British Journal of Medicine & Medical Research 4(31): 5062-5073, 2014 SCIENCEDOMAIN international www.sciencedomain.org Antinociceptive Effect of Gabapentin in Visceral Pain is not Mediated by GABAA or Opioid Receptors Atefeh Parhizgar1, Manzumeh Shamsi Meymandi1,2*, Gholamreza Sepehri1,2, Ehsan Sepehri1 and Gioia Heravi1,2 1Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran. 2Department of Physiology and Pharmacology, Kerman University of Medical Sciences, Kerman, Iran. Authors’ contributions This work was carried out in collaboration between all authors. Author AP conducted the study and literature searches. Author MSM designed the study, performed the statistical analysis, and wrote the first draft of the manuscript. Author GS wrote the protocol and the final manuscript. Authors ES and GH managed the analyses and experimental part of study. All authors read and approved the final manuscript. Received 14th February 2014 rd Original Research Article Accepted 23 March 2014 Published 10th July 2014 ABSTRACT Aims: There is evidence supporting the antinociceptive effect of gabapentin in visceral pain, however, the underlying mechanism(s) is not determined yet. So this study was performed to evaluate probable involvement of opioid and GABAergic receptors in the gabapentin effects on acetic acid-induced visceral pain in mice. Place and Duration of Study: Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran, between June 2012 and March 2013. Methodology: The acetic acid test was induced by intraperitoneal injection (i.p.) of acetic acid 0.6% (10ml/kg of body weight) in male mice. Writhing reflex was measured as the number of abdominal contractions in 45min. Animals received saline (as control) or gabapentin (1, 5, 10, 25, 50, 100 or 200mg/kg/ i.p.) 40min before acetic acid. The least effective dose of gabapentin (50mg/kg i.p.) was selected for further assessments and mice were pretreated either picrotoxin (0.75, 1 and 1.5mg/kg/i.p.) as chloride ___________________________________________________________________________________________ *Corresponding author: Email: [email protected], [email protected]; British Journal of Medicine & Medical Research, 4(31): 5062-5073, 2014 channel blocker, bicuculline (0.5, 0.75, 1.5 and 2mg/kg) as GABAA receptor antagonist or naloxone (2mg/kg/i.p.) as opioid receptor antagonist, 10min before gabapentin (50mg/kg i.p.) treatment. Separate groups received naloxone or picrotoxin (1.5mg/kg) or bicuculline (2mg/kg) alone 10min before saline in acetic acid induced visceral contractions test. Results: Gabapentin reduced acetic acid-induced writhing in dose-dependent manner. Visceral contractions following naloxone, picrotoxin and bicuculline administration prior to gabapentin was not significantly different from that of gabapentin alone. Only toxic doses of picrotoxin and bicuculline (1.5 and 2mg/kg, respectively) abolished the inhibitory effect of gabapentin. Conclusion: The effect of gabapentin on visceral nociception is not mediated through opioid and/or GABAA receptors. Keywords: Gabapentin; visceral pain; mice; opioid receptor; GABAergic receptor. 1. INTRODUCTION Pain is one of the most common features of many diseases. Due to the complexity and diverse mechanism(s) of pain conduction, the control of visceral pain remains among the medical difficulties. Various drugs are used to ameliorate the visceral pain. Gabapentin, a well tolerated anticonvulsant drug structurally related to gamma-aminobutyric acid, showed analgesic effect in a wide variety of pain states [1]. Substantial experimental data have been provided on the antinociceptive effects of gabapentin in different models of visceral pain. Although the exact mechanism of analgesic effect of gabapentin is not fully determined yet, but it appears that gabapentin modulates neurotransmitter release by selectively binding to α(2)δ subunit of voltage-dependent Ca2+ channels [2]. Gabapentin has shown to reduce defecation rates and colorectal distension [3] and is also effective in chronic pancreatitis induced by dibutyltin dichloride in rats [4]. In acetic acid-induced writhing, gabapentin decreased the number of contractions in dose dependent manner [5-7]. Intraperitoneal administration of acetic acid solution produces direct activation of visceral and somatic nociceptors and induces a strong component of inflammation [8]. The efficacy of gabapentin in inflammatory responses can be explained by the fact that in neuronal cultures gabapentin inhibited the activation of the transcription factor NF-κB evoked by substance P [9] and reducing abnormal hypersensitivity induced by inflammatory responses [2]. It has also been demonstrated that gabapentin effectively reversed the acetic acid-induced writhing responses through the inhibition of the release of substance P and excitatory amino acids glutamate and aspartate, which are the likely neuromodulators candidates for inflammatory nociceptive transmission [2,7,10]. In neuropathic pain, gabapentin efficacy is partially due to presynaptic inhibition of GABAergic system [11], however, several lines of evidence have shown that GABAergic receptors are not involved in the modulation antinociceptive effect of gabapentin, since it does not directly bind to GABAA or GABAB receptors [12]. GABAA-receptor antagonist bicuculline did not reversed anti-inflammatory and antinociceptive effect of gabapentin [2,13]. Neither GABAB receptors are considered the target of analgesic action of gabapentin in inflammatory and postoperative pain [2,13,14]. However, the involvement of GABAergic system in the antinociceptive effect gabapentin in visceral pain has not been investigated yet. 5063 British Journal of Medicine & Medical Research, 4(31): 5062-5073, 2014 In contrast to GABAergic transmission, several studies showed a synergistic interaction between opioidergic system and analgesic effect of gabapentin in experimental animal models of pain and also in clinical setting [5,10,15-17]. In tail flick model of acute pain as well as writhing test of visceral pain gabapentin enhanced antinociceptive effect of sub-analgesic doses of morphine [5,15]. Moreover gabapentin produced a linear dose-response relationship with similar slope as like morphine in writhing test, however this was not proved same site of action and mechanism of interaction [18]. So the aim of this study is to test the probable involvement of the opioid and GABAergic receptors in the analgesic effects of systemic gabapentin on acetic acid-induced visceral pain in mice. 2. MATERIALS AND METHODS 2.1 Animals The total numbers of 120 albino male mice, weighing 25-30g, were supplied from Kerman Neuroscience Research Center (Kerman, Iran). Animals were housed in standard plastic cages with wood chip bedding and were kept in temperature 22±2ºC and 12h light-dark cycle with free access to food and water. The experimental protocols used in this study were approved by the ethics committee of Kerman Neuroscience Research Center (No EC/KNRC/85–12), in accordance with the internationally accepted principles for laboratory animal use and care, as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). All experimental groups consisted of 6–12 mice that were initially allowed to habituate to laboratory surroundings. Each animal was used only once and was sacrificed humanly under anesthesia with diethyl-ether immediately after the experiment. 2.2 Drugs The drugs used were: Gabapentin (Park Davis Company, Italy), picrotoxin as chloride channel blocker related to GABAA receptor and bicuculline as GABAA receptor antagonist (Sigma, USA), and naloxone hydrochloride as opioid receptor antagonist (Tolidaru, Iran). All drugs were freshly dissolved in normal saline and were injected intraperitoneally. 2.3 Writhing Reflex The acetic acid-induced visceral pain model is widely used in experimental research. It can produce not only abdominal contractions but also gastrointestinal ileus [19]. Visceral pain was induced by acetic acid 0.6% (10ml/kg of body weight) and the nociceptive response were characterized by the presence of abdominal contractions, which consisted of the contraction of flank muscles associated with inward movement of hind limb, a hind paw reflex, or whole body stretching [5-7,19]. 2.4 Procedure mice received an intraperitoneal injection of freshly prepared 0.6% acetic acid 40min after saline (control) or gabapentin (1, 5, 10, 25, 50, 100 or 200mg/kg) administration. Picrotoxin (chloride channel blocker), bicuculline (GABAA receptor antagonist) or naloxone (opioid receptor antagonist) was used to evaluate the probable involvement of GABAergic and opioid receptors in gabapentin visceral pain antinociception. Pretreated groups received either picrotoxin (0.75, 1 and 1.5mg/kg), bicuculline (0.5, 0.75, 1.5 and 2mg/kg) or naloxone (2mg/kg) 10min before the least effective dose of gabapentin (50mg/kg i.p.) and then the 5064 British Journal of Medicine & Medical Research, 4(31): 5062-5073, 2014 abdominal contractions were recorded by the above mentioned protocol. Also 3 groups of mice received naloxone (2mg/kg), picrotoxin (1.5mg/kg) or bicuculline (2mg/kg) alone 10 min before saline in acetic acid induced visceral contractions test Table1. Table 1. Design of experimental animal groups Group Treatment Control Saline+acetic acid 40min after saline Gabapentin (1,5,10,25,50, Gabapentine+acetic acid 40min after gabapentin 100&200mg/kg) Naloxone (2mg/kg)