/ . Biochem., 80, 1165-1171 (1976)
A Correlation between Changes in 7--Aminobutyric Acid Metabolism and Seizures Induced by Antivitamin Be Miyako ABE and Makoto MATSUDA Department of Biochemistry, The Jikei University School of Medicine, Minato-ku, Tokyo 105 Received for publication, April 2, 1976
The effects of DL-penicillamine (DL-PeA), hydrazine and toxopyrimidine (TXP, 2-methyl6-amino-5-hydroxymethylpyrimidine) on 7--aminobutyric acid (GABA) metabolism in mouse brain were studied. All these compounds inhibited the activity of glutamate decarboxylase [EC 4.1.1.15] (GAD) and slightly inhibited that of 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 2.6.1.19] (GABA-T). In contrast, very different effects were observed on GABA levels; hydrazine caused a marked increase, DL-PeA had no effect, and TXP caused a slight decrease in the content of the amino acid. These results could be described by an equation which related the excitable state to changes in the flux of the GABA bypass. Since the values obtained from the equation clearly reflect the seizure activity, it is suggested that the decreased GABA flux might be a cause of convulsions induced by these drugs.
Antivitamin Bj is generally known to cause characteristic seizures in animals. Initial studies on the biochemical mechanism involved in the production of seizures induced by hydrazide, a kind of antivitamin B« .indicated that low GABA levels in the brain might be involved in the etiology of the seizures (1—3). Speculation regarding this possibility subsided, however, when various workers demonstrated the absence of a correlation between the onset of seizures and the decrease in content of brain GABA (4—6). Subsequent studies indicated that no correlation existed between the onset of seizures and any single parameter other than Abbreviations: DL-PeA, DL-penicillamine ; TXP, toxopyrimidine ; GABA, 4-aminobutyric acid; GAD, glutamate decarboxylase; GABA-T, GABA-2-oxoglutarate aminotransferase; PN, pyridoxine. Vol. 80, No. 5, 1976
GABA content, e.g., the activity of glutamate decarboxylase [EC 4.1.1.15] (GAD) or 4-aminobutyrate : 2-oxoglutarate aminotransferase [EC 2.6.1.19] (GABA-T) in the brain (7-9). Recent studies by Wood and Peesker (9) suggested that the state of excitation of the brain might, however, be related to a function involving two parameters of GABA metabolism, namely GAD activity and GABA content. The object of the present investigation was to determine whether a reduced metabolic rate of the so-called GABA bypass might be the cause of seizures. The parameters studied in the present investigation were the concentration of GABA and the activities of GAD and GABA-T, which are considered to regulate the metabolic rate of the GABA bypass.
1165
1166
M. ABE and M. MATSUDA
MATERIALS AND METHODS
Animals—Male DDY mice (weighing approx. 20 g) were used in the experiments. Administration of Drugs—The drugs used were PeA, pyridoxine (PN) and hydrazine from Nakarai Chemicals, and TXP from Takeda Pharmaceutical Co. Solutions of the drugs were prepared daily in 0.9% NaCl, the pH being adjusted to 7.0 immediately before use. The final concentration of each drug was adjusted so that the required dosage was administered in a volume equivalent to 1.0 percent of the body weight of the animals. All injections were intramuscular and the injected animals were kept in a laboratory with minimal background noise. Biochemical Analysis—The GABA level in the brain was estimated by a colorimetric method (10,11). The brain were removed as rapidly as possible and homogenized in 10 volumes of 75% ethanol. After centrifugation at 3,000 rpm for 10 min the supernatant was dried in vacuo and the residue obtained was suspended in 1.0 ml of water. The suspension was washed with 6 ml of chloroform saturated with water and centrifuged at 3,000 rpm for 10 min. 5 //I of the water layer obtained was applied to a filter paper for paper electrophoresis. GABA was separated from other amino acids by high-voltage paper electrophoresis (3000 V/ 60 cm, 1 hr) in formic acid, water, acetic acid= 1 : 16 : 3 (pH 1.9), and determined colorimetrically by the procedure described by Cocking and Yemm (11). The activity of GAD was determined by measuring GABA formation from glutamate. The brain was removed immediately after sacrifice of the mice and homogenized in 9 volumes of 1/15 M phosphate buffer, pH 6.2 using a Teflon homogenizer. After addition of the substrate (0.15 ml of 1.1 M sodium glutamate solution) to 1.5 ml of the homogenate, incubation was carried out at 37° for 20 min. GABA content in the reaction mixture was determined by the procedure described above. The activity of GABA-T was measured by determining glutamate production from 2oxoglutarate. Tissue was homogenized in 9
volumes of 0.1 M phosphate buffer, pH 8.0. The incubation mixture contained 0.15 ml of 0.5 M GABA solution, 0.45 ml of the same buffer, and 0.75 ml of the homogenate. After the addition of 0.15 ml of 0.1 M 2-oxoglutarate solution, incubation was carried out at 37° for 20 min. The reaction mixture was heated at 100° for 10 min and centrifuged at 12,000 rpm for 10 min. Glutamate in the supernatant was estimated using the enzymatic procedure described by Schousboe et al. (12). RESULTS GABA Metabolism at the Onset of Seizures Induced by Antivitamin Be—The effects of PeA, hydrazine, and TXP on GABA metabolism were studied using convulsant and non-convulsant dosage levels. Administration of DLPeA, hydrazine or TXP in convulsant amounts caused seizures in all animals (Table I). In contrast, D-PeA did not invoke seizure even when administered at 3.3 mmoles/kg body weight, corresponding to the convulsant dose for DL-PeA. The effects of convulsant doses of these drugs on GABA metabolism were determined at the onset of seizures (Table I). GAD activity was decreased by these convulsant drugs, but it was significantly lower at the onset of hydrazine-induced seizures than at the onset of DL-PeA- or TXP-induced seizures. In contrast, the GABA content was greatly elevated in the brain of hydrazine-treated mice but was unchanged in DL-PeA-induced and slightly lowered in TXP-induced animals at the onset of seizure. Since it has already been shown that hydrazine- (13), DL-PeA- (14), and TXPinduced seizures (15) in mice are prevented by the simultaneous administration of PN, experiments with this B8 vitamer were included in the present investigation. The administration of PN together with hydrazine, DLPeA or TXP prevented seizures induced in mice by these drugs and reduced the concomitant inhibition of GAD activity (Table I). Effect of DL-PeA on GABA Metabolism— The effect of DL-PeA on the levels of GABA in the brain of mice as a function of time after administration of the drug depended on the / . Biochem.
GABA METABOLISM AND ANTI-B, INDUCED SEIZURE TABLE I. and TXP.
1167
GABA metabolism in the brain of mice at the onset of seizures induced by DL-PeA, hydrazine,
Treatment (mmole/kg)
Time of decapitation (min)
Control DL-PeA (1.65)
130b
DL-PeA (3.3)
At seizures (130±20)» (116)
DL-PeA ( 3 . 3 ) + P N (1.8)
130b
DL-PeA (6.6)
At seizures (76±11)» (15)
D-PeA (1.65)
130"
D-PeA (3.3)
130"
Hydrazine (3.0) Hydrazine (3.0)+ PN (1.8) TXP (2.0)
At seizures (25±7)» (15) 25» At seizures (136±19)* (22)
TXP (2.0)+PN (1.8)
136 b
PN (1.8)
130 b
1
(fimo\e/g)
GAD (/imole/g/hr)
GABA-T (/imole/g/hr)
2.82±0.08 + (43) 2.79 ±0.14 ( 5) 2.71 ±0.07 (44) 2.98 ±0.15 (10) 2.71 ±0.13 ( 5) 2.85±0.10 ( 5) 3.45 ±0.12 ( 5) 4.14±0.21 ( 5) 4.97±0.59 ( 5) 2.34±0.08 ( 7) 2.89±0.05 ( 6) 2.84±0.12 ( 5)
18.4 ±0.6 (34) 17.2±1.5 ( 5) 12.7±0.5 (34) 18.5±0.4 (10) 13.2±0.6 ( 5) 18.0±0.9 ( 5) 18.6±0.6 ( 5) 6.9±1.9 ( 5) 8.0±1.4 ( 5) 14.9±0.6 (8) 17.9±0.7 (6) 20.4±0.7 ( 5)
52.7±1. 2 (34) 48.5±2.2 (5) 46.8 ±0.9 (38) 47.4±1.3 (11) 47.0±1.4 (5) 53.3±2.3 (5) 51.6±2.0 (5) 40.2±1.9 ( 5) 40.7±3.8 • ( 5) 52.5±1.7 (7) 53.7±2.2 (6) 53.2±1.3 (5)
GABA
Mean±S.D. for groups, +mean ±S.E.M.; numbers of animals in parentheses. observed up to the time of decapitation.
amount administered (Fig. 1A). When the amount of DL-PeA administration was 3.3 mmoles/kg, the initial rise in GABA was followed by a reversal such that the level of GABA at the onset of seizures fell to normal. At a dose of 1.65 mmoles/kg there was a very slight increase in the GABA level following the injection and a subsequent reversal to the normal level. All three dosage levels of DL-PeA produced significant decreases in GAD activity, although the pattern of inhibition varied somewhat with the amount administered (Fig. IB). Injection of DL-PeA, 1.65 mmoles/kg, produced a slow decline in GAD activity over the 130-min experimental period, whereas administration of 3.3 or 6.6 mmoles/kg produced a rapid decrease in the activity. The activity of GAD at the Vol. 80, No. 5, 1976
b
No seizures were
onset of seizures was about 13 //moles/hr/g. The inhibition of GABA-T on administration of DL-PeA was slower and smaller in extent than that of GAD (Fig. 1C). Activity of GABA-T remained at about 90 percent of the control even at the onset of seizures. Effect of PN on DL-PeA-induced Changes in GABA Metabolism—Mice treated with PN were also included in the study for comparative purposes. The administration of PN (1.8 mmoles/kg) with DL-PeA had no significant effect on the DL-PeA-induced changes with respect to GABA levels (Fig. 2A). PN administration together with DL-PeA counteracted the inhibition of GAD produced by DL-PeA alone, but did not change the slight inhibition of GABA-T brought about by DLPeA alone (Fig. 2B, C).
•. M. ABE and M. MATSUDA
1168 ,-.4.0
20 40
60 80 100 120 TIME (min)
Fig. 1. Effects of intramuscularly administered DLPeA on brain levels of GABA (A), GAD activity (B), and GABA-T activity (C) in mice. X, 1.65 mmoles/ kg; O, 3.3 mmoles/kg; • , 6.6 mmoles/kg. Each value is the mean±S.E.M. for five experiments.
DISCUSSION
Since there is increasing evidence indicating a neurotransmitter role for GABA (16—18), and since various antivitamine Bj compounds obviously affect GABA metabolism, deranged GABA metabolism induced by antivitamin B6 is expected to cause changes in brain excitability. However, no simple relationship existed between the onset of antivitamin B8-induced seizures and GABA levels, GAD activity, or GABA-T activity (Table I). A similar conclusion was previously reached by Wood and Peesker (
A Correlation between Changes in 7--Aminobutyric Acid Metabolism and Seizures Induced by Antivitamin Be Miyako ABE and Makoto MATSUDA Department of Biochemistry, The Jikei University School of Medicine, Minato-ku, Tokyo 105 Received for publication, April 2, 1976
The effects of DL-penicillamine (DL-PeA), hydrazine and toxopyrimidine (TXP, 2-methyl6-amino-5-hydroxymethylpyrimidine) on 7--aminobutyric acid (GABA) metabolism in mouse brain were studied. All these compounds inhibited the activity of glutamate decarboxylase [EC 4.1.1.15] (GAD) and slightly inhibited that of 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 2.6.1.19] (GABA-T). In contrast, very different effects were observed on GABA levels; hydrazine caused a marked increase, DL-PeA had no effect, and TXP caused a slight decrease in the content of the amino acid. These results could be described by an equation which related the excitable state to changes in the flux of the GABA bypass. Since the values obtained from the equation clearly reflect the seizure activity, it is suggested that the decreased GABA flux might be a cause of convulsions induced by these drugs.
Antivitamin Bj is generally known to cause characteristic seizures in animals. Initial studies on the biochemical mechanism involved in the production of seizures induced by hydrazide, a kind of antivitamin B« .indicated that low GABA levels in the brain might be involved in the etiology of the seizures (1—3). Speculation regarding this possibility subsided, however, when various workers demonstrated the absence of a correlation between the onset of seizures and the decrease in content of brain GABA (4—6). Subsequent studies indicated that no correlation existed between the onset of seizures and any single parameter other than Abbreviations: DL-PeA, DL-penicillamine ; TXP, toxopyrimidine ; GABA, 4-aminobutyric acid; GAD, glutamate decarboxylase; GABA-T, GABA-2-oxoglutarate aminotransferase; PN, pyridoxine. Vol. 80, No. 5, 1976
GABA content, e.g., the activity of glutamate decarboxylase [EC 4.1.1.15] (GAD) or 4-aminobutyrate : 2-oxoglutarate aminotransferase [EC 2.6.1.19] (GABA-T) in the brain (7-9). Recent studies by Wood and Peesker (9) suggested that the state of excitation of the brain might, however, be related to a function involving two parameters of GABA metabolism, namely GAD activity and GABA content. The object of the present investigation was to determine whether a reduced metabolic rate of the so-called GABA bypass might be the cause of seizures. The parameters studied in the present investigation were the concentration of GABA and the activities of GAD and GABA-T, which are considered to regulate the metabolic rate of the GABA bypass.
1165
1166
M. ABE and M. MATSUDA
MATERIALS AND METHODS
Animals—Male DDY mice (weighing approx. 20 g) were used in the experiments. Administration of Drugs—The drugs used were PeA, pyridoxine (PN) and hydrazine from Nakarai Chemicals, and TXP from Takeda Pharmaceutical Co. Solutions of the drugs were prepared daily in 0.9% NaCl, the pH being adjusted to 7.0 immediately before use. The final concentration of each drug was adjusted so that the required dosage was administered in a volume equivalent to 1.0 percent of the body weight of the animals. All injections were intramuscular and the injected animals were kept in a laboratory with minimal background noise. Biochemical Analysis—The GABA level in the brain was estimated by a colorimetric method (10,11). The brain were removed as rapidly as possible and homogenized in 10 volumes of 75% ethanol. After centrifugation at 3,000 rpm for 10 min the supernatant was dried in vacuo and the residue obtained was suspended in 1.0 ml of water. The suspension was washed with 6 ml of chloroform saturated with water and centrifuged at 3,000 rpm for 10 min. 5 //I of the water layer obtained was applied to a filter paper for paper electrophoresis. GABA was separated from other amino acids by high-voltage paper electrophoresis (3000 V/ 60 cm, 1 hr) in formic acid, water, acetic acid= 1 : 16 : 3 (pH 1.9), and determined colorimetrically by the procedure described by Cocking and Yemm (11). The activity of GAD was determined by measuring GABA formation from glutamate. The brain was removed immediately after sacrifice of the mice and homogenized in 9 volumes of 1/15 M phosphate buffer, pH 6.2 using a Teflon homogenizer. After addition of the substrate (0.15 ml of 1.1 M sodium glutamate solution) to 1.5 ml of the homogenate, incubation was carried out at 37° for 20 min. GABA content in the reaction mixture was determined by the procedure described above. The activity of GABA-T was measured by determining glutamate production from 2oxoglutarate. Tissue was homogenized in 9
volumes of 0.1 M phosphate buffer, pH 8.0. The incubation mixture contained 0.15 ml of 0.5 M GABA solution, 0.45 ml of the same buffer, and 0.75 ml of the homogenate. After the addition of 0.15 ml of 0.1 M 2-oxoglutarate solution, incubation was carried out at 37° for 20 min. The reaction mixture was heated at 100° for 10 min and centrifuged at 12,000 rpm for 10 min. Glutamate in the supernatant was estimated using the enzymatic procedure described by Schousboe et al. (12). RESULTS GABA Metabolism at the Onset of Seizures Induced by Antivitamin Be—The effects of PeA, hydrazine, and TXP on GABA metabolism were studied using convulsant and non-convulsant dosage levels. Administration of DLPeA, hydrazine or TXP in convulsant amounts caused seizures in all animals (Table I). In contrast, D-PeA did not invoke seizure even when administered at 3.3 mmoles/kg body weight, corresponding to the convulsant dose for DL-PeA. The effects of convulsant doses of these drugs on GABA metabolism were determined at the onset of seizures (Table I). GAD activity was decreased by these convulsant drugs, but it was significantly lower at the onset of hydrazine-induced seizures than at the onset of DL-PeA- or TXP-induced seizures. In contrast, the GABA content was greatly elevated in the brain of hydrazine-treated mice but was unchanged in DL-PeA-induced and slightly lowered in TXP-induced animals at the onset of seizure. Since it has already been shown that hydrazine- (13), DL-PeA- (14), and TXPinduced seizures (15) in mice are prevented by the simultaneous administration of PN, experiments with this B8 vitamer were included in the present investigation. The administration of PN together with hydrazine, DLPeA or TXP prevented seizures induced in mice by these drugs and reduced the concomitant inhibition of GAD activity (Table I). Effect of DL-PeA on GABA Metabolism— The effect of DL-PeA on the levels of GABA in the brain of mice as a function of time after administration of the drug depended on the / . Biochem.
GABA METABOLISM AND ANTI-B, INDUCED SEIZURE TABLE I. and TXP.
1167
GABA metabolism in the brain of mice at the onset of seizures induced by DL-PeA, hydrazine,
Treatment (mmole/kg)
Time of decapitation (min)
Control DL-PeA (1.65)
130b
DL-PeA (3.3)
At seizures (130±20)» (116)
DL-PeA ( 3 . 3 ) + P N (1.8)
130b
DL-PeA (6.6)
At seizures (76±11)» (15)
D-PeA (1.65)
130"
D-PeA (3.3)
130"
Hydrazine (3.0) Hydrazine (3.0)+ PN (1.8) TXP (2.0)
At seizures (25±7)» (15) 25» At seizures (136±19)* (22)
TXP (2.0)+PN (1.8)
136 b
PN (1.8)
130 b
1
(fimo\e/g)
GAD (/imole/g/hr)
GABA-T (/imole/g/hr)
2.82±0.08 + (43) 2.79 ±0.14 ( 5) 2.71 ±0.07 (44) 2.98 ±0.15 (10) 2.71 ±0.13 ( 5) 2.85±0.10 ( 5) 3.45 ±0.12 ( 5) 4.14±0.21 ( 5) 4.97±0.59 ( 5) 2.34±0.08 ( 7) 2.89±0.05 ( 6) 2.84±0.12 ( 5)
18.4 ±0.6 (34) 17.2±1.5 ( 5) 12.7±0.5 (34) 18.5±0.4 (10) 13.2±0.6 ( 5) 18.0±0.9 ( 5) 18.6±0.6 ( 5) 6.9±1.9 ( 5) 8.0±1.4 ( 5) 14.9±0.6 (8) 17.9±0.7 (6) 20.4±0.7 ( 5)
52.7±1. 2 (34) 48.5±2.2 (5) 46.8 ±0.9 (38) 47.4±1.3 (11) 47.0±1.4 (5) 53.3±2.3 (5) 51.6±2.0 (5) 40.2±1.9 ( 5) 40.7±3.8 • ( 5) 52.5±1.7 (7) 53.7±2.2 (6) 53.2±1.3 (5)
GABA
Mean±S.D. for groups, +mean ±S.E.M.; numbers of animals in parentheses. observed up to the time of decapitation.
amount administered (Fig. 1A). When the amount of DL-PeA administration was 3.3 mmoles/kg, the initial rise in GABA was followed by a reversal such that the level of GABA at the onset of seizures fell to normal. At a dose of 1.65 mmoles/kg there was a very slight increase in the GABA level following the injection and a subsequent reversal to the normal level. All three dosage levels of DL-PeA produced significant decreases in GAD activity, although the pattern of inhibition varied somewhat with the amount administered (Fig. IB). Injection of DL-PeA, 1.65 mmoles/kg, produced a slow decline in GAD activity over the 130-min experimental period, whereas administration of 3.3 or 6.6 mmoles/kg produced a rapid decrease in the activity. The activity of GAD at the Vol. 80, No. 5, 1976
b
No seizures were
onset of seizures was about 13 //moles/hr/g. The inhibition of GABA-T on administration of DL-PeA was slower and smaller in extent than that of GAD (Fig. 1C). Activity of GABA-T remained at about 90 percent of the control even at the onset of seizures. Effect of PN on DL-PeA-induced Changes in GABA Metabolism—Mice treated with PN were also included in the study for comparative purposes. The administration of PN (1.8 mmoles/kg) with DL-PeA had no significant effect on the DL-PeA-induced changes with respect to GABA levels (Fig. 2A). PN administration together with DL-PeA counteracted the inhibition of GAD produced by DL-PeA alone, but did not change the slight inhibition of GABA-T brought about by DLPeA alone (Fig. 2B, C).
•. M. ABE and M. MATSUDA
1168 ,-.4.0
20 40
60 80 100 120 TIME (min)
Fig. 1. Effects of intramuscularly administered DLPeA on brain levels of GABA (A), GAD activity (B), and GABA-T activity (C) in mice. X, 1.65 mmoles/ kg; O, 3.3 mmoles/kg; • , 6.6 mmoles/kg. Each value is the mean±S.E.M. for five experiments.
DISCUSSION
Since there is increasing evidence indicating a neurotransmitter role for GABA (16—18), and since various antivitamine Bj compounds obviously affect GABA metabolism, deranged GABA metabolism induced by antivitamin B6 is expected to cause changes in brain excitability. However, no simple relationship existed between the onset of antivitamin B8-induced seizures and GABA levels, GAD activity, or GABA-T activity (Table I). A similar conclusion was previously reached by Wood and Peesker (
