Relationships between pyridoxal phosphate availability, activity of vitamin B6-dependent enzymes and convulsions

BRAIN RESEARCH

111

RELATIONSHIPS BETWEEN PYRIDOXAL PHOSPHATE AVAILABILITY, ACTIVITY OF VITAMIN Bt-DEPENDENT ENZYMES AND CONVULSIONS

RICARDO TAPIA AND HERMINIA PASANTES Departamento de Bioquimica, blstituto de Biologia, Universidad Nacional de Mdxico, Mexico 20, D.F. (Mexico)

(Accepted December 16th, 1970)

INTRODUCTION In previous work it was shown that maximal inhibition of brain glutamate decarboxylase (EC 4.1.1.15) 23, as well as a decreased rate of synthesis of y-aminobutyric acid (GABA) from labelled glutamic acid in vivo 2°, occur at the moment of appearance of convulsions produced by pyridoxal phosphate-y-glutamyl hydrazone (PLPGH), by i-glutamic acid-y-hydrazide (GAH) and by some other drugs. These changes are independent of the total brain GABA levels, and they have been interpreted to mean that the inhibition of glutamate decarbo×ylase is causally involved in the production of certain types of convulsion, because it would result in a decrease of GABA concentration at the synaptic cleft20,25. It has also been shown that with some convulsants, such as PLPGH and other pyridoxal phosphate hydrazones, the inhibition of glutamate decarboxylase is secondary to a decrease in the concentration of pyridoxal phosphate (PLP) in brain zS. These results pose the question of the possible participation of other Bt-enzymes, which could also be inhibited as a consequence of the deficiency of PLP, in the mechanisms of production of convulsions. The present work was undertaken with the aim of investigating the possible influence of the main B6 enzymes present in nervous tissue, besides glutamate decarboxylase, in such mechanisms. Furthermore, the results obtained permit us to draw some conclusions on the extent of the dependence of the B6 enzymes upon PLP, which would give some indication of the strength of binding between the different apoenzymes and the coenzyme in vivo. The concentration of PLP in the brain was diminished by the administration of PLPGH and GAH to mice25. The activity of GABA (EC 2.6.1.19), aspartate (EC 2.6.1.1.) and alanine (EC 2.6.1.2) aminotransferases, and that of DOPA (EC 4.1.1.26) and histidine (EC 4.1.1.22) decarboxylases, were measured in the brain of these animals. The former three enzymes, particularly aspartate aminotransferase2,9, are responsible for the oxidation of amino acids through the tricarboxylic acid cycle; therefore, a decrease in their activity could result in a deficient supply of the energy required to fulfil the demands arising from neuronal activityz7. The two decarboxylases Brain Research, 29 (1971) 111-122

112

R . T A P I A A N D H . PASANTES

are responsible for the synthesis of some synaptic transmitter suspects - - dopamine, norepinephrine, 5-hydroxytryptamine, histamine - - and therefore they could be directly involved in the production of convulsive states, as postulated for glutamate decarboxylase. MATERIALS A N D M E T H O D S

Adult mice (local strain) were used in all experiments. In the experiments in vivo they were injected intraperitoneally with PLPGH (80 mg/kg), GAH (2 g/kg) or amethyl-DOPA (400 mg/kg). At the time indicated in Results they were killed by a blow on the back of the neck and immediate decapitation. Animals treated with PLPGH were decapitated during the maximal phase of the tonic terminal convulsions observed 30-50 min after treatment. PLPGH was synthesized from GAH and PLP as previously described 24. The coenzymes and enzymes used for the enzymic assays (lactic, malic and glutamic dehydrogenases) were purchased from Sigma Chemical Co. (St. Louis, Mo.), GAH was obtained from Calbiochem (Los Angeles, Calif.), and L-amethyl-DOPA was kindly provided by Merck, Sharp and Dohme de M6xico. Radioactive substrates were obtained as follows: DL-[1-14C]glutamic acid (spec. act. 2.64 mCi/mmole) from International Chemical and Nuclear Corp. (City of Industry, Calif.) and DL-[carboxyl-14C]DOPA (2.45 mCi/mmole) and L-[carboxyl-14C]histidine (13.7 mCi/mmole) from New England Nuclear Corp. (Boston, Mass.). Measurement of enzymic activity For the determination of aspartate and alanine aminotransferases the incubation mixture was as described by Bergmeyer and Bernt a,5 with brain homogenates in phosphate buffer (pH 7.6) (0.5 ml of 5 ~ (w/v) homogenate for the former enzyme and 1 ml of a 30 ~ homogenate for the latter) as source of the enzyme, in a final volume of 3 ml. Both enzymes were preincubated at 37°C for 5 rain before the addition of aketoglutaric acid; the incubation time was 5 min for aspartate aminotransferase and 10 min for alanine aminotransferase. The reactions were stopped with 0.2 ml of 100 K TCA. After centrifugation of the precipitate the supernatant was neutralized to about pH 7.6. For aspartate aminotransferase, 0.5 ml of the supernatant was used for the measurement of the oxaloacetic acid tbrmed during transamination, following the enzymic method of Bergmeyer and Bernt4. For alanine aminotransferase, 1 ml of the supernatant was used, and the amount of pyruvic acid formed during transamination was also measured enzymicallyL The substrate concentrations used in these methods were equal to or higher than those used in other published methods for measuring aspartate and alanine aminotransferases in brain tissuO,3, 2~ and they have proved to be adequate both with homogenates and mitochondrial preparations from brain 2,3. The incubation mixture for the determination of GABA aminotransferase activity was as previously described s, using 1 ml of a 10~ (w/v) homogenate in buffer (pH 8.2) as source of the enzyme. After 30 min of incubation, neutralized supernatants were obtained as described for the other aminotransferases studied, and 0.15 ml of the Brain Research, 29 (1971) 111-122

VITAMIN

B6, B6 ENZYMES

AND CONVULSIONS

113

supernatant was used for the determination of the glutamic acid formed during transamination, according to the enzymic procedure of Bernt and Bergmeyer6, in a final volume of 1 ml. Standard curves with L-glutamic acid were made in the presence of both a-ketoglutaric acid and neutralized TCA in concentrations comparable to those present in the neutralized supernatant. This permitted the correction of the error given by the excess of the keto acid, which is a product of the glutamate dehydrogenase reaction and in the conditions employed decreased the reaction rate. Since a small inhibitory effect of trichloroacetate on glutamate dehydrogenase was observed, its presence in the standard samples was also necessary. Glutamic decarboxylase was determined by measuring the 14C02 formation from DL-[1-14C]glutamic acid, as previously described 21, with 0.4 ml of a 20 % (w/v) homogenate in water as source of the enzyme. DOPA and histidine decarboxylases were also measured by the rate of decarboxylation of the corresponding carboxyl[14C]substrates. For DOPA decarboxylase the incubation mixture was as described by Pscheidt and Haber a3, with 0.5 ml of a 20 % (w/v) homogenate in water as source of the enzyme, in a final volume of 1.5 ml; the incubation time was 30 rain. When the effect of a-methyl-DOPA on DOPA decarboxylase was studied in vivo, the incubation medium contained only labelled DOPA as substrate (2/zCi), and the incubation time was 5 rain. These conditions avoided the possible reversal of the inhibition by excess of substrate in the assay system. To obtain a measurable activity of histidine decarboxylase, 1 ml of a 50 % (w/v) brain homogenate in buffer (pH 6.8) had to be used, and the incubation time had to be extended to 2 h, in a medium containing 16.6 # M L-histidine and 0.2 /zCi of L[carboxyl-~4C]histidine, at p H 6.8; the total volume was 1.5 ml. The labelled histidine solution was previously acidified with a small amount of 6 N HCI, heated in a boiling water bath for a few minutes and neutralized to p H 6 with NaOH. This procedure reduced the value of the blank, which was usually high owing to volatile a4C-containing contaminants ~1. The procedure for trapping the 14CO2 liberated from labelled DOPA and histidine and for counting the radioactivity was as previously described for the measurement of glutamate decarboxylase activity 21. A Nuclear Chicago liquid scintillation counter was used for the radioactivity measurements. The activity of all the enzymes studied, with the exception of histidine decarboxylase, which was not analyzed in this respect and which was not affected at all in any of the experimental conditions used, was linear with time up to at least the incubation time used in each experiment. The concentrations of G A H and P L P G H used in the experiments in vitro are indicated in the corresponding Tables. Neither of the substances studied had any effect on the enzymes used in the enzymic determinations of the activities of aminotransferases. RESULTS

As previously reported24, 25, P L P G H at the dose used produced fatal tonic conBrain Research, 29 (1971) 111-122

I 14

R. TAPIA AND H. PASANTES

TABLE I EFFECTSOF PLPGH AND GAH ON BRAINASPARTATEAMINOTRANSFERASEACTIVITY The values are/*moles of oxaloacetic acid formed/g of tissue/h. Mean ± S.E.M. Number of experiments in parentheses.

Control Treated

P L P G H in vivo (80 mg/kg, 40 min)

P L P G H in vitro (10 -4 M )

No P L P

No addition

PLPGH

144.0 ± 4.7 (4) 141.8 ± 9.2 (4)

122.3 ± 5.1 (4)

118.8 ± 3.6 (4)

G A H in vivo (2 glkg, 1 h) No P L P

G A H in vitro with PLP*

No addition

Control 97.8 -L 7.5 (4)

103.9± 7.3 112.5 ± 22 (4) (4)

Treated

104.6± 7.5 ~o (4) inhibition

94.7 ± 8.8 (4)

GAH (3.2 " 10 -a M )

GAH ± PLP*

PLP*

2.6 4.9 114.2 ± 19 (0-7.8)** (0-15.7)** (4)

(4)

(4)

98

96

--

GAH (10 -4 M )

107.0 ± 6.5 (4) 5

* PLP was added to the incubation mixture to a final concentration of 10 4 M. ** Range of values. The zero values were obtained in 2 experiments.

vulsions in 30-50 rain, a n d G A H - t r e a t e d mice showed depression a n d occasional clonic convulsions at the m o m e n t of killing (1 h after treatment). The a d m i n i s t r a t i o n of a - m e t h y l - D O P A p r o d u c e d n o appreciable effect o n the b e h a v i o u r o f the mice. Aminotransferases

P L P G H p r o d u c e d n o effect at all on aspartate aminotransferase either when administered to mice or when added in vitro. The a d m i n i s t r a t i o n of G A H did n o t affect the enzyme, b u t when this hydrazide was added at a c o n c e n t r a t i o n of 3.2 • 10 -3 M in vitro a n almost total i n h i b i t i o n was observed; however, n o i n h i b i t i o n was obtained with 10 4 M G A H ; P L P (10-4 M ) did n o t activate aspartate aminotransferase (Table

I). As with aspartate aminotransferase, P L P G H had n o detectable effect on alanine aminotransferase, either in vivo or in vitro. I n contrast, G A H inhibited the enzyme a b o u t 70 ~ b o t h in vivo a n d when added to the i n c u b a t i o n m e d i u m to a concentration o f 3.2 • 10 -3 M. The i n h i b i t i o n observed #~ vivo was n o t modified by the a d d i t i o n o f P L P to the i n c u b a t i o n mixture; 10 -4 M G A H or P L P did n o t modify a l a n i n e a m i n o t r a n s f e r a s e activity (Table II). W h e n the action o f P L P G H o n G A B A aminotransferase was studied in vivo only a slight non-significant i n h i b i t o r y effect was observed at the m o m e n t of appearBrain Research, 29 (1971) 111-122

VITAMIN B6, B6 ENZYMES AND CONVULSIONS

1] 5

TABLE II EFFECTS OF P L P G H AND G A H ON BRAINALANINZAMINOTRANSF~RASEACTIVITY The values are/tmoles of pyruvic acid formed/g of tissue/h. Mean ~_ S.E.M. Number of experiments in parentheses.

Control Treated

Control Treated Inhibition

P L P G H in vivo (80 mg/kg, 40 min)

P L P G H in vitro (10 -4 M )

No P L P

No addition

PLPGH

62.3 :k 2.7** (10) 63.1 :k 1.5 (4)

52.8 zk 2.1 (4)

50.2 ~ 2.2 (4)

G A H in vivo (2 g/kg~ 1 h)

G A H in vitro

No P L P

With PLP*

No addition

GAH (3.2" 10 s M )

G A H 4- PLP*

62.3 zk 2.7** (10) 21.3 zk 2.2 (6) 66

47.0 J- 1.2 (4) 13.8 zk 0.8 (4) 70

59.5 ~ 1.7 (4) % inhibition

15.6 ~ 1.9 (4) 74

16.5 dz 2.8

No addition

GAH (10-4 M ) 54.6 ± 2.1 (4)

G A H + PLP*

53.8 zL 2.1 (4)

(4) 72

50.2 ± 2.2 (4)

* PLP was added to the incubation mixture to a final concentration of l0 -4 M. ** Same controlgroup.

TABLE III EFFFCTS OF P L P G H ON BRAIN GABA AMINOTRANSFERASEACTIVITY The values are/~moles of glutamic acid formed/g of tissue/h. Mean ~ S.E.M. Number of experiments in parentheses.

Control Treated

In viva (80 mg/kg, 40 mitt)

In vitro (10 -4 M )

No P L P

No addition

PLP

PLPGH

26.1 zk 1.4 (6) 22.5 ± 1.7" (6)

31.4 ~: 1.8 (4)

35.4 zk 2.0* (4)

34.4 i 1.4" (4)

* Not significantly different from control values (P < 0.1).

anceofconvulsions.Invitro, PLPorPLPGH(lO-4 M) produced a small non-significant activation of the enzyme (Table III). The inhibitory action of GAH on GABA aminotransferase, both in vivo and in vitro, has been reported22, 2s. Brain Researeh, 29 (1971) 111-122

116

R . T A P I A A N D H . PASANTES

TABLE IV EFFECTSOF PLPGH AND GAH ON BRAINDOPA DECARBOXYLASEACTIVITY The values are counts/min - 10 3 in the 14CO2 produced in 30 rain of incubation in the conditions described in Materials and Methods. Mean 4- S.E.M. Number of experiments in parentheses.

Control

P L P G H in vivo (80 mg/kg, 40 rain)

P L P G H in vitro (10 -4 M )

No PLP

No addition

With PLP*

27.9 :k 6.2 (5) 39.1 ± 1.2 (5)

PLP*

29.6 4- 1.6 (5) 40.4 4- 2.4**

PLPGH

39.1 4- 2.7 (5)

(5) Treated Inhibition

10.1 + 5.7 (5) 40.2 4- 2.0 (5) 64 0 G A H in vivo (2 g/kg, 1 h) No PLP

Control

24.4 4- 1.5 (6) Treated 13.1 4- 1.2 (5) Inhibition 46

~ activation

36

32

GAH in vitro No addition

PLP*

With PLP*

34.94- 0.5 (6) 33.24- 1.2 (5) 5

GAH (3.2 • 10 -8 M)

GAIt + PLP*

32.5 4- 1.7 40.4 4- 2.4** 18.4 4- 0.6 (5) (5) (5) ~ inhibition - 43

28.4 4- 1.1 (5) 30

No addition PLP*

GAH + PLP* 31.1 4- 1.9 (5)

23.8 4- 1.7 (5)

33.44- 1.2 (3)

GAH (10 4 M) 21.0 4- 1.1 (5)

* PLP was added to the incubation mixture to a final concentration of 10-4 M. ** Same group.

Decarboxylases

Table IV shows that a d m i n i s t r a t i o n of P L P G H resulted in a considerable inhibition o f b r a i n D O P A decarboxylase, which was completely reversed by the a d d i t i o n o f P L P to the i n c u b a t i o n m e d i u m . However, as h a d been observed with glutamate decarboxylase21, 24, when the h y d r a z o n e was added in vitro at a c o n c e n t r a t i o n o f 10 -4 M, a n activation o f the enzyme, similar to that observed in the presence o f equim o l a r a m o u n t s of PLP, was obtained. The injection o f G A H also resulted in a n inhibition o f D O P A decarboxylase, which was reversed by P L P added to the i n c u b a t i o n m e d i u m . A b o u t the same i n h i b i t i o n of the enzyme as that observed in vivo was observed in the presence o f 3.2 • 10 -a M G A H , whereas 10 -4 M G A H hardly inhibited it (Table IV). A t the time o f appearance o f convulsions, a d m i n i s t r a t i o n of P L P G H did n o t modify the activity o f b r a i n histidine decarboxylase, either in the absence or the presence of 10 -4 M PLP. The m e a n control value of 5 experiments in the absence of P L P was 3500 c o u n t s / m i n in the 14CO2 p r o d u c e d in the reaction, while the m e a n value o f 3 experiments with treated animals was 3800 c o u n t s / m i n . N o activation at all was Brain Research, 29 (1971) 111-122

VITAMIN

B6, B6

117

ENZYMES A N D C O N V U L S I O N S

TABLE V a-METHYL-DOPA (400 mg/kg, 1 h) ON BRAINGLUTAMATEAND DOPA DECARBOXYLASES ACTIVITYin vivo

EFFECT OF

The values are means ± S.E.M. Number of experiments in parentheses.

Control Treated Inhibition

DOPA deearboxylase*

Glutamate decarboxylase**

30.3 ± 0.8 (7) 8. 6 :~ 0.7 (7) 72

4.7 ~z 0.26 (5) 4.7 ± 0.13 (6) 0

* Counts/min • 10 8 in the 14CO2 produced in 5 rain of incubation in the conditions described in Materials and Methods. ** Counts/min • 10-3 in the 14CO2 produced in 30 min of incubation in the conditions described in Materials and Methods.

o b s e r v e d when P L P (10 -4 M ) was a d d e d to b r a i n h o m o g e n a t e s f r o m c o n t r o l o r t r e a t e d mice. I n one e x p e r i m e n t no effect on histidine d e c a r b o x y l a s e activity was observed 1 h after the a d m i n i s t r a t i o n o f G A H (2 g/kg).

Effects of a-methyl-DOPA One o f the aims o f the p r e s e n t study was to o b t a i n i n f o r m a t i o n o f the specificity o f g l u t a m a t e d e c a r b o x y l a s e inhibition, as c o m p a r e d with other B6 enzymes, in relation to the a p p e a r a n c e o f convulsions. Therefore, since the results shown a b o v e indicate t h a t D O P A d e c a r b o x y l a s e is inhibited b y P L P G H at least to the same extent as glutam a t e d e c a r b o x y l a s e at the m o m e n t o f convulsions, in o t h e r experiments it was intended to inhibit the f o r m e r enzyme w i t h o u t affecting the latter. F o r this p u r p o s e am e t h y l - D O P A was a d m i n i s t e r e d to mice at a dose o f 400 mg/kg. This c o m p o u n d is k n o w n to inhibit competitively D O P A d e c a r b o x y l a s e in vitro a n d to lower b o t h the level o f u r i n a r y d o p a m i n e f o r m e d f r o m exogenous D O P A a n d the c a t e c h o l a m i n e c o n c e n t r a t i o n in brain14,as,17-1L T h e results are shown in T a b l e V. One h o u r after t r e a t m e n t with a - m e t h y l - D O P A , D O P A d e c a r b o x y l a s e was 72 ~ inhibited, while no effect on g l u t a m a t e d e c a r b o x y l a s e activity was observed. O t h e r animals were injected with the same dose o f the d r u g a n d o b s e r v e d for m o r e t h a n 24 h to ascertain the presence or absence o f convulsions. As m e n t i o n e d previously, no a p p a r e n t a l t e r a t i o n o f b e h a v i o u r was observed. DISCUSSION

Specificity of glutamate decarboxylase inhibition in relation to convulsions T h e results o b t a i n e d in the experiments with P L P G H in vivo indicate t h a t neither the a m i n o t r a n s f e r a s e s studied n o r histidine d e c a r b o x y l a s e are affected by the action o f this c o n v u l s a n t h y d r a z o n e . H o w e v e r , D O P A d e c a r b o x y l a s e was even m o r e inhibited

Brain Research, 29 (1971) 111-122

1 18

R. TAPIA AND H. PASANTES

than glutamate decarboxylase at the moment of occurrence of convulsions. Since DOPA decarboxylase is directly involved in the synthesis of dopamine and norepinephfine, this inhibition could be related to the production of" convulsions through a consequent decrease in the concentration of such transmitter suspects. The results obtained with a-methyl-DOPA, however, seem to rule out this possibility, since " r.m the brain of mice treated with this drug, DOPA decarboxylase was notably inhibited (Table V) and the animals did not convulse. Futhermore, it is known that treatment with a-methyl-DOPA actually reduces the cerebral dopamine and norepinephrine levels 15A9. The fact that under these conditions glutamate decarboxylase activity was not affected strongly supports the conclusion that, although other B6 enzymes are inhibited by P L P G H and other convulsants, there is specificity of glutamate decarboxylase participation with regard to the biochemical mechanisms involved in the production of convulsions by certain drugs. The possible participation of serotonin~'also seems to be ruled out by the present results, since it is known that the serotonin concentration in brain decreases in similar manner to that of dopamine after the administration of a-methyl-DOPA 1°, and there is much evidence substantiating the concept that the same enzyme is responsible for the decarboxylation of DOPA and 5-hydroxytryptophan in mammalian tissues18, z6. Nor, probably, is histamine metabolism involved in the production of PLPGH-induced convulsions, since histidine decarboxylase was not affected by the administration of this hydrazone (see Results). As for GAH-induced convulsions, its mechanism is possibly similar to that of P L P G H convulsions 25, but the metabolic effects of G A H are apparently more complex than those of PLP hydrazones (see below). B6 enzyme activity in relation to P L P availability

The administration of P L P G H results in a decrease of about 60 ~ in the PLP content of mouse brain, and no PLP is detectable in the brain of GAH-treated animals 1 h after the injection of this drug 25. Therefore, these experimental conditions seem to be adequate to evaluate the extent of dependence of the different B6 enzymes upon the available PLP in brain tissue, in vivo, provided that the drugs do not directly affect the activity of the enzymes. The fact that the aminotransferases studied, as well as histidine decarboxylase, are not affected by P L P G H treatment, whereas DOPA and glutamate decarboxylase are strongly inhibited (Table VI), suggests that in vivo the former 4 enzymes have tightly bound PLP, while the activity of the latter two enzymes depends largely on free, loosely bound coenzyme. Two findings support this suggestion : first, that the addition of PLP to the incubation mixture completely reverses the inhibition of DOPA and glutamate decarboxylases; second, that neither the aminotransferases nor histidine decarboxylase are activated by PLP or P L P G H in vitro, whereas DOPA and glutamate decarboxylases are considerably activated (Table VI). P L P G H activates B6-enzymes in vitro most probably because PLP is liberated by hydrolysis 21. The decrease of PLP concentration in brain produced by P L P G H treatment seems to be due to a diminished rate of synthesis of the coenzyme as a consequence of Brain Research, 29 (1971) 111-122

VITAMIN B6, B6 ENZYMES AND CONVULSIONS

1 19

TABLE VI SUMMARYOF EFFECTSOF PLPGH AND GAH ON BRAINAMINOTRANSFERASESAND DECARBOXYLASES Unless otherwise indicated below, the experimental conditions were as in the present study. Enzyme

P L P G H #t vivo

G A H in "vivo

% Inhibition

% Inhibition

No PLP

With PLP

No P L P

With PLP

14 2 0

61",34"* 3 66

25** 0 70

64 42 0

46 82*** 0

5 61"**

Aminotransferase

GABA Aspartate Alanine Decarboxylase

DOPA Glutamate Histidine

P L P G H in vitro

G A H in vitro

% Activation (10 -4 M )

% Inhibition ( 3 . 2 . 1 0 -3 M )

(10 -4 34)

PLPGH

PLP

No P L P

With P L P

No P L P

With PLP

11 0 0

11 6 0

46§§ 98 74

29§§ 96 72

-5 0

--6

32 78§ --

36 80§ 0

43 80§§ .

30 40§§ .

12 35§

7 0§

Aminotransferase

GABA Aspartate Alanine Decarboxylase

DOPA Glutamate Histidine

.

.

References and notes for the previously reported data: * Ref. 23. ** 3.5 h after treatment with GAH (160 mg/kg)2L *** 3.0 h after treatment with GAH (2 g/kg) 28. § Ref. 24. §§ Ref. 22.

pyridoxal kinase i n h i b i t i o n21, since a linear relationship between pyrJdoxal kinase activity a n d P L P c o n t e n t has been observed zS. Thus, the decrease in P L P levels p r o b a b l y occurs m a i n l y at the expense o f free PLP, at least initially, a n d it would be expected that u n d e r these c o n d i t i o n s only the enzymes whose activity depends at least partially u p o n loosely b o u n d coenzyme would be affected, while those with strongly b o u n d P L P would be m o r e resistant. This is in agreement with the a b o v e - m e n t i o n e d e x p l a n a t i o n for the greater susceptibility to P L P G H t r e a t m e n t of D O P A a n d glutamate decarboxylase in c o m p a r i s o n with that o f the other enzymes studied. These results, taken together, s u p p o r t the conclusion that the effects o f P L P G H on b r a i n enzymes are only a consequence of the decrease o f P L P concentration. Brain Research, 29 (1971) 111-122

120

R. TAPIA AND H. PASANTES

A decrease of some unphosphorylated forms of vitamin B6 in rat brain, produced by a B6-deficient diet, caused only small non-significant changes in the activity of brain aspartate and alanine amino transferases, whereas in other tissues these enzymes were considerably inhibitedL In contrast, it is known that glutamate decarboxylase activity is inhibited proportionally to the decrease in brain PLP concentration produced by a B6-deficient diet 12. These results are consistent with the conclusion that in brain tissue the aminotransferases have more affinity for PLP than glutamate decarboxylase in vivo. The results of the experiments with GAH, which produces a decrease of PLP in the brain to values not detectable by an enzymic method 25, are difficult to explain on the basis of the effect of this drug on PLP levels. In fact, alanine and GABA aminotransferases and glutamate decarboxylase, are notably inhibited in vivo by GAH, but this inhibition is not reversed by the addition of PLP to the incubation medium (Table VI). The results of the experiments in vitro show that the inhibition of alanine and aspartate aminotransferases produced by GAH was not reversed by PLP (Table VI). This is in agreement with the postulate that GAH acts independently of PLP availability. These effects of GAH on the aminotransferases could be involved in the production of convulsions by some of the metabolic mechanisms cited by Watkins 27, but they do not seem to be related to PLP availability. SUMMARY

The activity of GABA, aspartate and alanine aminotransferases, as well as that of DOPA and histidine decarboxylases, was measured in brains of mice treated with L-glutamic acid-y-hydrazide (GAH) and with pyridoxal phosphate-v-glutamyl hydrazone (PLPGH). At the doses used both substances produced a marked decrease in the concentration of brain pyridoxal phosphate, and PLPGH produced fatal convulsions. PLPGH did not affect the aminotransferases or histidine decarboxylase, either in vivo or in vitro. DOPA decarboxylase, like glutamate decarboxylase, was activated by PLPGH in vitro and inhibited in vivo at the time of occurrence of convulsions; the inhibition in vivo was completely reversed by the subsequent addition of pyridoxal phosphate to the incubation mixture. Treatment with GAH produced an inhibition of alanine aminotransferase quantitatively similar to that previously reported for GABA aminotransferase, and the inhibition was not reversed by pyridoxal phosphate added to the assay system. DOPA decarboxylase was also inhibited, but the addition of the coenzyme reversed the inhibition. In vitro, GAH inhibited the aminotransferases studied, as well as DOPA decarboxylase; the inhibition of the latter, like that of glutamate decarboxylase, was partially reversed by pyridoxal phosphate, whereas the inhibition of the aminotransferases was practically unaffected by an excess of the coenzyme. Treatment with a-methyl-DOPA produced a strong inhibition of DOPA decarboxylase, but glutamate decarboxylase was not affected under these conditions. Brain Research, 29 (1971) 111-122

VITAMIN B6, Be ENZYMES AND CONVULSIONS

121

W e conclude t h a t : (a) with r e g a r d to the Be enzymes studied in the p r e s e n t p a p e r , the i n h i b i t i o n o f g l u t a m a t e d e c a r b o x y l a s e is a p p a r e n t l y specifically related to the p r o d u c t i o n o f certain d r u g - i n d u c e d convulsions; (b) in vivo, the a m i n o t r a n s f e r a s e s a n d histidine d e c a r b o x y l a s e p r o b a b l y possess f i r m l y b o u n d p y r i d o x a l p h o s p h a t e , b u t the D O P A a n d g l u t a m a t e d e c a r b o x y l a s e s depend, at least partially, u p o n free, loosely b o u n d c o e n z y m e ; a n d (c) the effects o f P L P G H on Be enzymes in vivo seem to be due solely to the decreased c o n c e n t r a t i o n o f p y r i d o x a l p h o s p h a t e , whereas G A H a p p a r e n t ly acts i n d e p e n d e n t l y o f the availability o f the coenzyme. ACKNOWLEDGEMENTS

This w o r k was s u p p o r t e d in p a r t by a g r a n t f r o m the Brown H a z e n F u n d Research C o r p o r a t i o n . The a u t h o r s wish to t h a n k D r G. H. Massieu for c o n t i n u o u s e n c o u r a g e m e n t t h r o u g h o u t this investigation.

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