www.elsevier.com / locate / bres
Short communication
Effects of MK-801, dantrolene, and FK506 on convulsive seizures and
brain nitric oxide production in seizure-susceptible EL mice
*
Itsugi Nagatomo , Wataru Hashiguchi, Masataka Tominaga, Yasuaki Akasaki,
Masahiro Uchida, Morikuni Takigawa
Department of Neuropsychiatry, Faculty of Medicine, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima 890-8520, Japan
Accepted 10 October 2000
Abstract
To clarify the role of nitric oxide (NO) in the pathogenesis of seizures in susceptible EL mice, we investigated effects of three drugs potentially related to NO production, MK-801, dantrolene, and FK506, on convulsive seizures and brain NO metabolites (NOx). MK-801 or dantrolene, but not FK506, suppressed convulsive seizures in EL mice; only MK-801 reduced NOx in the brain. Our results suggested involvement of the N-methyl-D-aspartate receptor–channel complex and intracellular calcium mobilization, but not calcineurin, in the
convulsions of EL mice. 2001 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Epilepsy: basic mechanisms
Keywords: Nitric oxide; EL mouse; Convulsion; MK-801; Dantrolene; FK506
The EL mouse is an inbred mutant strain of ddY mouse [5,15,17], and we have shown that lower NO production in that is susceptible to convulsive seizures. It was developed the brain may be related to the susceptibility of the EL in 1954 [8], registered internationally in 1964, and estab- mouse to convulsive seizures [15,25]. However, the role of lished as an authentic model of epilepsy by electroence- NO in epileptic convulsion in the EL mouse is still unclear. phalography in 1976 [20]. This strain exhibits seizures in MK-801, a potent and selective noncompetitive antago-response to rhythmic vestibular stimuli such as tossing, nist at the NMDA receptor, dose-dependently suppresses rocking, to-and-fro horizontal swinging, pendular move- generalized tonic–clonic convulsions and increases levels ment, or alternating rotation. Repetition of such stimulation of glycine in the hippocampus [19]. FK506, a contrasting induces generalized convulsions. The EL mouse is an drug originally developed as a potential
immunosuppres-21
excellent model of complex partial seizures, or temporal sant, was found to inhibit a Ca -and calmodulin-depen-lobe epilepsy. dent protein phosphatase, calcineurin [13]. Dawson et al. Nitric oxide (NO) has been regarded as a neuronal [6] demonstrated that FK506 enhanced phosphorylation of messenger in the central nervous system (CNS) and a neuronal NOS (nNOS), which reduced NO production, and modular of several brain functions [4]. Activation of thus attenuated glutamate neurotoxicity in cultured neu-excitatory amino acid (EAA) receptors, particularly the rons. To clarify the role of NO in the pathogenesis of
N-methyl-D-aspartate (NMDA) subtypes, causes an influx seizures in EL mice, we investigated the effects of
MK-21
of Ca into neurons leading to calmodulin-dependent 801, FK506, and dantrolene (a compound that prevents
21
activation of NO synthase (NOS) [3,7]. NO in the CNS is mobilization of Ca from intracellular stores) on convul-involved in the pathogenesis of convulsive seizures sive seizures and NO metabolites in mouse brain.
This study was carried out after receiving permission from the Committee of Animal Experimentation, Faculty *Corresponding author. Tel.: 181-99-275-5346; fax: 1
81-99-265-of Medicine, Kagoshima University. EL mice were inbred 7089.
at our animal center. They housed in individual cages in a
E-mail address: nagatomo@m2.kufm.kagoshima-u.ac.jp
(I. Nagatomo). room at 23628C, constant humidity (50610%) and a 12
h / 12 h light / dark cycle (lights on 7.00 a.m.). The received EICOM). Nitrite and nitrate in the brain was separated by cages were cleaned once weekly to avoid excessive a reverse-phase separation column packed with polystyrene stimulation. The rhythmic vestibular stimulation is known polymer (NO-PAK, 4.6350 mm, EICOM), and nitrate was to provoke seizures in the EL mouse [21], and ‘tossing-up’ reduced to nitrite in a reduction column packed with stimulation method [1,9] was used in this study. EL mice copper-plated cadmium filings (NO-RED, EICOM). Nitrite were tossed up once a week from the age of 42 days. Each was mixed with a Griess reagent to form a purple azo dye mouse carefully taken from its plastic cages, placed in a in a reaction coil. The separation and reduction columns box (3033037 cm), and observed 3 min as it freely and the reaction coil were placed in a column oven that moved about. It then was tossed 50 times to a height of 10 was set at 358C. The absorbance of the color of the product to 15 cm. If the mouse displayed seizures within this dye at 540 nm was measured by a flow-through spec-period, tossing was halted. Mice developing tonic–clonic trophotometer (NOD-10, EICOM). The mobile phase, convulsions in over five consecutive weekly sessions were which was delivered by a pump at a rate 0.33 ml / min, was chosen for this study. 10% methanol containing 0.15 M NaCl / NH Cl and 0.54
The severity of seizures was scored according to our g / l 4Na-EDTA. The Griess reagent, which was 1.25% HCl previous study [16] as: grade 0, no seizures with 50 tossing containing 5 g / l sulfanilamide with 0.25 g / l N-naphthyl-stimulations; grade 1, abortive seizures within 50 such ethylenediamine, was delivered at a rate of 0.1 ml / min. stimulations; grade 2, abortive seizures within 25 such Differences were tested for statistical significance using stimulations; grade 3, tonic–clonic seizures within 50 such the Kruskal–Wallis test followed by the Mann–Whitney stimulations; grade 4, tonic–clonic seizures within 25 such test and one-way analysis of variance (ANOVA) followed stimulations. Abortive seizures were characterized by by Fisher’s PLSD. A P-value of ,0.05 was accepted as immobility with fixed staring. Mice with grade 4 seizures statistically significant.
were used in this study. Since the EL mouse shows a MK-801 suppressed seizure scores in an essentially refractory period between convulsions, we evaluated the dose-dependent manner, while all doses of dantrolene had time-course of the convulsive seizure. No seizures this effect. On the other hand, FK506 did not inhibit occurred 15 min following the first stimulation. The seizure significantly (Fig. 1). Brain NOx levels of mice seizure scores at 15 and 30 min after the first stimulation treated with MK-801 at doses of 0.25 and 0.5 mg / kg were were significantly lower than the grade 4, while the scores significantly lower than in controls, but dantrolene and at 1 and 2 h after the first stimulation approached grade 4. FK506 did not influence brain NOx levels at any dose We therefore evaluated seizure scores at 1 h after the first (Fig. 2).
stimulation. Details of the time-course of convulsive Recent advances in understanding epileptic phenomena seizures in the EL mouse have been described elsewhere have emphasized the importance of EAAs. The EAA
[6]. receptor agonist NMDA is known to be a potent
convul-MK-801 and dantrolene were dissolved in saline. FK506 sant, and NMDA receptor activation has been implicated in was dissolved in mannitol and polyoxyethylene hydro- development of epilepsy. NO can regulate NMDA receptor genated castor oil 60. With the mouse gently restrained by activation [10], presumably via a feedback effect on the the experimenter’s hands, an injection needle attached by a oxidation–reduction modulatory site of the NMDA re-polyethylene tube to a microsyringe was inserted intraperi- ceptor, that results in decreased NMDA receptor activity toneally for administration of MK-801 (0.05, 0.1, 0.25, or and protects neurons from effects of excessive receptor 0.5 mg / kg), dantrolene (20, 40, or 80 mg / kg) and FK506 stimulation [12].
(5, 10, or 20 mg / kg). One hour after administration, the Although MK-801, dantrolene, and FK506 all might be mouse was subjected to tossing stimulation and the related to NO production, the present results of these drugs severity of the resulting seizure was scored. differed concerning convulsive seizures in EL mice and in The mouse was killed by cervical dislocation, and the effects on brain NO metabolites; both MK-801 and dan-brain was rapidly excised. Since the half-life of NO is very trolene, but not FK506, suppressed convulsive seizures in short, NOS activities were determined by measuring levels EL mice, while only MK-801 reduced brain NOx levels. of nitrite plus nitrate (NOx). The brain with twice amount Doses of the three drugs used in the present study were of methanol were homogenized for 10 s with an ultrasonic comparable to those in previous experiments cell disruptor (Sonifier, Branson, USA). Homogenates [2,14,19,22,23].
Fig. 1. Dose-dependent effects of MK-801, dantrolene, and FK506 on seizure scores in EL mice. Each points is the mean6S.E.M. (Left) MK-801 (saline,
n514; 0.05 mg / kg, n510; 0.1 mg / kg, n510; 0.25 mg / kg, n510; 0.5 mg / kg, n510). (Middle) Dantrolene (saline, n514; 20 mg / kg, n58; 40 mg / kg,
n58; 80 mg / kg, n58). (Right) FK506 (vehicle, n58; 5 mg / kg, n58; 10 mg / kg, n512; 20 mg / kg, n59). Seizure scores of EL mice treated with MK-801 at four doses were significantly lower than the control (all, P,0.01). Seizure scores of mice treated with MK-801 at doses of 0.25 and 0.5 mg / kg were significantly lower than those with MK-801 at doses of 0.05 and 0.1 mg / kg (0.5 mg / kg vs. 0.1 mg / kg, P,0.05; others, P,0.01). Seizure scores of mice treated with dantrolene at all doses were significantly lower than the control (all, P,0.01). There were no significant differences among seizure scores of mice treated with FK506.
Fig. 2. NOx levels in the brain of EL mouse treated with MK-801, dantrolene, and FK506. Each value is the mean6S.E.M. (Left) MK-801 (saline, n59; 0.05 mg / kg, n510; 0.1 mg / kg, n510; 0.25 mg / kg, n510; 0.5 mg / kg, n510). (Middle) Dantrolene (saline, n59; 20 mg / kg, n58; 40 mg / kg, n58; 80 mg / kg, n58). (Right) FK506 (vehicle, n58; 5 mg / kg, n58; 10 mg / kg, n58; 20 mg / kg, n59). Brain NOx levels of mice treated with MK-801 at a dose of 0.25 mg / kg was significantly lower than those of the control and treated with MK-801 at doses of 0.05 and 0.1 mg / kg (control, P,0.01; others,
systemic injection of MK-801 potently suppressed general- Acknowledgements
ized tonic–clonic convulsions in a dose-dependent manner,
We thank the Yamanouchi Pharmaceutical Co. Ltd. and suggested that the NMDA system may be essential in
(Tokyo, Japan) and the Fujisawa Pharmaceutical Co. Ltd. triggering seizures in EL mice. Present results, like those
(Tokyo, Japan) for providing dantrolene and FK506, of Sato et al. [19], indicated that MK-801 reduced brain
respectively. NO metabolite levels as well as seizure scores. This
suggests that the NMDA system is central to both convul-sive seizures and changes in NO production, since
MK-References
801 suppressed brain NO production.
Reported studies concerning the effects of dantrolene on
[1] Y. Akasaki, The effect of phenobarbital on the multiple neuronal convulsive seizures have had conflicting results. Tizzano
activity in the hippocampus of El mouse, Jpn. J. Psychiat. Neurol. and colleagues [22,23] reported that limbic seizures
in-47 (1993) 121–129.
duced by 3,5-dihydroxyphenylglycine or by (1S,3R)-1- [2] K.K. Borowicz, M. Gasior, Z. Kleinrok, S.J. Czuczwar, Influence of aminocyclopentane-1,3-dicarboxylic acid, a selective isradipine, niguldipine and dantrolene on the anticonvulsive action metabotrophic glutamate receptor agonist, were attenuated of conventional antiepileptics in mice, Eur. J. Pharmacol. 323
(1997) 45–51. by injection of dantrolene (62.5 to 500 mg / kg, i.p.). On the
[3] D.S. Bredt, S.H. Snyder, Isolation of nitric oxide synthetase, a other hand, Borowicz et al. [2] reported that dantrolene (5
calmodulin-requiring enzyme, Proc. Natl. Acad. Sci. USA 87 (1990) to 20 mg / kg, i.p.) did not influence the electroconvulsive 682–685.
threshold. Our results supported those of Tizzano and [4] D.S. Bredt, S.H. Synder, Nitric oxide, a novel neuronal messenger, colleagues [22,23]; furthermore, even smaller doses (20 Neuron 8 (1992) 3–11.
[5] A. Bussion, N. Lakhmeche, C. Verrecchia, M. Plotkine, R.G. Boulu, and 40 mg / kg) were effective in suppressing seizures in
Nitric oxide: an endogenous anticonvulsant substance, NeuroReport EL mice. Doses of dantrolene that were effective in
4 (1993) 444–446.
suppressing seizures did not alter brain NOx levels. [6] T.M. Dawson, J.P. Steiner, V.L. Dawson, J.L. Danierman, G.R. Uhl, Dantrolene prevents intracellular calcium mobilization S.H. Synder, Immunosuppressant FK506 enhances phosphorylation subsequent to metabotropic glutamate receptor agonist of nitric oxide synthase and protects against glutamate neurotoxicity,
Proc. Natl. Acad. Sci. USA 90 (1993) 9808–9812. activation in primary neuronal cultures [23]. In this
[7] J. Garthwaite, G. Garthwaite, R.M.J. Palmer, S. Moncada, NMDA context, our results suggested that the anticonvulsive effect
receptor activation induces nitric oxide synthesis from arginine from of dantrolene in EL mice may occur via a pathway not rat brain slices, Eur J. Pharmacol. 172 (1989) 413–416.
involving NO metabolites. [8] K. Imaizumi, T. Nakano, Mutant stocks, strain; EL, Mouse News FK506 previously has been shown to have significant Lett. 31 (1964) 57.
[9] M. Ito, H. Tsuda, Role of noradrenergic system in the seizures of EL neuroprotective properties. In primary cortical cultures,
mouse, Neurosciences 18 (Suppl. 2) (1992) 93–103. this agent strongly reduced NMDA, but not non-NMDA,
[10] Y. Izumi, D.B. Clifford, C.F. Zorumski, Inhibition of long-term neurotoxicity [6]. Moia et al. [14] reported that FK506 potentiation by NMDA-mediated nitric oxide release, Science 257 reversibly inhibited the progression of the kindling stage, (1992) 1273–1276.
and suggested that calcineurin may be essential to epi- [11] H. Kawasaki, M. Itoh, T. Nakahara, M. Fukahori, K. Akazawa, 3
[ H]MK-801 binding to forebrain of EL (epileptic) and non-epileptic leptogenesis in kindling. FK506 inhibits the function of
21 strains of mice, Epilepsy Res. 8 (1991) 220–225. calcineurin, thereby inhibiting Ca -dependent
dephos-[12] S.A. Lipon, Prospects for clinically tolerated NMDA antagonists: phorylation and activation of nNOS [24]. Despite the use open-channel blockers and alternative redox states of nitric oxide, of a larger dose of FK506 than that required to block Trends Neurosci. 16 (1993) 527–532.
progression of the kindling stage [14], we found that [13] J. Liu, J.O. Farmer, W.S. Lane, J. Friedman, I. Weissman, S.L. Schreiber, Calcineurin is a common target of cyclophilin–cyclos-FK506 neither suppressed convulsive seizures of EL mice
polin A and FKBP-FK506 complexes, Cell 66 (1991) 807–815. nor reduced brain NOx levels.
[14] L.J.M.P. Moia, H. Matsui, G.A.M. de Barros, K. Tomizawa, K. The present results supported the suggestions of Miyamoto, Y. Kuwata, M. Tokuda, T. Itano, O. Hatase, Immuno-Kawasaki et al. [11] and Sato et al. [19] that functional suppressants and calcineurin inhibitors, cyclosporin A and FK506, changes of the NMDA receptor–channel complex are reversibly inhibit epileptogenesis in amygdaloid kindled rat, Brain
Res. 648 (1994) 337–341. important to mechanisms of convulsions in EL mice.
3 [15] I. Nagatomo, Y. Akasaki, S. Kuchiiwa, S. Nakagawa, M. Takigawa,
However, Kawasaki et al. [11], who investigated [
H]MK-Sparse distribution of NADPH diaphorase neurons in the hippocam-801 binding activities in the forebrains of EL mice, pal formation of the inbred mutant strain EL mouse, Brain Res. 730 concluded that seizure susceptibility of EL mice could not (1996) 223–226.
be completely explained by changes in affinity of the [16] I. Nagatomo, Y. Akasaki, F. Nagase, M. Nomaguchi, M. Takigawa, Relationships between convulsive seizures and serum and brain NMDA receptor–channel complex. Our results concerning
concentrations of phenobarbital and zonisamide in mutant inbred the effects of dantrolene and FK506 on convulsive seizures
strain EL mouse, Brain Res. 731 (1996) 190–198.
[18] K. Sato, K. Morimoto, M. Okamoto, Anticonvulsant action of acid (1S,3R-ACPD) produces limbic seizures that are not blocked by neurocompetitive antagonist of NMDA receptors (MK-801) in the ionotropic glutamate receptor antagonists, Neurosci. Lett. 162 kindling model of epilepsy, Brain Res. 463 (1988) 12–20. (1993) 12–15.
[19] K. Sato, K. Morimoto, M. Hiramatsu, A. Mori, S. Otsuki, Effect of a [23] J.P. Tizzano, K.I. Griffey, D.D. Schoepp, Induction or protection of noncompetitive antagonist (MK-801) of NMDA receptors on con- limbic seizures in mice by mGluR subtype selective agonists, vulsions and brain amino acid level in EL mice, Neurochem. Res. 14 Neuropharmacology 34 (1995) 1063–1067.
(1989) 741–744. [24] T.J. Toung, A. Bhardwaj, V.L. Dawson, T.M. Dawson, R.J. [20] J. Suzuki, Y. Nakamoto, Seizures patterns and electroencephalog- Traysman, P.D. Hurn, Neuroprotective FK506 does not alter in vivo rams of EL mouse, Electroencephal. Clin. Neurophysiol. 43 (1977) nitric oxide production during ischemia and early reperfusion in rats,
299–311. Stroke 30 (1999) 1279–1285.
[21] J. Suzuki, Y. Nakamoto, Sensory precipitating epilepsy focus in EL [25] M. Uchida, I. Nagatomo, Y. Akasaki, M. Tominaga, W. Hashiguchi, mice and Mongolian gerbils, Folia Psychiat. Neurol. 32 (1978) S. Kuchiiwa, S. Nakagawa, M. Takigawa, Nitric oxide production is 349–359. decreased in the brain of the seizure susceptible EL mouse, Brain [22] J.P. Tizzano, K.I. Griffey, J.A. Johnson, A.S. Fix, D.R. Helton, D.D. Res. Bull. 50 (1999) 223–227.
