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The role of nitric oxide in chemically- and electrically-induced seizures in mice
ELSEVIER
Neuroscience Letters 217 (1996) 145-148
The role of nitric oxide in chemically- and electrically-induced seizures in mice E d m u n d Przegalifiski*, L e o k a d i a Baran, Joanna S i w a n o w i c z Institute of Pharmacology, Polish Academy of Sciences, 12 Smgtna Street, 31-343 Krak6w, Poland
Received 19 August 1996; revised version received 13 September 1996; accepted 13 September 1996
Abstract
The effect of the nitric oxide synthase (NOS) inhibitors N-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) on seizures induced by N-methyl-D-aspartate (NMDA), pilocarpine (PIL) and pentylenetetrazol (PTZ), as well as on the electroconvulsive threshold was studied in mice. It was found that L-NAME and 7-NI decreased the dose of NMDA necessary to produce clonic convulsions in 50% of animals (CDs0). Such a proconvulsant effect was not observed in mice pretreated with N-nitro-D-arginine methyl ester (o-NAME), an inactive isomer of L-NAME. Neither L-NAME nor 7-NI affected the convulsions induced by PIL (clonic seizures) or PTZ (clonic and tonic seizures), having no effect on their CDs0 values. Similarly, neither NOS inhibitor affected the electroshock threshold. These results, together with some literature data, indicate that nitric oxide (NO) may be regarded as an anticonvulsant substance in relation to seizures induced by NMDA and other excitatory amino acids, but not by other agents, in mice. Keywords: Nitric oxide; Nitric oxide synthase inhibitors; N-Methyl-D-aspartate; Pilocarpine; Pentylenetetrazol; Electroconvulsive
threshold; Seizures; Mice
Nitric oxide (NO) is a small, membrane-diffusible molecule, synthesized from L-arginine by the Ca2+-calmodulin-dependent NO synthase (NOS) in various tissues including the brain [1]. The major action of NO is activation of the soluble guanylate cyclase which in turn increases the level of an intracellular second messenger, guanosine 3',5"-cyclic monophosphate (cGMP), in target cells [24]. NO is regarded as either a novel neuronal messenger or a neurotransmitter in the central nervous system and is involved in neuronal functions [ 1,24]. There are at least two arguments supporting a possible role of NO in convulsive phenomena: (1) excitatory amino acids, such as N-methyl-o-aspartate (NMDA) and kainate, are known to be potent convulsants [ 15], and the activation of NMDA receptors is accompanied by the formation of NO [6], (2) various convulsants, including excitatory amino acids, pentylenetetrazol (PTZ) and pilocarpine (PIL), increase the cGMP level in the cerebellum, whereas anticonvulsant drugs decrease it [26]. In fact, the role of
* Corresponding author. Tel.: +48 12 374022; fax: +48 12 374500.
NO in epileptogenesis has been examined in a number of studies, yet the obtained results are contradictory. NO has been suggested to be either an anticonvulsive [2,21] or a proconvulsive [4,19] endogenous substance. This apparent contradiction may be explained by different experimental conditions (species, convulsants, type of seizures), including pharmacological tools used to modify the NO pathway. Regarding the latter, it is noteworthy that the conclusions of most of the studies are based on results obtained with the use of NOS inhibitors. Moreover, practically all the NOS inhibitors used in those studies were arginine-based, affecting not only neuronal but also endothelial NOS. The inhibition of endothelial NOS leads to increased blood pressure [16], which, in turn, may affect the excitability of central neurons [5]. Furthermore, the specificity of these NOS inhibitors is limited by their muscarinic receptor blocking properties [3]. Therefore in the present study we examined the NMDA-, PTZ-, PIL- and electrically-induced seizures in mice treated with N-nitro-L-arginine methyl ester (LNAME) or 7-nitroindazole (7-NI), the latter being an in vivo selective neuronal NOS inhibitor without cholinolytic
0304-3940/96/$12.00 © 1996Elsevier ScienceIreland Ltd. All fights reserved PII S0304-3940(96)13085-8
146
E. Przegalihski et al. / Neuroscience Letters 217 (1996) 145-148
properties or significant cardiovascular effects in mice [17,18]. The experiments were carried out on male Albino Swiss mice weighing 26-33 g, which were obtained from a licensed dealer. The animals were housed in groups of 40 to a cage (50 × 38 × 20 cm) and kept at a room temperature (21 + I°C) on a 12:12 h light/dark cycle (light on at 0600 h), with food (granular standard diet, Bacutil) and water ad libitum. The tests were carried out between 1000 and 1500 h, and the mice were randomly assigned to experimental groups. The animals were injected with NMDA (RBI), PIL (Hestag) and PTZ (Sigma), placed separately in plastic cages and observed for 30 min for the occurrence of clonic (induced by NMDA and PIL), or clonic and tonic (after PTZ) seizures. The doses of NMDA, PIL and PTZ, necessary to induce seizure responses in 50% of mice (CDs0, convulsant dose 50), were. calculated. To determine CDs0, three to five doses of the convulsants and 8 - 1 0 mice per dose were used. Electroshocks were delivered to the animals through ear clip electrodes (ECT unit 7801, Ugo Basile, Milan, Italy), with saline (0.9% NaC1) applied to the ears to ensure good contact. Stimulation parameters consisted of 100 rectangular pulses/s, 0.5 ms in width, for a duration of 1 s. The electroshock threshold, defined as the current necessary to cause the hindleg tonic extensor component of seizures in 50% of animals (CC50, convulsant current 50), was calculated. To determine CC50, three to five currents were tested on groups of 8 - 1 0 mice each. L-NAME (Sigma) and N-nitro-D-arginine methyl ester (D-NAME; Sigma) were injected in doses of 3, 10 and 30
mg/kg, and 7-NI (RBI) in doses of 10 and 30 mg/kg, at 30 min before the convulsants or electroshocks. All the substances were dissolved in bidistilled water, except for 7-NI which was suspended in a 1% aqueous solution of Tween80, and injected intraperitoneally (i.p.). The CDs0 and CC50 values (with 95% confidence limits) and their statistical evaluations were calculated by fitting the data by a probit analysis according to Litchfield and Wilcoxon [12]. As shown in Table 1, L-NAME and 7-NI reduced the convulsive threshold for NMDA. A significant decrease in the CDs0 of NMDA was observed after 10 and 30 mg/kg of L-NAME (by 37 and 41%, respectively) and after 30 mg/ kg of 7-NI (by 32%). The CDs0 value of NMDA was not changed by D-NAME. In contrast to the NMDA-induced seizures, neither L-NAME nor 7-NI affected the convulsions induced by PIL (clonic seizures) and PTZ (clonic and tonic seizures), having no effect on their CDs0 values. Similarly, L-NAME and 7-NI did not affect the electroshock threshold, as the CC50 values were not changed in mice pretreated with those NOS inhibitors. The present data show that pharmacological interference in the NO pathway does not affect in a similar way the susceptibility to seizures evoked by various chemical convulsions or electroshocks in mice. Actually, we observed that the NOS inhibitors, L-NAME or 7-NI, reduced the dose of NMDA necessary to produce clonic convulsions (CDs0), which indicates an increased seizure susceptibility to the excitatory amino acid. This observation, together with the findings that the CDs0 of NMDA was not affected by o-NAME, an inactive isomer which does not inhibit the NOS activity [10], seem to indicate
Table 1 Effect of N-nitro-L-arginine methyl ester (L-NAME), N-nitro-D-arginine methyl ester (D-NAME) and 7-nitroindazole (7-NI) on N-methylt~-aspartate (NMDA), pilocarpine (P1L), pentylenetetrazol (PTZ) and electrically (ECS)-induced seizures in mice Treatment and dose (mg/kg, i.p.)
CDso (mg/kg, i.p.; 95% confidence limits) NMDA
Vehicle L-NAME 3 L-NAME 10 L-NAME 30
116.8 (107.8-126.7) 108.2 (91.9-127.3) 73.5 (65.7-82.4)* 68.8 (60.6--78.0)*
Vehicle D-NAME 3 D-NAME 10 D-NAME 30
110.0 100.6 100.4 116.5
Vehicle 7-NI 10 7-NI 30
116.4 (105.8-128.1) 108.7 (91.6-129.0) 79.1 (64.5-97.8)*
PIL
205.1 172.4 200.4 208.7
CC50 (mA; 95% confidence limits) PTZ
(171.0-245.9) (135.3-219.7) (172.9-232.2) (168.3-258.8)
ECS
Clonus
Tonus
53.4 53.1 53.1 53.1
76.9 76.9 77.2 67.8
(46.3--61.5) (48.1-58.5) (48.1-58.5) (48.1-58.1)
(67.2-88.0) (67.2-88.0) (73.3-81.4) (57.7-79.7)
10.6 9.6 10.8 11.7
(9.5-11.7) (8.5-10.7) (10.0-11.6) (10.9-12.5)
(93.6-129.2) (91.2-111.1) (86.2-116.9) (100.3-135.3) 172.5 (141.9-209.6) 161.7 (122.7-213.1) 200.8 (163.5-246.1)
56.7 (49.9-64.5) 42.1 (32.0-55.4) 52.2 (44.1~1.9)
78.4 (72.5-84.9) 79.0 (71.5-87.3) 75.2 (70.6--80.0)
11.0 (9.9-12.2) 10.8 (9.8-12.1) 11.7 (10.0-13.7)
Mice treated with NMDA, PIL or PTZ were individually observed for 30 min for the occurrence of seizures. Three to five doses of the convulsants or three to five currents, and 8-10 mice/dose or electroshock were used. L-NAME, D-NAME and 7-NI were injected 30 min before convulsants or electroshocks. *P < 0.05 versus respective vehicle.
E. Przegalihski et al. / Neuroscience Letters 217 (1996) 145-148
that endogenous NO may play the role of an anticonvulsant substance in the NMDA-induced seizures. Such a conclusion is in agreement with recent reports which also implicate the anticonvulsant activity of NO in convulsions induced by excitatory amino acids. In fact, NOS inhibition has been shown to prolong the duration of the seizure activity or to increase mortality, evoked respectively by intacerebroventricular (i.c.v.) or systemic injection of NMDA to mice [2,9], as well as to potentiate the seizures induced by peripheral or intra-amygdala administration of kainate to mice and/or rats [13,21,22], or by i.c.v, administration of quinolinate to rats [8]. Importantly, in some of those studies, proconvulsant effects of NOS inhibitors were found to be prevented by co-administration of the NO-generating drugs molsidomine or Larginine [2,8]. The anticonvulsant role of NO in the seizures induced by excitatory amino acids has been suggested [2,8,22] to be related to its formation in response to activation of NMDA receptors [7] and to the negative feedback exerted by NO on the activity of these receptors through different mechanisms including a competitive blockade of the NMDA recognition site [14], or an interaction with the redox modulatory site of the NMDA receptor [ 11 ]. Consequently, the inhibition of NOS is likely to suppress this negative feedback and enhance the excitability of postsynaptic neurons. It should be mentioned, however, that in contrast to the results presented here, there are data showing that NOS inhibitors exert a protective action against excitatory amino acid-induced convulsions. Accordingly, N-monomethyl-L-arginine was found to prevent the motor and electrocortical seizures elicited by administration of NMDA or kainate into the deep prepiriform cortex of rats [4], while 7-NI was shown to reduce the kainateinduced behavioral seizures and NO formation in different rat brain structures [19]. On the other hand, the lack of effect of N-nitro-L-arginine on the NMDA-evoked seizures in mice was also reported [9]. In contrast to the results on NMDA we found that neither the PIL-induced clonic seizures, the PTZ-evoked clonic and tonic convulsions, nor the electrically-induced tonic seizures in mice were affected by either L-NAME or 7-N1. These findings, together with the proconvulsive action of L-NAME and 7-NI on the NMDA-induced seizures, indicate that these two NOS inhibitors do not differ between themselves in their effect on the chemically- or electrically-induced seizures in mice, and that the cardiovascular effects and muscarinic blocking properties of arginine-based NOS inhibitors [3,5], or at least of LNAME, are not involved in its activity in the convulsion models used in the present study. Moreover, these results show that the anticonvulsant role of endogenous NO is not a general phenomen, but seems to be related only to seizures induced by NMDA and/or other excitatory amino acids. The latter suggestion would agree with the above-
147
discussed putative mechanism of the anticonvulsant activity of NO, i.e. the feedback inhibition of the NMDA receptor function. As far as PTZ- and PIL-induced seizures are concerned, other authors have reported different effects. For example, Osonoe et al. [20] and Hara et al. [9] found in rats and mice, respectively, that NOS inhibitors attenuated the tonic, but not the clonic, seizures produced by PTZ. However, the former authors observed such an effect after considerably higher doses of NOS inhibitors, e.g. after 500 mg/kg of L-NAME, which in comparison with much lower doses of the inhibitor necessary to inhibit the NOS activity [23], indicates that their effect may be non-specific. At the same time, Hara et al. [9] examined the effect of N-nitro-L-arginine versus a constant dose of PTZ. In our opinion, a comparison of the CDs0 values of PTZ between control and the NOS inhibitor-pretreated animals (used in the present study) is a more reliable index. The PILinduced seizures were found to be attenuated or potentiated by 7-NI or N-nitro-L-arginine, respectively [ 13,25]. However, again, the effect of 7-NI in mice was studied versus a constant dose of PIL, and weak attenuation of seizures was observed after a dose as high as 100 mg/kg of the NOS inhibitor [25], whereas a potentiating effect of N-nitro-L-arginine was demonstrated in rats [13]. In conclusion, our results together with some literature data, indicate that endogenous NO may play the role of an anticonvulsant substance, only in the case of seizures induced by NMDA and/or other excitatory amino acids, though. This work was supported by grant 4 PO5A 073 08 from the Committee for Scientific Research. [1] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [2] Buisson, A., Lakhmeche, N., Verrecchia, C., Plothine, M. and Boulu, R.G., Nitric oxide: an endogenous anticonvulsant substance, NeuroReport, 4 (1993) 444-446. [3] Buxton, I.L., Cheek, D.J., Eckman, D., Wertfall, D.P., Sanders, K.M. and Keef, K.D., NC-Nitro-L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists, Circ. Res., 72 (1993) 387-395. [4] De Sarro, G.B., Di Paola, E.D., De Sano, A. and Vidal, M.J., Role of nitric oxide in the genesis of excitatory amino acid-induced seizures from the deep prepiriform cortex, Fundam. Clin. Pharmacol., 5 (1991) 503-511. [5] Fewell, J.E. and Johnson, P., Acute increases in blood pressure cause arousal from sleep in lambs, Brain Res., 311 (1994l 259265. [6] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [7] Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endotheliumderived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-387. [8] Haberny, K.A., Pou, S. and Eccles, C.U., Potentiation of quinolinate-induced hippocampal lesions by inhibition of NO synthesis, Nettrosci. Lett., 146 (1992) 187-190. [9] Hara, S., Kuriiawa, F., Iwata, N., Mukai, T., Kano, S. and Endo, T.,
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Distinct effects of N~-nitro-L-arginine on seizures induced by several drugs in mice, Pharmacol. Biochem. Behav., 53 (1996) 673677. Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble quanylate cyclase, Prec. Natl. Acad. Sci. USA, 86 (1989) 51595162. Lei, S.Z., Pan, Z.-H., Aggarwal, S.K., Chen, H.-S.V., Hartman, J., Sucher, N.J. and Lipton, S.A., Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex, Neuron, 8 (1992) 1087-1099. Litchfield, J.T. and Wilcoxon, F., A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99-103. Maggio, R., Fumagalli, F., Donati, E., Barbier, P., Racagni, G., Corsini, G.U. and Riva, M., Inhibition of nitric oxide synthase dramatically potentiates seizures induced by kainic acid and pilocarpine in rats, Brain Res., 679 (1995) 184-187. Manzoni, O., Prezeau, L., Marin, P., Deshager, S., Bockaert, J. and Fagni, L., Nitric oxide-induced blockade of NMDA receptors, Neuron, 8 (1992) 653-662. Meldrum, B., Amino acid neurotransmitters and new approaches to anticonvulsant drug action, Epilepsia, 25 (1984) S140-S149. Moncada, S., Palmer, R.M.J. and Higgs, S.A., Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev., 43 (1991) 109-142. Moore, P.K,, Babbedge, R.C., Wallace, P., Gaffen, Z.A. and Hart, S.L., 7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure, Br. J. Pharmacol., 108 (1993) 296-297.
[18] Moore, P,K., Wallace, P., Gaffen, Z., Hart, S.L. and Babbedge, R.C., Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects, Br. J. Pharmacol., 110 (1993) 219-224. [19] Mtilsch, A., Busse, R., Mordvintcev, P.I., Vanin, A.F., Nielsen, E.O., Scheel-Krtiger, J. and Olesen, S.-P., Nitric oxide promotes seizure activity in kainate-treated rats, NeuroReport, 5 (1994) 2325-2328. [20] Osonoe, K., Mori, N., Suzuki, K. and Osonoe, M., Antiepileptic effects of inhibitors of nitric oxide synthase examined in pentylenetetrazol-induced seizures in rats, Brain Res., 663 (1994) 338340. [21] Przegalinski, E., Baran, L. and Siwanowicz, J., The role of nitric oxide in the kainate-induced seizures in mice, Neurosci. Lett., 170 (1994) 74-76. [22] Rondouin, G., Bockaert, J. and Lerner-Natoli, M., L-Nitroarginine, an inhibitor of NO synthase, dramatically worsens limbic epilepsy in rats, NeuroReport, 4 (1993) 1187-1190. [23] Salter, M., Duffy, C. and Hazelwood, R., Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results, Neurophannacology, 34 (1995) 327-334. [24] Schuman, E.M. and Madison, D.V., Nitric oxide and synaptic function, Annu. Rev. Neurosci., 17 (1994) 153-183. [25] Van Leeuven, R., De Vries, R. and Dzoljic, M.R., 7-Nitro indazole, an inhibitor of neuronal nitric oxide synthase, attenuates pilocarpine-induced seizures, Ear. J. Pharmacol., 287 (1995) 211-213. [26] Wood, P.L., Pharmacology of the second messenger, cyclic quanosine 3', 5'-monophosphate, in the cerebellum, Pharmacol. Rev., 43 (1991) 1-25.
Neuroscience Letters 217 (1996) 145-148
The role of nitric oxide in chemically- and electrically-induced seizures in mice E d m u n d Przegalifiski*, L e o k a d i a Baran, Joanna S i w a n o w i c z Institute of Pharmacology, Polish Academy of Sciences, 12 Smgtna Street, 31-343 Krak6w, Poland
Received 19 August 1996; revised version received 13 September 1996; accepted 13 September 1996
Abstract
The effect of the nitric oxide synthase (NOS) inhibitors N-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) on seizures induced by N-methyl-D-aspartate (NMDA), pilocarpine (PIL) and pentylenetetrazol (PTZ), as well as on the electroconvulsive threshold was studied in mice. It was found that L-NAME and 7-NI decreased the dose of NMDA necessary to produce clonic convulsions in 50% of animals (CDs0). Such a proconvulsant effect was not observed in mice pretreated with N-nitro-D-arginine methyl ester (o-NAME), an inactive isomer of L-NAME. Neither L-NAME nor 7-NI affected the convulsions induced by PIL (clonic seizures) or PTZ (clonic and tonic seizures), having no effect on their CDs0 values. Similarly, neither NOS inhibitor affected the electroshock threshold. These results, together with some literature data, indicate that nitric oxide (NO) may be regarded as an anticonvulsant substance in relation to seizures induced by NMDA and other excitatory amino acids, but not by other agents, in mice. Keywords: Nitric oxide; Nitric oxide synthase inhibitors; N-Methyl-D-aspartate; Pilocarpine; Pentylenetetrazol; Electroconvulsive
threshold; Seizures; Mice
Nitric oxide (NO) is a small, membrane-diffusible molecule, synthesized from L-arginine by the Ca2+-calmodulin-dependent NO synthase (NOS) in various tissues including the brain [1]. The major action of NO is activation of the soluble guanylate cyclase which in turn increases the level of an intracellular second messenger, guanosine 3',5"-cyclic monophosphate (cGMP), in target cells [24]. NO is regarded as either a novel neuronal messenger or a neurotransmitter in the central nervous system and is involved in neuronal functions [ 1,24]. There are at least two arguments supporting a possible role of NO in convulsive phenomena: (1) excitatory amino acids, such as N-methyl-o-aspartate (NMDA) and kainate, are known to be potent convulsants [ 15], and the activation of NMDA receptors is accompanied by the formation of NO [6], (2) various convulsants, including excitatory amino acids, pentylenetetrazol (PTZ) and pilocarpine (PIL), increase the cGMP level in the cerebellum, whereas anticonvulsant drugs decrease it [26]. In fact, the role of
* Corresponding author. Tel.: +48 12 374022; fax: +48 12 374500.
NO in epileptogenesis has been examined in a number of studies, yet the obtained results are contradictory. NO has been suggested to be either an anticonvulsive [2,21] or a proconvulsive [4,19] endogenous substance. This apparent contradiction may be explained by different experimental conditions (species, convulsants, type of seizures), including pharmacological tools used to modify the NO pathway. Regarding the latter, it is noteworthy that the conclusions of most of the studies are based on results obtained with the use of NOS inhibitors. Moreover, practically all the NOS inhibitors used in those studies were arginine-based, affecting not only neuronal but also endothelial NOS. The inhibition of endothelial NOS leads to increased blood pressure [16], which, in turn, may affect the excitability of central neurons [5]. Furthermore, the specificity of these NOS inhibitors is limited by their muscarinic receptor blocking properties [3]. Therefore in the present study we examined the NMDA-, PTZ-, PIL- and electrically-induced seizures in mice treated with N-nitro-L-arginine methyl ester (LNAME) or 7-nitroindazole (7-NI), the latter being an in vivo selective neuronal NOS inhibitor without cholinolytic
0304-3940/96/$12.00 © 1996Elsevier ScienceIreland Ltd. All fights reserved PII S0304-3940(96)13085-8
146
E. Przegalihski et al. / Neuroscience Letters 217 (1996) 145-148
properties or significant cardiovascular effects in mice [17,18]. The experiments were carried out on male Albino Swiss mice weighing 26-33 g, which were obtained from a licensed dealer. The animals were housed in groups of 40 to a cage (50 × 38 × 20 cm) and kept at a room temperature (21 + I°C) on a 12:12 h light/dark cycle (light on at 0600 h), with food (granular standard diet, Bacutil) and water ad libitum. The tests were carried out between 1000 and 1500 h, and the mice were randomly assigned to experimental groups. The animals were injected with NMDA (RBI), PIL (Hestag) and PTZ (Sigma), placed separately in plastic cages and observed for 30 min for the occurrence of clonic (induced by NMDA and PIL), or clonic and tonic (after PTZ) seizures. The doses of NMDA, PIL and PTZ, necessary to induce seizure responses in 50% of mice (CDs0, convulsant dose 50), were. calculated. To determine CDs0, three to five doses of the convulsants and 8 - 1 0 mice per dose were used. Electroshocks were delivered to the animals through ear clip electrodes (ECT unit 7801, Ugo Basile, Milan, Italy), with saline (0.9% NaC1) applied to the ears to ensure good contact. Stimulation parameters consisted of 100 rectangular pulses/s, 0.5 ms in width, for a duration of 1 s. The electroshock threshold, defined as the current necessary to cause the hindleg tonic extensor component of seizures in 50% of animals (CC50, convulsant current 50), was calculated. To determine CC50, three to five currents were tested on groups of 8 - 1 0 mice each. L-NAME (Sigma) and N-nitro-D-arginine methyl ester (D-NAME; Sigma) were injected in doses of 3, 10 and 30
mg/kg, and 7-NI (RBI) in doses of 10 and 30 mg/kg, at 30 min before the convulsants or electroshocks. All the substances were dissolved in bidistilled water, except for 7-NI which was suspended in a 1% aqueous solution of Tween80, and injected intraperitoneally (i.p.). The CDs0 and CC50 values (with 95% confidence limits) and their statistical evaluations were calculated by fitting the data by a probit analysis according to Litchfield and Wilcoxon [12]. As shown in Table 1, L-NAME and 7-NI reduced the convulsive threshold for NMDA. A significant decrease in the CDs0 of NMDA was observed after 10 and 30 mg/kg of L-NAME (by 37 and 41%, respectively) and after 30 mg/ kg of 7-NI (by 32%). The CDs0 value of NMDA was not changed by D-NAME. In contrast to the NMDA-induced seizures, neither L-NAME nor 7-NI affected the convulsions induced by PIL (clonic seizures) and PTZ (clonic and tonic seizures), having no effect on their CDs0 values. Similarly, L-NAME and 7-NI did not affect the electroshock threshold, as the CC50 values were not changed in mice pretreated with those NOS inhibitors. The present data show that pharmacological interference in the NO pathway does not affect in a similar way the susceptibility to seizures evoked by various chemical convulsions or electroshocks in mice. Actually, we observed that the NOS inhibitors, L-NAME or 7-NI, reduced the dose of NMDA necessary to produce clonic convulsions (CDs0), which indicates an increased seizure susceptibility to the excitatory amino acid. This observation, together with the findings that the CDs0 of NMDA was not affected by o-NAME, an inactive isomer which does not inhibit the NOS activity [10], seem to indicate
Table 1 Effect of N-nitro-L-arginine methyl ester (L-NAME), N-nitro-D-arginine methyl ester (D-NAME) and 7-nitroindazole (7-NI) on N-methylt~-aspartate (NMDA), pilocarpine (P1L), pentylenetetrazol (PTZ) and electrically (ECS)-induced seizures in mice Treatment and dose (mg/kg, i.p.)
CDso (mg/kg, i.p.; 95% confidence limits) NMDA
Vehicle L-NAME 3 L-NAME 10 L-NAME 30
116.8 (107.8-126.7) 108.2 (91.9-127.3) 73.5 (65.7-82.4)* 68.8 (60.6--78.0)*
Vehicle D-NAME 3 D-NAME 10 D-NAME 30
110.0 100.6 100.4 116.5
Vehicle 7-NI 10 7-NI 30
116.4 (105.8-128.1) 108.7 (91.6-129.0) 79.1 (64.5-97.8)*
PIL
205.1 172.4 200.4 208.7
CC50 (mA; 95% confidence limits) PTZ
(171.0-245.9) (135.3-219.7) (172.9-232.2) (168.3-258.8)
ECS
Clonus
Tonus
53.4 53.1 53.1 53.1
76.9 76.9 77.2 67.8
(46.3--61.5) (48.1-58.5) (48.1-58.5) (48.1-58.1)
(67.2-88.0) (67.2-88.0) (73.3-81.4) (57.7-79.7)
10.6 9.6 10.8 11.7
(9.5-11.7) (8.5-10.7) (10.0-11.6) (10.9-12.5)
(93.6-129.2) (91.2-111.1) (86.2-116.9) (100.3-135.3) 172.5 (141.9-209.6) 161.7 (122.7-213.1) 200.8 (163.5-246.1)
56.7 (49.9-64.5) 42.1 (32.0-55.4) 52.2 (44.1~1.9)
78.4 (72.5-84.9) 79.0 (71.5-87.3) 75.2 (70.6--80.0)
11.0 (9.9-12.2) 10.8 (9.8-12.1) 11.7 (10.0-13.7)
Mice treated with NMDA, PIL or PTZ were individually observed for 30 min for the occurrence of seizures. Three to five doses of the convulsants or three to five currents, and 8-10 mice/dose or electroshock were used. L-NAME, D-NAME and 7-NI were injected 30 min before convulsants or electroshocks. *P < 0.05 versus respective vehicle.
E. Przegalihski et al. / Neuroscience Letters 217 (1996) 145-148
that endogenous NO may play the role of an anticonvulsant substance in the NMDA-induced seizures. Such a conclusion is in agreement with recent reports which also implicate the anticonvulsant activity of NO in convulsions induced by excitatory amino acids. In fact, NOS inhibition has been shown to prolong the duration of the seizure activity or to increase mortality, evoked respectively by intacerebroventricular (i.c.v.) or systemic injection of NMDA to mice [2,9], as well as to potentiate the seizures induced by peripheral or intra-amygdala administration of kainate to mice and/or rats [13,21,22], or by i.c.v, administration of quinolinate to rats [8]. Importantly, in some of those studies, proconvulsant effects of NOS inhibitors were found to be prevented by co-administration of the NO-generating drugs molsidomine or Larginine [2,8]. The anticonvulsant role of NO in the seizures induced by excitatory amino acids has been suggested [2,8,22] to be related to its formation in response to activation of NMDA receptors [7] and to the negative feedback exerted by NO on the activity of these receptors through different mechanisms including a competitive blockade of the NMDA recognition site [14], or an interaction with the redox modulatory site of the NMDA receptor [ 11 ]. Consequently, the inhibition of NOS is likely to suppress this negative feedback and enhance the excitability of postsynaptic neurons. It should be mentioned, however, that in contrast to the results presented here, there are data showing that NOS inhibitors exert a protective action against excitatory amino acid-induced convulsions. Accordingly, N-monomethyl-L-arginine was found to prevent the motor and electrocortical seizures elicited by administration of NMDA or kainate into the deep prepiriform cortex of rats [4], while 7-NI was shown to reduce the kainateinduced behavioral seizures and NO formation in different rat brain structures [19]. On the other hand, the lack of effect of N-nitro-L-arginine on the NMDA-evoked seizures in mice was also reported [9]. In contrast to the results on NMDA we found that neither the PIL-induced clonic seizures, the PTZ-evoked clonic and tonic convulsions, nor the electrically-induced tonic seizures in mice were affected by either L-NAME or 7-N1. These findings, together with the proconvulsive action of L-NAME and 7-NI on the NMDA-induced seizures, indicate that these two NOS inhibitors do not differ between themselves in their effect on the chemically- or electrically-induced seizures in mice, and that the cardiovascular effects and muscarinic blocking properties of arginine-based NOS inhibitors [3,5], or at least of LNAME, are not involved in its activity in the convulsion models used in the present study. Moreover, these results show that the anticonvulsant role of endogenous NO is not a general phenomen, but seems to be related only to seizures induced by NMDA and/or other excitatory amino acids. The latter suggestion would agree with the above-
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discussed putative mechanism of the anticonvulsant activity of NO, i.e. the feedback inhibition of the NMDA receptor function. As far as PTZ- and PIL-induced seizures are concerned, other authors have reported different effects. For example, Osonoe et al. [20] and Hara et al. [9] found in rats and mice, respectively, that NOS inhibitors attenuated the tonic, but not the clonic, seizures produced by PTZ. However, the former authors observed such an effect after considerably higher doses of NOS inhibitors, e.g. after 500 mg/kg of L-NAME, which in comparison with much lower doses of the inhibitor necessary to inhibit the NOS activity [23], indicates that their effect may be non-specific. At the same time, Hara et al. [9] examined the effect of N-nitro-L-arginine versus a constant dose of PTZ. In our opinion, a comparison of the CDs0 values of PTZ between control and the NOS inhibitor-pretreated animals (used in the present study) is a more reliable index. The PILinduced seizures were found to be attenuated or potentiated by 7-NI or N-nitro-L-arginine, respectively [ 13,25]. However, again, the effect of 7-NI in mice was studied versus a constant dose of PIL, and weak attenuation of seizures was observed after a dose as high as 100 mg/kg of the NOS inhibitor [25], whereas a potentiating effect of N-nitro-L-arginine was demonstrated in rats [13]. In conclusion, our results together with some literature data, indicate that endogenous NO may play the role of an anticonvulsant substance, only in the case of seizures induced by NMDA and/or other excitatory amino acids, though. This work was supported by grant 4 PO5A 073 08 from the Committee for Scientific Research. [1] Bredt, D.S. and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8 (1992) 3-11. [2] Buisson, A., Lakhmeche, N., Verrecchia, C., Plothine, M. and Boulu, R.G., Nitric oxide: an endogenous anticonvulsant substance, NeuroReport, 4 (1993) 444-446. [3] Buxton, I.L., Cheek, D.J., Eckman, D., Wertfall, D.P., Sanders, K.M. and Keef, K.D., NC-Nitro-L-arginine methyl ester and other alkyl esters of arginine are muscarinic receptor antagonists, Circ. Res., 72 (1993) 387-395. [4] De Sarro, G.B., Di Paola, E.D., De Sano, A. and Vidal, M.J., Role of nitric oxide in the genesis of excitatory amino acid-induced seizures from the deep prepiriform cortex, Fundam. Clin. Pharmacol., 5 (1991) 503-511. [5] Fewell, J.E. and Johnson, P., Acute increases in blood pressure cause arousal from sleep in lambs, Brain Res., 311 (1994l 259265. [6] Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-67. [7] Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endotheliumderived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain, Nature, 336 (1988) 385-387. [8] Haberny, K.A., Pou, S. and Eccles, C.U., Potentiation of quinolinate-induced hippocampal lesions by inhibition of NO synthesis, Nettrosci. Lett., 146 (1992) 187-190. [9] Hara, S., Kuriiawa, F., Iwata, N., Mukai, T., Kano, S. and Endo, T.,
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