544
Selected Papers: Experimental Pharmacology
Effects of Antiepileptic Drugs on Sodium Channel in Rat Brain Hiroshi Tamai, M.D., Takashi Mimaki, M.D., Tohru Ogihara, M.D. and Makoto Mino, M.D.
Department of Pediatrics, Osaka Medical College, Takatsuki Introduction Voltage-sensitive sodium channels mediate increases in Na' permeability that are responsible for the rising phase of the action potential in neurons. Both diphenylhydantoin (PHT) and carbamazepine (CBZ) have proven to decrease the early, transient sodium currents in mammalian myelinated nerve fibers3 In the present study, we examined the effects of antiepileptic drugs (AEDs) on the sodium channel by measuring L3H] saxitoxin (SAX) binding to the rat brain membrane preparation. Subjects and Methods
Brain Membrane Preparation The brain was rapidly removed from a male SD rat (1 50-200 g) and homogenized with 10 strokes of a glass-Teflon homogenizer in ice-cold 0.32 M sucrose with 0.1 mM PMSF, and sedimented at 1,OOO X g for 10 min. The supernatant (Sl) was saved and the pellet ( P l ) was resuspended in 20 volumes of 0.32 M sucrose with 0.1 mM PMSF, and centrifuged as before. The pellet (P2) was discarded and the two supernatants (S1 +S2) were combined and sedimented at 27,OOOXg for 40 min. The supernatants were discarded and the pellet (P3) was resuspended in 20 volumes of 200 mM KCl, 10 mM Hepes/Tris buffer and stored at -8OOC. Specific L3H] SAX Binding Assay Specific binding of L3H] SAX to the brain membrane preparation was measured by a rapid filtration procedure at 0°C in the presence of a saturating concentration
of ['HI SAX ( 5 nM as a final concentration) in standard medium (130 mM choline chloride, 50 mM HepedTris, 5.4 mM KCl, 1.6 mM MgSO,, pH 7.4). The reaction was stopped by addition of 3.0 ml wash medium after 4 sec incubation. Nonspecific binding was determined in the presence of 1 p M tetrodotoxin and subtracted from the total binding to determine specific binding'. Protein determinations were formed using the method of Lowry. In order to examine the effects of AEDs, the brain membrane preparation was preincubated with 0.1 mM PHT, CBZ, phenobarbital (PB), sodium valproate (VPA) and zonisamide (ZNS). Results 1. Preincubation with 0.1 mM PHT inhibited the specific [3H] S A X binding to the brain membrane preparation by 23.2zk 2.0% of control (meankSE, n=4, p < 0.01) (Fig. 1). On the other hand, no effect was seen on the specific L3H] SAX binding by pretreatment with CBZ, VPA, PB or ZNS. 2. This inhibition by PHT was reversible since the decreased specific L3HI SAX binding was recovered after washing out PHT from the incubation medium (Fig. 2). Discussion Catterall et al. have reported the molecular structure of the sodium channel in the
PHT
CBZ
VPA
PB
ZNS
Fig. 1: Effects of antiepileptic drugs on specific ['HI saxitoxin binding in rat brain. Each bar represents percent of control.
The Japanese Journal of Psychiatry and Neurology, Vol. 46, No. 2, 1992
p4.01
T
T
Y t BE 13-4
CONTROL
PHT
PHT BUFFER
+
PHT BUFFER + PHT
+
Fig. 2: Decrement of specific [jH] saxitoxin binding in response to phenytoin reversed by washing.
central nervous system by using neurotoxins as probes.2 He described that the sodium channel could be measured by SAX binding to synaptosomes. We demonstrated in this paper that the early phase, but not the late phase, of [3H] SAX binding to the brain membrane preparation was significantly inhibited by pretreatment with 0.1 mM PHT. Other AEDs (CBZ, VPA, PB, ZNS) did not attenuate the L3H] SAX binding when measured after preincubation
545
with these drugs. PHT might be effective for epilepsy probably due to its effect on sodium channel, which corresponds to inhibition of Na' influx into the nerve. This effect of PHT seems harmless, because its inhibition was reversible. Decreased specific 13H] S A X binding was recovered after washing out PHT, or after incubating for over 30 sec. The present study demonstrated the transient inhibitory effect of PHT on the sodium channel neuropharmacologically by measuring r3H] S A X binding to the rat brain membrane preparation. References Catterall, W.A., Morrow, C.S. and Hartshome, R.P.: Neurotoxin binding to receptor sites associated with voltage-sensitive sodium channels in intact, lysed, and detergentsolubilized brain membranes. J Biol Chem 2 5 4 11379-1 1387, 1979.
Hartshorne, R.P. and Catteral, W.A.: The sodium channel from rat brain. J Biol Chem 259(3): 1667-1675, 1984.
Schwarz, J.R. and Grigat, G.: Phenytoin and carbamazepine: Potential- and frequency-dependent block of Na currents in mammalian myelinated nerve fibers. Epilepsia 30(3): 286-294, 1989.
Selected Papers: Experimental Pharmacology
Effects of Antiepileptic Drugs on Sodium Channel in Rat Brain Hiroshi Tamai, M.D., Takashi Mimaki, M.D., Tohru Ogihara, M.D. and Makoto Mino, M.D.
Department of Pediatrics, Osaka Medical College, Takatsuki Introduction Voltage-sensitive sodium channels mediate increases in Na' permeability that are responsible for the rising phase of the action potential in neurons. Both diphenylhydantoin (PHT) and carbamazepine (CBZ) have proven to decrease the early, transient sodium currents in mammalian myelinated nerve fibers3 In the present study, we examined the effects of antiepileptic drugs (AEDs) on the sodium channel by measuring L3H] saxitoxin (SAX) binding to the rat brain membrane preparation. Subjects and Methods
Brain Membrane Preparation The brain was rapidly removed from a male SD rat (1 50-200 g) and homogenized with 10 strokes of a glass-Teflon homogenizer in ice-cold 0.32 M sucrose with 0.1 mM PMSF, and sedimented at 1,OOO X g for 10 min. The supernatant (Sl) was saved and the pellet ( P l ) was resuspended in 20 volumes of 0.32 M sucrose with 0.1 mM PMSF, and centrifuged as before. The pellet (P2) was discarded and the two supernatants (S1 +S2) were combined and sedimented at 27,OOOXg for 40 min. The supernatants were discarded and the pellet (P3) was resuspended in 20 volumes of 200 mM KCl, 10 mM Hepes/Tris buffer and stored at -8OOC. Specific L3H] SAX Binding Assay Specific binding of L3H] SAX to the brain membrane preparation was measured by a rapid filtration procedure at 0°C in the presence of a saturating concentration
of ['HI SAX ( 5 nM as a final concentration) in standard medium (130 mM choline chloride, 50 mM HepedTris, 5.4 mM KCl, 1.6 mM MgSO,, pH 7.4). The reaction was stopped by addition of 3.0 ml wash medium after 4 sec incubation. Nonspecific binding was determined in the presence of 1 p M tetrodotoxin and subtracted from the total binding to determine specific binding'. Protein determinations were formed using the method of Lowry. In order to examine the effects of AEDs, the brain membrane preparation was preincubated with 0.1 mM PHT, CBZ, phenobarbital (PB), sodium valproate (VPA) and zonisamide (ZNS). Results 1. Preincubation with 0.1 mM PHT inhibited the specific [3H] S A X binding to the brain membrane preparation by 23.2zk 2.0% of control (meankSE, n=4, p < 0.01) (Fig. 1). On the other hand, no effect was seen on the specific L3H] SAX binding by pretreatment with CBZ, VPA, PB or ZNS. 2. This inhibition by PHT was reversible since the decreased specific L3HI SAX binding was recovered after washing out PHT from the incubation medium (Fig. 2). Discussion Catterall et al. have reported the molecular structure of the sodium channel in the
PHT
CBZ
VPA
PB
ZNS
Fig. 1: Effects of antiepileptic drugs on specific ['HI saxitoxin binding in rat brain. Each bar represents percent of control.
The Japanese Journal of Psychiatry and Neurology, Vol. 46, No. 2, 1992
p4.01
T
T
Y t BE 13-4
CONTROL
PHT
PHT BUFFER
+
PHT BUFFER + PHT
+
Fig. 2: Decrement of specific [jH] saxitoxin binding in response to phenytoin reversed by washing.
central nervous system by using neurotoxins as probes.2 He described that the sodium channel could be measured by SAX binding to synaptosomes. We demonstrated in this paper that the early phase, but not the late phase, of [3H] SAX binding to the brain membrane preparation was significantly inhibited by pretreatment with 0.1 mM PHT. Other AEDs (CBZ, VPA, PB, ZNS) did not attenuate the L3H] SAX binding when measured after preincubation
545
with these drugs. PHT might be effective for epilepsy probably due to its effect on sodium channel, which corresponds to inhibition of Na' influx into the nerve. This effect of PHT seems harmless, because its inhibition was reversible. Decreased specific 13H] S A X binding was recovered after washing out PHT, or after incubating for over 30 sec. The present study demonstrated the transient inhibitory effect of PHT on the sodium channel neuropharmacologically by measuring r3H] S A X binding to the rat brain membrane preparation. References Catterall, W.A., Morrow, C.S. and Hartshome, R.P.: Neurotoxin binding to receptor sites associated with voltage-sensitive sodium channels in intact, lysed, and detergentsolubilized brain membranes. J Biol Chem 2 5 4 11379-1 1387, 1979.
Hartshorne, R.P. and Catteral, W.A.: The sodium channel from rat brain. J Biol Chem 259(3): 1667-1675, 1984.
Schwarz, J.R. and Grigat, G.: Phenytoin and carbamazepine: Potential- and frequency-dependent block of Na currents in mammalian myelinated nerve fibers. Epilepsia 30(3): 286-294, 1989.
