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Effect of certain drugs on the γ-aminobutyric acid system of the central nervous system
Biochemical Pharmacology, 1966, Vol. 15,pp.49-54.Pergamon PressLtd.,Printedin GreatBritain.
EFFECT OF CERTAIN DRUGS ON THE r-AMINOBUTYRIC ACID SYSTEM OF THE CENTRAL NERVOUS SYSTEM I. A. Neuro-biochemistry
SYTINSKII
and T. N.
PRIYATKINA
Laboratory, A. A. Ukhtomskii University of Leningrad
Physiological Institute,
(Received 19 July 1965; accepted 7 October 1965)
Abstract-The central nervous system stimulants caffeine and centedrin increase the y-aminobutyric acid content of the brain by 25-30 per cent. Administration of andaxin, which caused a general depression, results in a 23 per cent decrease in the brain ~-aminobutyric acid level. Intraperitoneal injection in rats of picrotoxin, metrazol, caffeine, centedrin, hept-amyl, 20-28 M.D., barbamil and andaxin does not produce changes in the enzymatic activities of L-glutamic acid decarboxylase and y-aminobutyric acida-ketoglutaric acid transaminase. Addition of these pharmacological preparations to the incubation mixture of brain homogenates of rats also does not alter the activity of the enzyme systems connected with y-aminobutyric acid in the brain. Administration in vitro of caffeine (600 mg/lOO g brain tissue) suppressed L-glutamic acid decarboxylase by about 10 per cent.
THE DISCOVERY
that y-aminobutyric acid (r-ABA) has an inhibitory effect on the nervous system of animals has led to the suggestion that it participates in regulating the physiological activity of the brain .1-7 The appearance of certain convulsive states is connected with a decrease in the y-ABA content of the brain by decreasing the activity of L-glutamic acid decarboxylase (GAD).s-11 The increase of y-ABA in the brain after administration of hydroxylamine or amino-oxyacetic acid may be due to an inhibition of y-aminobutyric acid-a-ketoglutaric acid transaminase (Y-ABA-T). This enzyme catalyzes the reversible transamination of y-ABA with a-ketoglutarate to give glutamate and succinic semialdehyde, which can be oxidized further to succinic acid and so enter the tricarboxylic acid cycle. 12-14 Many extremely contradictory papers concerning the quantitative determination of y-ABA in the brain during different functional states of the central nervous system have been published.15~2s A number of investigators assume that yABA is a specific ‘inhibitory agent’ affecting the activity of neurones and synaptic transmission and participating in the regulation of physiological activity in the brain, the excitability of which is an inverse relation to the concentration of y-ABA.49 5922-25 The purpose of the present investigation was to study the effect of a number of pharmacological agents on the y-ABA content of the brain and the activities of the enzymes involved in the synthesis and destruction of this amino acid (GAD and Y-ABA-T) in order to determine whether the y-ABA system is an index ofthe functional activity of the central nervous system. BP.-D
49
so
I. A, SY*~;NSKII and T, N. PRIYATICJNA
~~~~~~~
Albino F&3 wej~ti~~ a~~~a~y 150 g were tfsed and braim tissue was prepared acoording to our modificationas, of the method of Roberts.2a The rats were dipped into liquid nitrogen, the frozen cortex was quickly removed and stirred with liquid ~i~~gen~ the ~rot~~~s were then ~~~~~~tated by 75 per cent ethanol (f :7) and ~ntr~~~~~ The ~~~at~~t &id was ~~~~ and the residue re-extracted dth ice-cold 75 pr cent ~~~ano~ and ~va~ora~~d to denim The residue was ~~~~~~~~d in water and ~~~trifu~ed, and the ~~~~a~~~~ was again evaporated to dryness, The amount of $LBA- was deter~~~~d by one-d~~~~siona~ ~~om~to8ra~hy on Wh&= man No. 1 p&per using butaWl*acetic acid- water [4:1:5 (v/v)], The chromat~~~ms were dipped into a 05% ninhydrin solution in acetone. Then thy were heated at 80” in a dr$ug eaWmet for 20-25 E&L The port.ians carrying the pABA spots were out 4ut and eh&ed tit& W305 “i, CWQ in 75 ;{ aeon, _&IIs~tro~~~~met~ ~~~d~~~s were made w% s~~~ophoto~~~~~ model S&&S. at 512 mp.
The activity of GAD in brain ~orn~e~tes ws measured by the d~~~~~~t~~~~ crF &e inmease in the blent of yA3A dur&g ~~~~~~o~ of brain ~ornogen~~~~ ~6th ~~~~~& acid. The a~~~~s were killed by ~~~p~tat~o~ and the brahzs disseoted as rapidly as possible. The tissue samples were homogenized immediately in i~~+&i @05M ~hos~~~t~ bufber pH 63 (X:2> with gSa% ~orno~eni~r at 4”. The in~~b~tio~ mixture was comprised of I*0 ml of the brain homogenate and 1.0 ml of WI5 M gh&amic acid (neutralized to pH &S-6=7). Samples were incubated in closed tubes for 30 min aS37” in anaer0bZG ~o~~t~~~s (Ns$ At the ertrt of the ~~~~~t~on periezd the reaction was stopped %ij?beatin for 18 m& at TOi3”,Five m%of %W%F RE%Sthen added and after shakirrg the samples were centrifuged. The controf s&mples were heated immediately till incubation. The samples were assayed for r_ABA in the usual rn~nner which is based on on~~rnens~o~~ ~hromato~~~~~s.se Activity is expressed as m&r+ m&es of r_ABA formed per g of fresh t&rue per hosr.
Effect of certain drugs on the r-aminobutyllc acid system of the central nervous system
51
Hungarian manufacture, methyl ether L-phenyk-piperidyl (2) hydrochloride] increased spontaneous motor capacity along with a sharply increased orienting reaction. 20-28 M.D. [4’-fluoro-4(142 methoxy) phenyl-piperazinc-butyrophenone of Delalande manufacture-Paris] and andaxin (of Hungarian manufacture)-2 methyl2n-propyl-l,3-propanediol dicarbamate) produced a state of depression. After the injection of barbamil (sodium amytol, sodium salt of ethyl-isoamylbarbiturate acid) a state of deep sleep of the animals was detected. The animals were killed 5-10 min after the injection of picrotoxin, metrazol, caffeine, centedrin, hept-amyl; 15 min after 20-28 MD and 40 min after the administration of andaxin and barbamil. The doses of these drugs in experiments in vitro (expressed as mg/lOO g of brain tissue) were picrotoxind, metrazol-60, caffeine-600, centedrin-30, hept-amyl-15, 20-28 M.D.-6.6, andaxin-300 and barbamil-60. The data obtained were examined statistically by the t-test.29 RESULTS
In the normal state of relative physiological quiescence the individual variations in the r-ABA level in the brain were not large (14.4-17.8 mg%). The y-ABA content of the hemispheres of rat brains in a state of centedrin or caffeine excitation was 25-30 % higher (see Table 1). No alteration in YABA- concentration of the mammalian brain was observed during 5 min seizures induced by picrotoxin and metrazol. The effect of andaxin, seen as muscular weakness and a state of depression in the animals, decreased the amount of y-ABA present in the brain by 23 per cent. These changes in r-ABA content as compared with the normal are statistically signifkant : P < 0.01. Barbamil depresses cortical activity but its effect is not accompanied by the change of y-ABA level in the brain. The activity GAD of brain rats showed normal activity in vivo and in vitro (Table 2). Only caffeine in vitro (600 mg/lOO g) suppressed GAD, but there was only about a 10 per cent inhibition. No significant inhibition was noted of y-ABA-T either in vitro or in vivo by administering of some of the pharmacological agents (Table 3). DISCUSSION Our results indicate that pABA must not be considered an ‘inhibitory agent’ and that neither increase nor decrease in its content in the brain serve as an index of the TABLE 1. r-ABA CONTENTOFRATBRAIN(IN MEAN VALUES f No. of em-
Experimental conditions
periments
control
15
pkr~~l’ caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
:; :; 13
+ Musaelyan experiments (SO).
mg/lOOg WETWT. 0~~1ssu~; S.E.M.) Dose (mg/lOO g)
y-ABA 16.2 f O-08
10-O G 80.0 :8:X K&X 10.0
168 f 17.6 20.4 f $‘; : . 15.8 f 12.5 f 16.3 f
0.46 0.9 0.56 8’;; . 0.39 0.08 O-57
52
I. A. SYRNSIUIand T. N. PRIYA-
TABLE2. EFFECTOF
PHARMACOLOGICAL
DECARBOXYLASE (ACTIVITY~MOLF54
SUBSTANCES UPON GLUTAMIC ACID
IN RAT BRAIN in vivo AND ADDITION OF IT in Vitro
rABA
PER g TISSUE PER HR; hiEAN VALUES &
Experiments
in uitfo
Experiments
Experimental
conditions
No. of experiments 44
control $icr$to;; caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
TABLE
3.
EFFECT
TRANSAMINASE
; 14 6
Dose (mg/lOO g)
1;0
?
6z.i 30.0
16.2 f 0.25 18.8 037 17.7 f 0.54
: 12
80-O 10.0 100 10.0 10.0 100.0 10.0
8’ lo”
OF PHAR~~O~GICAL
SUBSTANCES
AND
Experiments
caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
Dose (mg/ 100 g)
18.1 f 0.31 027
GLUTAMIC ACID PER g TISSUE
control piz;gin
No. of experiments
TO
IN RAT BRAIN in ViVO
Expeximental conditions
Azt%y
No. of expeximenta
Dose
40
11 If
8’ 10 1:
70 g:; 30.0 15.0 32.: 60.0
ADDITION
UPON
PER hr; MEAN VALUES f
29.4 f 0.6 :;*; : :‘;
:
No. of ewe+ ments
activity GAD . . :;.; r ;.:; 18.2 18.7 17.8 18.0 18.0 18.7 18.6
f f f & f f
0.71 0.67 0.31 0.15 0.19 0.22 0.23
ACID
S.E.M.)
Dose (ms/
in vivo
activity y-ABA-T
loog)
s
::
:7;. 2 :‘i* 28.6 f l-3 29.2 &- 1.3 28-l 31.2 f 2.0 1.2
oivo
(ACWITY-@OLES
Experiments
activity y-ABA-T
in
y-AMINOBUTYRIC
OF IT in Vitro
in vitro
S.E.M.)
1’0 9 ::
10.0
28.4 f 0.9
;3*;
g;
1010 1.0 1000 10.0
; 8’;
3010 f 1.3 29.3 i 0.8 30.4 29.2 f 0.9 0.8
functional state of the nervous system. The effect of andaxin, seen as a state of depression in the animals decreased the amount of TABA present in the brain by 23 per cent. In the present experiments with barbamyl, which caused intracortical inhibition, no alteration was noted 40 min after injection. But as has been found earlier 31 a 30 per cent drop in the y-ABA content of the brain was noted by 80 min after injection of the preparation instead of the conjectured rise. A similar contradiction was seen in the experiments with stimulants (caffeine and centedrin)-the y-ABA content of the brain increased by 25-30 per cent, instead of decreasing as expected. Our results show that the changes of levels of y-ABA in the brains of rats were not the results of the changes of GAD and r-ABA-T activities while their activity in the experimental conditions practically did not change. Earlier we showed that it is necessary to immerse the animals in liquid nitrogen for obtaining values for y-ABA
Effect of certain drugs on the y-aminobutyric acid system of the central nervous system
53
that represent true in vim values more closely95. The results of our methodic investigation@ indicated the necessity of keeping the shortest time intervals between decapitation of the rat and homogenization of the brain tissue. In the attempt to obtain the true values of enzymes activities special physiological precautions were taken in our experiments. The activities of the enzymes were determined only in brain homogenates without pyridoxal-phosphate and salts of metals. The extracts of brain acetone powder were not used in our experiments. However, the reaction conditions for the enzymes were optimal (pH, temp., Ns-anaerobic conditions). According to Maslovass the y-ABA level in brain is changed in conformity with the phases of neuronal excitability. Contradictory data about the y-ABA content in the brain during different functional states of the central nervous system were explained by the absence of a true appropriate control of the state of whole body and especially of the states of the blood circulation and respiration. Chickvaidzesr reported that r-ABA content in brain and its alterations which were seen in the experiments with stimulants and depressants are not always connected with the degrees of activity of enzymes of r-ABA metabolism. Results of Roberts= and Ersting34 indicate that pharmacological substances do not greatly affect the activity of the enzyme systems connected with y-ABA metabolism in the brain. At the present time we have no data about the biochemical mechanisms involved in phenomena such as seizure or sleep. The nature of drug effects on the enzymatic activities of r-ABA metabolism remains to be determined. The results discussed in this paper have been limited to a consideration of absence of congruence between the ABA level in brain and its enzymes. At the same time it should be acknowledged that a stable r-ABA level is an essential factor in normal brain activity, which indicates the great metabolic plasticity of the nervous system. y-ABA is continuously formed in neuron cells and influences modulatory effect in the central nervous system. The decrease in its production in synapses would suggest an increase in neuronal excitability because r-ABA plays a regulatory role influencing excitatory and inhibitory states by means of the formation of a general inhibitory background to the cortex. REFERENCES 1. K. A. C. ELLIOTTand H. H. JASPER,Physiol. Reu. 39, 383 (1959). 2. E. ROBERTS,Inhibition in the neroous system and gamma-aminobutyric acid, p. 144, Pergamon Press, Oxford (1960). 3. J. CROSSLAND, J. Pharm. Pharmac. 12, I (1960). 4. G. E. VLALIIMIROV and I. A. SYTIN~KU.Usp. soot-em. biol. 51, 3 (1961). 5. M. N. MASLOVAand I. A. SYTINSKII,Farmakol. toksikol. 625, (1961). 6. E. ROBERTS,J. WEIN and D. G. SIMON~JXN, Vitamins and Hormones, 22, 503 (1964). 7. A. S. BATIJEVand I. A. SYTIN~KII.Usp. soot-em. biol. 59, 134 (1965). 8. K. F. KILLAM,S. R. DA~GUPTAand E. K. KILLAM,Inhibition in the neroous system andgammaaminobutyric acta, p. 302, Pergamon Press, Oxford (1960). 9. E. ROBERTS, C. F. BAXTERand E. RIDELBERG, Proc. 2nd Intern. meet. Neurobiol. p. 392, Pergamon
Press, Oxford (1959). 10. C. F. BAXTERand E. ROBWTS.Proc. Sot. exp. Biol. Med. 104,426 (1960). 11. I. B. OSTRETSOVA and I. A. SYTIN-, Ukr. biokhim. zh. 34,456 (1962). 12. C. F. BAXTERand E. ROBERTS, J. biol. Chem. 236, 3287 (1961). 13. C. K. CHAI, E. ROBERTS and R. L. SIDMAN,Proc. Sot. biol. Med. 109,491 (1962). 14. P. D. ROA, J. K. TEWSand W. E. STONE,Biochem. Pharmac. 13,447 (1964). 15. S. BERL,D. P. PURFWRA,M. GIRADOand H. WAELSCH.J. Neurochem. 4,311 (1959).
54
I. A. SY~NSKIIand T. N. PRIYATKINA
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
E. W. STONE,S. K. TEWSand E. MITCHELL,NeuroZogy10,241 (1960). R. P. KAMRIN and A. A. I(AMRIN, J. Neurochem.6,219 (1961). R. A. FERRARI and A. ARNOLD, Biochem. biophys. Acta 52,361 (1961). H. M. HXICKINEN and E. KYLONEN.Biochem. J. 78,588 (1961). S. S. MU~AELYANand I. A. SYTINSKII,Dokl. Akad. Nauk SSSR 139,994 (1961). V. N. CHIKVAIDZE,Tr. IZZAllunion Conf. of biochemistry nervous system p. 181, Erevan (1963). M. N. MASLOVAand I. A. Smxn, Neuropatol. andpsychiatr. II (1963). K. F. KILLAM,Fed Proc. 17, 1028 (1958). D. M. WOODBURYand A. VERNADAKIS, Fed. Proc. 17,420 (1958). V. BONAVITA,Archiv. ital. biol. 99, 191 (1961). N. F. SHATIJNOVA and I. A. S~TINSKII,Nervous system, 3, 12 (1962). E. ROBERTS and S. FRANKEL,J. biol. Chem. 187, 55 (1950). I. A. SYTIN~KU,V. A. BERNSHTAM and T. N. PRNATIUNA,Nervous system (in press) (1965). P. A. l+%xER,Statistical Research Methods, MOSCOW(1958). S. S. MUSAELYAN,Nervous system 3, 17 (1962).
27.
28.
29. 30. 31. I. A. SYTINSKII,E. L. AVENIROVA,S. P. DEMENTEVA, I. B. OSTRET~~VA and T. N. PRIYATKINA. Tr. ZZZAll-union cot& of biochemistry nervous system. p. 163, Erevan (1963). 32. M. N. MASLOVA,7%e role of GABA in the nervous system activity F. 49, Leningrad (1964). 33. C. F. BAXTERand E. ROBERTS, J. biol. Chem. 233, 1135 (1958). 34. M. I. E. ERSTING,W. F. KA~FE, W. TH. NAIJTA,H. K. OUSTERNI~ and C. DEWART. J. Neurochem.
5, 121 (1960). 35. I. A. S-M and N. T. THINH,J. Neurochem. 11,551 (1964). 36. E. L. AVENIROVA,M. N. MASUIVA, V, I. R~~ENGARTand I. A. SYTINSKII,Vopr. med. them. (in press 1965).
EFFECT OF CERTAIN DRUGS ON THE r-AMINOBUTYRIC ACID SYSTEM OF THE CENTRAL NERVOUS SYSTEM I. A. Neuro-biochemistry
SYTINSKII
and T. N.
PRIYATKINA
Laboratory, A. A. Ukhtomskii University of Leningrad
Physiological Institute,
(Received 19 July 1965; accepted 7 October 1965)
Abstract-The central nervous system stimulants caffeine and centedrin increase the y-aminobutyric acid content of the brain by 25-30 per cent. Administration of andaxin, which caused a general depression, results in a 23 per cent decrease in the brain ~-aminobutyric acid level. Intraperitoneal injection in rats of picrotoxin, metrazol, caffeine, centedrin, hept-amyl, 20-28 M.D., barbamil and andaxin does not produce changes in the enzymatic activities of L-glutamic acid decarboxylase and y-aminobutyric acida-ketoglutaric acid transaminase. Addition of these pharmacological preparations to the incubation mixture of brain homogenates of rats also does not alter the activity of the enzyme systems connected with y-aminobutyric acid in the brain. Administration in vitro of caffeine (600 mg/lOO g brain tissue) suppressed L-glutamic acid decarboxylase by about 10 per cent.
THE DISCOVERY
that y-aminobutyric acid (r-ABA) has an inhibitory effect on the nervous system of animals has led to the suggestion that it participates in regulating the physiological activity of the brain .1-7 The appearance of certain convulsive states is connected with a decrease in the y-ABA content of the brain by decreasing the activity of L-glutamic acid decarboxylase (GAD).s-11 The increase of y-ABA in the brain after administration of hydroxylamine or amino-oxyacetic acid may be due to an inhibition of y-aminobutyric acid-a-ketoglutaric acid transaminase (Y-ABA-T). This enzyme catalyzes the reversible transamination of y-ABA with a-ketoglutarate to give glutamate and succinic semialdehyde, which can be oxidized further to succinic acid and so enter the tricarboxylic acid cycle. 12-14 Many extremely contradictory papers concerning the quantitative determination of y-ABA in the brain during different functional states of the central nervous system have been published.15~2s A number of investigators assume that yABA is a specific ‘inhibitory agent’ affecting the activity of neurones and synaptic transmission and participating in the regulation of physiological activity in the brain, the excitability of which is an inverse relation to the concentration of y-ABA.49 5922-25 The purpose of the present investigation was to study the effect of a number of pharmacological agents on the y-ABA content of the brain and the activities of the enzymes involved in the synthesis and destruction of this amino acid (GAD and Y-ABA-T) in order to determine whether the y-ABA system is an index ofthe functional activity of the central nervous system. BP.-D
49
so
I. A, SY*~;NSKII and T, N. PRIYATICJNA
~~~~~~~
Albino F&3 wej~ti~~ a~~~a~y 150 g were tfsed and braim tissue was prepared acoording to our modificationas, of the method of Roberts.2a The rats were dipped into liquid nitrogen, the frozen cortex was quickly removed and stirred with liquid ~i~~gen~ the ~rot~~~s were then ~~~~~~tated by 75 per cent ethanol (f :7) and ~ntr~~~~~ The ~~~at~~t &id was ~~~~ and the residue re-extracted dth ice-cold 75 pr cent ~~~ano~ and ~va~ora~~d to denim The residue was ~~~~~~~~d in water and ~~~trifu~ed, and the ~~~~a~~~~ was again evaporated to dryness, The amount of $LBA- was deter~~~~d by one-d~~~~siona~ ~~om~to8ra~hy on Wh&= man No. 1 p&per using butaWl*acetic acid- water [4:1:5 (v/v)], The chromat~~~ms were dipped into a 05% ninhydrin solution in acetone. Then thy were heated at 80” in a dr$ug eaWmet for 20-25 E&L The port.ians carrying the pABA spots were out 4ut and eh&ed tit& W305 “i, CWQ in 75 ;{ aeon, _&IIs~tro~~~~met~ ~~~d~~~s were made w% s~~~ophoto~~~~~ model S&&S. at 512 mp.
The activity of GAD in brain ~orn~e~tes ws measured by the d~~~~~~t~~~~ crF &e inmease in the blent of yA3A dur&g ~~~~~~o~ of brain ~ornogen~~~~ ~6th ~~~~~& acid. The a~~~~s were killed by ~~~p~tat~o~ and the brahzs disseoted as rapidly as possible. The tissue samples were homogenized immediately in i~~+&i @05M ~hos~~~t~ bufber pH 63 (X:2> with gSa% ~orno~eni~r at 4”. The in~~b~tio~ mixture was comprised of I*0 ml of the brain homogenate and 1.0 ml of WI5 M gh&amic acid (neutralized to pH &S-6=7). Samples were incubated in closed tubes for 30 min aS37” in anaer0bZG ~o~~t~~~s (Ns$ At the ertrt of the ~~~~~t~on periezd the reaction was stopped %ij?beatin for 18 m& at TOi3”,Five m%of %W%F RE%Sthen added and after shakirrg the samples were centrifuged. The controf s&mples were heated immediately till incubation. The samples were assayed for r_ABA in the usual rn~nner which is based on on~~rnens~o~~ ~hromato~~~~~s.se Activity is expressed as m&r+ m&es of r_ABA formed per g of fresh t&rue per hosr.
Effect of certain drugs on the r-aminobutyllc acid system of the central nervous system
51
Hungarian manufacture, methyl ether L-phenyk-piperidyl (2) hydrochloride] increased spontaneous motor capacity along with a sharply increased orienting reaction. 20-28 M.D. [4’-fluoro-4(142 methoxy) phenyl-piperazinc-butyrophenone of Delalande manufacture-Paris] and andaxin (of Hungarian manufacture)-2 methyl2n-propyl-l,3-propanediol dicarbamate) produced a state of depression. After the injection of barbamil (sodium amytol, sodium salt of ethyl-isoamylbarbiturate acid) a state of deep sleep of the animals was detected. The animals were killed 5-10 min after the injection of picrotoxin, metrazol, caffeine, centedrin, hept-amyl; 15 min after 20-28 MD and 40 min after the administration of andaxin and barbamil. The doses of these drugs in experiments in vitro (expressed as mg/lOO g of brain tissue) were picrotoxind, metrazol-60, caffeine-600, centedrin-30, hept-amyl-15, 20-28 M.D.-6.6, andaxin-300 and barbamil-60. The data obtained were examined statistically by the t-test.29 RESULTS
In the normal state of relative physiological quiescence the individual variations in the r-ABA level in the brain were not large (14.4-17.8 mg%). The y-ABA content of the hemispheres of rat brains in a state of centedrin or caffeine excitation was 25-30 % higher (see Table 1). No alteration in YABA- concentration of the mammalian brain was observed during 5 min seizures induced by picrotoxin and metrazol. The effect of andaxin, seen as muscular weakness and a state of depression in the animals, decreased the amount of y-ABA present in the brain by 23 per cent. These changes in r-ABA content as compared with the normal are statistically signifkant : P < 0.01. Barbamil depresses cortical activity but its effect is not accompanied by the change of y-ABA level in the brain. The activity GAD of brain rats showed normal activity in vivo and in vitro (Table 2). Only caffeine in vitro (600 mg/lOO g) suppressed GAD, but there was only about a 10 per cent inhibition. No significant inhibition was noted of y-ABA-T either in vitro or in vivo by administering of some of the pharmacological agents (Table 3). DISCUSSION Our results indicate that pABA must not be considered an ‘inhibitory agent’ and that neither increase nor decrease in its content in the brain serve as an index of the TABLE 1. r-ABA CONTENTOFRATBRAIN(IN MEAN VALUES f No. of em-
Experimental conditions
periments
control
15
pkr~~l’ caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
:; :; 13
+ Musaelyan experiments (SO).
mg/lOOg WETWT. 0~~1ssu~; S.E.M.) Dose (mg/lOO g)
y-ABA 16.2 f O-08
10-O G 80.0 :8:X K&X 10.0
168 f 17.6 20.4 f $‘; : . 15.8 f 12.5 f 16.3 f
0.46 0.9 0.56 8’;; . 0.39 0.08 O-57
52
I. A. SYRNSIUIand T. N. PRIYA-
TABLE2. EFFECTOF
PHARMACOLOGICAL
DECARBOXYLASE (ACTIVITY~MOLF54
SUBSTANCES UPON GLUTAMIC ACID
IN RAT BRAIN in vivo AND ADDITION OF IT in Vitro
rABA
PER g TISSUE PER HR; hiEAN VALUES &
Experiments
in uitfo
Experiments
Experimental
conditions
No. of experiments 44
control $icr$to;; caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
TABLE
3.
EFFECT
TRANSAMINASE
; 14 6
Dose (mg/lOO g)
1;0
?
6z.i 30.0
16.2 f 0.25 18.8 037 17.7 f 0.54
: 12
80-O 10.0 100 10.0 10.0 100.0 10.0
8’ lo”
OF PHAR~~O~GICAL
SUBSTANCES
AND
Experiments
caffeine centedrin hept-amyl 20-28 M.D. andaxin barbamil
Dose (mg/ 100 g)
18.1 f 0.31 027
GLUTAMIC ACID PER g TISSUE
control piz;gin
No. of experiments
TO
IN RAT BRAIN in ViVO
Expeximental conditions
Azt%y
No. of expeximenta
Dose
40
11 If
8’ 10 1:
70 g:; 30.0 15.0 32.: 60.0
ADDITION
UPON
PER hr; MEAN VALUES f
29.4 f 0.6 :;*; : :‘;
:
No. of ewe+ ments
activity GAD . . :;.; r ;.:; 18.2 18.7 17.8 18.0 18.0 18.7 18.6
f f f & f f
0.71 0.67 0.31 0.15 0.19 0.22 0.23
ACID
S.E.M.)
Dose (ms/
in vivo
activity y-ABA-T
loog)
s
::
:7;. 2 :‘i* 28.6 f l-3 29.2 &- 1.3 28-l 31.2 f 2.0 1.2
oivo
(ACWITY-@OLES
Experiments
activity y-ABA-T
in
y-AMINOBUTYRIC
OF IT in Vitro
in vitro
S.E.M.)
1’0 9 ::
10.0
28.4 f 0.9
;3*;
g;
1010 1.0 1000 10.0
; 8’;
3010 f 1.3 29.3 i 0.8 30.4 29.2 f 0.9 0.8
functional state of the nervous system. The effect of andaxin, seen as a state of depression in the animals decreased the amount of TABA present in the brain by 23 per cent. In the present experiments with barbamyl, which caused intracortical inhibition, no alteration was noted 40 min after injection. But as has been found earlier 31 a 30 per cent drop in the y-ABA content of the brain was noted by 80 min after injection of the preparation instead of the conjectured rise. A similar contradiction was seen in the experiments with stimulants (caffeine and centedrin)-the y-ABA content of the brain increased by 25-30 per cent, instead of decreasing as expected. Our results show that the changes of levels of y-ABA in the brains of rats were not the results of the changes of GAD and r-ABA-T activities while their activity in the experimental conditions practically did not change. Earlier we showed that it is necessary to immerse the animals in liquid nitrogen for obtaining values for y-ABA
Effect of certain drugs on the y-aminobutyric acid system of the central nervous system
53
that represent true in vim values more closely95. The results of our methodic investigation@ indicated the necessity of keeping the shortest time intervals between decapitation of the rat and homogenization of the brain tissue. In the attempt to obtain the true values of enzymes activities special physiological precautions were taken in our experiments. The activities of the enzymes were determined only in brain homogenates without pyridoxal-phosphate and salts of metals. The extracts of brain acetone powder were not used in our experiments. However, the reaction conditions for the enzymes were optimal (pH, temp., Ns-anaerobic conditions). According to Maslovass the y-ABA level in brain is changed in conformity with the phases of neuronal excitability. Contradictory data about the y-ABA content in the brain during different functional states of the central nervous system were explained by the absence of a true appropriate control of the state of whole body and especially of the states of the blood circulation and respiration. Chickvaidzesr reported that r-ABA content in brain and its alterations which were seen in the experiments with stimulants and depressants are not always connected with the degrees of activity of enzymes of r-ABA metabolism. Results of Roberts= and Ersting34 indicate that pharmacological substances do not greatly affect the activity of the enzyme systems connected with y-ABA metabolism in the brain. At the present time we have no data about the biochemical mechanisms involved in phenomena such as seizure or sleep. The nature of drug effects on the enzymatic activities of r-ABA metabolism remains to be determined. The results discussed in this paper have been limited to a consideration of absence of congruence between the ABA level in brain and its enzymes. At the same time it should be acknowledged that a stable r-ABA level is an essential factor in normal brain activity, which indicates the great metabolic plasticity of the nervous system. y-ABA is continuously formed in neuron cells and influences modulatory effect in the central nervous system. The decrease in its production in synapses would suggest an increase in neuronal excitability because r-ABA plays a regulatory role influencing excitatory and inhibitory states by means of the formation of a general inhibitory background to the cortex. REFERENCES 1. K. A. C. ELLIOTTand H. H. JASPER,Physiol. Reu. 39, 383 (1959). 2. E. ROBERTS,Inhibition in the neroous system and gamma-aminobutyric acid, p. 144, Pergamon Press, Oxford (1960). 3. J. CROSSLAND, J. Pharm. Pharmac. 12, I (1960). 4. G. E. VLALIIMIROV and I. A. SYTIN~KU.Usp. soot-em. biol. 51, 3 (1961). 5. M. N. MASLOVAand I. A. SYTINSKII,Farmakol. toksikol. 625, (1961). 6. E. ROBERTS,J. WEIN and D. G. SIMON~JXN, Vitamins and Hormones, 22, 503 (1964). 7. A. S. BATIJEVand I. A. SYTIN~KII.Usp. soot-em. biol. 59, 134 (1965). 8. K. F. KILLAM,S. R. DA~GUPTAand E. K. KILLAM,Inhibition in the neroous system andgammaaminobutyric acta, p. 302, Pergamon Press, Oxford (1960). 9. E. ROBERTS, C. F. BAXTERand E. RIDELBERG, Proc. 2nd Intern. meet. Neurobiol. p. 392, Pergamon
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