Effects of GAD and GABA-T inhibitors on GABA metabolism in vivo

GABA Nrun)frcrnsmissi~,,l Brain Rrwurch Bulldn,

Vol. 5, Suppl. 2, pp.

621-625.

Printed

in the U.S.A

Effects of GAD and GABA-T Inhibitors on GABA Metabolism In Vivo P. V. TABERNER, University

TABERNER,

C. B. CHARINGTON

AND J. W. UNWIN

Department of Pharmacology of Bristol Medical School, University Bristol BS8 1 TD, U.K.

P. V., C. B. CHARINGTON

AND J. W. UNWIN. Effius

Walk

of GAD and GABA-T

inhihifors

on GABA

mrfabotism in viva. BRAIN RES. BULL. 5: Suppl. 2, 621-625, 1980.-Methods are described for the investigation of GABA metabolism in vivo following the administration of convulsant or anticonvulsant drugs to rats and mice. The ability of penicillins to inhibit glutamate decarboxylate did not correlate with their convulsant potency and no detectable change occurred in GABA metabolism in vivo following a dose of penicillin G sufficient to produced epileptiform spiking of the EEG. Ethanolamine-0-sulphate and sodium valproate both increased the relative incorporation of “‘C from glucose into GABA at minimally anticonvulsant doses. L-penicillamine, which was convulsant at high doses, inhibited GAD at equivalent concentrations and reduced the incorporation of 14Cinto GABA. The role of the GABA system in the action of these drugs is discussed. GABA-T inhibitors

GAD

GABA

Penicillin G

THE principal aim of this paper is to describe means for correlating the effects of drugs which are used to manipulate the GABA system with the neurochemical changes occuring in viva. Neurochemical methods have several disadvantages compared to continuous electrical recording or behavioral observation, since they are generally destructive and require the removal of tissue for analysis. Animals can therefore only be used either in a control or in an experimental capacity and the biological variation between animals adds to the variability of the results obtained. Many metabolites, including GABA, have fairly short half-lives and any postmortem changes have to be taken into account in assaying the level of the metabolite in the brain. Enzyme levels, measured in vitro, often given misleading information about the activity of the enzyme in viva, particularly when a reversible enzyme inhibitor has been administered, since the inhibitor is effectively diluted by the enzyme extraction procedure [ 11. It is with the intention of overcoming this last problem that irreversible catalytic enzyme inhibitors have been developed [ll]. These agents also have the advantage of being longer acting and, in the case of GABA-T inhibitors, are potentially useful as anti-epileptic drugs. With regard to glutamate decarboxylase (GAD) inhibitors, it has been found that the onset of convulsions as a result of the administration of such drugs, correlates well with a reduction in GABA synthesis and presynaptic inhibition as well as an eventual fall in brain GABA level [ 1, 10, 171. From the rapid onset of convulsions induced by GAD inhibitors, it can be concluded that newly synthesized GABA is preferentially released by the inhibitory nerve terminals and that the overall tissue level of GABA may not

Copyright

‘; 1980 ANKHO

International

Ethanolamine

sulfate

Sodium valproate

necessarily reflect the functional capacity of GABA-mediated inhibitory neurones. In the present work, the inhibitory potency of a range of penicillins against GAD has been compared with their convulsant potency when administered directly into the brain. Also, GABA synthesis has been measured in vivo in the cerebral cortex of rats which have received a unilateral dose of penicillin, placed stereotaxically in one hemisphere. Since the penicillin thus administered does not penetrate to the contralateral hemisphere, it has proved possible to examine GABA metabolism under control conditions in the same animal. This procedure eliminates the major disadvantage of requiring a separate animal as control. With regard to GABA metabolism by GABA-aminotransferase (GABA-T), there is now strong evidence that the inhibition of this enzyme potentiates GABAmediated inhibition and has consequently an anticonvulsant action [2, 11, 151. There is still some doubt, however, concerning the site of action of the anti-epileptic agent sodium valproate, which was originally believed to act by inhibiting GABA-T, but which is now known to be less specific in its actions [5,7]. However, the GABA released by inhibitory neurones has to be retaken up into the neurone or into glial cells before it can be metabolized, and the inhibition of the GABA uptake process should provide an alternative means of potentiating the synaptic actions of endogenously released GABA [5,7]. L-2,4_Diaminobutyric acid (L-DABA), which inhibits GABA uptake into neurones has been shown to be anticonvulsant against drugs which act by blocking GABA synthesis [20]. The effects of GABA-T inhibitors and GABA uptake blockers on GABA metabolism in vivo have been examined in an attempt to correlate the concentration of

Inc.-0361-9230/80/080621-05$01.00/O

TABERNER,

622

CHARINGTON

AND UNWIN

drug required to produce a measurable anticonvulsant effect with that producing inhibition of GABA metabolism. A preliminary account of some of these findings has already been given [ 191.

3 hr in Formalin, coronal sections 0.5 mm thick were ohserved by light microscopy. The D-(U-Y) glucose (2.5 PCi, 1 pCi/mmol) was injected directly into the 3rd ventricle through a hole made on the

METHOD

injected slowly (1 pi/s for 10 set). Rats were killed by decapitation 5 min after the injection and the head dropped immediately into liquid N2. Equivalent areas of cortex (45-55 mg) were taken from the site of the penicillin injection and the contralaieral (control) hemisphere. The determination of the incorporation of ‘YZ into glutamate and GABA was determined as described previously [14]. Sodium valproate was kindly donated by Reckitt and Colman Ltd., Hull, England. Ethanolamine-0-sulphate (EOS) was synthesized by the method of Lloyd et al. [ 121. All other drugs and reagents were obtained from commercial sources.

midline

The rats used were adult male Wistar albinos weighing between 300 and 350 g. Mice were adult LACG of either sex weighing between 28 and 34 g. All animals were bred in this department. The convulsant potency of the penicillin was determined from CD,,, values obtained by the intracerebroventricular injection of the drugs using methods described previously [ 181, The criterion for convulsions was taken as a full tonic extension of both hind limbs. Glutamate decarboxylase activity was assayed in extracts of whole mouse brain purified to the level of Step 3 of the method of Wu et al. 1231(representing a 24-fold purification), and assayed by the isotopic method of Roberts and Simonsen 1161. Each assay was performed in triplicate. GABA-T was partially purified from whole mouse brain by the methods of Charington [3], to approximately 0.3 ulmg protein (1 unit=1 pmole product produced per min at 37”), and assayed by the isotopic method of Hall and KravitzrB]. GABA metabolism was assessed in mice with the intracerebroventricular injection of 5 &!i of D-(U-‘*C) glucose (22.5 @i/,umole) by the methods described previously [ 1,141. For the measurement of GABA metabolism in the rat, the animals were anaesthetised with urethane (1.85 glkg given IP in divided doses over 30 min). The trachea was cannulated and the head placed in a stereotaxic frame. The skull was exposed and burr holes made at the appropriate positions using a dental drill. The occurrence of epileptiform spike discharges in EEG was monitored using one channel of a George Washington 400 MD12 oscillograph with stainless steel pin recording electrodes. Stereotaxic injections of 1-s ~1 of the penicillins were made 2 mm below the surface of the cortex 4 mm lateral to the sagital suture and 5 mm caudal to the coronal suture. The injection site and penetration of the injected solutions were verified by the injection of 2 ~1 of pontamine sky blue dye. After 20 min the rat was perfused with 40% paraformaldehyde in 0.9% saline directly into the aorta. After fixing for

POTENCIES

Amoxycillin Ampicillin 6-Amino~niciUanic Penicillin G Penicillin V L-Penicillamine Flucloxacillin D-Penicillamine Cloxacillin

Acid

6.5 10 1.6 7.6 8.5 2.9 3.1 2.6 2.6

AND PENICILLAMINE

CD,,, (nmoIes/mouse)

Mean latency to convulsion (min)

>229 >219 >369 51.5 f 5.15 74.0 k 19.6 1737 + 76.5 72.0 k 13.8 2139 k 71.1 77.0 -c 10.8

1.94 r 0.50 1.62 ?z 0.27 2.24 t 0.28 2.12 t 0.22 43.5 tt 8.6 2.80 ” 0.20

CD,,, values ( -c 95% confidence limits) were calculated for drugs administered intracerebroventricularly. Ki values were measured with respect to L-glutamate (see Fig.

I).

The glucose

was

AND DISCUSSION

1

inhibition (&,

Drug

suture.

Although penicillin (usually penicillin G) has been very widely used as a means of producing focal epileptogenic lesions in the cortex, its mechanism of action is still unclear. Several workers have reported reduced GABA-mediated pre- or postsynaptic inhibition in the foci (6, 8, 131, but the precise mechanism whereby this effect is brought about has not been conclusively demonstrated [21]. Although several penicillin derivatives have been shown to be fairly potent inhibitors of GAD in vitro [4], this does not appear, from the results obtained here, to be a si~~cant factor in the generation of seizures. Several penicilIms (amoxycillin, ampicillin and 6-amino-penicillanic acid) were relatively insoluble at physiological pH and could only be tested up to the limit of their solubility, at which point they did not produce convulsions, see Table 1. Of the remaining drugs tested, all except D-penicillamine had similar short latencies of action. The delay before the onset of seizures following D-penicillamine possibly suggests a different mechanism of action from the other drugs tested. Inhibitor constant (K,) values were determined from Dixon plots of l/v against I with varying concentrations of L-glutamate and the nature of the i~ibition determined from

OF THE PENICILLINS

GAD

to the coronal

RESULTS

TABLE COMPARATIVE

2 mm rostral

623

DRUGS AND GABA METABOLISM TABLE 2 INCOKPORATIONOF'4CINTOGLUTAMATEANDGABAINVIVOINKATCOKTEX GABA 300 i”g

Penicillin G

Rat no.

2 3 4 5 6 7 Mean & SEM

2.4 4.3 0.8 4.3 0.7 0.9 3.0 2.34 + 0.60

Rat no. I 2 3 4 5 6 7 Mean i SEM

L-penicillamine 0.7 3.7 1.6 2.0 0.5 0.9 1.1 *1.50 + 0.41

1

Glutamate 300 I*g

Control

Penicillin G

Control

0.8

7.8

7.1 2.8 10.9 2.4 4.7 4.6 5.76 -+ 1.14

4.3 4.1 11.0 2.2 2.5 4.2 4.2 4.64 + I.11

60 a L-penicillamine 6.1 4.0 3.2 2.1 1.6 1.2 1.8 “2.86 i 0.65

Control 8.1 7.0 4.2 2.8 2.3 1.3 3.2 4.13 + 0.95

0.9 1.6 I.7 0.9 0.5 3.6 1.43 + 0.40

60 /-G Control 1.1

6.0 4.3 4.5 0.7 0.6 2.0 2.74 t 0.82

The data represent the incorporation of 14C into GABA and glutamate expresed as a percentage of the total soluble supernatant counts. Differences between the drug-treated and control hemispheres were analyzed using the Wilcoxon Matched-pairs signed ranks test. *experimental,
It-GLUI ,mMi

5

ID-PEN1

lmMl

FIG. I. Inhibition of Glutamate Decarboxylase by D-penicillamine. Each point is the mean of 3 observations. The lines were plotted by the method of least squares and the inhibitor constant (K,) calculated from the extrapolation of the point of intersection of the lines onto the abscissa of the Dixon plot (l/v against S). K,=2.6 mM.

Lineweaver-Burk double reciprocal plots of l/v against l/s. A typical example, for D-penicillamine, is shown in Fig. 1 which indicates that D-penicillamine, like all the other drugs tested, is a competitive inhibitor of GAD with respect to glutamate. The results are summarized in Table 1, From the CD,,, values, it is apparent that D- and L-peni~illamine are considerably less potent as convulsants than the other penicillins and, apart from these two drugs, the wide discrepancy between the Ki and the far lower concentration required to produce convulsions suggests that GAD inhibition is not likely to contribute to their convulsant action. The lack of effect of penicillin G compared to Lpeni~iuamine on the inco~oration of ‘*C glucose into glutamate and GABA is shown in Table 2 in which the paired data from individual rats is tabulated. The incorporation of 14C into glutamate, expressed as a percentage of the total soluble supematant counts, is very much less than that found in the mouse experiments. This presumably reflects the distance required for the **Cglucose to diffuse from the CSF to the cerebral cortex in the rat. Following a unilateral dose of 300 ,ug of L-penicillamine, both the incorporation of 14Cinto glutamate and GABA were reduced in relation to the untreated hemisphere. In all the pencillin G-treated rats, spiking was apparent in the EEG during the period of 14C metabolism, but following the administration of 300 pg L-penicill~ine a temporary depression of the EEG signal occurred which was not observed with the D-isomer. This unexpected phenomenon is being investigated further, Effects of Sodium Valproate and EOS on GABA Metabolism Mice were used throughout

these experiments

and the

624

TABERNER.

CHARINGTON

AND UNWlN

TABLE 3 EFFECT

OF EOS ON ‘Y INCORPORATION c/c of total supernatant

Time

0

12 hr 24 hr 48 hr

Glutamate

Aspartate

15.6 17.0 11.6 20.3

25.1 26.4 15.7 23.5

-t 2 5 +

2.2 1.9 1.5 1.3

-c t f -t

INTO AMINO ACIDS

3.4 3.2 1.8 2.9

38.2 39.5 47.2 37.6

GABA ~__ Glutamate (Mean ratio)

dpm

Neutral

-r t f ?

GABA

2.6 3.2 2.1 3.2

2.15 *4.40 2.51 3.36

t 0.38 + 0.80 t 0.47 -+ 0.53

0. I64 0.262 0.211 0.161

Results are expressed as the means 2 SEM of at least 6 observations. The values given represent the incorporation expressed as the % of the total supematant counts recovered from the brain tissue. The neutral faction includes glutamine, serine and alanine as well as unchanged glucose. *value>zero

time value, ~~0.05

(Student’s

f-test).

TABLE 4 EFFECT

OF VALPROATE

ON 14C INCORPORATION

Glutamate

% of total supematant dpm Neutral Aspartate

18.7 ? 2.0 16.4 + 2.4 24.8 2 2.1

25.1 2 3.4 15.4 2 3.1 27.9 -t 4.5

INTO AMINO ACIDS

GABA GABA

Glutamate (Mean ratio)

2.15 + 0.38 *4.08 2 0.21 3.03 c 0.42

0.164 0.321 0.192

Dose (m moles/kg) 0

0.54 1.08

34.0 t 2.6 37.4 t 3.8 42.2 i 5.1

For legend details see Table 3. *values>control value, pcO.05.

doses of sodium valproate and EOS chosen as those which had previously been shown to have an anticonvulsant effect against 3-mercaptopropionate-induced seizures [ 191. The results for EOS and sodium valproate are shown in Tables 3 and 4, respectively. EOS, which is a relatively specific inhibitor of GABA-T, produced a significant increase in the relative incorporation of 14C into GABA at 12 hr postinjection (0.4 pmole, ICV). The GABA incorporation was still raised at 24 and 48 hr postinjection, although incorporation into glutamate was unaltered. This indicates that there is an increase in GABA production relative to its breakdown following EOS. Sodium valproate produced a similar pattern of effects (Table 4) although, in this case, the maximum effect was observed 1 hr after injection of 0.54 mmoles/kg IP. Sodium valproate, although it may not act directly by inhibiting GABA-T, does appear to produce a relative increase in the rate of production of GABA. It should perhaps be

pointed out that the dose of valproate used in these experiments was only just sufficient to increase the CD,, of 3-mercaptopropionate [ 191. The concentration of valproate likely to be present in the brain after IP doses of this level will be well below those necessary to produce any significant inhibition of GABA-T [22], thus reinforcing the suggestion that valproate may be acting elsewhere on the GABA system. In conclusion, the use of pulse labelling with 14C precursors (in this case, glucose) can provide useful information on the relative rate of GABA synthesis occurring in vivo. The correlation of the doses of drug required to produce changes in GABA metabolism with those having detectable effects on the convulsive threshold are probably more valid in terms of discovering a mechanism of action than are results derived from individual enzyme assays in vitro.

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2. Anlezark, G., R. W. Horton, B. S. Meldrum and M. C. B. Sawaya. Anticonvulsant action of ethanolamine-0-sulphate and di-n-propylacetate and the metabolism of aminobutyric acid (GABA) in mice with audiogenic seizures. Biochem. Pharmac. 25: 413-417, 1976. 3. Charington, C. B. Ph. D. Dissertation, University of Saskatoon, Saskatchewan, Canada, 1977.

4. Charington, C. B. and P. V. Tabemer. Penicillin-induced conv&ions and inhibition of glutamate decarboxylase. Br. J. Pharmac. 66: 72P, 1979. 5. Curtis, D. R., A. W. Duggan and G. A. R. Johnston.

The inactivation of extracellularly administered amino acids in the feline spinal cord. Expf Brain Res. 10: 447-456, 1970. 6. Curtis, D. R., C. J. A. Game, G. A. R. Johnston, R. M. McCulloch and R. M. MacLachlan. Convulsive action of penicillin. Brain Rcs. 43: 242-245,

1972.

DRUGS AND GABA METABOLISM 7. Curtis, D. R., C. J. A. Game and D. Lodge. The in viva inac-

tivation of GABA and other inhibitory amino acids in the cat nervous system. Exp/ Brain Res. 25: 413-428, 1976. 8. Davidoff, R. A. Penicillin and inhibition in the cat spinal cord. Brain Res. 45: 638-642,

1972.

9. Hall, Z. W. and E. A. Kravitz. Metabolism of y-aminobutyric acid (GABA) in the lobster nervous system. I. GABA-glutamate transaminase. J. Neurochem. 14: 45-54, 1%7. 10. Horton, R. W. and B. S. Meldrum. Seizures induced by allylglycine, 3-mercaptopropionic acid and 4-deoxypyridoxine in mice and photosensitive baboons and different modes of inhibition of cerebral glutamic acid decarboxylase. Br. J. Pharmac. 49: 52-63, 1973. 11. Jung, M. J., B. Lippert, B. W. Metcalf, P. J. Schechter, P. Bohlen and A. Sjoerdsma. The effect of 4-amino hex-S-ynoic acid (y-acetylenic GABA, y-ethynyl GABA), a catalytic inhibitor of GABA transaminase, on brain GABA metabolism in viva. J. Neurochem.

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1977.

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1961.

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625

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1978.

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