Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice

Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice

Epilepsy Research 68 (2006) 103–113 Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice Noushin Yahyavi-Firou...

286KB Sizes 0 Downloads 67 Views

Epilepsy Research 68 (2006) 103–113

Involvement of nitric oxide pathway in the acute anticonvulsant effect of melatonin in mice Noushin Yahyavi-Firouz-Abadi a,b,c,1 , Pouya Tahsili-Fahadan a,b,1 , Kiarash Riazi a , Mohammad Hossein Ghahremani b , Ahmad Reza Dehpour a,∗ a

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran b Department of Pharmacology and Toxicology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran c Department of Biology, Faculty of Science, Tehran University, Tehran, Iran Received 20 June 2005; received in revised form 16 September 2005; accepted 23 September 2005 Available online 10 January 2006

Abstract Melatonin, the major hormone produced by the pineal gland, is shown to have anticonvulsant effects. Nitric oxide (NO) is a known mediator in seizure susceptibility modulation. In the present study, the involvement of NO pathway in the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced clonic seizures was investigated in mice. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) significantly increased the clonic seizure threshold induced by intravenous administration of PTZ. This effect was observed as soon as 1 min after injection and lasted for 30 min with a peak effect at 3 min after melatonin administration. Combination of per se non-effective doses of melatonin (10 and 20 mg/kg) and nitric oxide synthase (NOS) substrate l-arginine (30, 60 mg/kg) showed a significant anticonvulsant activity. This effect was reversed by NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 30 mg/kg), implying an NO-dependent mechanism for melatonin effect. Pretreatment with l-NAME (30 mg/kg) and N(G)-nitro-l-arginine (l-NNA, 10 mg/kg) inhibited the anticonvulsant property of melatonin (40 and 80 mg/kg) and melatonin 40 mg/kg, respectively. Specific inducible NOS (iNOS) inhibitor aminoguanidine (100 and 300 mg/kg) did not affect the anticonvulsant effect of melatonin, excluding the role of iNOS in this phenomenon, while pretreatment of with 7-NI (50 mg/kg), a preferential neuronal NOS inhibitor, reversed this effect. The present data show an anticonvulsant effect for melatonin in i.v. PTZ seizure paradigm, which may be mediated via NO/l-arginine pathway by constitutively expressed NOS. © 2005 Elsevier B.V. All rights reserved. Keywords: Melatonin; Nitric oxide; Pentylenetetrazole; Clonic seizure threshold; Mice

1. Introduction ∗ Corresponding author. Tel.: +98 21 66112802; fax: +98 21 66402569. E-mail address: [email protected] (A.R. Dehpour). 1 These authors have had equal contribution to this work.

0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2005.09.057

Melatonin, or N-acetyl-5-methoxytryptamine, the major hormone produced by the pineal gland, has a number of documented functions in mammals,

104

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

including reproductive changes, hypothermic responses, hypnotic effects, and induction of phase shifts of the circadian system. Claims have also been made for immuno-enhancing, anti-cancer, anti-aging, and antioxidant effects of melatonin (see review, Brzezinski, 1997; Czeisler, 1997). Melatonin has important regulatory effects on the central nervous system. By acting through its own plasma membrane receptors, melatonin modulates the electrical activity of the neurons (AcunaCastroviejo et al., 1995) and facilitates the inhibitory ␥-aminobutyric acid (GABA)-ergic neurotransmitter function (Wan et al., 1999), which is suggested to produce its CNS depressant effects such as sedative, analgesic, anticonvulsive, hypnotic, and anxiolytic effects (Golombek et al., 1996; Dawson and Encel, 1993). Melatonin exerts anticonvulsant effect on kindled seizures in rats (Albertson et al., 1981; Mevissen and Ebert, 1998) and elevates the electroconvulsive threshold in mice (Borowicz et al., 1999). Chronic melatonin treatment in male gerbils has been shown to reduce the pentylenetetrazole (PTZ)-induced seizure incidence and mortality rate (Champney et al., 1996). Moreover, it is suggested that the anticonvulsant property of melatonin involves a modulation of both brain amino acids and nitrite level (Bikjdaouene et al., 2003). Melatonin depresses brain excitability through inhibition of the glutamate-mediated response of the striatum to motor cortex stimulation (Escames et al., 2001) and inhibition of neuronal excitation produced by either NMDA or l-arginine (Escames et al., 2004). The GABAergic effect of melatonin possibly depends on the combined effect on membrane ion permeability with the increasing chloride ion influx through GABAA -dependent chloride channel (Rosenstein et al., 1990). In humans, evidence is limited to a few experiences. Melatonin production in untreated patients with active epilepsy is increased (Schapel et al., 1995) and its diurnal variation in epileptic children is changed (Molina-Carballo et al., 1994). Moreover, it is administered as adjunctive anticonvulsant therapy in intractable seizures (MolinaCarballo et al., 1997). Nitric oxide (NO), a short-lived molecule synthesized from l-arginine by activation of nitric oxide synthase (NOS), acts as a neuronal messenger or neurotransmitter in the central and peripheral nervous system (Bredt et al., 1990). The role of NO in seizure has been widely investigated, using various NOS inhibitors and NO donors and it is a known modulator of seizure

susceptibility with either anticonvulsant (Buisson et al., 1993; Starr and Starr, 1993; Theard et al., 1995) or proconvulsant (Mulsch et al., 1994; Nidhi et al., 1999; Osonoe et al., 1994) effects in different seizure paradigms. Increasing evidence suggests that both neuronal and inducible isoforms of NOS participate in several important brain processes (Licinio et al., 1999). Epileptic seizures can induce a significant increase in NO production, which may be involved in seizureinduced enhancement of neurogenesis (Bashkatova et al., 2000; Kaneko et al., 2002). PTZ-induced clonic seizure paradigm represents an animal model of myoclonic seizures and is very sensitive to changes in seizure susceptibility (Swinyard and Kupferberg, 1985). Disinhibition of inhibitory neurotransmitter GABA, through specific interaction with the GABAA -gated chloride ionophores and/or activation of NMDA receptors appear to be factors involved in the initiation and generalization of PTZ-induced seizures (L¨oscher et al., 1991). In the present study, we examined the effect of acute melatonin administration on the threshold of clonic seizures induced by intravenous injection of PTZ in mice. Regarding the functional interactions of melatonin with NO signaling pathway, we further investigated the possible involvement of NO in the modulatory effect of melatonin on seizure susceptibility using NO precursor l-arginine, different NOS inhibitors including NG -nitro-l-arginine methyl ester (l-NAME), NG nitro-l-arginine (l-NNA), 7-nitroindazole (7-NI), and selective inducible NOS (iNOS) inhibitor, aminoguanidine.

2. Materials and methods 2.1. Drugs The following drugs were used: melatonin, larginine, l-NAME, PTZ (Sigma, Germany), 7-NI, l-NNA and aminoguanidine (Tocris, UK). To make a solubilization of various doses of melatonin, indolamine was initially dissolved in ethanol until the adequate mixture (2.5%, v/v, ethanol–saline) was reached. The procedure was performed under constant shaking. 7-NI was suspended in 1% solution of twin 80. All other drugs were dissolved in sterile isotonic saline solution to such concentrations that the requisite doses

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

were administered intraperitoneally (i.p.) in a volume of 10 ml/kg of the mice body weight. Appropriate vehicle controls were performed for each experiment. PTZ was prepared in saline as 1% solution. 2.2. Subjects Male NMRI mice (Pasteur Institute of Iran, Tehran) weighing 24–30 g at the time of experiments were used throughout the study. The animals were housed in standard polycarbonate cages in groups of six in a temperature-controlled room (23 ± 2 ◦ C) on a 12h light/12-h dark cycle (lights on at 08:00) with free access to food and water. All animals were na¨ıve and acclimatized at least 3 days before experiments. Experiments were conducted during the period between 9:00 a.m. and 3:00 p.m. All procedures were carried out in accordance with institutional guidelines for animal care and use and possible measures were undertaken to minimize the number of animals used and also to minimize animals’ discomfort including immediate euthanasia after acute experiments. Each mouse was used only once and each treatment group consisted of six to nine animals. 2.3. Determination of seizure threshold The clonic seizure threshold (CST) was determined by inserting a 30-gauge dental needle into the lateral tail vein of mouse. The needle was then secured to the tail by a narrow piece of adhesive tape. With mouse moving freely, the PTZ solution was infused into the tail vein at a constant rate of 0.5 ml/min using a Hamilton microsyringe, which was connected to the dental needle by polyethylene tubing. Infusion was halted when forelimb clonus followed by full clonus of the body was observed. Minimal dose of PTZ (mg/kg of mouse weight) needed to induce clonic seizure was measured as an index of CST. The CST examiner was blind to groups and treatments. 2.4. Treatments In experiment 1, melatonin (80 mg/kg) was administered at 1, 3, 6, 9, 15, 30, and 60 min prior to PTZ to distinct groups of mice. This dose was chosen according to our pilot studies and previous experiments (Bikjdaouene et al., 2003; Mevissen and Ebert, 1998).

105

Experiment 2 examined the effects of a wide doserange of melatonin (5, 10, 20, 40, and 80 mg/kg) on CST. Animals in this experiment received acute i.p. injections of appropriate vehicle (2.5% ethanol/saline solution, 10 ml/kg) or melatonin 3 min before determination of CST. This time point was selected according to experiment 1. Based on these two experiments melatonin doses of 10, 20, 40, and 80 mg/kg with the pre-test injection interval of 3 min were used in subsequent experiments. Experiment 3, 4, 5, and 6 were carried out to examine the possible involvement of NO pathway in melatonin-induced modulation of CST. In experiment 3, the NOS substrate, l-arginine (15, 30, and 60 mg/kg), or saline (10 ml/kg) was administered 42 min before melatonin (10 and 20 mg/kg) and 45 min before PTZ. In three further groups, animals were pretreated with lNAME (30 mg/kg), an inhibitor of NOS, 15 min prior to l-arginine (60 mg/kg) and 57 min before melatonin (10 and 20 mg/kg) or its vehicle. CST was determined 3 min after the administration of melatonin. Based on the results of this section, experiment 4 was carried out to examine the effect of concomitant administration of l-NAME or l-NNA with melatonin on CST. l-NAME (10 and 30 mg/kg) or saline was administered 57 min prior to melatonin (40 and 80 mg/kg) and was compared to the corresponding saline/vehicle, l-NAME/vehicle or saline/melatonin groups. l-NNA (10 mg/kg) was administered 57 min prior to melatonin (40 mg/kg) and was compared to saline/vehicle, saline/melatonin, or l-NNA/melatonin groups. In experiment 5, we examined the effect of irreversible inducible NOS inhibitor (iNOS) aminoguanidine, instead of the non-specific NOS inhibitors, on melatonin modulation of CST. Mice received aminoguanidine (100 and 300 mg/kg) 57 min before melatonin (80 mg/kg) or vehicle administration. Experiment 6 assessed the effect of 7-NI, a preferential nNOS inhibitor (Babbedge et al., 1993; Zagvazdin et al., 1996) on melatonin-induced modulation of seizure threshold. 7-NI (50 mg/kg) was injected 27 min before melatonin (40 mg/kg) and was compared to 1% twin 80/melatonin, 7-NI/vehicle and vehicle/vehicle control groups. Doses and time intervals were chosen based upon our previous study (Homayoun et al., 2002) and pilot experiments. Doses of l-NAME, l-NNA, and l-arginine are all subthreshold doses that have been proved in our previous study to be ineffective on CST (Homayoun et

106

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

al., 2002). Corresponding saline and melatonin vehicle controls were used in all experiments. 2.5. Statistical analysis Data are expressed as mean ± S.E.M. of CST in each experimental group. Two-way analysis of variance (ANOVA), and one-way ANOVA followed by post hoc Tukey multiple comparisons were used to analyze the data. P-value less than 0.05 was considered statistically significant.

3. Results 3.1. The effect of melatonin on CST Fig. 1 shows the time-course of the anticonvulsant effect of melatonin (80 mg/kg). One-way ANOVA revealed a significant effect (F7,42 = 17.819, P < 0.001). Post hoc analysis showed that melatonin exerted powerful anticonvulsant effect as soon as 1 min after administration (P < 0.001), with maximal effect at 3 min (P < 0.001) in comparison with saline-treated controls. This effect decreased thereafter but remained significant at 30 min (P = 0.029) but not at 1 h after injection. Fig. 2 shows the effect of acute i.p. administration of different doses of melatonin (5, 10, 20,

Fig. 2. The effect of different doses of melatonin on PTZ-induced seizure threshold in mice. Melatonin was injected 3 min before PTZ. 0 mg/kg stands for vehicle injection. Data are presented as mean ± S.E.M. ** P ≤ 0.01 and *** P < 0.001 compared to vehicle control group. +++ P < 0.001 compared with melatonin 40 mg/kg. Each group consisted of six to eight mice.

40, and 80 mg/kg) on PTZ-induced CST. One-way ANOVA revealed a significant effect (F5,32 = 29.927, P < 0.001). Post hoc analysis showed a significant anticonvulsant effect for acute melatonin at doses of 40 mg/kg (P < 0.01) and 80 mg/kg (P < 0.001) with maximal effect at 80 mg/kg. Melatonin (80 mg/kg) showed a significantly stronger effect than melatonin 40 mg/kg (P < 0.001). Melatonin (10 and 20 mg/kg), which did not produce a significant anticonvulsant effect, was chosen for experiment 3 to allow better detection of possible additive effects, while melatonin (40 and 80 mg/kg) that induced a significant anticonvulsant effect compared to their controls were chosen for experiment 4 to examine possible inhibitory effects. 3.2. The effect of co-administration of l-arginine and melatonin on CST

Fig. 1. The time-course of the anticonvulsant effect of melatonin (80 mg/kg) on PTZ-induced seizure threshold. Melatonin was administered intraperitoneally. Data are presented as mean ± S.E.M. * P < 0.05, *** P < 0.001 compared to vehicle control group. Each group consisted of six to eight mice.

Fig. 3 shows that co-administration of NO precursor l-arginine with melatonin (10 and 20 mg/kg) exerts significant anticonvulsant activity. Two-way ANOVA including l-arginine (0 (saline), 15, 30, and 60 mg/kg) plus vehicle or melatonin showed a significant effect [l-arginine factor (F3,63 = 22.213, P < 0.001), melatonin factor (F2,63 = 30.197, P < 0.001), and l-arginine × melatonin factor (F6,63 = 8.812, P < 0.001)]. Further analysis with One-way ANOVA

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

107

implies that the excitatory property of l-arginine may be related to its effect on NO synthesis. 3.3. The effect of l-NAME and l-NNA on the anticonvulsant effect of melatonin

Fig. 3. Effect of pretreatment with l-arginine on the anticonvulsant effect of melatonin in mice. l-Arginine or saline was injected 42 min before melatonin (10, 20 mg/kg) or its vehicle. CST was determined 3 min after the administration of melatonin. In groups that pretreated with l-NAME, the drug was administered 15 min prior to l-arginine. Data are presented as mean ± S.E.M. *** P < 0.001 in comparison with saline/melatonin or saline/vehicle controls. +++ P < 0.001 in comparison with l-arginine 60 mg/kg/melatonin 10 mg/kg, ++ P < 0.01 in comparison with larginine 60 mg/kg/melatonin 20 mg/kg. Each group consisted of six to nine mice.

(F11,63 = 16.668, P < 0.001) followed by post hoc comparisons showed that l-arginine did not alter the CST in control animals. However, l-arginine (30 and 60 mg/kg) in combination with melatonin (20 mg/kg), also l-arginine (60 mg/kg) in combination with melatonin (10 mg/kg) showed a significant anticonvulsant effect in comparison with saline/ melatonin (P < 0.001) and saline/vehicle controls (P < 0.001). To examine whether the observed excitatory effect of l-arginine is related to NO synthesis, we administered l-NAME (30 mg/kg) 15 min prior to l-arginine (60 mg/kg) and followed by melatonin (vehicle, 10, or 20 mg/kg). One-way ANOVA, analyzing the effect of l-NAME on the l-arginine/melatonin combination groups and relevant control vehicles showed a significant effect (F8,48 = 12.464, P < 0.001). Based on this analysis, the co-administration of the mentioned doses of l-NAME and l-arginine did not alter the seizure threshold in control animals. However, lNAME (30 mg/kg) reversed the enhancing effect of l-arginine (60 mg/kg) on the anticonvulsant effect of melatonin (10, 20 mg/kg), as shown in Fig. 3. This

Experiment 4 was carried out to examine the possible inhibitory effect of two non-selective NOS inhibitors, l-NAME and l-NNA, on the anticonvulsant effect of melatonin. Fig. 4A shows the results of the effect of l-NAME on the anticonvulsant effect of melatonin. Two-way ANOVA showed a significant effect [l-NAME factor (F2,52 = 23.523, P < 0.001), melatonin factor (F2,52 = 89.941, P < 0.001), lNAME × melatonin factor (F4,52 = 9.943, P < 0.001)]. Further analysis with one-way ANOVA followed by Tukey post hoc comparisons (F8,52 = 38.351, P < 0.001) showed that l-NAME (10 and 30 mg/kg) did not alter the CST in control groups. Nevertheless, l-NAME at dose of 30 mg/kg blocked the anticonvulsant effect induced by melatonin (40 and 80 mg/kg) compared to their control saline/melatonin groups (For both P < 0.001). l-NAME (10 mg/kg) was not able to show any significant effect on the anticonvulsant effect of melatonin at doses of 40 and 80 mg/kg. Statistical analysis indicated that l-NNA inhibited melatonin anticonvulsant properties (one-way ANOVA, F3,24 = 16.243, P < 0.001). Tukey post hoc comparisons showed that l-NNA (10 mg/kg) did not alter the CST in l-NNA/saline control group (P = 0.88) but inhibited the anticonvulsant property of melatonin (40 mg/kg) (P = 0.002) (Fig. 4B). 3.4. Aminoguanidine did not affect the anticonvulsant effects of melatonin We examined the effect of iNOS inhibitor, aminoguanidine, on anticonvulsant effects of melatonin in order to examine the probable involvement of iNOS in this regard. No significant interaction was seen [two-way ANOVA; aminoguanidine factor (F2,33 = 0.471, P > 0.05), melatonin factor (F1,33 = 250.298, P < 0.001), and aminoguanidine × melatonin factor (F2,33 = 0.803, P > 0.05)]. Pretreatment with aminoguanidine (100 and 300 mg/kg) did not affect the CST neither in vehicle nor in melatonin (80 mg/kg) groups (Fig. 5).

108

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

Fig. 5. The effect of pretreatment with aminoguanidine on the anticonvulsant effect of melatonin in mice. Aminoguanidine (100, 300 mg/kg) or saline was administered 57 min prior to melatonin (40, 80 mg/kg). CST was determined 3 min after the administration of melatonin. Data are presented as mean ± S.E.M. *** P < 0.001 in comparison with corresponding saline-treated controls. Each group consisted of six to nine mice.

7-NI (50 mg/kg) did not alter the CST in 7-NI/vehicle control group (P = 0.782) but inhibited the anticonvulsant effect of melatonin (40 mg/kg) (P = 0.03, compared to vehicle/melatonin control group).

Fig. 4. (A) l-NAME inhibited the anticonvulsant effect of melatonin in mice. l-NAME (10, 30 mg/kg) or saline was administered 57 min prior to melatonin (40, 80 mg/kg). CST was determined 3 min after the administration of melatonin. * P < 0.05, ** P < 0.01, *** P < 0.001 in comparison with saline/vehicle controls. +++ P < 0.001 in comparison with corresponding saline/melatonin group. (B) l-NNA inhibited the anticonvulsant effect of melatonin in mice. l-NNA (10 mg/kg) or saline was administered 57 min prior to melatonin (40 mg/kg). CST was determined 3 min after the administration of melatonin. *** P < 0.001 in comparison with saline/vehicle controls and ++ P < 0.01 in comparison with saline/melatonin group. Data are presented as mean ± S.E.M. Each group consisted of six to nine mice.

3.5. The effect of 7-NI on the anticonvulsant effects of melatonin Fig. 6 shows the effect of 7-NI on melatonininduced modulation of seizure threshold. One-way ANOVA revealed a significant effect (F3,21 = 17.178, P < 0.001). Tukey’s post hoc comparisons showed that

Fig. 6. The effect of pretreatment with 7-NI on the anticonvulsant effect of melatonin in mice. 7-NI (50 mg/kg) or its vehicle was administered 57 min prior to melatonin (40 mg/kg). CST was determined 3 min after the administration of melatonin. 0 represents 7-NI vehicle injection. Data are presented as mean ± S.E.M. *** P < 0.001, ## P < 0.01 in comparison with vehicle/vehicle control group, + P < 0.05 in comparison with vehicle/melatonin group. Each group consisted of six to nine mice.

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

4. Discussion The present work shows that acute i.p. administration of melatonin increases the threshold of clonic seizure induced by PTZ. The maximum response was observed at 3 min after administration of melatonin. Concomitant administration of l-arginine with low and per se non-effective doses of melatonin induced a potent anticonvulsant effect. Pretreatment with lNAME and l-NNA, non-specific NOS inhibitors, diminished the anticonvulsant effect of melatonin completely. Pretreatment with 7-NI, a preferential nNOS inhibitor, blocked this effect while aminoguanidine, an irreversible iNOS inhibitor did not. These findings imply the involvement of l-arginine/NO pathway in melatonin-induced modulation of seizure susceptibility in mice. The induction of seizure by intravenous infusion of PTZ is a standard experimental model of clinical myoclonic seizures with both face and construct validity (Swinyard and Kupferberg, 1985; L¨oscher et al., 1991). This model is proved to be more sensitive than i.p. PTZ administration method, and allows better detection of modulatory effects on convulsive tendency (L¨oscher et al., 1991). PTZ increases activity in major epileptogenic centers of forebrain like amygdale and piriform cortex (Gale, 1992). Neurochemical evidence suggests that PTZ binds to the picrotoxin site of the GABA receptor complex and blocks the GABA-mediated inhibition. Disinhibition of inhibitory neurotransmitter, GABA and/or activation of NMDA receptor appear to be factors involved in the initiation and generalization of the PTZ-induced seizures (Kaputlu and Uzbay, 1997). The present paper is the first report of the anticonvulsant effect of melatonin in intravenous PTZ paradigm of clonic seizures in mice. Consistent with our results, lines of evidence in different animal models of epilepsy suggest the anticonvulsant effect for melatonin. Melatonin (50 mg/kg) significantly raises the electroconvulsive threshold in female Swiss mice (Borowicz et al., 1999). Also pinealectomy (which reduces plasma melatonin level) in previously parathyroidectomized rats induces seizure (Reiter and Morgan, 1972). It has been shown that intracerebroventricular (i.c.v.) administration of melatonin attenuates the convulsant effect of i.c.v. administered kainate, quinolinate, glutamate, NMDA, and PTZ but it is ineffective against i.p. admin-

109

istered PTZ (Lapin et al., 1998). Bikjdaouene et al. (2003) have shown that administration of melatonin 30 min before i.p. injection of PTZ increases the latency and decreases the duration of first seizure and also reduces the PTZ-induced mortality from 87.5 to 25% in this model of epilepsy in rats. It has been reported that chronic but not acute melatonin treatment reduces the PTZ-induced seizure incidence and mortality rate in male gerbils (Champney et al., 1996). However, there is a discrepancy in the findings probably due to differences in animal species (gerbil versus mouse), the approach used to measure seizure (seizure threshold versus seizure incidence) and the dose of melatonin (0.1–10 mg/kg versus 5–80 mg/kg). In humans, evidence is limited to a few experiments. Melatonin treatment suppresses epileptiform seizures in both adults and children (Munoz-Hoyos et al., 1998) and chronic high dose melatonin is used as adjunctive anticonvulsant therapy in intractable seizures (Molina-Carballo et al., 1997). Also significant changes are found in day–night melatonin levels during convulsions in normal children and in children with febrile or epileptic convulsions (Molina-Carballo et al., 1994). In the central nervous system, NO acts as a diffusible intercellular signaling molecule (Dawson and Snyder, 1994). Nitric oxide exerts a significant inhibitory effect on NMDA receptor function (Manzoni et al., 1992). It has been proposed that this effect is mediated via the redox site on the NMDA receptor, but interaction with the relevant cysteines in the NMDA receptor subunits has been ruled out (Aizenman and Potthoff, 1999). NO is synthesized from l-arginine in an NADPH-dependent reaction by NO synthase. Three different isoforms of NOS have been identified including one inducible (iNOS) and two constitutively expressed (endothelial (eNOS) and neuronal (nNOS)) forms (Moncadam and Higgs, 1993). In seizure susceptibility regulation, NOS substrates or NO donors exert various anticonvulsant (Buisson et al., 1993; Starr and Starr, 1993; Theard et al., 1995) or proconvulsant (Mulsch et al., 1994; Nidhi et al., 1999; Osonoe et al., 1994) effects in different models of seizure; this apparent contradiction may be explained by different experimental conditions (species, convulsive agents, and type of model seizures), including pharmacological tools used to modify the NO pathway. Moreover, the effect of different NOS inhibitors varies with the model of seizure, type of NOS inhibitor and other

110

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

experimental conditions and it is suggested that some of their effects are not necessarily due to decrease in NO level (Borowicz et al., 2000). For instance, lNAME and l-NG -monomethylarginine attenuate PTZinduced seizure in rats (Osonoe et al., 1994). In contrast, l-NAME enhances NMDA-induced convulsions in mice (Buisson et al., 1993; Przegalinski et al., 1996; Tutka et al., 1996). l-NNA is shown to impair the protective activity of euthosuximide but not Phenobarbital, diazepam and sodium valproate against the clonic phase of PTZ-induced seizures in mice (Czuczwar et al., 1999). l-NNA has protective effect against i.c.v. administered glutamate but is in effective against tonic–clonic seizures induced by systemic bicucullin, PTZ and pilocarpine (Tutka et al., 1996). It potentiates aminophylline-induced seizures but is ineffective against electroconvulsive, aminooxyacetic acid and PTZ-induced convulsions in mice (Urbanska et al., 1996). l-NAME and l-NNA are alkyl esters of arginine and affect both eNOS and nNOS. So far 7-NI, a preferential but not selective (Babbedge et al., 1993; Zagvazdin et al., 1996) neuronal NOS inhibitor, has been examined in various models of epilepsy. Although the drug tends only to delay the onset of seizures provoked by i.c.v. administration of NMDA in mice (Eblen et al., 1996), it also significantly decreases the dose of NMDA necessary to produce clonic convulsions in 50% of mice (Przegalinski et al., 1996). Conversely, it markedly suppresses sound-induced seizures and enoxacin evoked convulsions in mice and displays protective properties in genetically seizure prone rats (Van Leeuwen et al., 1995; Smith et al., 1996; Masukawa et al., 1998). 7-NI enhances the protective activity of Phenobarbital in maximal electroshock (Borowicz et al., 1997) but not in PTZ-induced seizures (Borowicz et al., 2000). Also it potentiates the protective action of clonazepam and euthosuximide against PTZ-induced clonic seizures in mice (Borowicz et al., 2000). In the light of diverse effects of NOS inhibitors upon the susceptibility to different types of seizure, we have decided to assess the role of four different NOS inhibitors (l-NAME, l-NNA, aminoguanidine, 7-NI) on melatonin-induced modulation of seizure threshold. However, consistent with our results the inhibition or stimulation of NO synthesis at low to moderate doses does not affect the clonic seizures induced by PTZ (Tsuda et al., 1997; Buisson et al., 1993; Przegalinski et al., 1996). In the present study, concomitant admin-

istration of per se non-effective doses of l-arginine and sub-protective doses of melatonin resulted in the inhibition of seizure activity. Moreover, non-effective doses of l-NAME and l-NNA inhibited the melatonininduced anticonvulsive effects, suggesting that the protective action of melatonin is mediated via increasing NO production through l-arginine/NO pathway. Accumulating evidence has shown that NO production is increased in hippocampus, cerebral cortex and some other brain regions of PTZ-treated rats owing to activation of various isoforms of NOS (Bashkatova et al., 2000; Kaneko et al., 2002). It still remains unclear whether the increase of NO production in brain after seizure is anticonvulsive or proconvulsive (Jalenkovic et al., 2002). It is found that NO produced by both nNOS and iNOS after seizures could contribute to the increased precursor cell proliferation induced by seizures (Jiang et al., 2004). On the other hand, it is shown that melatonin partially counteracts the PTZinduced increase in nitrite content in parieto-temporal, striatum and brain stem without changes in other brain regions after PTZ-induced tonic-clonic seizure in rats (Bikjdaouene et al., 2003). This may be due to its scavenger effect (Noda et al., 1999); however, there is no conclusive data available about the role of NO pathway in melatonin-induced modulation of seizure threshold. In the nervous system, nNOS is largely responsible for NO production (Bredt et al., 1990), while iNOS is also reported to be present in normal adult brain and to contribute to the pathophysiology of many neuronal diseases (Licinio et al., 1999). Also, iNOS produces large amounts of NO continuously for long periods, a feature that is responsible for the cytotoxicity of NO (Garthwaite and Boulton, 1995). Consequently, eNOS and nNOS produce NO in small and highly regulated bursts that are well suited for the molecular messenger function of NO. Therefore, to examine the probable contribution of iNOS in melatonin-induced protection against seizure, we used aminoguanidine, which is an irreversible specific inhibitor of iNOS (Al-Shabanah et al., 2000). Aminoguanidine did not alter the seizure susceptibility in melatonin- or vehicletreated subjects, excluding the involvement of iNOS in this phenomenon and suggesting a role for constitutively expressed NOS (cNOS). Moreover, 7-NI a preferentially nNOS inhibitor reversed the effect of melatonin implying the involvement of nNOS in this phenomenon. On the other hand, there is the possibility

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

of pharmacokinetic interaction of drugs used in the present study. However, there is limited evidence in the literature in favour of such possibility and several previous studies using different combination of these agents in various experimental settings have not found any such interactions (Regrigny et al., 1999; Brzozowska et al., 2002; Sanchez-Campos et al., 2001; Homayoun et al., 2002; Khavandgar et al., 2002). These evidences would not rule out the possible pharmacokinetic interactions or modulation of brain penetration of pentylenetetrazole but makes it unlikely. Regarding the reports that suggest an inhibitory effect of melatonin on NOS (Bettahi et al., 1996; Pozo et al., 1994; Chang et al., 2002), interactions of melatonin with larginine/NO pathway in different processes warrant further investigation. In summary, acute melatonin administration increases the PTZ-induced clonic seizure threshold in mice and this effect might be due to increase in constitutive nitric oxide activity. References Acuna-Castroviejo, D., Escames, G., Macias, M., Munoz Hoyos, A., Molina Carballo, A., Arauzo, M., Montes, R., 1995. Cell protective role of melatonin in the brain. J. Pineal Res. 19, 57–63. Aizenman, E., Potthoff, W.K., 1999. Lack of interaction between nitric oxide and the redox modulatory site of the NMDA receptor. Br. J. Pharmacol. 126, 296–300. Albertson, T.E., Peterson, S.L., Stark, L.G., Lakin, M.L., Winters, W.D., 1981. The anticonvulsant properties of melatonin on kindled seizures in rats. Neuropharmacology 20, 61–66. Al-Shabanah, O.A., Alam, K., Nagi, M.N., Al-Rikabi, A.C., AlBekairi, A.M., 2000. Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice. Life Sci. 66, 265–270. Babbedge, R.C., Bland-Ward, P.A., Hart, S.L., Moore, P.K., 1993. Inhibition of rat cerebellar nitric oxide synthase by 7nitroindazole and related substituted indazoles. Br. J. Pharmacol. 110, 225–229. Bashkatova, V., Vitskova, G., Narkevich, V., Vanin, A., Mikoyan, V., Rayevsky, K., 2000. Nitric oxide content measured by ESR-spectroscopy in the rat brain is increased during pentylenetetrazole-induced seizures. J. Mol. Neurosci. 14, 183–190. Bettahi, I., Pozo, D., Osuna, C., Reiter, R.J., Acuna-Castroviejo, D., Guerrero, J.M., 1996. Melatonin reduces nitric oxide synthase activity in rat hypothalamus. J. Pineal Res. 20, 205–210. Bikjdaouene, L., Escames, G., Leon, J., Ferrer, J.M., Khaldy, H., Vives, F., Acuna-Castroviejo, D., 2003. Changes in brain amino acids and nitric oxide after melatonin administration in rats with pentylenetetrazole-induced seizures. J. Pineal Res. 35, 54–60.

111

Borowicz, K.K., Kaminski, R., Gasior, M., Kleinrok, Z., Czuczwar, S.J., 1999. Influence of melatonin upon the protective action of conventional anti-epileptic drugs against maximal electroshock in mice. Eur. Neuropsychopharmacol. 9, 85–90. Borowicz, K.K., Kleinrok, Z., Czuczwar, S.J., 1997. Influence of 7-nitroindazole on the anti-convulsive action of conventional antiepileptic drugs. Eur. J. Pharmacol. 331, 127–132. Borowicz, K.K., Luszczki, J., Kleinrok, Z., Czuczwar, S.J., 2000. 7-nitroindazole, a nitric oxide inhibitor, enhances the anticonvulsive action of ethosuximide and clonazepam against pentylenetetrazol-induced convulsions. J. Neural Transm. 107, 1117–1126. Bredt, D.S., Hwang, P.M., Snyder, S.H., 1990. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347 (6295), 768–770. Brzezinski, A., 1997. Melatonin in humans. New Engl. J. Med. 336, 186–195. Brzozowska, I., Konturek, P.C., Brzozowski, T., Konturek, S.J., Kwiecien, S., Pajdo, R., Drozdowicz, D., Pawlik, M., Ptak, A., Hahn, E.G., 2002. Role of prostaglandins, nitric oxide, sensory nerves and gastrin in acceleration of ulcer healing by melatonin and its precursor, l-tryptophan. J. Pineal Res. 32, 149–162. Buisson, A., Lakhmeche, N., Verrecchia, C., Plotkine, M., Boulu, R.G., 1993. Nitric oxide: an endogenous anticonvulsant substance. Neuroreport 4, 444–446. Champney, T.H., Hanneman, W.H., Legare, M.E., Appel, K., 1996. Acute and chronic effects of melatonin as an anticonvulsant in male gerbils. J. Pineal Res. 20, 79–83. Chang, H.M., Ling, E.A., Chen, C.F., Lue, H., Wen, C.Y., Shieh, J.Y., 2002. Melatonin attenuates the neuronal NADPH-d/NOS expression in the nodose ganglion of acute hypoxic rats. J. Pineal Res. 32, 65–73. Czeisler, C.A., 1997. Commentary: evidence for melatonin as a circadian phase shifting agent. J. Biol. Rhythms 12, 618–626. Czuczwar, S.J., Tutka, P., Klonowski, P., Kleinrok, Z., 1999. NG -nitro-l-arginine impairs the anticonvulsive action of ethosuximide against pentylenetetrazole. Eur. J. Pharmacol. 366, 137–142. Dawson, D., Encel, N., 1993. Melatonin and sleep in humans. J. Pineal Res. 15, 1–12. Dawson, T.M., Snyder, S.H., 1994. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J. Neurosci. 14, 5147–5159. Eblen, F., L¨oschmann, P.A., Wullner, U., Turski, L., Klockgether, T., 1996. Effects of 7-nitroindazole, NG -nitro-l-arginine, and D-CPPene on harmaline-induced postural tremor, N-methyl-daspartate-induced seizures and lisuride-induced rotations in rats with nigral 6-hydroxydopamine lesions. Eur. J. Pharmaol. 299, 9–16. Escames, G., Leon, J., Lopez, L.C., Acuna-Castroviejo, D., 2004. Mechanisms of N-methyl-d-aspartate receptor inhibition by melatonin in the rat striatum. J. Neuroendocrinol. 16, 929– 935. Escames, G., Macias, M., Leon, J., Garcia, J., Khaldy, H., Martin, M., Vives, F., Acuna-Castroviejo, D., 2001. Calcium-dependent effects of melatonin inhibition of glutamatergic response in rat striatum. J. Neuroendocrinol. 13, 459–466.

112

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113

Gale, K., 1992. Subcortical structures and pathways involved in convulsive seizure generation. J. Clin. Neurophysiol. 9, 264–277. Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the central nervous system. Annu. Rev. Physiol. 57, 683–706. Golombek, D.A., Pevet, P., Cardinali, D.P., 1996. Melatonin effects on behavior: possible mediation by the central GABAergic system. Neurosci. Biobehav. Rev. 20, 403–412. Homayoun, H., Khavandgar, S., Namiranian, K., Gaskari, S.A., Dehpour, A.R., 2002. The role of nitric oxide in anticonvulsant and proconvulsant effects of morphine in mice. Epilepsy Res. 48, 33–41. Jalenkovic, A., Jovankovic, M., Ninkovic, M., Maksimovic, D., Bokonjic, D., Boskovic, B., 2002. Nitric oxide (NO) and convulsions induced by pentylenetetrazol. Ann. N. Y. Acad. Sci. 962, 296–305. Jiang, W., Xiao, L., Wang, J., Huang, Y., Zhang, X., 2004. Effects of nitric oxide on dentate gyrus cell proliferation after seizures induced by pentylenetetrazol in the adult rat brain. Neurosci. Lett. 367, 344–348. Kaneko, K., Itoh, K., Berliner, L.J., Miyasaka, K., Fujii, H., 2002. Consequences of nitric oxide generation in epileptic-seizure rodent models as studied by in vivo EPR. Magn. Reson. Med. 48, 1051–1056. Kaputlu, I., Uzbay, T., 1997. l-NAME inhibits pentylenetetrazole and strychnine-induced seizures in mice. Brain Res. 753, 98–101. Khavandgar, S., Homayoun, H., Dehpour, A.R., 2002. The role of nitric oxide in the proconvulsant effect of delta-opioid agonist SNC80 in mice. Neurosci. Lett. 329, 237–239. Lapin, I.P., Mirzaev, S.M., Ryzov, I.V., Oxenkrug, G.F., 1998. Anticonvulsant activity of melatonin against seizures induced by quinolinate, kainate, glutamate, NMDA, and pentylenetetrazole in mice. J. Pineal Res. 24, 215–218. Licinio, J., Prolo, P., McCann, S.M., Wong, M.L., 1999. Brain iNOS: current understanding and clinical implications. Mol. Med. Today 5, 225–232. L¨oscher, W., Honack, D., Fassbender, C.P., Nolting, B., 1991. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazole seizure models. Epilepsy Res. 8, 171–189. Manzoni, O., Prezeau, L., Marin, P., Dehager, S., Bockaert, J., Fagni, L., 1992. Nitric oxide-induced blockade of NMDA receptors. Neuron 8, 653–662. Masukawa, T., Nakanishi, K., Natsuki, R., 1998. Role of nitric oxide in the convulsions following the coadministration of enoxacin with fenbufen in mice. Jpn. J. Pharmacol. 76, 425–429. Mevissen, M., Ebert, U., 1998. Anticonvulsant effects of melatonin in amygdale-kindeled rats. Neurosci. Lett. 257, 13–16. Molina-Carballo, A., Munoz-Hoyos, A., Reiter, R.J., Sanchez-Forte, M., Moreno-Madrid, F., Rufo-Campos, M., Molina-Font, J.A., Acuna-Castroviejo, D., 1997. Utility of high doses of melatonin as adjunctive anticonvulsant therapy in a child with severe myoclonic epilepsy: two years’ experience. J. Pineal Res. 23, 97–105. Molina-Carballo, A., Munoz-Hoyos, A., Rodriguez-Cabezas, T., Acuna-Castroviejo, D., 1994. Day–night variations in melatonin secretion by the pineal gland during febrile and epileptic convulsions in children. Psychiatry Res. 52, 273–283.

Moncadam, S., Higgs, A., 1993. The l-arginine-nitric oxide pathway. New Engl. J. Med. 329, 2002–2012. Mulsch, A., Busse, R., Mordvintcev, P.I., Vanin, A.F., Nielsen, E.O., Scheel-Kruger, J., Olesen, S.P., 1994. Nitric oxide promotes seizure activity in kainate-treated rats. Neuroreport 5, 2325–2328. Munoz-Hoyos, A., Sanchez-Forte, M., Molina-Carballo, A., Escames, G., Martin-Medina, E., Reiter, R.J., Molina-Font, J.A., Acuna-Castroviejo, D., 1998. Melatonin’s role as an anticonvulsant and neural protector: experimental and clinical evidence. J. Child Neurol. 13, 501–509. Nidhi, G., Balakrishnan, S., Pandhi, P., 1999. Role of nitric oxide in electroshock and pentylenetetrazole seizure threshold in rats. Methods Find. Exp. Clin. Pharmacol. 21, 609–612. Noda, Y., Mori, A., Liburdy, R., Packer, L., 1999. Melatonin and its precursors scavenge nitric oxide. J. Pineal Res. 27, 159–163. Osonoe, K., Mori, N., Suzuki, K., Osonoe, M., 1994. Antiepileptic effects of inhibitors of nitric oxide synthase examined in pentylenetetrazol-induced seizures in rats. Brain Res. 663, 338–340. Pozo, D., Reiter, R.J., Calvo, J.R., Guerrero, J.M., 1994. Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum. Life Sci. 55, PL455–PL460. Przegalinski, E., Baran, L., Swianowicz, J., 1996. The role of nitric oxide in chemically and electrically-induced seizures in mice. Neurosci. Lett. 217, 145–148. Regrigny, O., Delagrange, P., Scalbert, E., Lartaud-Idjouadiene, I., Atkinson, J., Chillon, J.M., 1999. Effects of melatonin on rat pial arteriolar diameter in vivo. Br. J. Pharmacol. 127, 1666–1670. Reiter, R.J., Morgan, W.W., 1972. Attempts to characterize the convulsive response of parathyroidectomized rats to pineal gland removal. Physiol. Behav. 9, 203–208. Rosenstein, R.E., Chuluyan, H.E., Diaz, M.C., Cardinali, D.P., 1990. GABA as a presumptive paracrine signal in the pineal gland. Evidence on an intrapineal GABAergic system. Brain Res. Bull. 25, 339–344. Sanchez-Campos, S., Arevalo, M., Mesonero, M.J., Esteller, A., Gonzalez-Gallego, J., Collado, P.S., 2001. Effects of melatonin on fuel utilization in exercised rats: role of nitric oxide and growth hormone. J. Pineal Res. 31, 159–166. Schapel, G.J., Beran, R.G., Kennaway, D.L., McLoughney, J., Matthews, C.D., 1995. Melatonin response in active epilepsy. Epilepsia 36, 75–78. Smith, S.E., Man, C.E., Yip, P.K., Tang, E., Chapman, A.G., Meldrum, B.S., 1996. Anticonvulsant effect of 7-nitroindazole in rodents with reflex epilepsy may result from l-arginine accumulation or reduction in nitric oxide or l-citrulline formation. Br. J. Pharmacol. 119, 165–173. Starr, M.S., Starr, B.S., 1993. Paradoxical facilitation of pilocarpineinduced seizures in the mouse by MK-801 and the nitric oxide synthesis inhibitor l-NAME. Pharmacol. Biochem. Behav. 45, 321–325. Swinyard, E.A., Kupferberg, H.J., 1985. Antiepileptic drugs: detection, quantification, and evaluation. Fed. Proc. 44, 2629–2633. Theard, M.A., Baughman, V.L., Wang, Q., Pelligrino, D.A., Albrecht, R.F., 1995. The role of nitric oxide in modulating brain activity and blood flow during seizure. Neuroreport 6, 921–924.

N. Yahyavi-Firouz-Abadi et al. / Epilepsy Research 68 (2006) 103–113 Tsuda, M., Suzuki, T., Misawa, M., 1997. Aggravation of DMCMinduced seizure by nitric oxide synthase inhibitors in mice. Life Sci. 60, PL339–PL343. Tutka, P., Klonowski, P., Dziecivch, J., Kleinork, Z., Czuczwar, S.J., 1996. NG -nitro-l-arginine differentially affects glutamate or kainite-induced seizures. Neuroreport 7, 1605–1608. Urbanska, E.M., Drewleska, E., Borowicz, K., Blaszczak, P., Kleinork, Z., Czuczwar, S.J., 1996. NG -nitro-l-arginine and seizure susceptibility in four seizure models in mice. J. Neural Transm. 103, 1145–1152. Van Leeuwen, R., De Vries, R., Dzoljik, M.R., 1995. 7Nitroindazole, an inhibitor of neuronal nitric oxide synthase,

113

attenuates pilocarpine-induced seizures. Eur. J. Pharmacol. 287, 211–213. Wan, Q., Man, H.Y., Liu, F., Braunton, J., Niznik, H.B., Pang, S.F., Brown, G.M., Wang, Y.T., 1999. Differential modulation of GABAA receptor function by Mel1a and Mel1b receptors. Nat. Neurosci. 2, 401–403. Zagvazdin, Y., Sancesario, G., Wang, W-X., Share, L., Fitzgerald, M.E.C., Reiner, A., 1996. Evidence from its cardiovascular effect that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo. Eur. J. Pharmacol. 303, 61–69.