Research ReportAlpha-conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurones
Introduction
Conotoxins are small peptides of 12 to 19 amino acids, that act as highly selective antagonists of ion channels in animal cells. A subgroup of these, the 4, 7 loop class of α-conotoxins, interacts selectively with neuronal-type nAChRs [8] and the degree of selectivity for different nAChR subtypes is remarkable [23]. This renders α-conotoxins attractive as prospective drug leads. We recently identified and characterized α-conotoxin Vc1.1 from a tropical marine snail, Conus victoriae, found in waters off the coast near Broome, Western Australia. A molecular approach has been used to identify α-conotoxin Vc1.1, whose amino acid sequence was deduced by sequencing a cDNA library obtained by RT-PCR from the mRNA isolated from a total RNA extract from the venom ducts of a Conus victoriae[30]. The coding sequence was 201 base pairs, which translates into 66 amino acids. The predicted pre-region of the protein precursor was 21 amino acids, the pro-region 29 amino acids, and the mature peptide 16 amino acids in length. The peptide Vc1.1, as synthesized by solid-phase techniques, had 16 amino acids with a C-terminal amide and with two disulfide bonds connected in a 4, 7 loop structure. Purification by RP-HPLC gave a single major peak (>70% pure) whose mass was confirmed by ESI-MS as 1806.6. The peptide was soluble in water and stable in solution when tested after 8 weeks. The characteristics of Vc1.1 are those of a specific antagonist of neuronal nAChRs [30].
We showed that Vc1.1 can specifically antagonize neuronal but not muscle-type nAChRs [30]. This conotoxin was also shown to suppress the vascular response to selective stimulation of sensory nerves in rats [30] suggesting that antagonists of nAChRs had the potential to suppress pain transmission in these animals. The analgesic effect of strong nicotinic agonists, such as epibatidine, nicotine and analogues has been well documented and supports the involvement of nicotinic transmission in pain perception [7], [34]. There is, however, no precedent for the use of nicotinic antagonists to suppress pain transmission in animals in vivo.
Previous studies using N-type calcium channel blockers suggest that voltage-dependent calcium channels (VDCCs) mediate pain [35]. In particular, the N-type VDCC has been implicated in pain-related behaviors and calcium channel blockers such as the ω-conotoxins have attracted attention as antinociceptives [4]. Specific antagonists for neuronal N-type calcium channels have been shown to reduce heat hyperalgesia and mechanical allodynia in the CCI model when administered directly to the site of nerve injury [39], and subcutaneous administration of an N-type, but not P- or Q-type, Ca2+ channel antagonist attenuated mechanical hyperalgesia in the PNL model of pain [38].
In this study, we nevertheless demonstrate the ability of the neuronal nAChR antagonist Vc1.1 to modulate neuropathic pain in rats. We have used two commonly used models of neuropathic pain, the chronic constriction injury (CCI) model [3] and the partial nerve ligation (PNL) model [33] of the rat sciatic nerve. These models reflect pain behaviors exhibited by different populations of human neuropathic pain in patients [18].
We have also assessed the ability of Vc1.1 to modulate functional recovery of the injured neurones. As an indicator of the functional activity of C-fibres, we used an established blister model combined with laser Doppler flowmetry to assess the ability of the injured nerves to mount a peripheral inflammatory response upon perfusion of the blister base with substance P [2], [15], [16].
Section snippets
Animals
Outbred, young (3 to 4 months old), male Sprague–Dawley rats with an average weight of 250–350 g were housed in groups of 4, in a constant temperature room (21 ± 0.5 °C), under a 12/12-h light/dark cycle and had free access to food and water ad libitum. Anesthesia was induced with sodium pentobarbitone (Nembutal 60 mg/kg i.p.) and additional doses of pentobarbitone 15 mg/kg were given throughout the experiment. Body temperature was maintained at 37 °C. Animals were killed by anesthetic overdose
Development of mechanical hyperalgesia in CCI and PNL rats
Rats subjected to surgery developed mechanical hyperalgesia between 5–7 days and 8–10 days postoperatively in CCL and PNL rats respectively. Mechanical paw withdrawal thresholds decreased from 278 ± 9.7 g prior to surgery to 115 ± 8.6 g after CCI surgery and from 276 ± 8.8 g prior to surgery to 116 ± 8.3 g after PNL surgery. These are within the ranges previously established for mechanical hyperalgesia with this technique [16].
Effect of α-conotoxin Vc1.1 on mechanical hyperalgesia
The capacity of Vc1.1 to attenuate mechanical hyperalgesia once
Discussion
Neuropathic pain presents a continuing management problem as current treatments are in the main ineffective or not well tolerated [9], [25], [32]. The present study reports that α-conotoxin Vc1.1, a neuronal-type nicotinic receptor antagonist [30], is an effective and long-lasting analgesic in two rat models (CCI and PNL) of human neuropathic pain.
Changes in mechanical hyperalgesia in rats with CCI or PNL were used to determine the effectiveness of Vc1.1 as an analgesic. Vc1.1 administered i.m.
Acknowledgment
The authors thank Ms. Vi Pham for her assistance in this project.
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