CORRELATIVE CHANGES O F PYRIDOXAL KINASE PYRIDOXAL-5'-PHOSPHATE AND GLUTAMATE DECARBOXYLASE

IN BRAIN, DURING DRUG-INDUCED CONVULSIONS

Ricardo Tapia, Miguel PCrez de la Mora and Guillermo H. Massieu Deportamento de Bioquimica, lnstituto de Biologla

Universidad Nacional de Mexico MGxico D. F., Mexico

Numerous recent articles and reviews have been published on the y- aminobutyric acid (GABA) system,14 which in mammals is present only in the central nervous system due to the unique occurrence of L-glutamic acid decar- boxylase (GAD) in this tissue.5*6 The reaction catalyzed by this enzyme is the only known route of synthesis of GABA. On the other hand, the GABA-a- ketoglutaric aminotransferase (ABAT) , another key enzyme in the metabolism of GABA, is present in brain and also in other tissues.

In recent years, with the use of neurophysiological methods, it has been shown that GABA has a definite inhibitory action on several types of synapse^.^*^-'^ Furthermore, some recent work on motor neurons of the cerebral cortex" and other neuronal nuclei12 is in agreement with the idea that GABA is a natural inhibitory transmitter in the nervous system of vertebrates.

These data, together with the finding that the convulsions induced by several drugs occur at the moment when GAD activity and GABA concentration are decreased,l3-I7 could indicate that the unique occurrence of GAD and GABA in the nervous tissue of mammals is related to a specific function of this metabolic system in this particular tissue.

On the other hand, GAD is a B6-dependent enzyme which, in contrast to other Bs-enzymes, is particularly sensitive to the absence of pyridoxal phosphate (PALP), both in vitro6J8 and in vivo.lO Certain reports have shown that during the convulsions induced by some substances there is a decreased PALP con- centration in brain.ls22

This paper describes some of the results obtained in experiments carried out in our laboratory, designed to obtain some information on the role of GAD and GABA in the mechanism of the production of convulsions by some drugs and on the role of PALP in relation to GAD modifications during the convulsions.

The experimental approach to this problem has been to employ substances with convulsant action and to try to correlate this action with some biochemical modifications that can be demonstrated to occur at the same time as the ap- pearance of the convulsions. Some carbonyl-trapping agents, mainly the L-glu- tamic acid-y-hydrazide (glutamyl hydrazide) and amino-oxyacetic acid (AOAA) , and several derivatives of PALP, mainly hydrazones (PALP-hydra- zones), have been used.

Studies on GAD Activity and GABA Concentration During Convulsions

Both the glutamyl hydrazide and AOAA greatly increase the concentration of GABA in brainZ3az4 by inhibiting ABAT and they induce con- vulsions when administered at high d o ~ ~ . ~ , ~ ~ However, at small doses, the hydrazide is not an anticonvulsant, whereas AOAA possesses a powerful anti- convulsant action against convulsions induced by thiosemicarbazide28 and pentylenetetrazol.20 Another important difference between glutamyl hydrazide

251

258 Annals New York Academy of Sciences and AOAA is that the former inhibits GAD activity in V ~ V O , ' ~ , ~ ~ whereas AOAA and hydroxylamine, a compound with anticonvulsant and biochemical effects similar to those of AOAA,26jS0 do not affect GAD activity in vivo when adminis- tered at anticonvulsant doses.26 On the other hand, some PALP-hydrazones pro- duce convulsions and diminish GAD activity and GABA c o n ~ e n t r a t i o n . ~ ~ * ~ ~ , ~ ~

All these data were the basis for a study of GAD activity during convulsions produced in different experimental conditions, with the working hypothesis that an inhibition of this enzyme could be involved in the mechanisms of production of some types of convulsions, independently of the total concentration of GABA in the whole brain.

Accordingly, GAD activity was measured in the brain of mice treated with convulsant doses of glutamyl hydrazide and of AOAA. In the case of the hy- drazide, the enzyme was measured at various times after the administration of the The results are shown in FIGURE 1. GAD activity is progressively inhibited with time, reaching its lowest value at the moment of convulsions. This enzymatic activity is also inhibited during the convulsions produced by AOAA. In both cases, GABA levels were increased, while in animals treated with pyridoxal phosphate-y-glutamyl hydrazone (PALP-glutamyl hydrazone) the convulsions occurred simultaneously with a decrease of both GAD activity and GABA concentration (FIGURE 1 ) .

These results have been interpreted to mean that GAD-inhibition is indeed an important factor in the production of certain types of convulsions, the inhibition of ABAT activity accounting for the observed increase in GABA levelsS2 (see also TABLE 4, columns 6 and 7). In this regard, it is pertinent to mention that convulsions induced by anthranilic hydroxamic acid occur when the inhibition of GAD produced by this compound reaches its maximal value.I5 Furthermore, 2,4-diaminobutyric acid also produces convulsions when GABA levels are in- creased and GAD activity is inhibited.lg

If the decrease of GAD activity results in convulsions because of a diminished rate of formation of GABA, the possible inhibitory transmitter, it would seem paradoxical that convulsions occur with very high levels of this amino acid in brain. This fact may be explained by the different subcellular distribution of the enzymes of GABA metabolism. ABAT is a mitochondrial enzyme, while the particulated GAD is mainly in the axoplasm of the nerve Therefore, the level of GABA present at the synapses (presumably inhibitory synapses) would be diminished if GAD activity were decreased, although the total GABA concentration may be increased, since it would not be metabolized by the ABAT in the mitochondria, which would not seem important for the physiological effect of GABA. Some experiments were made in order to test the validity of this hypothesis under conditions in which the tissue has not been destroyed as it is in the determination of GAD activity.

At different times after the administration of glutamyl hydrazide or PALP- glutamyl hydrazone to mice, labeled glutamic acid ( 3,4-I4C) was injected intra- cranially. Two minutes later the mice were sacrificed and the percentage radio- activity in GABA in relation to that in glutamic acid, as well as the concentration of these amino acids, was measured in brain.36 The results showed a progressive decrease in the rate of GABA formation from glutamic acid, independent of the total brain GABA concentration, which was increased by the glutamyl hydraz-. ide and decreased by the PALP-glutamyl hydrazone. The decrease in the rate of GABA formation was maximal at the moment when convulsions occurred (FIGURE 1). From FIGURE 1 it is also apparent that in the experimental condi-

Tapia et al. : Changes with Drug-Induced Convulsions 400r

259

I I I I I I 2 3 4 TIME (hr)

FIGURE 1. Changes with time of total GABA, GAD activity and rate of formation of GABA from labeled glutamic acid (see text), in brain of mice treated with some convulsant substances. Symbols: 0 and 0, after treatment with L-glutamyl-y-hydrazide (2 g/kg); X, treated with AOAA (400 mg/kg) ; A, treated with PALP-glutamylhydrazone (205 pnoles/ kg): 1, GABA; 2, GABA formation; 3, GAD. Convulsions occurred 2 to 3.5 hr after the administration of the hydrazide, 10 to 20 min after AOAA, and 30 to 45 min after the hydra- zone. Data from References 32 and 36.

tions described, the measurenlent of GAD activity actually indicates the rate of GABA synthesis in vivo.

These results, therefore, support the hypothesis that the mechanism of certain convulsions involves a decrease of GABA concentration at the synaptic cleft, due to an inhibition of GAD activity. This idea has been suggested also by other authors on different ground^,^' and the data supporting it are therefore in full agreement with the postulate that GABA is a natural inhibitory transmitter in mammal brain.

Studies on Brain Pyridoxal Phosphate in Relation to GAD Activity During Convulsions

The deficiency of GAD in regard to PALP has been already mentioned. The activity of this enzyme is almost doubled by the addition of its coenzyme to the incubation medium, whereas the ABAT, for example, is not activated, but rather slightly inhibited, by PALP added in ~ i t r o . ~ ~ Furthermore, it has been shown that the activity of GAD is an almost linear function of the concentration of PALP

260 Annals New York Academy of Sciences

in brain.19 On the other hand, convulsions produced by isoniazid in the mouse,2o by PALP-isonicotinyl hydrazone in the cat,21 and by thiosemicarbazide and semicarbazide in the rat22 are accompanied by a decrease of PALP concentra- tion. Therefore, the participation of this coenzyme in the mechanism of inhibi- tion of GAD in vivo seems to be an important factor (see Reference 38 for a review). Some of the work made recently in our laboratory along this line is described in this section,

Pyridoxal hydrazones inhibit brain pyridoxal kinase in vitrqse and it has been postulated that the inhibitory effect of the PALP-glutamyl hydrazone on GAD activity in vivo is due to an inhibition of pyridoxal kinase." In order to ob- tain further information on these possible mechanisms of GAD inhibition, some other pyridoxal phosphate derivatives - the unsubstituted hydrazone, mono- methylhydrazone, isonicotinylhydrazone and thiosemicarbazone - were synthe- sized, and the activity of GAD and pyridoxal kinase was measured in the brain of mice treated with these drugs. Similar experiments were done with convulsant and nonconvulsant doses of glutamyl hydrazide at different times after treatment and also with convulsant doses of AOAA. For comparison, pentylentetrazol, which is different in chemical structure and possibly in mechanism of action, was also assayed. The effect of most of the PALP hydrazones on pyridoxal kinase has been reported previouslys1 (see TABLE 4) , but the action of the other substances on this enzyme is presented in detail here. The methods for measuring pyridoxal kinase and GAD activities were as previously d e s ~ r i b e d . ~ ~

TABLE 1 shows the activity of pyridoxal kinase in brain of mice treated with PALP-methylhydrazone, glutamyl hydrazide and AOAA. The methylhydrazone produced convulsions and inhibited the kinase, although to a lesser extent than the other convulsant hydrazones (TABLE 4). At the time of occurrence of con- vulsions after the administration of the glutamyl hydrazide (2 g/kg), the en- zyme was found to be 50.8% inhibited, while 6.5 hr after a nonconvulsant dose

TABLE 1 EFFECT OF THE ADMINISTRATION OF SOME CONVULSANT SUBSTANCES ON

PYRIDOXAL KINASE ACTIVITY IN BRAINS OF MICE

Treatment Pyridoxal Kinas* % Inhibition Timet

Control (NaCI 0.9%)

6.03 f 0.23 - 2 to 2.5 hr (8)

Glutamyl hydrazide 2.97 2 0.13$ 50.8 2 to 2.5 hr (2 g/kg) (8) (convulsions) Control 7.05 rfr 0.35 - 6.5 hr

Glutamyl hydrazide 4.62 rfr 0.24$ 34.5 6.5 hr (NaClO.9%) (4)

(160 mg/kg) (6)

(NaCI 0.9%) (8) Control 8.44 f 0.18 - 30 to 45 min

PALP-methylhydrazone 6.86 f 0.32$ 19 30 to 45 min (220 pmoles/kg) (9) (convulsions)

AOAA 8.19 f 0.35 2 5 to 15 min (400 mg/kg) ( 5 ) (convulsions)

pg of PALP produced/hr/80 mg of tissue. Mean f standard error. Number of animals in parentheses. t Time to death in convulsions (when indicated) or sacrifice of the animals. $ p < 0.001 (difference from control values).

Tapia et al.: Changes with Drug-Induced Convulsions 261

of the hydrazide (160 mg/kg), a smaller inhibition was observed. AOAA at a convulsant dose did not affect the activity of pyridoxal kinase (TABLE 1 ) .

These and previous results show, in general, that, among the substances studied, those that produce convulsions considerably inhibited both GAD and pyridoxal kinase activities, with two exceptions. AOAA did not affect the latter enzyme, and pentylenetetrazol had no effect on GAD. With the nonconvulsant substances or nonconvulsant doses, only a slight effect, or no effect, was observed (see TABLE 4) . On these grounds, it has been suggested that the inhibition of GAD activity produced by the convulsant PALP-hydrazones is secondary to their inhibitory effect on pyridoxal kinase activity. This postulate would apply also to the glutamyl hydrazide but not to AOAA, which did not inhibit the kinase. A relationship between GAD inhibition and the occurrence of convulsions, however, could be observed with the hydrazones, with the hydrazide, and with AOAA (TABLE 4) .

An interesting result was that the inhibition of GAD produced by the ad- ministration of the convulsant PALP-hydrazones was completely reversed by the addition of PALP to the incubation mixture (TABLE 4 ) . These data support the conclusion that the inhibition of GAD produced by these substances is due to a decreased amount of PALP available to this enzyme due to the inhibition of pyridoxal kinase. Apparently in the case of the glutamyl hydrazide, this mecha- nism is also present. However, the fact that the inhibition of GAD produced by this drug was only partially reversed by PALP suggests that a direct effect on the GAD protein also occurs. Since AOAA did not inhibit pyridoxal kinase, it was expected that the decrease in GAD activity after AOAA administration would only be partially reversed by the addition of PALP to the incubation mixture. Thus, its effect on GAD is probably a direct one, a conclusion that agrees with a theoretical approach on the fitness of this molecule on the active site of the enzyme.2 In this regard, it is of interest that the derivative formed between AOAA and PALP, the oxime-0-acetic acid, is an inhibitor of GAD in vitro in such a way that the addition of PALP does not reverse the inhibition?l

According to the foregoing discussion, it would be expected that substances that inhibit pyridoxal kinase should decrease PALP levels. Therefore, the con- centration of PALP was measured in brain of mice treated with the PALP- hydrazones and the other substances studied. For the extraction of PALP, the method used by BilodeauZ2 with perchloric acid was employed, with slight modifications. PALP was measured in the clear supernatants after centrifuging the perchlorate formed during neutralization, by the apotryptophanase method of Wada and coworkers,40 as modified by Minard;lQ the apotryptophanase was obtained by dialysis of the holoenzyme supplied by Sigma Chemical Company (Grade 11).

The results of these determinations are shown in TABLES 2 and 3. The three convulsant hydrazones decreased PALP concentration by more than 50% at the time of occurrence of convulsions, while the nonconvulsant hydrazones prac- tically did not modify it. Convulsant doses of the glutamyl hydrazide ( 2 g/kg) decreased PALP levels to a value not detectable by the method used. This lack of PALP in brain was observed both one hour after treatment, when only clonic convulsions occurred, and at the time of appearance of tonic convulsions (two to three hr after treatment). At 6.5 hr after the administration of a noncon- vulsant dose of the same hydrazide (160 mg/kg), a clear but much smaller effect on PALP concentration was observed. Convulsant doses of AOAA de creased it slightly, while pentylenetetrazol, which does not have any effect on

262

GAD activity, did not modify PALP levels when administered at a convulsant dose (TABLE 3).

Annals New York Academy of Sciences

Correlation among Pyridoxal Kinuse, Pyridoxal Phosphate, GAD and Convulsions

The results presented in this paper permit an attempt to correlate the parame- ters measured. In TABLE 4, the percent of the control value of some enzymes

TABLE 2 EFFECT OF THE ADMINISTRATION OF SOME PALP-HYDRAZONES ON

PALP CONCENTRATION IN BRAIN OF MICE

Treatment

Control (NaClO.9%)

PALP-glutamylhydrazone (205 pmoles/kg)

PALP-unsubstituted hydrazone (220 pmoles/kg)

PALP-methylhydrazone (220 pmoles/kg)

PALP-isonicotin ylhydrazone (220 pmoles/kg)

PALP-thiosemicarbazone (220 umoles/kn)

PALP' ( re /e of ussue)

I decrease Timet

1.80 2 0.04 (28)

0.79 f 0.04$ (8)

0.68 f 0.06% ( 5 )

0.81 f 0.03$ ( 5 )

1.68 f 0.08 ( 5 )

1.68 2 0.15 1 5 )

-

56

60

55

7

7

30 to 45 rnin (convulsions) 30 to 45 rnin (convulsions) 30 to 45 min (convulsions) 40 to 60 rnin

40 to 60 rnin . _ . r r -I

Figures are means f standard error. Number of animals is in parentheses. t Time of death in convulsions (when indicated) or sacrifice of the animals. t p < 0.001 (diflerence from control values).

TABLE 3 E~FFECT OF THE ADMINISTRATION OF SOME CONVULSANT SUBSTANCES

ON PALP CONCENTRATION IN BRAIN OF MICE

Treatment ( re /g of tissue) decrease Timet PALP' 46

Control 1.80 f 0.04 - - (NaClO.9%) (28)

Glutamyl hydradde 0 100 2 to 2.5 hr (2 g / W (4) (tonic conv.)

Glutamyl hydrazide 0 100 1 hr (sacr. in (2 g / b ) ( 5 ) clonic conv.)

Glutamyl hydrazide 1.68 If: 0.13 7 2.5 hr

Glutamyl hydrazide 1.27 f 0.07% 29 6.5 hr (160 mg/ke) ( 5 )

(160 W k e ) ( 5 ) AOAA 1.53 f 0.06s 15 5 to 20 min

(400 me/&) (4) (convulsions) Pentylenetetrazol 2.04 f 0.17 0 2 to 30 min

(75 me/ks) ( 5 ) (convulsions)

Figures are means f standard error. Number of animals is in parentheses. t Time of death in convulsions (when indicated) or sacrifice of the animals. The values of

control animals were pooled because no difference waa found among the mice sacrificed at different times.

t p < 0.001 (diflerence from control values). J p < 0.05 (Mererice from control values).

Tapia et al.: Changes with Drug-Induced Convulsions 263

and of PALP and GABA, in brain of mice treated with the substances studied, is illustrated, as well as the presence or absence of convulsant action. These data will now be discussed.

Convulsant PALP-hydrazones produced convulsions when pyridoxal kinase, PALP, and GAD were decreased, while the nonconvulsant hydrazones did not modify any of these parameters. Since ABAT activity is not altered under these conditions, the result of the GAD inhibition is a decrease in GABA concentra- tion. The level of this amino acid did not change after the administration of the nonconvulsant hydrazones. Convulsant doses of glutamyl hydrazide had even more notable effects on the kinase, PALP, and GAD, but the level of GABA was increased because of the inhibition of ABAT activity (see also Reference 32). Low doses of the hydrazide required a longer time to produce effects on the measured enzymes, and apparently the changes did not reach the threshold value for producing convulsions.

AOAA behaves differently from the PALP-hydrazones: it did not affect pyridoxal kinase and only diminished PALP concentration slightly; however, GAD activity was also notably diminished. GABA levels were increased, due to a strong inhibition of ABAT activity (TABLE 4) .

Finally, the convulsions induced by pentylenetetrazol apparently have a com- pletely different mechanism, which does not involve the GABA system or the synthesis of PALP, since neither PALP concentration nor GAD activity were modified by this compound. In rats, it has been reported that this convulsant decreased PALP levels in brain by about 50% ; this decrease has been interpre- ted to be probably not a cause but an effect of the convulsant state.22

TABLE 4 CORRELATION AMONG SOME METABOLIC EFFECTS in vivo OF SEVERAL SUBSTANCES

AND THEIR CONVULSANT ACTION'

Substance GAD

P ridoxal Without With Convul- kinase PALP PALP PALPt GABA ABAT sions

1

2

3

4

5

6

1

8

9 10

PALP-glutamyl- hydrazone PALP-unsubstituted hydrazone PALP-methylhydra- zone PALP-isonicontinyl- hydrazone PALP-thiosemi- carbazone Glutamyl hydrazide (2 g/kg, 2 to 3.5 hr) Glutamyl hydrazide (160 mg/kg, 2.5 hr.) Glutamyl hydrazide (160 mg/kg, 6.5 hr) AOAA Pentylenetetrazol

64 44 58 96

68 40 52 93

81 45 60 100

100 93 88 95

100 93 99 100

49 0 18 61

- 93 100 -

66 71 70 91 85 40 78 - 100 100 -

-

66 100 Yes

68 - Yes

- Yes

No

No

570 51 Yes

No

429 67 No 140 0 Yes

- Yes

-

- 96

- -

- -

- -

* The figures are percent of control value in all cases. For doses and time to convulsions

t Added to the incubation mixture. see TABLES 2 and 3. References for the previously reported data: 23, 25, 31, 32, 36.

264 Annals New York Academy of Sciences

According to the correlative changes shown in TABLE 4, and from the evidence of the inhibition of GABA synthesis during convulsions summarized in FIGURE 1, the mechanism of action of the convulsant PALP-hydrazones is postulated to be as follows: pyridoxal kinase inhibition + decrease in PALP concentration --* GAD inhibition + decrease in the rate of synthesis of GABA (possibly in certain inhibitory nerve endings) + convulsions. This sequence of events ap- parently occurs also after the administration of convulsant doses of the glutamyl hydrazide, in addition to its direct effect on GAD discussed previously.

When the percent of concentration of PALP is plotted against the percent of activity of pyridoxal kinase, using the value obtained in the different experi- mental conditions tested, a straight line with an intercept at about 50% of pyridoxal kinase activity is obtained (FIGURE 2) . This value corresponds to that in the animals treated with a convulsant dose of the glutamyl hydrazide, and it suggests that the rate of PALP degradation is probably greater than that of its synthesis, since at 50% of activity of the kinase there is no detectable PALP in brain. The rate of PALP degradation is probably not altered under any of the conditions tested, since with the exception of point 8 a straight line can be drawn through all the points, Another possibility that could account for the observed intercept on pyridoxal kinase activity is that the available amount of ATP, which is a substrate for the enzyme, would be diminished as a consequence of the hyperactivity of neurons. However, on these grounds it would be difficult to explain the linearity of the graph.

The activity of GAD as a function of PALP concentration, plotted again using the percent of control values, is shown in FIGURE 3. With the exception of the animals treated with AOAA, in which a notable inhibition of GAD and only a slight change in PALP was observed (point 9), a linear relationship between these two parameters was observed. The agreement of this graph with that obtained by Minard with BB-deficient or methylhydrazine-treated micelB is noteworthy. In this regard, it is of interest to note that the change in threshold for the isoniazid-induced convulsions is parallel to the change in brain PALP concen-

401 20

PYRIDOXAL KINASE % of control FIGURE 2. PALP concentration as a function of pyridoxal kinase activity. Symbols: 0, no convulsions; 0, convulsions. The experimental conditions for each point are indicated by the corresponding numbers in TABLE 4.

Tapia et al.: Changes with Drug-Induced Convulsions 265

‘y I I 1 I

20 40 60 80 100 PALP per cent of control

FIGURE 3. GAD activity as a function of PALP Concentration. Symbols: 0, no con- vulsions; e, convulsions. The experimental conditions for each point are indicated by the corresponding numbers in TABLE 4.

tration during ontogeny in the rat.41 From the value of the intercept in FIGURE 3 (point 6) it can be tentatively concluded that about 20% of the GAD present in brain has bound PALP and therefore can not be affected by substances that diminish the concentration of the coenzyme.

In regard to the convulsant action of the substances employed, FIGURES 2 and 3 allow a comprehensive view: pentylenetetrazol causes convulsions in short periods of time and has no effect whatsoever on PALP concentration or GAD activity. Therefore, its convulsant effect, as generally accepted, does not involve either the GABA system or PALP metabolism (points 10 in FIGURES 2 and 3 ). AOAA- induced convulsions are probably due to the inhibition of GAD activity not in- volving a decrease in PALP concentration (point 9 in FIGURE 3). Convulsions produced by the glutamyl hydrazide and by the three convulsant PALP- hydrazones result apparently from the inhibition of GAD produced as a conse- quence of the low levels of PALP, a condition determined by the inhibition of pyridoxal kinase activity (points 1 to 3 and 6 in FIGURES 2 and 3 ) .

Although a critical level for the production of convulsions seems to exist at about 60% of PALP concentration and GAD activity, convulsions do not occur in some conditions in which a lower amount of PALP and G A D is observed, for instance after a BB-deficient dietI9 or after the administration of o-methyl- p y r i d o ~ i n e . ~ ~ Many factors thus seem to be involved in the mechanism of con- vulsions besides those implied in this paper. However, in the case of the PALP- hydrazones studied, the decrease of endogenous PALP by inhibition of pyridoxal kinase seems to be the initial event in the mechanism of production of this type of convulsions. At least in this case and in the case of glutamyl hydrazide and AOAA, convulsions probably occur as a result of the inhibition of GAD and the consequent lack of GABA at the synapses, possibly certain inhibitory synapses.

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