a direct action of crotalphine on the NO–cGMP pathway or K+ channels has not been assessed and cannot be ruled out.
Other studies have indicated that activation of the NO–cGMP–PKG pathway causes nociceptive effects in neuropathic pain (Yoon et al., 1998; LaBuda et al., 2006). This discrepancy may be due to differences in the peripheral and central effects of NO or to the different subsets of nociceptive primary sensory neurons in which the NO–cGMP pathway plays opposing roles (Vivancos et al., 2003). To our knowledge, this is the first report on antinociception induced by peripheral activation of the NO–cGMP–PKG pathway in a model of neuropathic pain induced by CCI.
In conclusion, crotalphine induces antinociception in neuropathic pain, mediated by the activation of peripheral δ-opioid and κ-opioid receptors. In addition, the effect of the peptide seems to be mediated by activation of the NO–cGMP–PKG pathway, followed by the opening of ATP-sensitive K+ channels. The present data contribute to a better understanding of the antinociceptive effect of crotalphine, as well as to the control of neuropathic pain, and point to the therapeutic potential of this peptide for the treatment of chronic pain.
This work was supported by funds from the Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil (FAPESP, grants number 1998/14307-9, 2002/04918-8, 2007/02478-4, 2007/00135-2, 2007/03404-4), of Instituto Nacional de Ciencia e Tecnologia em Toxinologia (INCTTOX PROGRAM) of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)/FAPESP (Grant number 2008/57898-0) and Financeira de Estudos e Projetos (FINEP, grant number 2050/04). We are indebted to Dr. Marcelo L. Santoro, from Instituto Butantan, for statistical analysis assistance.
Conflicts of interest
There are no conflicts of interest.
Bennett GJ, Xie YK. A peripheral mononeuropathy in rat
that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107
Bushlin I, Rozenfeld R, Devi LA. Cannabinoid–opioid interactions during neuropathic pain and analgesia. Curr Opin Pharmacol. 2010;10:80–86
Cahill CM, Morinville A, Hoffert C, O'Donnell D, Beaudet A. Up-regulation and trafficking of delta opioid receptor in a model of chronic inflammation: implications for pain control. Pain. 2003;101:199–208
Caviedes BE, Herranz JL. Advances in physiopathology and the treatment of neuropathic pain. Rev Neurol. 2002;35:1037–1048
Chacur M, Longo I, Picolo G, Gutierrez JM, Lomonte B, Guerra JL, et al. Hyperalgesia induced by Asp49 and Lys49 phospholipases A(2) from Bothrops asper snake venom: pharmacological mediation and molecular determinants. Toxicon. 2003;41:667–678
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat
paw. J Neurosci Methods. 1994;53:55–63
Cichewicz DL. Synergistic interactions between cannabinoid and opioid analgesics. Life Sci. 2004;74:1317–1324
Cunha FQ, Lorenzetti BB, Poole S, Ferreira SH. Interleukin-8 as a mediator of sympathetic pain. Br J Pharmacol. 1991;104:765–767
Dworkin RH, Backonja M, Rowbotham MC, Allen RR, Argoff CR, Bennett GJ, et al. Advances in neuropathic pain: diagnosis, mechanisms, and treatment recommendations. Arch Neurol. 2003;60:1524–1534
Dworkin RH, O'Connor AB, Audette J, Baron R, Gourlay GK, Haanpaa ML, et al. Recommendations for the pharmacological management of neuropathic pain: an overview and literature update. Mayo Clin Proc. 2010;85:S3–14
Eisenberg E, McNicol ED, Carr DB. Efficacy and safety of opioid agonists in the treatment of neuropathic pain of nonmalignant origin: systematic review and meta-analysis of randomized controlled trials. JAMA. 2005;293:3043–3052
Furlan AD, Sandoval JA, Mailis-Gagnon A, Tunks E. Opioids for chronic noncancer pain: a meta-analysis of effectiveness and side effects. Cmaj. 2006;174:1589–1594
Gad SW, Weil CSHayer A. Statistics for toxicologists. Principles and methods of toxicology. 19892nd ed. New York Raven Press LTD:435–483
Guan Y, Johanek ML, Hartke VT, Shim B, Tao XY, Ringkamp M, et al. Peripherally acting mu-opioid receptor agonist attenuates neuropathic pain in rats after L5 spinal nerve injury. Pain. 2008;138:318–329
Gutierrez V, Konno K, Chacur M, Sampaio S, Picolo G, Brigate P, et al. Crotalphine
induces potent antinociception
in neuropathic pain by acting at peripheral opioid receptors
. Eur J Pharmacol. 2008;594:84–92
Harvey LO. Efficient estimation of sensory thresholds. Behav Res Methods Instrum Comput. 1986;18:623–632
Hassan AH, Ableitner A, Stein C, Herz A. Inflammation of the rat
paw enhances axonal transport of opioid receptors
in the sciatic nerve qand increases their density in the inflamed tissue. Neuroscience. 1993;55:185–195
Heimann AS, Gomes I, Dale CS, Pagano RL, Gupta A, de Souza LL, et al. Hemopressin is an inverse agonist of CB1 cannabinoid receptors. Proc Natl Acad Sci USA. 2007;104:20588–20593
Jarvis MF, Boyce-Rustay JM. Neuropathic pain: models and mechanisms. Curr Pharm Des. 2009;15:1711–1716
Jordan BA, Devi LA. G-protein-coupled receptor heterodimerization modulates receptor function. Nature. 1999;399:697–700
Kabli N, Cahill CM. Anti-allodynic effects of peripheral delta opioid receptors
in neuropathic pain. Pain. 2007;127:84–93
Karaki H, Kuwahara M, Sugano S, Doi C, Doi K, Matsumura N, et al. Oral administration of peptides derived from bonito bowels decreases blood pressure in spontaneously hypertensive rats by inhibiting angiotensin converting enzyme. Comp Biochem Physiol C. 1993;104:351–353
Konno K, Picolo G, Gutierrez VP, Brigatte P, Zambelli VO, Camargo AC, et al. Crotalphine
, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus.
LaBuda CJ, Koblish M, Tuthill P, Dolle RE, Little PJ. Antinociceptive activity of the selective iNOS inhibitor AR-C102222 in rodent models of inflammatory, neuropathic and post-operative pain. Eur J Pain. 2006;10:505–512
Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stewart I, Park J, Schulz S, et al. Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev. 2000;52:375–414
Milligan ED, Mehmert KK, Hinde JL, Harvey LO, Martin D, Tracey KJ, et al. Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120. Brain Res. 2000;861:105–116
Milligan ED, O'Connor KA, Nguyen KT, Armstrong CB, Twining C, Gaykema RP, et al. Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines. J Neurosci. 2001;21:2808–2819
Milligan ED, Twining C, Chacur M, Biedenkapp J, O'Connor K, Poole S, et al. Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci. 2003;23:1026–1040
Nozaki-Taguchi N, Yamamoto T. Involvement of nitric oxide in peripheral antinociception
mediated by kappa- and delta-opioid receptors
. Anesth Analg. 1998;87:388–393
Obara I, Parkitna JR, Korostynski M, Makuch W, Kaminska D, Przewlocka B, et al. Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain. Pain. 2009;141:283–291
Oluyomi AO, Poole S, Smith TW, Hart SL. Antinociceptive activity of peptides related to interleukin-1 beta-(193–195), Lys-Pro-Thr. Eur J Pharmacol. 1994;258:131–138
Ortiz MI, Castaneda-Hernandez G, Granados-Soto V. Possible involvement of potassium channels in peripheral antinociception
induced by metamizol: lack of participation of ATP-sensitive K+ channels. Pharmacol Biochem Behav. 2003;74:465–470
Ownby CL, Selistre De Araujo HS, White SP, Fletcher JE. Lysine 49 phospholipase A2 proteins. Toxicon. 1999;37:411–445
Picolo G, Cury Y. Peripheral neuronal nitric oxide synthase activity mediates the antinociceptive effect of Crotalus durissus terrificus
snake venom, a delta- and kappa-opioid receptor agonist. Life Sci. 2004;75:559–573
Picolo G, Giorgi R, Cury Y. Delta-Opioid receptors
and nitric oxide mediate the analgesic effect of Crotalus durissus terrificus
snale venom. Eur J Pharmacol. 2000;391:55–62
Portenoy RK. Chronic opioid therapy in nonmalignant pain. J Pain Symptom Manage. 1990;5:S46–S62
Portenoy RK, Hagen NA. Breakthrough pain: definition, prevalence and characteristics. Pain. 1990;41:273–281
Przewlocki R, Przewlocka B. Opioids in chronic pain. Eur J Pharmacol. 2001;429:79–91
Randall LO, Selitto JJ. A method for measurement of analgesia activity on inflamed tissue. Arch Inst Pharmacodyn. 1957;111:209–219
Rodrigues AR, Duarte ID. The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K(+) channels. Br J Pharmacol. 2000;129:110–114
Sachs D, Cunha FQ, Ferreira SH. Peripheral analgesic blockade of hypernociception: activation of arginine/NO/cGMP/protein kinase G/ATP-sensitive K+ channel pathway. Proc Natl Acad Sci USA. 2004;101:3680–3685
Seppo L, Jauhiainen T, Poussa T, Korpela R. A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am J Clin Nutr. 2003;77:326–330
Song XJ, Wang ZB, Gan Q, Walters ET. cAMP and cGMP contribute to sensory neuron hyperexcitability and hyperalgesia in rats with dorsal root ganglia compression. J Neurophysiol. 2006;95:479–492
Treutwein B, Strasburger H. Fitting the psychometric function. Percept Psychophys. 1999;61:87–106
Vivancos GG, Parada CA, Ferreira SH. Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues. Br J Pharmacol. 2003;138:1351–1357
Waldhoer M, Fong J, Jones RM, Lunzer MM, Sharma SK, Kostenis E, et al. A heterodimer-selective agonist shows in vivo relevance of G protein-coupled receptor dimers. Proc Natl Acad Sci USA. 2005;102:9050–9055
Wang CX, Olschowka JA, Wrathall JR. Increase of interleukin-1beta mRNA and protein in the spinal cord following experimental traumatic injury in the rat
. Brain Res. 1997;759:190–196
Wang J, Zhang LC, Lv YW, Ji Y, Yan XJ, Xue JP. Involvement of the nitric oxide–cyclic GMP–protein kinase G-K+ channel pathway in the antihyperalgesic effects of bovine lactoferrin in a model of neuropathic pain. Brain research. 2008;1209:1–7
Wessendorf MW, Dooyema J. Coexistence of kappa- and delta-opioid receptors
spinal cord axons. Neurosci Lett. 2001;298:151–154
Yonehara N, Takiuchi S. Involvement of calcium-activated potassium channels in the inhibitory prejunctional effect of morphine on peripheral sensory nerves. Regul Pept. 1997;68:147–153
Yoon YW, Sung B, Chung JM. Nitric oxide mediates behavioral signs of neuropathic pain in an experimental rat
model. Neuroreport. 1998;9:367–372
Yuen KC, Baker NR, Rayman G. Treatment of chronic painful diabetic neuropathy with isosorbide dinitrate spray: a double-blind placebo-controlled cross-over study. Diabetes Care. 2002;25:1699–1703
Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983;16:109–110