GABA metabolism in O2-induced convulsions

GABA

Nrurorrunsmission Bulldin.

Brain R~wwd~

Vol. 5, Suppl. 2, pp. 789-792. Printed in the U.S.A.

GABA Metabolism in O,-Induced Convulsions MORRIS D. FAIMAN, Department

KATSUJI

of Pharmacology

HAYA,

JOHN A. ZEMPEL

and Toxicology,

AND RICHARD

University of Kansas,

Lawrence,

L. SCHOWEN KS 66045

FAIMAN, M. D., K. HAYA, J. A. ZEMPEL AND R. L. SCHOWEN. GAB.4 metabolism in Orinduced convulsions. BRAIN RES. BULL. 5: Suppl. 2,789-792, 1980.-The effect of increased oxygen pressure on cortical GABA metabolism

was investigated. Increased oxygen pressure decreased cortical GABA, GAD, and glutamate, and increased glutamine. Marked increases in cortical GABA over controls were found in mice pretreated with gabaculine, a specific GABA-T inhibitor, and exposed to either 4% or 106% of 6 atm of oxygen. The increase appears to be a pressure effect. Gabaculine failed to protect mice from oxygen convulsions although brain GABA was increased approximately 208%. Disulfiram, protective against oxygen convulsions did not prevent the oxygen-induced decrease in cortical GAD and GABA. The decrease in GABA by oxygen appears to be due to the inhibition of GAD. Oxygen

GABA metabolism

ConvuIsions

THE toxicity of hyperbaric oxygen (OHP) to the mammalian nervous system is characterized by overt clonic-tonic sei-

zures. Although this effect on the CNS has been known for over 100 years, the biochemical mechanism(s) by which these convulsions occur remain(s) unknown. Recent evidence suggests that ~-~inobuty~c acid (GABA) is an inhibitory neurotransmitter in mammalian brain (71. As such, decreased brain GABA has been implicated as a mechanism by which many drugs induce seizures. Unfortunately not all convulsants decrease brain GABA, and therefore its role in seizures remains controversial [I8]. Although the role of GABA as a mechanism by which drugs induce seizures is inconsistent for a number of convulsive agents, oxygeninduced seizures appear to correlate well with lowered brain GABA [ 151. Suggestions have therefore been made that oxygen convulsions occur as a result of decreased brain GABA. It has further been proposed that OHP not only decreases brain GABA, but also glutamic acid decarboxylase (GAD), and that these together influence the effectiveness of the GABA system with respect to its modulation of brain excitability [ 161. In view of the controversial role of brain GABA in oxygen convulsions, studies were carried out to further study the relationship between changes in brain GABA and the susceptibility of animals to oxygen convulsions. METHOD

Male Swiss Webster albino mice (WA/ICT) (A.R.S. Sprague-Dawley, Madison, WI., USA) weighing 25-38 g, were used in all experiments. Oxygen exposures were carried out in a hyperbaric chamber in which mice could be sacrificed inside the chamber without the necessity of decompression [9]. The cerebral cortex was chosen as the brain area for investigation. This region freezes rapidly in the intact unshaven mouse [41. Furthermore, electrical recordings from oxygen-induced convulsions suggest there is no consistent origin in the brain during paroxysmal electrical discharges [61. Mice were exposed to 6 atm of 100% oxygen for 0 or 16 min. These exposure times reflected a percentage of the time taken for 50% of the mice to convulse (CT,,,), and corresponded to 0 and 100% of the CT,,,. Mice were sacrificed by immersion in liquid nitrogen, cere-

Copyright

0 1980 ANKHO

International

bra1 cortex excised, and the various amino acids and enzymes determined. GABA was determined by the method of Collins et al. [2], GAD and gamma aminobutyric acid transaminase (GABA-T) by the method of Van Felder [ 111, glutamate by the method of Folbergrova et al. 1.51and glutamine by the method of Buttery and Rowsell [l]. RESULTS

The effect of 6 atm of 100% oxygen on cerebral GABA, GAD, glutamate, GABA-T and glutamine is shown in Table 1. A statistically significant decrease in cerebral GABA and glutamate was found at all time periods investigated. GAD decreased approximately 14% in mice sacrificed after exposure to 6 atm of oxygen for 16 min. OHP also inhibited GABA-T, but this was found only initially, and during the onset of seizures. Glut~ine levels increased with the greatest increase found during seizure onset. Gabaculine, a specific inhibitor of GABA-T [8,10] was used to study GABA turnover. Gabaculine had no effect on GAD activity, but inhibited GABA-T with a concommitant increase in GABA. The effect of 6 atm of 4% and 100% oxygen on brain GAB A after pretreatment with gabaculine is shown in Fig. 1. Gabaculine alone increased brain GABA by approximately 300%. Exposure of gabaculine-treated mice to oxygen further increased brain GABA. Of interest was the observation that similar increases were observed at both oxygen pressures. This would appear to suggest that the increase in brain GABA may be due to pressure per se and not the oxygen tension. The effect of gabaculine as an oxygen anticonvulsant is shown in Table 2. Mice given gabaculine showed marked increases in brain GABA, yet, no protection from oxygen convulsions was observed. DISCUSSION

In considering the role for GABA in oxygen-induced convulsions, two questions must be addressed. These are, the role of brain GABA in oxygen-induced convufsions, and the mechanism by which oxygen decreases brain GABA. Exposure of mice to 6 atm of 100% oxygen produced approximately a 23% decrease in cortical GABA. This decrease occurred as soon as the 6 atm pressure was reaehed (0% of the CT&, and remained at this lowered level through-

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790

TABLE EFFECT OF OXYGEN

Treatment

GABA (PmoYg)

1

ON GABA METABOLISM

Glu (pmolg)

Gin (PmoUg)

GAD @moVg/hr)

GABA-T (pmoilgihr)

Control

4.32 + 0.8

15.59 r 0.33

5.02 + 0.25

22.5 rt 1.8

70.6 -r 3.3

0% CT,,, (% of con)

3.33 k 0.16t (77.1)

14.03 -t 0.56* PO)

5.88 + 0.41 (117.1)

21.6 t 1.3 (96)

62.6 -c 3.4 (88.7)

lOQ% CT,,, f% of con)

3.32 2 0.131 (76.9)

13.92 t 0.21* (89.3)

5.38 t 0.1 i$: (107.2)

19.3 L 1.2 (85.8)

67.2 f 2.6 (95.2)

Convulsed (% of con)

3.30 2 0.141 (76.4)

13.24 r 0.3W (84.9)

6.55 + 0.331 f130.5)

21.7 t 0.9 (96.4)

61.4 t 2.2 (87)

*Significant @<0.05) from control (SNK). tSignificant (~~0.01) from control (SNK). SSignificant (p
and 100% CT,, (SNK).

TABLE 2 EFFECT OF GABACULINE

Dose

level @&kg)

Median time to hy~~tivity (see)

ON HYPERACTIVITY AND TONIC CONVULSIONS EXPOSED TO 6 ATA 100% 02

Median time to tonic convulsions (see)

Control

614 (n= 12)

908 (n= 10)

50

738 (n=ll)

1184 (n= 12)

Control

641 (n=7)

1065 (n=8)

75*

936 (n=6)

t

IN MICE

% of control GABA content

GAB A-T activity

234

46

108

253

38

100

GAD activity

*Animals exhibited sedation. 16 out of 8 animah did not convulse prior to the stop time of 60 min.

out the oxygen exposure

period

including

the onset

of sei-

zures. Cortical GAD decreased approximately 14% but this decrease was found only when mice were exposed to oxygen for 16 min (100% of the CT,,) (Table 1). Although the 14% decrease in GAD was not statistically significant, the same quantitative decrease was found in earlier studies [3]. In those previous studies, statistical significance was found because of the larger sample size employed. Gabaculine specifically inhibits GABA-T, resulting in increased GABA. Of interest was the observation that in mice pretreated with gabaculine and exposed to 6 atm of either 4% or 100% oxygen, cerebral GABA increased above levels found in mice treated only with gabaculine (Fig. 1). This finding suggests that GABA turnover is increased. However, since similar increases were observed at both 6 atm of 4% and 100% oxygen, it appears that the observed increase in GABA is due to the pressure and not oxygen. This is suggested from studies by others in which decreased brain GABA was found to be a function of the partial pressure of oxygen [14]. The studies reported here do not appear to support the suggestion that decreased GABA is responsible for the onset

of oxygen convulsions. Several lines of evidence would seem to support this conclusion. For example, similar decreases in GABA were found at both (YTO and 100% of the CT,,. Yet, at 0% of the CT,, no evidence of central oxygen toxicity was apparent. It has been suggested that both GABA and GAD must be considered together in explaining seizure susceptibility. Thus, the expression REGABAwas developed to reflect the state of excitability of the brain with respect to changes in both GABA and GAD [ 171. However, in our present studies in mice exposed to 6 atm of 100% oxygen for X6min (100% of the CT,& a RI&W value of 86.3% was calculated, a number considerably higher than the proposed 65% critical REGARD value. Even during the onset of seizures, the REGABGC&Ulated was 97.5%. Additional evidence suggesting that GABA does not seem to play a role in the susceptibility of animals to oxygen convulsions stems from in vivo studies with both gabaculine and disulfiram. Pretreatment of mice with gabaculine increased brain GABA approximately 200%, yet no protection from oxygen convulsions was found (Table 2). Furthermore, pretreatment with disulkam did not prevent the oxygen-induced decrease in GABA and GAD, and yet protected against oxygen convulsions 133. Suggestions have

791

GABA IN 0, CONVULSIONS been made that the subcellular distribution of GABA, for example, the concentration of GABA in the synaptic cleft, may be more important in modulating excitability than overall brain GABA levels. However, we have found that in rats exposed to 6 atm of 100% oxygen, GABA uptake in cortical synaptosomes was markedly inhibited (unpublished data). Therefore, since inhibition of uptake should theoretically increase GABA in the synapse, oxygen should actually not produce convulsions. The mechanism by which oxygen at high pressure decreases brain GABA remains speculative. Oxygen has been shown previously to inhibit GAD activity both in vitro [13] and in vivo [3,12]. It has therefore been suggested that decreased brain GABA is due primarily to GAD inhibition. However, since oxygen also has been found to decrease brain glutamate (Table l), this could account for the lower GABA level. At steady-state, the change in brain GABA depends on the balance between GABA synthesis and metabolism. That is: dG _= dt

v

kc;,,, G~~(Ec.MJ _ kc;ABA-T G(ET.ABA-T) KGkBA-T+ G

Algebraic rearrangement final expression:

dG = G

and differentiation

results in the

VGABA-’ max

V,,4p

’

for the reaction for the

of the GAD/glutamate

reaction

of the

GABA-

EGA,)and Ec;ABA_l.=concentration of the GAD and GABA-T enzymes; K”*“=dissociation enlyme-substrate

constant complex;

K$,ABA-T=dissociation constant enzyme-substrate complex; G=concentration

for for

the

glutamate/GAD

the

GABMGABA-T

of glutamate;

percent change in (GABA).

e V”i\D and nldX

qGabaculine

+ lDD% 02 + 4% 02

a lODmg/kg @ 1 Gabxuline Prior to O2 Exposure. b

&og:

in 0.9% Saline 4 hrs.

%%N2

Significantly Different from Gabacullne Treated and from Control (p I 0.05) + Significantly Different From Control (p 1. 0.05) i+ S.E.M. (n =3)

l

FIG. 1. Mice were pretreated with gabaculine in a dose of 100 mgikg IP, four hours before exposure to 6 ata of either 4% or 100% 0,. After exposure for 0,4, 8, 12, and 16 min, which corresponded to 0, 25, 50, 75 and 100% of the CT,,,, the animals were sacrificed and cortical GABA determined.

of GABA;

GLu=concentration dG 7f=

0 Gabaculine m Control

G m

KGABA-T

Definitions

kCAHA_T=rate constant TiGABA complex;

Percent d CT,,

c!I Gabaculine

-. dEc.m EM,,

kc;AI)=rate constant complex;

CORTl CAL GABA FOLLOWI NG PRETREATMENT WITH GABACULINE’ AND SUBSEQUENT EXPOSURE TO 6 ATA OF EITHER 4% 0: OR looA O2

= percent change in [GAD] VGABA-T max

enzymatic processes metabolism.

are the maximal velocities for the two concerned with GABA synthesis and

From the equation it is apparent that the percentage change in (GABA) is a function of the percentage change in (GAD) since the concentration of glutamate does not appear in the final equation. The relationship between the change in (GABA) and the change in (GAD) is not a direct one since

other parameters such as KGgB”-‘r the V,,, on (GAD) and (GABA-T) and the concentration of (GABA) are found in the equation. In conclusion, decreased brain GABA does not appear to play a major role in oxygen-induced convulsions. It also seems that decreased GABA is due mainly to the inhibition of GAD by oxygen, and that lowered levels of glutamate do not contribute to decreased brain GABA. ACKNOWLEDGEMENTS

This research was supported in part by a grant from the National Institute of General Medical Sciences, grant number POl-22357 and the National Institute of Neurological and Communicative Disorders and Stroke, grant number NS-07797.

792

FAlMAN

!-.‘I .‘\I..

REFERENCES I. Buttery, P. J. and E. V. Rowsell. Enzymic assays for ammonia and L-glutamine in tissue extracts. Anaiyr. Birtchem. 39: 297310, 1971. 2. Collins, G. G. S. GABA-

3.

4.

5.

r 6.

oxoghttarate transaminase, glutamate decarboxylase, and the half-life of GABA in different areas of rat brain. Biochem. Pharmac. 21: 2849-2859. 1972. Faiman, M. D., R. J. Nolan, C. F. Baxter and D. E. Dodd. Brain ~-~inobuty~c acid, ghuarnic acid decarboxylase, glutamate, and ammonia in mice during hyperbaric oxygenation. J. Neurochem. 28: 861-865, 1977. Ferrendelli, J. A., M. H. Gay, W. G. Sedgwick and M. M. Chang. Quick freezing of the murine CNS: Comparison of regional cooling rates and metabolite levels when using liquid nitrogen or Freon-12. J. Nerrruchem. 19: 979-987. 1972. Folbergrova, J., J. V. Passonneau, 0. H. Lowry and D. W. Schulz. Glycogen, ammonia and related metabohtes in the brain during seizures evoked by methionine sulphoximine. J. Neurochem. 16: 191-203, 1%9. Hare]. D., D. Kerem and S. Lavy. The influence of high oxygen pressure on the electrical activity of the brain. Electroenceph.

Clin. Nrurophysid. 26: 310-317, 1%9. 7. Kmjevic, K. Glutamate and ~-~inobuty~c

10. Rondo, R. R. and F. W. Bangerter. The in 13irciinhibiiton of gaba-transaminase by gabaculine. Bioclzem. hiophys. Re\. C’omtrzzrrz.76: 12761281, 1977. Il. Van Gelder. N. M. Glutamate dehydrogenase, glutamic acid decarboxvlase. and GABA amino transferase in epilentic mouse cortex. C&r. J: Ph.v.siol. Phurmoc. 52: 952-959, i974. 12. Wood, J. D.. W. J. Watson and N. E. Stacey. A eomparative study of hyperbaric oxygen-induced and drug-induced convulsions with particular reference to y-aminobutyric acid metabolism. J. Nwvchem. 13: 361-370. 1966. 13. Wood, J. D., W. J. Watson and A. J. Ducker. Oxygen poisoning in various mammalian species and the possible role of gammaaminobutyric acid metabolism. .I. Nrurochcnr. 14: 1067-1074, 1967. 14. Wood, J. D., W. J. Watson and G. W. Murray. Correlation between decreases in brain y-aminobutyric acid levels and susceptibility to convulsions induced by hyperbaric oxygen. J. N~wwhcwz.

Ncrcrochem.

acid in brain. Nnt-

ure 228: 119-124, 1970. 8. Matsui, Y. and T. Deguchi. Effects of gabacuhne, a new potent inhibitor of gamma-aminobutyrate transaminase, on the brain pamma-aminobutyrate content and convulsions in mice. I& Sci. 20: 1291-1295, 1977. 9. Nolan, R. J. And M. D. Faiman. Brain energetics in oxygeninduced convulsions. J. Neurochem. 22: 645-650, 1974.

16: 281-287,

1969.

15. Wood, J. D. Seizures induced by hyperbaric oxygen and cerebral y-aminobutyric acid in chicks during development. J. 17: 573-579,

1970.

16. Wood, J. D. and S. J. Peesker. A correlation between changes in GABA metaboIism and isonicotinic acid hvdrazide-induced seizures. Bruin Res. 45: 489-498, 1972. 17. Wood, J. D. and S. J. Peesker. Development of an expression which relates the excitable state of the brain to the level of GAD activity and GABA content with particular reference to the action of hydrazine and its derivatives. J. Neurwhem. 23: 703712, 1974.

18. Wood, J. D. The role of ~-aminobuty~c acid in the mechanism of seizures. PI-c~~.Ne~ro~i(}~. 5: 77-95, 1975.