European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Neuropharmacology and analgesia
Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide Mona Dastgheib a, Leila Moezi a,b,n a b
Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
art ic l e i nf o
a b s t r a c t
Article history: Received 27 November 2013 Received in revised form 23 April 2014 Accepted 24 April 2014
Agomelatine is a potent MT1 and MT2 melatonin receptor agonist as well as a 5-HT2C serotonin receptor antagonist. It was approved by the European Medicines Agency as an antidepressant drug in year 2009. On the other hand, the involvement of melatonin and serotonin receptors in the modulation of seizure threshold has been demonstrated previously. The purpose of this study was to investigate the effect of agomelatine on penthylenetetrazol-induced seizure threshold in male mice. Therefore, we evaluated the effect of acute (12.5, 25, 50, 75 and 100 mg/kg, p.o. and chronic (25, 50 and 75 mg/kg, p.o., once a day, for 7 days) agomelatine administration on mouse model of intravenous penthylenetetrazol-induced seizure. For evaluation of nitrergic system involvement in the anticonvulsant effect of agomelatine, co-administration of multiple nitric oxide synthase (NOS) inhibitors [L-NAME, a non-selective NOS inhibitor, (5 mg/kg, p.o.), aminoguanidine, a selective iNOS inhibitor, (100 mg/kg, p.o.) or 7-nitroindazol, a selective nNOS inhibitor, (60 mg/kg, p.o.)] and agomelatine (50 and 75 mg/kg) were examined. In acute study, agomelatine (50 and 75 mg/kg) increased clonic seizure threshold compared to control group (P o0.05 and 0.01, respectively). In chronic study, agomelatine had no effect on clonic seizure threshold compared with control mice. Co-administration of L-NAME, aminoguanidine or 7-nitroindazol with agomelatine (50 and 75 mg/kg) prevented a agomelatine-induced anti-convulsant effect. Our results suggest that agomelatine has anticonvulsant activity in intravenous penthylenetetrazol-induced seizure in acute therapy and this effect can be at least in part due to iNOS or nNOS induction. & 2014 Published by Elsevier B.V.
Keywords: Agomelatine Seizure Penthylenetetrazol Nitric oxide Mice
1. Introduction The demand for new drugs for the treatment of epilepsy, one of the most common brain disorders, is still very high. Conventional antiepileptic drugs fail to control the occurrence of seizures in about 30% of the patients (Brodie and Dichter, 1996). Moreover, the use of these drugs is often precluded by the occurrence of untoward side-effects (such as behavioural problems and cognitive impairment) or of interactions with other drugs, especially in children (Mattson, 1992). Melatonin is secreted by the pineal gland in a suprachiasmatic nuclei-driven circadian fashion and is involved in the regulation of the circadian rhythm of several biological functions (Cagnacci, 1996; Reiter, 1991). Melatonin has an anticonvulsant effect on kindled seizures in rats (Albertson et al., 1981; Mevissen and Ebert, 1998) and
n Corresponding author at: Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Tel./fax: þ 98 711 2307591. E-mail address: [email protected] (L. Moezi).
elevates the electroconvulsive threshold in mice (Borowicz et al., 1999). Chronic melatonin treatment in male gerbils has been shown to reduce the incidence and mortality rate of pentylenetetrazoleinduced seizures (Champney et al., 1996). Melatonin's anticonvulsant effect appears to be mediated by melatonin ML1/2 receptors. GABAergic and serotonergic mechanisms may also play an important role in mediating the anticonvulsant activity of melatonin (Ray et al., 2004). Moreover, it has been suggested that the anticonvulsant property of melatonin involves modulation of both brain amino acids and nitrite levels (Bikjdaouene et al., 2003). Agomelatine, a naphthalene analog of melatonin, is a newly developed selective agonist of the human cloned melatonergic MT1 and MT2 receptors (Millan et al., 2003). In comparison with melatonin, agomelatine shows a longer half-life and a comparatively greater affinity for MT1 and MT2 melatonin receptors in different brain areas (Delagrange and Boutin, 2006). Agomelatine also shows serotonin 5-HT2C receptor antagonist activity (Millan et al., 2003). The antidepressant activity of agomelatine has been demonstrated in several animal models of depression including chronic mild stress (Papp et al., 2003) and learned helplessness
http://dx.doi.org/10.1016/j.ejphar.2014.04.039 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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(Bertaina-Anglade et al., 2006) and has been successfully used in humans to treat depression (Loo et al., 2002). It is an antidepressant which was approved by the European Medicine Agency in year 2009 (Sansone and Sansone, 2011). The first report on the anticonvulsant activity of agomelatine was published in 2012 by Aguiar et al. (2012). The results of their study indicated that acute agomelatine administration exert an anticonvulsant activity in female mice induced by intraperitoneal pilocarpine or pentylenetetrazol (PTZ), and this action may be related to GABAertic mechanisms. Nitric oxide (NO) is regarded as both neuronal messenger and/or neurotransmitter in the central nervous system (Bredt et al., 1990). NO is generated from L-arginine by the nitric oxide synthase (NOS) family (Snyder and Bredt, 1991). NO is also a known modulator of seizure susceptibility with diverse anticonvulsant (Starr and Starr, 1993; Tsuda et al., 1997) or proconvulsant (De Sarro et al., 1991; Osonoe et al., 1994) properties in different seizure settings. Melatonin inhibits constitutive NOS (cNOS) activity via complex formation with calmodulin, thereby decreasing NO production in various brain regions (Bettahi et al., 1996; Pozo et al., 1997). In addition, melatonin controls mitochondrial NOS isoform and regulates mitochondrial respiration and bioenergetics and protects mitochondria from excess NO (Acuna-Castroviejo et al., 2005). On the other hand, the induction of l-arginine/NO pathway through neuronal NOS (nNOS) has been reported in melatonin-induced modulation of seizure susceptibility of mice (Yahyavi-Firouz-Abadi et al., 2006). In the present study, we examined the effect of acute agomelatine administration on the threshold of clonic seizures induced by intravenous injection of PTZ in male mice. Regarding the functional interactions of melatonin with NO signaling pathway and the fact that agomelatine is a melatonin receptor agonist, we further investigated the possible involvement of NO in the modulatory effect of agomelatine on seizure susceptibility using different NOS inhibitors including L-NAME, 7-nitroindazole (7-NI), and aminoguanidine.
2. Material and methods 2.1. Chemicals The drugs used were as follows: Pentylenetetrazol (PTZ), agomelatine (MT1 and MT2 receptor agonist and 5-HT2C antagonist), N-nitro-L-arginine methyl ester (L-NAME) (a non-selective NOS inhibitor), aminoguanidine (a selective iNOS inhibitor) and 7-nitroindazole (7-NI) (a selective nNOS inhibitor). All drugs named above except agomelatine were purchased from Sigma (St.Louis, Mo, USA). Agomelatine was purchased from Servier (Suresnes/France). PTZ was prepared in saline as a 0.5% solution and administered as intravenous infusion. Agomelatine was prepared at concentration 12.5, 25, 50, 75 and 100 mg in 10 cm3carboxymethylcellulose (CMC, 0.5%) as a solvent agent and administered orally by gavage. Other drugs were dissolved in normal saline solution at concentration such that the requisite dose could be administered in a volume of 10 ml/kg and administered per orally. 2.2. Animals Male NMRI mice 20–30 g were used throughout this study. Animals were housed in groups of 5–6 and were allowed free access to food and water. All animals were acclimated at least 3 days before all experiments were conducted during the period between 10.00 AM and 14.00 PM with normal room light (12 h regular light/dark cycle) and room temperature (23 72 1C).
All procedures were carried out in accordance with the institutional guidelines for animal care and use and all possible measures were taken to minimize the number of animals used and their suffering. Each mouse was used only once and each treatment group consisted of 6–8 animals. 2.3. PTZ-induced seizure Syringe pump (Harvard, USA) was used to deliver precise amount of PTZ at specific time intervals. Rate of infusion was 0.5 cm3/min and catheter diameter which connected to the animal tail through 30 gauge dental needle was 4.6 mm. Infusion was halted were forelimb clonus followed by full clonus of the body was observed. Minimal dose of PTZ (mg/kg of mice weight) needed to induce clonic seizure was considered as an index of seizure threshold. 2.4. Rotarod test The effect of agomelatine on coordinated motor movements was assessed using the rotarod test. A day before the test, the mice were trained to stay on the rotating wheel (3 cm in diameter, 20 rpm) for more than one min. On the test day, the animals were tested on the rotarod (Model 7600, UGO Basile, Italy) 60 min after the administration of different doses of agomelatine. The number of seconds each mouse remained on the rotating wheel was recorded for a maximum of 300 s. 2.5. Treatment In experiment 1, for evaluation of acute effects of agomelatine on intravenous PTZ-induced seizure, mice received different doses of agomelatine (12.5, 25, 50, 75 and100 mg/kg, p.o.), 60 min before PTZ–induced seizure. Control groups received diazepam (1 mg/kg) as a positive control and CMC (0.5%) as a negative control. In experiment 2, for evaluation of chronic effects of agomelatine on PTZ-induced seizure, animals received different doses of agomelatine (25, 50 and75 mg/kg, once in a day for 7 days, p.o.) and control groups received ethosuxamide (150 mg/kg) as a positive control and CMC (0.5%) as a negative control. In experiment 3, for evaluation of time course of agomelatine anticonvulsant effect, animals received agomelatine at dose of 75 mg/kg 30, 45, 60 and 90 min before PTZ-induced seizure. In experiments 4–6, for evaluation of NOS inhibitors effects on PTZ-induced seizure, animals received L-NAME (5 mg/kg, p.o.) as a non-selective NOS inhibitor, aminoguanidine (100 mg/kg, p.o.) as a selective iNOS inhibitor and 7-nitroindazole (7-NI) (60 mg/kg, p.o.) as a selective nNOS inhibitor, 15 min before agomelatine (50 and 75 mg/kg) and 75 min before PTZ administrations. Control groups received normal saline 15 min before CMC administration and 75 min before PTZ-induced seizure. The PTZ threshold of control group which received vehicle was obtained separate for each experiment, and then the data were combined. In experiment 7, for evaluation of agomelatine on motor coordination, rotarod test was done and mice were trained 24 h before agomelatine (25, 50 and 75 mg/kg) administration. Trained mice were capable of staying at least 120 s on 20 rpm-rotating rotarod. 2.6. Statistical analysis Data are expressed as Mean7S.E.M. The one way analysis of variance (ANOVA) followed by the Tukey test was employed to analyze the data. P o0.05 was considered as the significance level. Statistical software was SPSS Version 16; (SPSS Inc., Chicago, IL)
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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3. Results
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3.1. Acute effect of agomelatine different doses on clonic seizure threshold
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3.2. Evaluation of time course of agomelatin anticonvulsant effect Fig. 2 shows time course of agomelatine (75 mg/kg) anticonvulsant effect. One-way ANOVA revealed a significant effect (F[4,34] ¼2.949, P o0.05). Post hoc analysis indicated that agomelatin exerted an anticonvulsant effect 60 min after administration (P o0.01). 3.3. Chronic effects of agomelatine different doses on chronic seizure threshold
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Agomelatine (mg/kg) Fig. 1. The effect of different acute doses of agomelatine on intravenous PTZinduced seizure threshold in mice. Agomelatine was injected 60 min before PTZ. Data are presented as mean7 S.E.M. nPo 0.05 and nnPo 0.01 compared to vehicle control group. Each group consisted of six to eight mice.
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Fig. 1 shows the effect of acute administration of agomelatine different doses (12.5, 25, 50, 75 and 100 mg/kg, p.o.) on clonic seizure threshold. One-way ANOVA revealed a significant effect (F[5,43] ¼4.625, P o0.01). Post hoc analysis indicated that agomelatine (50 and 75 mg/kg) increased clonic seizure threshold compared to control group (Po0.05 and 0.01, respectively). During the time of PTZ infusion (2 min), convulsion did not appear in diazepam (1 mg/kg) group which shows the anti-convulsant effect of diazepam, as a positive control in this experiment.
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Fig. 3 shows the effect of chronic administration of agomelatine different doses (25, 50 and 75 mg/kg) on clonic seizure threshold. One-way ANOVA did not reveal a significant effect (F[3,31] ¼ 11.448, P 40.05). Therefore, chronic agomelatine administration had no effect on clonic seizure threshold compared to control group. During the time of PTZ infusion (2 min), convulsion did not appear in ethosuxamide (150 mg/kg) group which shows the anticonvulsant effect of ethosuxamide, as a positive control in this experiment.
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3.5. Effect of aminoguanidin on clonic latency in the presence of agomelatine Fig. 5 demonstrates results from aminoguanidine (100 mg/kg) and agomelatine (50 or 75 mg/kg) co-treatments. One One-way ANOVA revealed a significant effect (F[5, 48]¼3.944, P o0.01) for aminoguanidine administration before agomelatine (50 or 70 mg/kg). According to results this co-administration prevented the anticonvulsant effect of agomelatine (50 or 75 mg/kg). 3.6. Effect of 7-nitroindazole (60 mg/kg) on clonic seizure threshold in the presence of agomelatine Fig. 6 shows results from 7-nitroindazole (60 mg/kg) and agomelatine (50 or 75 mg/kg) co-administrations. One-way ANOVA demonstrated a significant effect (F[5, 46]¼ 5.154, P o0.01). Post hoc analysis indicated that 7-nitroindazole prevented the anticonvulsant effect of agomelatine (50 or 75 mg/kg).
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Fig. 2. The time-course of the anticonvulsant effect of acute agomelatine (75 mg/ kg) on PTZ-induced seizure threshold. Agomelatine was administered orally. Data are presented as mean 7 S.E.M. nnP o 0. 01 compared to vehicle control group. Each group consisted of six to eight mice.
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3.4. Effect of L-NAME on clonic latency in the presence of agomelatine Fig. 4 shows results from L-NAME (5 mg/kg) and agomelatine (50 or 75 mg/kg) co-administration. One-way ANOVA revealed a significant effect (F[5, 47] ¼5.761, P o0.001) for L-NAME administration before agomelatine. Post hoc analysis indicated that L-NAME prevented the anti-convulsant effect of agomelatine (50 or 75 mg/kg).
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Fig. 3. The effect of different doses of chronic agomelatine administration on intravenous PTZ-induced seizure threshold in mice. Animals received different doses of agomelatine (25, 50 and 75 mg/kg, once in a day for 7 days). Data are presented as mean 7 S.E.M. Each group consisted of six to eight mice.
3.7. Acute effect of agomelatine on motor coordination Fig. 7 shows acute effect of agomelatine different doses (25, 50 and 75 mg/kg) on motor coordination. According to results agomelatine did not decrease motor coordination.
4. Discussion Melatonin or N-acetyl–methoxytryptamine is a hormone synthesized from tryptophan mainly by the pineal gland of mammals and remains one of the most mysterious substances
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Fig. 4. L-NAME inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. L-NAME was administered 15 min before agomelatine and 75 min before PTZ administration. nPo 0.05 and nnP o 0.01 in comparison with controls. ♯♯P o 0.01 in comparison with corresponding agomelatine group. Data are presented as mean 7S.E.M. Each group consisted of six to eight mice.
Fig. 5. Aminoguanidine inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. Aminoguanidine was administered 15 min before agomelatine and 75 min before PTZ administration. nPo 0.05 and nnP o 0.01 in comparison with controls. Data are presented as mean7 S.E.M. Each group consisted of six to eight mice.
Fig. 6. 7-nitroindazole inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. 7-nitroindazole was administered 15 min before agomelatine and 75 min before PTZ administration. nP o0.05 and nnP o0.01 in comparison with controls. ♯♯P o 0.01 in comparison with corresponding agomelatine group. Data are presented as mean 7S.E.M. Each group consisted of six to eight mice.
produced by the human body (Witt-Enderby et al., 2003). Indeed the hormone controls important physiological functions. It has been shown to have anxiolytic, sedative and anticonvulsant effects (Champney et al., 1996). Melatonin reduces mortality and severity of the convulsion and increases the latency period in pentylenetetrazole-induced seizure (Guardiola-Lemaitre, 1997). Pentylenetetrazole has been a classic GABA receptor antagonist (Aguiar et al., 2012). Intravenous infusion of PTZ is a standard experimental model of clinical myoclonic and absence seizures that has both face and constructs validities. This model is more sensitive than intraperitoneal PTZ administration and allows
better detection of modulatory effects in convulsive tendency (Durlach-Misteli and Van Ree, 1992). Agomelatine (ß-methyl-6-chlormelatonin) is a synthetic analog of melatonin that belongs to a new class of atypical antidepressants with a novel mechanism of action at melatoninergic and serotoninergic receptors. It acts as an agonist on MT1 and MT2 receptors and 5-HT2C antagonist (Akiyoshi et al., 1995). The results of the present research have demonstrated that acute administration of agomelatine (50 and 75 mg/kg) had anticonvulsant action against intravenous PTZ-induced seizure but chronic agomelatine therapy caused no significant alteration in
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Fig.7. The acute effect of agomelatine on motor coordination. Rotarod test was done and mice were trained 24 h before agomelatine (50 and 75 mg/kg) administration. Trained mice were capable of staying at least 120 s on 20 rpm-rotating rotarod. Data are presented as mean 7 S.E.M. Each group consisted of six to eight mice.
convulsion latency time. The time course of agomelatin anticonvulsant effects indicated that agomelatin exerted anticonvulsant effect 60 min after oral gavage; therefore in the rest of experiments we administered agomelatine 60 min before PTZ. According to the similarity of agomelatine and melatonin mechanisms of actions and according to studies which have shown anticonvulsant effects of melatonin on different types of seizure, it is acceptable that agomelatin may have anticonvulsant efficacy too. Aguiar et al. (2012) studied anticonvulsant effects of agomelatine in female mice. According to their results, in the intraperitoneal PTZ-induced seizure model, agomelatine (at 25 or 50 mg/kg) showed a significant increase in latency to convulsion, and agomelatine (at 50 or 75 mg/kg) also increased significantly time until death. In the pilocarpine-induced seizure model, only agomelatine in high doses (75 mg/kg) showed a significant increase in latency to convulsions and in time until death. In the strychnine-, electroshock- and picrotoxininduced seizure models, agomelatine caused no significant alterations in latency to convulsions and in time until death. Their results suggested that agomelatine had anticonvulsant activity in intraperitoneal PTZ- or pilocarpineinduced seizure models. These results are in line with our findings which showed anti-convulsant effects of acute agomelatine in intravenous model of PTZ. It seems that some adaptation occurs after chronic treatment with agomelatine which eradicate the anti-convulsant effect of agomelatine. The differences in results of acute and chronic experiments might be due to agomelatine receptors desensitization or internalization. Actually our findings are in direction with some of studies which show that melatonin can induce a concentration and time dependent receptor desensitization or internalization. These processes are involved in melatonin receptor signaling and indeed are dependent upon the duration of agonist exposure and the concentration of agonist being used (Durlach-Misteli and Van Ree, 1992; Gerdin et al., 2004; Guardiola-Lemaitre, 1997; Witt-Enderby et al., 2003). Some of other studies also show desensitization of 5-HT2C receptor system. The 5-HT2C receptor couples to multiple effector mechanisms and rapid desensitization of 5-HT2C receptor were reported due to some mediators release or accumulation. Therefore the loose of anti-convulsant effects in chronic use of agomelatine might be because of desensitization in melatonin or 5-HT2C receptors (Akiyoshi et al., 1995; Kostyalik et al., 2014; Stout et al., 2002). Further studies should be done to explore the real mechanisms of this adaptation. Nitric oxide is a gaseous free radical, which is synthesized from amino-acid L-arginine by different nitric oxide synthase (NOS) isoforms, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) and acts as a neuronal messenger and modulator of neurotransmitters in the brain. Several lines of evidence suggest nitric oxide as a modulator of seizure activity
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with diverse anticonvulsant and proconvulsant effects based on the type of seizure, source of nitric oxide and other neurotransmitter systems involvement (Stout et al., 2002). Several studies showed that melatonin and 5-HT2C receptors exert some of their actions through interaction with nitric oxide (Kostyalik et al., 2014; Miyamoto, 2009). Melatonin controls mitochondrial NOS isoform and regulates mitochondrial respiration and bioenergetics and protects mitochondria from excess NO (Acuna-Castroviejo et al., 2005). On the other hand, the induction of NO pathway through nNOS has been reported in melatonin-induced modulation of seizure susceptibility of mice (Yahyavi-Firouz-Abadi et al., 2006). The involvement of 5-hydroxytryptamine2c receptors in serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in rat cerebellum has been demonstrated previously (Sansone and Sansone, 2011) and trazodone behaves as a potent full agonist at the 5-HT2C receptor mediates inhibition of the cerebellar N-methylD-aspartate/nitric oxide/cyclic GMP system (Miyamoto, 2009). Our present data showed that co-administration of agomelatine (50 and 75 mg/kg) and L-NAME, a non-selective NOS inhibitor, prevented the anticonvulsant effect of agomelatine in the intravenous PTZ model of seizures. It shows that NO release may be at least part of the anticonvulsant effect of agomelatine. For finding more details about the responsible NOS isoforms in this pathway we examined co-administration of aminoguanidine, a selective iNOS inhibitor or 7-NL a selective nNOS inhibitor and agomelatine (50 and 75 mg/kg). In both experiments anticonvulsant effects of agomelatine decreased. These data demonstrated that both iNOS and nNOS are responsible in anti-convulsant effect of agomelatine. Minimal neurological deficits, such as sedation and impaired motor function which may be considered wrongly as an anticonvulsant effect are quantified by the rotarod test. According to our result, agomelatine different doses (25, 50 and 75 mg/kg) have no muscle relaxant activity these results are in agreement with previous studies indicating that mice treated with melatonin showed no impairment on rotarod performance (Miyamoto, 2009). These data for the first time illustrated that release of nitric oxide through nitric oxide synthase might be involved in the antiseizure effects of agomelatine. In conclusion, agomelatine can play a role as an anticonvulsant agent in acute therapy. This anticonvulsant effect can be at least in part due to iNOS or nNOS induction.
Acknowledgments The present article was financially supported by Shiraz University of Medical Sciences Grant no. 91-01-36-5207.
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Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide Mona Dastgheib a, Leila Moezi a,b,n a b
Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
art ic l e i nf o
a b s t r a c t
Article history: Received 27 November 2013 Received in revised form 23 April 2014 Accepted 24 April 2014
Agomelatine is a potent MT1 and MT2 melatonin receptor agonist as well as a 5-HT2C serotonin receptor antagonist. It was approved by the European Medicines Agency as an antidepressant drug in year 2009. On the other hand, the involvement of melatonin and serotonin receptors in the modulation of seizure threshold has been demonstrated previously. The purpose of this study was to investigate the effect of agomelatine on penthylenetetrazol-induced seizure threshold in male mice. Therefore, we evaluated the effect of acute (12.5, 25, 50, 75 and 100 mg/kg, p.o. and chronic (25, 50 and 75 mg/kg, p.o., once a day, for 7 days) agomelatine administration on mouse model of intravenous penthylenetetrazol-induced seizure. For evaluation of nitrergic system involvement in the anticonvulsant effect of agomelatine, co-administration of multiple nitric oxide synthase (NOS) inhibitors [L-NAME, a non-selective NOS inhibitor, (5 mg/kg, p.o.), aminoguanidine, a selective iNOS inhibitor, (100 mg/kg, p.o.) or 7-nitroindazol, a selective nNOS inhibitor, (60 mg/kg, p.o.)] and agomelatine (50 and 75 mg/kg) were examined. In acute study, agomelatine (50 and 75 mg/kg) increased clonic seizure threshold compared to control group (P o0.05 and 0.01, respectively). In chronic study, agomelatine had no effect on clonic seizure threshold compared with control mice. Co-administration of L-NAME, aminoguanidine or 7-nitroindazol with agomelatine (50 and 75 mg/kg) prevented a agomelatine-induced anti-convulsant effect. Our results suggest that agomelatine has anticonvulsant activity in intravenous penthylenetetrazol-induced seizure in acute therapy and this effect can be at least in part due to iNOS or nNOS induction. & 2014 Published by Elsevier B.V.
Keywords: Agomelatine Seizure Penthylenetetrazol Nitric oxide Mice
1. Introduction The demand for new drugs for the treatment of epilepsy, one of the most common brain disorders, is still very high. Conventional antiepileptic drugs fail to control the occurrence of seizures in about 30% of the patients (Brodie and Dichter, 1996). Moreover, the use of these drugs is often precluded by the occurrence of untoward side-effects (such as behavioural problems and cognitive impairment) or of interactions with other drugs, especially in children (Mattson, 1992). Melatonin is secreted by the pineal gland in a suprachiasmatic nuclei-driven circadian fashion and is involved in the regulation of the circadian rhythm of several biological functions (Cagnacci, 1996; Reiter, 1991). Melatonin has an anticonvulsant effect on kindled seizures in rats (Albertson et al., 1981; Mevissen and Ebert, 1998) and
n Corresponding author at: Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Tel./fax: þ 98 711 2307591. E-mail address: [email protected] (L. Moezi).
elevates the electroconvulsive threshold in mice (Borowicz et al., 1999). Chronic melatonin treatment in male gerbils has been shown to reduce the incidence and mortality rate of pentylenetetrazoleinduced seizures (Champney et al., 1996). Melatonin's anticonvulsant effect appears to be mediated by melatonin ML1/2 receptors. GABAergic and serotonergic mechanisms may also play an important role in mediating the anticonvulsant activity of melatonin (Ray et al., 2004). Moreover, it has been suggested that the anticonvulsant property of melatonin involves modulation of both brain amino acids and nitrite levels (Bikjdaouene et al., 2003). Agomelatine, a naphthalene analog of melatonin, is a newly developed selective agonist of the human cloned melatonergic MT1 and MT2 receptors (Millan et al., 2003). In comparison with melatonin, agomelatine shows a longer half-life and a comparatively greater affinity for MT1 and MT2 melatonin receptors in different brain areas (Delagrange and Boutin, 2006). Agomelatine also shows serotonin 5-HT2C receptor antagonist activity (Millan et al., 2003). The antidepressant activity of agomelatine has been demonstrated in several animal models of depression including chronic mild stress (Papp et al., 2003) and learned helplessness
http://dx.doi.org/10.1016/j.ejphar.2014.04.039 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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(Bertaina-Anglade et al., 2006) and has been successfully used in humans to treat depression (Loo et al., 2002). It is an antidepressant which was approved by the European Medicine Agency in year 2009 (Sansone and Sansone, 2011). The first report on the anticonvulsant activity of agomelatine was published in 2012 by Aguiar et al. (2012). The results of their study indicated that acute agomelatine administration exert an anticonvulsant activity in female mice induced by intraperitoneal pilocarpine or pentylenetetrazol (PTZ), and this action may be related to GABAertic mechanisms. Nitric oxide (NO) is regarded as both neuronal messenger and/or neurotransmitter in the central nervous system (Bredt et al., 1990). NO is generated from L-arginine by the nitric oxide synthase (NOS) family (Snyder and Bredt, 1991). NO is also a known modulator of seizure susceptibility with diverse anticonvulsant (Starr and Starr, 1993; Tsuda et al., 1997) or proconvulsant (De Sarro et al., 1991; Osonoe et al., 1994) properties in different seizure settings. Melatonin inhibits constitutive NOS (cNOS) activity via complex formation with calmodulin, thereby decreasing NO production in various brain regions (Bettahi et al., 1996; Pozo et al., 1997). In addition, melatonin controls mitochondrial NOS isoform and regulates mitochondrial respiration and bioenergetics and protects mitochondria from excess NO (Acuna-Castroviejo et al., 2005). On the other hand, the induction of l-arginine/NO pathway through neuronal NOS (nNOS) has been reported in melatonin-induced modulation of seizure susceptibility of mice (Yahyavi-Firouz-Abadi et al., 2006). In the present study, we examined the effect of acute agomelatine administration on the threshold of clonic seizures induced by intravenous injection of PTZ in male mice. Regarding the functional interactions of melatonin with NO signaling pathway and the fact that agomelatine is a melatonin receptor agonist, we further investigated the possible involvement of NO in the modulatory effect of agomelatine on seizure susceptibility using different NOS inhibitors including L-NAME, 7-nitroindazole (7-NI), and aminoguanidine.
2. Material and methods 2.1. Chemicals The drugs used were as follows: Pentylenetetrazol (PTZ), agomelatine (MT1 and MT2 receptor agonist and 5-HT2C antagonist), N-nitro-L-arginine methyl ester (L-NAME) (a non-selective NOS inhibitor), aminoguanidine (a selective iNOS inhibitor) and 7-nitroindazole (7-NI) (a selective nNOS inhibitor). All drugs named above except agomelatine were purchased from Sigma (St.Louis, Mo, USA). Agomelatine was purchased from Servier (Suresnes/France). PTZ was prepared in saline as a 0.5% solution and administered as intravenous infusion. Agomelatine was prepared at concentration 12.5, 25, 50, 75 and 100 mg in 10 cm3carboxymethylcellulose (CMC, 0.5%) as a solvent agent and administered orally by gavage. Other drugs were dissolved in normal saline solution at concentration such that the requisite dose could be administered in a volume of 10 ml/kg and administered per orally. 2.2. Animals Male NMRI mice 20–30 g were used throughout this study. Animals were housed in groups of 5–6 and were allowed free access to food and water. All animals were acclimated at least 3 days before all experiments were conducted during the period between 10.00 AM and 14.00 PM with normal room light (12 h regular light/dark cycle) and room temperature (23 72 1C).
All procedures were carried out in accordance with the institutional guidelines for animal care and use and all possible measures were taken to minimize the number of animals used and their suffering. Each mouse was used only once and each treatment group consisted of 6–8 animals. 2.3. PTZ-induced seizure Syringe pump (Harvard, USA) was used to deliver precise amount of PTZ at specific time intervals. Rate of infusion was 0.5 cm3/min and catheter diameter which connected to the animal tail through 30 gauge dental needle was 4.6 mm. Infusion was halted were forelimb clonus followed by full clonus of the body was observed. Minimal dose of PTZ (mg/kg of mice weight) needed to induce clonic seizure was considered as an index of seizure threshold. 2.4. Rotarod test The effect of agomelatine on coordinated motor movements was assessed using the rotarod test. A day before the test, the mice were trained to stay on the rotating wheel (3 cm in diameter, 20 rpm) for more than one min. On the test day, the animals were tested on the rotarod (Model 7600, UGO Basile, Italy) 60 min after the administration of different doses of agomelatine. The number of seconds each mouse remained on the rotating wheel was recorded for a maximum of 300 s. 2.5. Treatment In experiment 1, for evaluation of acute effects of agomelatine on intravenous PTZ-induced seizure, mice received different doses of agomelatine (12.5, 25, 50, 75 and100 mg/kg, p.o.), 60 min before PTZ–induced seizure. Control groups received diazepam (1 mg/kg) as a positive control and CMC (0.5%) as a negative control. In experiment 2, for evaluation of chronic effects of agomelatine on PTZ-induced seizure, animals received different doses of agomelatine (25, 50 and75 mg/kg, once in a day for 7 days, p.o.) and control groups received ethosuxamide (150 mg/kg) as a positive control and CMC (0.5%) as a negative control. In experiment 3, for evaluation of time course of agomelatine anticonvulsant effect, animals received agomelatine at dose of 75 mg/kg 30, 45, 60 and 90 min before PTZ-induced seizure. In experiments 4–6, for evaluation of NOS inhibitors effects on PTZ-induced seizure, animals received L-NAME (5 mg/kg, p.o.) as a non-selective NOS inhibitor, aminoguanidine (100 mg/kg, p.o.) as a selective iNOS inhibitor and 7-nitroindazole (7-NI) (60 mg/kg, p.o.) as a selective nNOS inhibitor, 15 min before agomelatine (50 and 75 mg/kg) and 75 min before PTZ administrations. Control groups received normal saline 15 min before CMC administration and 75 min before PTZ-induced seizure. The PTZ threshold of control group which received vehicle was obtained separate for each experiment, and then the data were combined. In experiment 7, for evaluation of agomelatine on motor coordination, rotarod test was done and mice were trained 24 h before agomelatine (25, 50 and 75 mg/kg) administration. Trained mice were capable of staying at least 120 s on 20 rpm-rotating rotarod. 2.6. Statistical analysis Data are expressed as Mean7S.E.M. The one way analysis of variance (ANOVA) followed by the Tukey test was employed to analyze the data. P o0.05 was considered as the significance level. Statistical software was SPSS Version 16; (SPSS Inc., Chicago, IL)
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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3. Results
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3.2. Evaluation of time course of agomelatin anticonvulsant effect Fig. 2 shows time course of agomelatine (75 mg/kg) anticonvulsant effect. One-way ANOVA revealed a significant effect (F[4,34] ¼2.949, P o0.05). Post hoc analysis indicated that agomelatin exerted an anticonvulsant effect 60 min after administration (P o0.01). 3.3. Chronic effects of agomelatine different doses on chronic seizure threshold
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Agomelatine (mg/kg) Fig. 1. The effect of different acute doses of agomelatine on intravenous PTZinduced seizure threshold in mice. Agomelatine was injected 60 min before PTZ. Data are presented as mean7 S.E.M. nPo 0.05 and nnPo 0.01 compared to vehicle control group. Each group consisted of six to eight mice.
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Fig. 1 shows the effect of acute administration of agomelatine different doses (12.5, 25, 50, 75 and 100 mg/kg, p.o.) on clonic seizure threshold. One-way ANOVA revealed a significant effect (F[5,43] ¼4.625, P o0.01). Post hoc analysis indicated that agomelatine (50 and 75 mg/kg) increased clonic seizure threshold compared to control group (Po0.05 and 0.01, respectively). During the time of PTZ infusion (2 min), convulsion did not appear in diazepam (1 mg/kg) group which shows the anti-convulsant effect of diazepam, as a positive control in this experiment.
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Fig. 3 shows the effect of chronic administration of agomelatine different doses (25, 50 and 75 mg/kg) on clonic seizure threshold. One-way ANOVA did not reveal a significant effect (F[3,31] ¼ 11.448, P 40.05). Therefore, chronic agomelatine administration had no effect on clonic seizure threshold compared to control group. During the time of PTZ infusion (2 min), convulsion did not appear in ethosuxamide (150 mg/kg) group which shows the anticonvulsant effect of ethosuxamide, as a positive control in this experiment.
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3.5. Effect of aminoguanidin on clonic latency in the presence of agomelatine Fig. 5 demonstrates results from aminoguanidine (100 mg/kg) and agomelatine (50 or 75 mg/kg) co-treatments. One One-way ANOVA revealed a significant effect (F[5, 48]¼3.944, P o0.01) for aminoguanidine administration before agomelatine (50 or 70 mg/kg). According to results this co-administration prevented the anticonvulsant effect of agomelatine (50 or 75 mg/kg). 3.6. Effect of 7-nitroindazole (60 mg/kg) on clonic seizure threshold in the presence of agomelatine Fig. 6 shows results from 7-nitroindazole (60 mg/kg) and agomelatine (50 or 75 mg/kg) co-administrations. One-way ANOVA demonstrated a significant effect (F[5, 46]¼ 5.154, P o0.01). Post hoc analysis indicated that 7-nitroindazole prevented the anticonvulsant effect of agomelatine (50 or 75 mg/kg).
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Fig. 2. The time-course of the anticonvulsant effect of acute agomelatine (75 mg/ kg) on PTZ-induced seizure threshold. Agomelatine was administered orally. Data are presented as mean 7 S.E.M. nnP o 0. 01 compared to vehicle control group. Each group consisted of six to eight mice.
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3.4. Effect of L-NAME on clonic latency in the presence of agomelatine Fig. 4 shows results from L-NAME (5 mg/kg) and agomelatine (50 or 75 mg/kg) co-administration. One-way ANOVA revealed a significant effect (F[5, 47] ¼5.761, P o0.001) for L-NAME administration before agomelatine. Post hoc analysis indicated that L-NAME prevented the anti-convulsant effect of agomelatine (50 or 75 mg/kg).
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Fig. 3. The effect of different doses of chronic agomelatine administration on intravenous PTZ-induced seizure threshold in mice. Animals received different doses of agomelatine (25, 50 and 75 mg/kg, once in a day for 7 days). Data are presented as mean 7 S.E.M. Each group consisted of six to eight mice.
3.7. Acute effect of agomelatine on motor coordination Fig. 7 shows acute effect of agomelatine different doses (25, 50 and 75 mg/kg) on motor coordination. According to results agomelatine did not decrease motor coordination.
4. Discussion Melatonin or N-acetyl–methoxytryptamine is a hormone synthesized from tryptophan mainly by the pineal gland of mammals and remains one of the most mysterious substances
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Fig. 4. L-NAME inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. L-NAME was administered 15 min before agomelatine and 75 min before PTZ administration. nPo 0.05 and nnP o 0.01 in comparison with controls. ♯♯P o 0.01 in comparison with corresponding agomelatine group. Data are presented as mean 7S.E.M. Each group consisted of six to eight mice.
Fig. 5. Aminoguanidine inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. Aminoguanidine was administered 15 min before agomelatine and 75 min before PTZ administration. nPo 0.05 and nnP o 0.01 in comparison with controls. Data are presented as mean7 S.E.M. Each group consisted of six to eight mice.
Fig. 6. 7-nitroindazole inhibited the anticonvulsant effect of agomelatine (50 or 75 mg/kg) in mice. 7-nitroindazole was administered 15 min before agomelatine and 75 min before PTZ administration. nP o0.05 and nnP o0.01 in comparison with controls. ♯♯P o 0.01 in comparison with corresponding agomelatine group. Data are presented as mean 7S.E.M. Each group consisted of six to eight mice.
produced by the human body (Witt-Enderby et al., 2003). Indeed the hormone controls important physiological functions. It has been shown to have anxiolytic, sedative and anticonvulsant effects (Champney et al., 1996). Melatonin reduces mortality and severity of the convulsion and increases the latency period in pentylenetetrazole-induced seizure (Guardiola-Lemaitre, 1997). Pentylenetetrazole has been a classic GABA receptor antagonist (Aguiar et al., 2012). Intravenous infusion of PTZ is a standard experimental model of clinical myoclonic and absence seizures that has both face and constructs validities. This model is more sensitive than intraperitoneal PTZ administration and allows
better detection of modulatory effects in convulsive tendency (Durlach-Misteli and Van Ree, 1992). Agomelatine (ß-methyl-6-chlormelatonin) is a synthetic analog of melatonin that belongs to a new class of atypical antidepressants with a novel mechanism of action at melatoninergic and serotoninergic receptors. It acts as an agonist on MT1 and MT2 receptors and 5-HT2C antagonist (Akiyoshi et al., 1995). The results of the present research have demonstrated that acute administration of agomelatine (50 and 75 mg/kg) had anticonvulsant action against intravenous PTZ-induced seizure but chronic agomelatine therapy caused no significant alteration in
Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Fig.7. The acute effect of agomelatine on motor coordination. Rotarod test was done and mice were trained 24 h before agomelatine (50 and 75 mg/kg) administration. Trained mice were capable of staying at least 120 s on 20 rpm-rotating rotarod. Data are presented as mean 7 S.E.M. Each group consisted of six to eight mice.
convulsion latency time. The time course of agomelatin anticonvulsant effects indicated that agomelatin exerted anticonvulsant effect 60 min after oral gavage; therefore in the rest of experiments we administered agomelatine 60 min before PTZ. According to the similarity of agomelatine and melatonin mechanisms of actions and according to studies which have shown anticonvulsant effects of melatonin on different types of seizure, it is acceptable that agomelatin may have anticonvulsant efficacy too. Aguiar et al. (2012) studied anticonvulsant effects of agomelatine in female mice. According to their results, in the intraperitoneal PTZ-induced seizure model, agomelatine (at 25 or 50 mg/kg) showed a significant increase in latency to convulsion, and agomelatine (at 50 or 75 mg/kg) also increased significantly time until death. In the pilocarpine-induced seizure model, only agomelatine in high doses (75 mg/kg) showed a significant increase in latency to convulsions and in time until death. In the strychnine-, electroshock- and picrotoxininduced seizure models, agomelatine caused no significant alterations in latency to convulsions and in time until death. Their results suggested that agomelatine had anticonvulsant activity in intraperitoneal PTZ- or pilocarpineinduced seizure models. These results are in line with our findings which showed anti-convulsant effects of acute agomelatine in intravenous model of PTZ. It seems that some adaptation occurs after chronic treatment with agomelatine which eradicate the anti-convulsant effect of agomelatine. The differences in results of acute and chronic experiments might be due to agomelatine receptors desensitization or internalization. Actually our findings are in direction with some of studies which show that melatonin can induce a concentration and time dependent receptor desensitization or internalization. These processes are involved in melatonin receptor signaling and indeed are dependent upon the duration of agonist exposure and the concentration of agonist being used (Durlach-Misteli and Van Ree, 1992; Gerdin et al., 2004; Guardiola-Lemaitre, 1997; Witt-Enderby et al., 2003). Some of other studies also show desensitization of 5-HT2C receptor system. The 5-HT2C receptor couples to multiple effector mechanisms and rapid desensitization of 5-HT2C receptor were reported due to some mediators release or accumulation. Therefore the loose of anti-convulsant effects in chronic use of agomelatine might be because of desensitization in melatonin or 5-HT2C receptors (Akiyoshi et al., 1995; Kostyalik et al., 2014; Stout et al., 2002). Further studies should be done to explore the real mechanisms of this adaptation. Nitric oxide is a gaseous free radical, which is synthesized from amino-acid L-arginine by different nitric oxide synthase (NOS) isoforms, endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) and acts as a neuronal messenger and modulator of neurotransmitters in the brain. Several lines of evidence suggest nitric oxide as a modulator of seizure activity
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with diverse anticonvulsant and proconvulsant effects based on the type of seizure, source of nitric oxide and other neurotransmitter systems involvement (Stout et al., 2002). Several studies showed that melatonin and 5-HT2C receptors exert some of their actions through interaction with nitric oxide (Kostyalik et al., 2014; Miyamoto, 2009). Melatonin controls mitochondrial NOS isoform and regulates mitochondrial respiration and bioenergetics and protects mitochondria from excess NO (Acuna-Castroviejo et al., 2005). On the other hand, the induction of NO pathway through nNOS has been reported in melatonin-induced modulation of seizure susceptibility of mice (Yahyavi-Firouz-Abadi et al., 2006). The involvement of 5-hydroxytryptamine2c receptors in serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in rat cerebellum has been demonstrated previously (Sansone and Sansone, 2011) and trazodone behaves as a potent full agonist at the 5-HT2C receptor mediates inhibition of the cerebellar N-methylD-aspartate/nitric oxide/cyclic GMP system (Miyamoto, 2009). Our present data showed that co-administration of agomelatine (50 and 75 mg/kg) and L-NAME, a non-selective NOS inhibitor, prevented the anticonvulsant effect of agomelatine in the intravenous PTZ model of seizures. It shows that NO release may be at least part of the anticonvulsant effect of agomelatine. For finding more details about the responsible NOS isoforms in this pathway we examined co-administration of aminoguanidine, a selective iNOS inhibitor or 7-NL a selective nNOS inhibitor and agomelatine (50 and 75 mg/kg). In both experiments anticonvulsant effects of agomelatine decreased. These data demonstrated that both iNOS and nNOS are responsible in anti-convulsant effect of agomelatine. Minimal neurological deficits, such as sedation and impaired motor function which may be considered wrongly as an anticonvulsant effect are quantified by the rotarod test. According to our result, agomelatine different doses (25, 50 and 75 mg/kg) have no muscle relaxant activity these results are in agreement with previous studies indicating that mice treated with melatonin showed no impairment on rotarod performance (Miyamoto, 2009). These data for the first time illustrated that release of nitric oxide through nitric oxide synthase might be involved in the antiseizure effects of agomelatine. In conclusion, agomelatine can play a role as an anticonvulsant agent in acute therapy. This anticonvulsant effect can be at least in part due to iNOS or nNOS induction.
Acknowledgments The present article was financially supported by Shiraz University of Medical Sciences Grant no. 91-01-36-5207.
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Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
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Please cite this article as: Dastgheib, M., Moezi, L., Acute and chronic effects of agomelatine on intravenous penthylenetetrazol-induced seizure in mice and the probable role of nitric oxide. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.04.039i
