Antinociceptive Effect of Gabapentin in Visceral Pain Is Not Mediated by GABAA Or Opioid Receptors

British Journal of Medicine & Medical Research 4(31): 5062-5073, 2014

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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

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*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.

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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) Naloxone 10min before saline+acetic 40min after saline Picrotoxin (1.5mg/kg) Picrotoxin 10min before saline+acetic acid 40min after saline Bicuculline (1.5mg/kg) Bicuculline 10min before saline+acetic acid 40min after saline Gabapentin (50mg/kg) Naloxone 10min before gabapentin+acetic acid 40min after +Naloxone (2mg/kg) gabapentin Gabapentin (50mg/kg) Picrotoxin 10min before gabapentin+acetic acid 40min +Picrotoxin (0.75 ,1 & after gabapentin 1.5mg/kg) Gabapentin (50mg/kg) Bicuculline10min before gabapentin+acetic acid 40min +Bicuculline ( 0.5 , 0.75 after gabapentin ,1.5 &2mg/kg) All drugs were injected intraperitoneally

The mice were then placed in individual 30×30×50cm plexiglass boxes for the observation of writhing reflex. The numbers of writhing contractions were counted by two separate behavioral investigators who were blind to the injected drug. To assure the accuracy of data collection the film of some experiment was taken and controlled by a third investigator. Abdominal contractions were recorded by visual observation of animals five minutes after acetic acid injection for 45 minutes. The time and doses of antagonists’ injection were chosen according to preliminary experiments and previous studies [13,20-22]. The control group received normal saline.

2.5 Statistical Analysis

Abdominal contractions were calculated as mean±SEM of 6 mice in each group except the control group (n=12) and 50mg/kg of gabapentin (n=8) dealing with multiple comparisons. One-way analysis of variance (ANOVA) followed by the Tukey’s post-hoc test was used to compare the number of contraction between treated groups. P=.05 was regarded as significant.

3. RESULTS

3.1 The Effect of Gabapentin on the Acetic Acid-induced Visceral Contractions

The mean number of acetic acid induced writhing reflexes in control mice was 111.8±6.6 contractions during 45min of observation Fig. 1.

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Gabapentin decreased number of writhing reflexes significantly in a dose-dependent manner. Gabapentin at dose of 25mg/kg or less (or 1, 5 and 10) had no significant effect on writhing reflexes, while the doses of 50, 100 and 200 mg/kg caused a significant reduction in the number of contractions compared to control group (p<0.001) Fig. 1. The least effective dose of gabapentin was 50mg/kg which reduced the number of contractions from 111.8±6.6 to 51.1±4.9 (almost 50% of inhibition). Therefore we used this dose for further assessments.

140

120

100

60 * * * 40

20 MeanNumber Contractionsof 0 Control 1 5 10 25 50 100 200 Gabapentin Dose (mg/kg)

Fig. 1. Gabapentin effect on acetic acid-induced visceral contractions. Gabapentin reduced number of abdominal contractions in dose dependent manner The data were expressed as mean±SEM of at least 6 mice.* p<0.001 compared to control group

3.2 The Effect of Pretreatment of GABAA Antagonists on the Gabapentin Antinociception

Bicuculline (0.5, 0.75 and 1.5mg/kg) injection 10min prior to 50mg/kg of gabapentin had no significant effect on writhing contractions compared to gabapentin (50mg/kg) treated group. The number of contractions were significantly decreased in these groups as compared to control (P<0.001). However, pretreatment of the highest dose of bicuculline (2mg/kg) increased significantly (P=.01) the number of writhing (95.6±6.3) compared to gabapentin Fig. 2. The data of intraperitoneal injection of 2mg/kg of bicuculline alone was not reported since it produced mortal convulsions in 80% of mice.

Pretreatment of 0.5, 0.75 and 1.5mg/kg of bicuculline did not reduce the number of abdominal contractions compared to gabapentin (50mg/kg), while pretreatment of 2mg/kg increased the number of abdominal contractions compared to gabapentin alone.

Intraperitoneal injection of picrotoxin (0.75, 1mg/kg) prior to gabapentin (50mg/kg) did not showed any significant effect on writhing reflexes in acetic acid induced visceral pain as compared to gabapentin treated group. However, higher dose of picrotoxin (1.5mg/kg) reversed the gabapentin effect on writhing reflexes in acetic acid induced visceral pain and the number of contractions(100.3±21.5) were increased significantly as compared to gabapentin group (P=.05). The number of contractions (100.3±21.5) following 1.5mg/kg of picrotoxin alone was not different compared to controls Fig. 3.

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140 120 # 100 80 60 * * * * 40 20 Mean Number of ContractionsNumberof Mean 0 Dose (mg/kg)

Fig. 2. The effect of bicuculline pretreatment on the gabapentin antinociception effect on acetic acid-induced visceral contractions The data were expressed as mean±SEM of at least 6 mice.* P<0.001 compared to control group.# P=.01 compared to gabapentin

140 # 120

100

60 * * * 40

Mean Number of ContractionsNumberof Mean 20

0 Dose (mg/kg)

Fig. 3. The effect of picrotoxin pretreatment on the gabapentin antinociceptive effect on acetic acid-induced visceral contractions The data were expressed as mean±SEM of at least 6 mice.* P=.05 compared to control group.# P=.05 compared to gabapentin

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Pretreatment of 1.5mg/kg of picrotoxin prior to gabapentin (50mg/kg) increased the number of abdominal contractions to control level while same dose had no effect alone.

3.3 The Effect of Pretreatment of Opioid Antagonist on the Gabapentin Antinociception

Our result showed that there no significant difference between the number of visceral contractions in naloxone (2mg/kg) treated group (87.5±7.7) as compared to controls Fig. 4. Also its pretreatment prior to gabapentin (50 mg/kg) did not change the effect of gabapentin in writhing reflexes and the number of contractions (45.2±8.7) was not significantly different compared to gabapentin Fig. 4.

140

120

100

60 * *

40 Mean Number of ContractionsNumberof Mean 20

0 Dose (mg/kg)

Fig. 4. The effect of naloxone pretreatment on the gabapentin antinociceptive effect on acetic acid-induced visceral contractions The data were expressed as mean±SEM of at least 6 mice.* P=.05 compared to control group

Pretreatment of 2mg/kg of naloxone prior to gabapentin (50mg/kg) did not change the number of abdominal contractions.

4. DISCUSSION

Gabapentin showed a dose-dependent effect in reducing the number of acetic acid-induced contractions in mice. Several previous studies confirm this finding in different models of visceral pain [2-4], including in acetic acid-induced writhing [5-7] without significant impairment on motor performance [3,6].

The result of this study showed that gabapentin analgesic effect was not reversed by pretreatment of either bicuculline (0.5, 0.75 and 1.5mg/kg) or picrotoxin(0.75 and 1mg/kg). In agreement, in formalin induced inflammatory pain, bicuculline (i.t.) did not affect the spinal antinociception of gabapentin [13]. Also in postoperative pain, the anti-allodynic effect of intrathecal injection of gabapentin was not reversed by GABAA antagonist [14]. Even in

5068 British Journal of Medicine & Medical Research, 4(31): 5062-5073, 2014 sciatic nerve injury in rats, bicuculline worsen neuropathic pain [23]. However, singular high dose of bicuculline (2mg/kg) reversed the analgesic effect of gabapentin but it also produced convulsion and mortality in 80% of animals. So it seems that this effect can be considered to its toxic effect rather than explaining the interaction of gabapentin on GABAA receptors.

Gabapentin structurally is related to gamma-aminobutyric acid, but there is no evidence of its binding to GABAA or GABAB receptor or its conversion to a GABA-receptor agonist [2,12]. GABAergic receptors instead are considered to be involved in anticonvulsant activity of gabapentin [24] and also in modulation of acute or chronic, neuropathic and inflammatory pain [25-26]. Indeed, baclofen (i.p.) as a GABAB agonist act like gabapentin in reducing abdominal contractions evoked by acetic acid in mice [20] and also reduced visceral pain with colorectal distention in rats [27]. Although gabapentin exert its analgesic effect in partial nerve ligation and postoperative pain through spinal site of action, nonetheless spinal GABAB receptors are considered the target of this effect [2,13-14].

In our study, picrotoxin at high dose of 1.5mg/kg was able to reverse the antinociceptive effect of gabapentin, while same dose injected alone did not show any analgesic effect. Picrotoxin is a convulsive substrate that bind at internal site of chloride channel related to GABAA receptors inhibiting the effect of GABA in increasing hyperpolarization [28]. Several studies showed the inhibitory effect of picrotoxin on analgesic effect of different drugs which indicated the involvement of GABAA receptors in pain response mechanism [21,29,30]. However, the picrotoxin interaction with gabapentin analgesic effect has not been investigated yet. The only interaction found between gabapentin with picrotoxin reported that picrotoxin inhibited the neuroprotective effect of gabapentin in an animal model of Huntington disease and oxidative stress induced damage in mice [31,32]. Picrotoxin at doses less or equal to 1mg/kg showed no involvement of GABAA receptors in antinociceptive effect of gabapentin. These finding are similar to the results of other studies [12,24]. However the dose of 1.5mg/kg reversed the gabapentin effect on visceral pain which could be related to picrotoxin toxic effect or through total blockage of chloride channel, rather than its effect on GABAA receptors. Indeed, afferent sensory input was changed during visceral pain [6,33].

The positive interaction between opioidergic system and gabapentin has been studied in several investigations [5,15-17,34]. Our finding showed that naloxone pretreatment did not have any effect in acetic acid-induced visceral pain and also did not affect antinociceptive effect of gabapentin. Naloxone was not able to reduce antinociceptive effect of pregabalin which was five times more potent than gabapentin in reducing abdominal contractions in writhing test [18,35]. As well, in trinitrobenzen sulfunic acid induced visceral pain naloxone was not able to reverse the allodynia [36]. Similarly, in acute thermal pain assessed by tail flick test, in inflammatory pain induced by formalin and in neuropathic pain naloxone did not reversed the effect of peripheral gabapentin [37,38], but reversed analgesic action of intrathecal gabapentin [13], sustaining an important involvement of opioid receptors at spinal site of anti-inflammatory effect of gabapentin. In visceral pain evoked by acetic acid, it has been shown that the opioid agonist, like morphine, increased the antinociception of gabapentin [5]. Also intrathecal gabapentin enhanced the analgesic effects of subtherapeutic dose of morphine in a rat experimental model of pancreatitis [39]. But interestingly, no study has investigated the effect of an opioid antagonist on antinociception of gabapentin in visceral pain.

The transcranial magnetic stimulation showed that visceral pain is concomitant with increased cortical activity and brain concentration of excitatory amino acids such as

5069 British Journal of Medicine & Medical Research, 4(31): 5062-5073, 2014 glutamate and aspartate [40]. It was also verified that the spinal concentration of excitatory amino acids are increased in acetic acid-induced visceral pain [7]. Indeed, gabapentin alleviate visceral pain through inhibition of spinal concentration of these excitatory amino acids [7]. On the other hand, it has been also established that the main mechanism of anticonvulsant and analgesic action of gabapentin is through binding to α(2)δ subunits of Ca+2 channels and decreasing calcium influx and the release of neurotransmitters [10]. Hereby, α(2)δ subunits of N- type Ca+2 localized on primary afferent neurons of intestine might be the other peripheral site of action of gabapentin [41].

The main mechanisms supposed for analgesic effect of gabapentin include reducing the concentration of excitatory amino acids [7], the inhibition of activation of transcription factor evoked by substance P [9] and its peripheral analgesic effect through activation of peripheral No-cyclic-G-protein kinase pathway [37]. However, our findings showed that GABAA antagonist or opioid antagonist did not changed the analgesic effect gabapentin in visceral pain , so it is implicated that the effect of gabapentin on acetic acid induced visceral pain is not mediated through GABAA or/ opioidergic receptors.

Although both of opioidergic and GABAergic receptors are involved in pain transmission, low doses of bicuculline as GABAA receptors antagonist, picrotoxin as chloride blocker of same receptor and naloxone as opioid receptor antagonist did not changed the dose dependent antinociceptive activity of gabapentin in acetic acid -induced model of visceral pain. However the highest doses used of picrotoxin and bicuculline reversed the inhibitory effect of gabapentin probably due to their toxic effect.

5. CONCLUSION

In summary, our results showed that gabapentin reduced acetic acid-induced writhing in dose-dependent manner. Visceral contractions following naloxone, picrotoxin and bicuculline administration prior to the least effective dose of 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. So the effect of gabapentin on visceral nociception is not mediated through opioid and/or GABAA receptors and further investigation is needed to elucidate the underlying mechanism(s).

CONSENT

Not applicable.

ETHICAL APPROVAL

All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) and the European Community guidelines (EEC Directive of 1986) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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