Journal o/ Neurochemislry Vol. 30. pp. 146-1465 Pergamon Press Ltd. 1978. Printed in Great Britain 0 International Society for Neurochemistry Ltd.
ELEVATION OF 7-AMINOBUTYRYLCHOLINE CONTENT IN CAT CEREBRAL CORTEX BY ELECTRICAL STIMULATION OF AFFERENT NERVES D. LICHTSHTEIN' and J. DOBKIN' Department of Physiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 30 August 1977. Accepted 28 October 1977)
Abstract-A method for the separation and determination of y-aminobutyrylcholine (GABACh) from trichloracetic acid treated brain extracts has been developed. It consists of the separation of the extracts on Dowex 1 x 8 columns, precipitation of the quaternary ammonium bases and their separation by paper chromatography. Using this technique it was found that electrical stimulation for 30s of one brachial plexus in the cat resulted in 4.6 fold increase in GABACh concentration in the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex. This change in GABACh concentration was not reversed within 5 s of cessation of the stimulus. The results are discussed in relation to the possible physiological role of GABACh: (a) as a neurotransmitter; (b) as a substance participating in the inactivation process of GABA. The possible connection of GABACh to the metabolism of GABA and acetylcholine is discussed.
~-AMINOBUTYRYLCHOLINE (GABACh), the choline ester of y-aminobutyric acid (GABA), was isolated by KURIAKI et a/.. (1958) from the trichloroacetate-soluble fraction of normal dog brain. It has been crystallized by KEWITZ(1959) from porcine brain. Even though GABACh is a reaction product of two important chemical components of the CNS, namely GABA (the inhibitory neurotransmitter), and choline (the precursor and product of acetylcholine), there have been only a limited number of reports directly concerned with GABACh. It would appear that this paucity of information stems from the lack of a specific method for its separation and determination. The possible physiological action of GABACh as a neurotransmitter was discussed several years ago (HOWLS, 1971; JOHNSTON & CURTIS, 1972; BOWERY & BROWN,1972). However, it is generally agreed that the electrophysiological and pharmacological data d o not support this possibility. & DOBKIN, We have demonstrated (LICHTSHTEIN 1976) that afferent electrical stimulation (AES) of one brachial plexus in the cat causes a reversible decrease in GABA concentration in the stimulated (contralateral) cerebral cortex, as compared with the control (ipsilateral) cortex. Since GABA is absent from the
venous blood collected from the transverse sinus under these conditions (LICHTSHTEIN & DOBKIN,unpublished data) and the amount of this transmitter which leaks to the CSF is a number of orders of magnitude smaller than that which disappears from 1969; the stimulated cortex (MITCHELL& SRINIVASAN, CUTLER & DUDZINSKI, 1975),these mechanisms cannot be responsible for the observed reduction of cortical GABA level by AES. Neither can the reduction be due to increased metabolism involving GABA-T (GABA a-ketoglutarate transaminase) because this is not prevented by aminooxyacetic acid, an inhibitor of GABA-T (LICHTSHTEIN & DOBKIN, 1976). In the search for an explanation for the observed decrease in GABA levels, the possibility was raised that the observed decrease in GABA levels is accompanied by an increase in the concentration of one or more of the GABA compounds present in the mammalian brain (for review on GABA metabolic pathways see BAXTER,1970). In this study we investigated the possibility that AES induces a change in the level of GABACh in the cerebral cortex. MATERIALS AND METHODS
The experiments were performed on adult (2.54kg) unfasted cats of both sexes. The preparation of the animal Present address: Department of Physiological Chemis- for monolateral AES and for the fixation and sampling try, Roche Institute of Molecular Biology, Nutley, NJ of cerebral cortical tissue in situ were carried out as pre071 10, U.S.A. viously described (LICHTSHTEIN & D ~ B K I1976). N , The tisTo the regret of his colleagues, Prof. J. Dobkin passed sue sample used included most of the sensomotor cortex away on 9 April 1977. and parts of the parietal cortex and weighed between 400 Abbreviations used: GABACh, y-aminobutyrylcholine; and 700mg. AES, afferent electrical stimulation; GABA, y-aminobuChemical analyses. Before analysis, extracts of 1-6 cortyric acid; GABA-T, y-aminobutyric acid-cc-ketoglutarate tices were combined, depending on the analysis they were transaminase. to undergo. 1461
1462
D. LICHTSHTEIN and J. DOBKIN
TCA remouul. This was carried out by passing the solution through Dowex 1 x 8 acetate column. 200 mesh, 1 x 2Ocm. followed by washing with a dilute acetic acid. The acidic effluent, containing neutral and basic compounds, was dried in a rotary evaporator. Separation of GABACh. After synthesizing GABACh, a method was developed for its separation from TCA extracts. Three grams of tissue are needed in order to carry out this procedure. Reineckate precipitation. This was carried out according to Click’s modification (GLICK,1944) of Beattie’s principle (BEATTIE,1936). The dry residue of the Dowex I x 8 column was resuspended in 3ml 0.1 N-HCI. To this was added 0.21111 of 2% Reinecke salt and the mixture was left for 30 min at 4°C in order to obtain maximal crystallization. The suspension was centrifuged (300 @ 10 min.) and the precipitate washed with the Reinecke solution followed by 3 washes with absolute n-propanol. The precipitate was redissolved in 3 ml acetone, the solution was centrifuged and the acetone supernatant was separated and evaporated to a volume of 1 ml. Separation of the bases from the Reinecke solution was achieved by passing the acetone solution, diluted with 3 volumes of 0.05 N-acetic acid, through a Dowex 1 x 8 column. The free bases were eluded from the column with dilute acetic acid. The effluent was evaporated and the residue redissolved in 0.1-0.2 ml acetone, and the whole volume was transferred to paper for chromotography. Paper chromotography of quaternary ammonium comet al. (1953) pounds. The method developed by BREGOFF was used. Whatman paper No. 1; the moving phase was: butanol-ethanol-acetic acid-water 8:2: 1:3. Color was developed either with Dragendorffs reagent (KBiI.,), or with Ninhydrin 0.1%. The GABACh standard gave a spot at R , = 0.18, and this was well separated from free choline (RF= 0.4) and other known choline esters. Elutiori of GABACh from the chromatogram. In these experiments, no color developing was used, the area between RF = 0.05 and 0.25 was cut out and washed with 30ml methanol, which was evaporated on a rotary evaporator, and transferred for determination of the ester. The separation procedure is illustrated in Fig. 1. Determination of GABACh and choline. These were determined by precipitation as Reineckates (as described above). The intensity of the respective Reinecke in the acetone solution was read in a Gilford spectrophotometer at either 526 nm [linear for Reineckate concentrations from 30 pM to 300 p~ (GLICK,1944)] or at 327 nni (linear for Reineckate concentrations from 3 PM to 3 0 [WINZLER ~ ~ & MESERV,19451). In some experiments for GABACh determination, the ester was separated from the Reinecke solution by passing the acetone solution through Dowex 1 x 8 column, the effluent was hydrolyzed (2 N HCL, 5 h, 100°C) and GABA or choline levels measured quantitatively. GABACh which passes through these procedures gives a recovery rate of 84%, and the results presented here were corrected accordingly. GABA determination. GABA was determined using the enzymatic method (JACOBY & S c m , 1959). The enzymatic kit GABase containing GABA-T and succinic semialdehyde dehydrogenase was obtained from Sigma (prod. No. 2006). Synthesis of GABACh. GABACh was synthesized from GABA and choline in dioxane as the chloride salt and purified by recrystallization from ethanol ether solution
(British Patent, 1961). The crystals had a melting point of 193°C. Analysis (in per cent) showed: C-41.31 H-8.39 N-11.07 (326.96; calculated: C-41.38 H-8.65 N-10.73 C1-27.15. RESULTS
Table 1 illustrates results of paper chromotography of stimulated and control TCA brain extracts, in which quaternary ammonium compounds were separated. In the stimulated samples a spot appears at R F = 0.18, which corresponds to the GABACh standard. This spot is absent in samples of control cortical extracts, presumably because here the quantities of ester present were below the sensitivity limit of the reagents used. In other experiments where the chromatograms were not developed, the ester was eluted and measured quantitatively. These results are given in Table 2. GABACh levels in extracts of stimulated cortex were 4.6-fold greater than in extracts of non-stimulated cortex. In some experiments, the ester was hydrolyzed following its determination, and levels of the products GABA or choline were measured. Table 2 indicates that equimolar values of GABA and choline were found corresponding to the GABACh concentrations found in the corresponding sample. These results further substantiate the reliability of this quantitative measurement of GABACh. From the control experiments shown in Table 3, it can be concluded that no difference in GABACh content in the cerebral cortex occurs because of the 30s delay which exists in our experiments between the freezing of the non-stimulated and the stimulated cortices. Therefore the whole difference can be attributed to the AES. We tested the possibility that GABACh levels may O O W ~ Xi
xa
.
AC-
0 05 N HAC
Nrulrrl and b a r i t compounds
l r i thl a r or tc ti c atid cilrrtl
I
Evaporation
Drr r r r i d u r
wash Rrinecke
roll
Solulion 01 Reinctkatrr Oowrr 1 x 8 Ac0.05N H A c
1
Choline, Choline erltrr k p r chrnatograpby, BuOH, EthOH. A c A , Watrr
I
Srparatrd quricrnrry rmincr Elution A b s o l u t r Methanol
1
Propunol
1
0 1 N HC1 Reinecke 1011 (4Oc. 3 0 r n i n l
Prctioitrtrd Kcinrtkrttr
Acetone
-
\
Devtlopin(
rilb Drrqrndorll or Ilinhydrinr rcr~mtr
P u r n t i t r t i w detrrminrtion o f GABACh
FIG.1. Flow sheet for separation of y-aminobutyrylcholine from trichloroacetic acid brain extracts.
y-Aminobutyrylcholine in cat cerebral cortex
1463
TABLE I. DESCENDING CHROMATOGRAPHY OF QUATERNARY AMMONIUM BASES, SEPARATED FROM BRAIN TCA EXTRACTS. RF of spots found
No. of Developing reagent analyses Dragendorff Ninhydrine
4* 4*
Control
Stimulated
0.293; 0.40§; 0.5411 0.437
0.18t; 0.29$; 0.408;0.541) 0.18t ; 0.431
* Each analysis was made on combined TCA extracts obtained from 6 cats. TCA was removed and Reinecke precipitation was performed. The bases were separated from the Reinecke and spotted on the chromatograms; Whatman No. 1. The moving phase: BuOH; EtOH; acetic acid; water. t Corresponds to y-aminobutyrylcholine standard R,. 1Corresponds to choline standard R,. 5 Corresponds to acetylcholine standard R,. I( Corresponds to GABA standard RF. Unidentified spot. return to control values 5 s following cessation of stimulation as was found for GABA (LICHTSHTEIN & DOBKIN,1976). Table 4 shows that no reversibility was apparent. Unfortunately, our preparation using the freezing technique does not permit us to examine this over longer periods of time.
DISCUSSION
Our results clearly demonstrate the presence of GABACh in the cat cerebral cortex (Table 1) and that unilateral AES of 30s duration produces a 4.6-fold increase in the ester content of the contralateral stimulated cortex (Table 2). The question raised by this observation concerns the physiological context in which this may occur. The relationship of GABACh to electrophysiological phenomena in the CNS is unclear. A few authors have demonstrated a significant inhibitory action showing that local application of the ester on the cerebral cortex surface TABLE2. EFFECT OF
of a cat causes a decrease of the evoked potentials recorded from this area after afferent electrical stimulation or direct stimulation of the brain (TAKAHASHI et al., 1969; ASANO1960; ASHIDAet al., 1965). Based on these observations and the structural similarity of GABACh to Bicuculline, it has been suggested that GABACh functions as a neurotransmitter (HOWELIS, 1971). Other electrophysiological investigations (HONOUR& MCLENNAN,1960; CURTIS& WATKINS, & CURTIS,1964, 1960; CURTISet al., 1961; CRANFORD KRNJEVIC,1964) show that GABACh has no specific inhibitory action. Injection of GABACh into the circulatory system of the cat produces a decrease in arterial blood pressure and causes inhibition at the neuromuscular junction (HOLMSTED& SJOQVIST, 1960). Application of the ester to cat sympathetic ganglion has shown a weak ‘acetylcholine-like’ and a strong ‘GABA-like’ activity (BOWERY& BROWN, 1972). The authors correlate these reactions with the hydrolysis of GABACh by the choline esterase present in the tissue. According to these investiga-
AFFERENT ELECTRICAL STIMULATION ON
GABACh
LEVEL IN THE CEREBRAL CORTEX
Direct determination Product determination-after hydrolysis GABACh pmo1/100g GABA pmo1/100g Choline pmo1/100 g Difference Ipsilateral Contralateral contralateral minus Ipsilateral Contralateral Ipsilateral Contralateral ipsilateral 2.11 1.14 1.05 0.96 1.31 1.09 -
kS.E.M.
6.80 5.84 5.31 5.07 5.82 6.21
4.69 4.70 4.26 4.11 4.51 5.12
1.91 0.74 0.87
5.90 4.17 5.05
-
-
-
-
-
-
-
-
1.32 1.50
5.12 7.03
-
-
-
1.27 k 0.17
5.84 k 0.25
4.56 f 0.14t
_ _ _ _ ~ ~ ~
-~
1.26
-
5.45 k 0.48
0.22 ~~~
~
-
-
-
1.16 1.15 0.80 1.73 2.30
4.65 5.25 5.82 6.21 8.20
1.43 k 0.26
6.03 f 0.60
~~
* The control cerebral cortex, ipsilateral to the electrically stimulated brachial plexus, was frozen before commencement of stimulation. After 30 s of stimulation the stimulated contralateral cortex was frozen while stimulation was still under way. Each value for ipsilateral and contralateral was obtained from 6 cats. GABACh levels were measured after elution of the ester from paper chromatograms (Direct determination).GABA or choline were measured after hydrolysis (2 N-HCI 5 h 100°C) of the ester following its determination (product determination). t Significantly greater than zero. P < 0.001 N.C. 3 0 / 6 4
D. LICHTSHTEIN and J. DOBKIN
I464
TABLE3. GABACh No. of
analyses
LEVELS IN THE CEREBRAL CORTEX OF NON-STIMULATED CATS
GABACh pmo1/100 g Hemisphere Difference frozen second first minus second
Hemisphere frozen first 1.02
4
+ 0.88
1.06
0.09
0.04 & 0.12*
A 30s interval was allowed to elapse between the application of the freezing containers to each hemisphere. The chemical procedure was as described in Table 2. Each analysis was made on combined extracts obtained from 6 cats. Data are means f S.E.M. * Not significantly different from zero.
TABLE 4. DIFFERENCE IN GABACh LEVELS IN THE CEREBRAL CORTEX 5 S
AFTER CESSATION
OF STlMlJLATlON
GABACh prno1/100g No. of
analyses 4
Ipsilateral 1.10
0.09
Contralateral 6.39
+ 0.42
Difference ipsilateral minus contralateral 5.29 f 0.35*
Experimental conditions were the same as in Table 2 except that after 30 s of stimulation the current was switched off and freezing of the contralateral (stimulated)cortex was initiated 5 s later. Each analysis was made on combined extracts obtained from 6 cats. Data are means f S.E.M. * Significantly greater than zero. P < 0.001. tions, studying the ester as a neurotransmitter would not seem to be a fruitful approach. The fact that GABACh concentration rises during electrical stimulation does not support the concept of its action as a neurotransmitter. If it were a neurotransmitter, one would expect a decrease in its concentration (if it is degraded as acetylcholine), or at least that it should remain constant (if it is recycled) during stimulation. Another approach might be that GABACh participates in the process of GABA inactivation. One possibility would be that following its action on the postsynaptic membrane, GABA combines with choline to form GABACh, which is inactive at the synapse. Such an inactivation mechanism, involvind an anabolic reaction, was proposed (WERMAN,1966) and it may occur along with other mechanisms of GABA inactivation. In this mechanism of inactivation, knowledge about the subcellular distribution of a GABACh synthesizing enzyme about which nothing is known, is of major importance. A second possibility is that the formation of GABACh is a second stage in the inactivation process. The process of high affinity uptake of GABA into the nerve or glial cells is generally considered to be the one responsible for GABA inactivation (for review see MARTIN,1976). GABA uptake cannot by itself explain the decrease found upon stimulation, unless it is related to other mechanisms such as degradation with GABA-T or an anabolic proccss such as GABACh synthesis. The aim of this study as presented in the Introduction was to try and elucidate the mechanism responsible for the reduction of GABA in the cerebral cortex by AES. Is the increase of GABACh concentration related to the GABA decrease? It must be emphasized
that although we found an increase in GABACh in our search for the missing GABA, we have no proof that it is the same GABA which is utilized in the synthesis of GABACh. The fact that following cessation of AES a return t o control GABACh levels does not take place (Table 4), in spite of the observed return of GABA concentrations to normal, suggests that there is not a simple buffer-like relationship between these two compounds. Even if we consider the increase in GABACh levels by AES to be due to a process which binds GABA and causes its decrease, this mechanism can account for only about 10% of the amount of GABA which disappears (4.6 pmol GABACh/100 g brain produced during 30s AES as compared to a decrease of 43 pmol GABA/100 g brain during 30 s AES). Since the levels of GABA peptide, Homocarnosine and Homoanserine d o not change during AES (LICHTSHTEIN & DOBKIN, unpublished data) it would be worthwhile to focus further studies on other GABA products in the brain such as y-butyrobetaine and Carnitine, y-Guanidinobutyric acid, and y-amino 8-hydroxybutyric acid, which have not yet been determined in our experiments. Finally, it might be suggested as an alternative hypothesis that GABACh functions as a metabolic bridge between the GABA-ergic and cholinergic systems. Although there is no evidence in the literature supporting such a role, this possibility is too important to be ignored. Unfortunately the paucity of information available concerning the enzymatic systems involved in GABACh metabolism prevents our describing a more factually based hypothesis for the physiological role of this compound.
y-Aminobutyrylcholine in cat cerebral cortex
1465
Acknowledgements-D.L. is indebted t o Prof. J. MAGNES GLICKD . J. (1944) J . biol. Chem. 156, 643-651. HOLMSTEDT B. 8c SJOQVISTF. (1960) Biochem. Pharmac. 3, for his interest and to Mr. M. MARTONfor skillful technical 297-304. assistance. HONOURA. J. & MCLENNANH. (1960) J. Physiol. 150, REFERENCES 30&3 18. HOWELLSD. J. (1971) J. Pharm. Pharmac. 23, 794795. ASANOM., NOROT. & KURIAKIK. (1960) Nature, Lond. JACOBYW. B. & SCOTTE. M. (1959) J. bid. Chem. 234, 185, 848-849. ASHIDAH., TAKEUCHI N., NORI A. & JINNAID. (1965) 937-953. JOHNSTON G . A. R. & CURTISD. R. (1972) J . Pharm. PharNature, Lond. 206, 514515. BAXTERC. F. (1970) in Handbook of Neurochemistry rnac. 24, 251-252. (LAJTHA,A. ed.) Vol. 3, pp. 289-353. Plenum Press, New KEWITZH. (1959) Arch. exp. Path. Pharmak. 237, 308-318. KRNJEVIC K. (1964) Int. Rev. Neurobiol. 7 , 41-98. York. KURIAKIK., YAKUSIJIT., NOROT., SHIMIZU T. & SMI S. BEATTIEF. J. R. (1936) Biochem. J . 30, 1554-1559. BOWERY N. G. & BROWND. A. (1972) J . Pharm. Pharmac. H. (1958) Nature, Lond. 181, 133&1337. 24, 663-666. LICHTSHTEIN D. & DOBKINJ. (1976) J. Neurochem. 26, BREGOFFH. M., ROBERTSE. & DELWICHE C. C. (1953) 135-139. J . bid. Chem. 205, 565-574. MARTIND. L. (1976) in GABA in Neruous System Function British Patent (1961) No. 842.843. Chem. Abst. 55, 6396C. (ROBERTS E., CHASE T. N., TOWER D. B. eds.) pp, CRANFORD J. M. & CURTISD . R. (1964) Br. J . Pharmac. 347-386. Raven Press, New York. Chemother. 23, 313-329. MITCHELL J. F. & SRINIVASAN V. (1969) Nature, Lond. 224, CURTISD. R., PHILLIS J. W. & WATKINSJ. C. (1961) J . 663-666. Physiol., Lond. 1 9 , 296-323. TAKAHASHI H., NAGASHIMA A. & KOSHIMOC. (1958) CURTISD . R. & WATKINS J. C. (1960) J . Neurochem. 6, Nature, Lond. 182, 143-1444, 117-141. WERMANR. (1966) Comp. Biochem. Physiol. 18, 745-766. CUTLERR. W. P. & DUDZINSKI D. S. (1975) Brain Res. WINZLER R. S. & MESERVE. R. (1945) J. bid. Chem. 159, 88, 415423. 395-397.
ELEVATION OF 7-AMINOBUTYRYLCHOLINE CONTENT IN CAT CEREBRAL CORTEX BY ELECTRICAL STIMULATION OF AFFERENT NERVES D. LICHTSHTEIN' and J. DOBKIN' Department of Physiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 30 August 1977. Accepted 28 October 1977)
Abstract-A method for the separation and determination of y-aminobutyrylcholine (GABACh) from trichloracetic acid treated brain extracts has been developed. It consists of the separation of the extracts on Dowex 1 x 8 columns, precipitation of the quaternary ammonium bases and their separation by paper chromatography. Using this technique it was found that electrical stimulation for 30s of one brachial plexus in the cat resulted in 4.6 fold increase in GABACh concentration in the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex. This change in GABACh concentration was not reversed within 5 s of cessation of the stimulus. The results are discussed in relation to the possible physiological role of GABACh: (a) as a neurotransmitter; (b) as a substance participating in the inactivation process of GABA. The possible connection of GABACh to the metabolism of GABA and acetylcholine is discussed.
~-AMINOBUTYRYLCHOLINE (GABACh), the choline ester of y-aminobutyric acid (GABA), was isolated by KURIAKI et a/.. (1958) from the trichloroacetate-soluble fraction of normal dog brain. It has been crystallized by KEWITZ(1959) from porcine brain. Even though GABACh is a reaction product of two important chemical components of the CNS, namely GABA (the inhibitory neurotransmitter), and choline (the precursor and product of acetylcholine), there have been only a limited number of reports directly concerned with GABACh. It would appear that this paucity of information stems from the lack of a specific method for its separation and determination. The possible physiological action of GABACh as a neurotransmitter was discussed several years ago (HOWLS, 1971; JOHNSTON & CURTIS, 1972; BOWERY & BROWN,1972). However, it is generally agreed that the electrophysiological and pharmacological data d o not support this possibility. & DOBKIN, We have demonstrated (LICHTSHTEIN 1976) that afferent electrical stimulation (AES) of one brachial plexus in the cat causes a reversible decrease in GABA concentration in the stimulated (contralateral) cerebral cortex, as compared with the control (ipsilateral) cortex. Since GABA is absent from the
venous blood collected from the transverse sinus under these conditions (LICHTSHTEIN & DOBKIN,unpublished data) and the amount of this transmitter which leaks to the CSF is a number of orders of magnitude smaller than that which disappears from 1969; the stimulated cortex (MITCHELL& SRINIVASAN, CUTLER & DUDZINSKI, 1975),these mechanisms cannot be responsible for the observed reduction of cortical GABA level by AES. Neither can the reduction be due to increased metabolism involving GABA-T (GABA a-ketoglutarate transaminase) because this is not prevented by aminooxyacetic acid, an inhibitor of GABA-T (LICHTSHTEIN & DOBKIN, 1976). In the search for an explanation for the observed decrease in GABA levels, the possibility was raised that the observed decrease in GABA levels is accompanied by an increase in the concentration of one or more of the GABA compounds present in the mammalian brain (for review on GABA metabolic pathways see BAXTER,1970). In this study we investigated the possibility that AES induces a change in the level of GABACh in the cerebral cortex. MATERIALS AND METHODS
The experiments were performed on adult (2.54kg) unfasted cats of both sexes. The preparation of the animal Present address: Department of Physiological Chemis- for monolateral AES and for the fixation and sampling try, Roche Institute of Molecular Biology, Nutley, NJ of cerebral cortical tissue in situ were carried out as pre071 10, U.S.A. viously described (LICHTSHTEIN & D ~ B K I1976). N , The tisTo the regret of his colleagues, Prof. J. Dobkin passed sue sample used included most of the sensomotor cortex away on 9 April 1977. and parts of the parietal cortex and weighed between 400 Abbreviations used: GABACh, y-aminobutyrylcholine; and 700mg. AES, afferent electrical stimulation; GABA, y-aminobuChemical analyses. Before analysis, extracts of 1-6 cortyric acid; GABA-T, y-aminobutyric acid-cc-ketoglutarate tices were combined, depending on the analysis they were transaminase. to undergo. 1461
1462
D. LICHTSHTEIN and J. DOBKIN
TCA remouul. This was carried out by passing the solution through Dowex 1 x 8 acetate column. 200 mesh, 1 x 2Ocm. followed by washing with a dilute acetic acid. The acidic effluent, containing neutral and basic compounds, was dried in a rotary evaporator. Separation of GABACh. After synthesizing GABACh, a method was developed for its separation from TCA extracts. Three grams of tissue are needed in order to carry out this procedure. Reineckate precipitation. This was carried out according to Click’s modification (GLICK,1944) of Beattie’s principle (BEATTIE,1936). The dry residue of the Dowex I x 8 column was resuspended in 3ml 0.1 N-HCI. To this was added 0.21111 of 2% Reinecke salt and the mixture was left for 30 min at 4°C in order to obtain maximal crystallization. The suspension was centrifuged (300 @ 10 min.) and the precipitate washed with the Reinecke solution followed by 3 washes with absolute n-propanol. The precipitate was redissolved in 3 ml acetone, the solution was centrifuged and the acetone supernatant was separated and evaporated to a volume of 1 ml. Separation of the bases from the Reinecke solution was achieved by passing the acetone solution, diluted with 3 volumes of 0.05 N-acetic acid, through a Dowex 1 x 8 column. The free bases were eluded from the column with dilute acetic acid. The effluent was evaporated and the residue redissolved in 0.1-0.2 ml acetone, and the whole volume was transferred to paper for chromotography. Paper chromotography of quaternary ammonium comet al. (1953) pounds. The method developed by BREGOFF was used. Whatman paper No. 1; the moving phase was: butanol-ethanol-acetic acid-water 8:2: 1:3. Color was developed either with Dragendorffs reagent (KBiI.,), or with Ninhydrin 0.1%. The GABACh standard gave a spot at R , = 0.18, and this was well separated from free choline (RF= 0.4) and other known choline esters. Elutiori of GABACh from the chromatogram. In these experiments, no color developing was used, the area between RF = 0.05 and 0.25 was cut out and washed with 30ml methanol, which was evaporated on a rotary evaporator, and transferred for determination of the ester. The separation procedure is illustrated in Fig. 1. Determination of GABACh and choline. These were determined by precipitation as Reineckates (as described above). The intensity of the respective Reinecke in the acetone solution was read in a Gilford spectrophotometer at either 526 nm [linear for Reineckate concentrations from 30 pM to 300 p~ (GLICK,1944)] or at 327 nni (linear for Reineckate concentrations from 3 PM to 3 0 [WINZLER ~ ~ & MESERV,19451). In some experiments for GABACh determination, the ester was separated from the Reinecke solution by passing the acetone solution through Dowex 1 x 8 column, the effluent was hydrolyzed (2 N HCL, 5 h, 100°C) and GABA or choline levels measured quantitatively. GABACh which passes through these procedures gives a recovery rate of 84%, and the results presented here were corrected accordingly. GABA determination. GABA was determined using the enzymatic method (JACOBY & S c m , 1959). The enzymatic kit GABase containing GABA-T and succinic semialdehyde dehydrogenase was obtained from Sigma (prod. No. 2006). Synthesis of GABACh. GABACh was synthesized from GABA and choline in dioxane as the chloride salt and purified by recrystallization from ethanol ether solution
(British Patent, 1961). The crystals had a melting point of 193°C. Analysis (in per cent) showed: C-41.31 H-8.39 N-11.07 (326.96; calculated: C-41.38 H-8.65 N-10.73 C1-27.15. RESULTS
Table 1 illustrates results of paper chromotography of stimulated and control TCA brain extracts, in which quaternary ammonium compounds were separated. In the stimulated samples a spot appears at R F = 0.18, which corresponds to the GABACh standard. This spot is absent in samples of control cortical extracts, presumably because here the quantities of ester present were below the sensitivity limit of the reagents used. In other experiments where the chromatograms were not developed, the ester was eluted and measured quantitatively. These results are given in Table 2. GABACh levels in extracts of stimulated cortex were 4.6-fold greater than in extracts of non-stimulated cortex. In some experiments, the ester was hydrolyzed following its determination, and levels of the products GABA or choline were measured. Table 2 indicates that equimolar values of GABA and choline were found corresponding to the GABACh concentrations found in the corresponding sample. These results further substantiate the reliability of this quantitative measurement of GABACh. From the control experiments shown in Table 3, it can be concluded that no difference in GABACh content in the cerebral cortex occurs because of the 30s delay which exists in our experiments between the freezing of the non-stimulated and the stimulated cortices. Therefore the whole difference can be attributed to the AES. We tested the possibility that GABACh levels may O O W ~ Xi
xa
.
AC-
0 05 N HAC
Nrulrrl and b a r i t compounds
l r i thl a r or tc ti c atid cilrrtl
I
Evaporation
Drr r r r i d u r
wash Rrinecke
roll
Solulion 01 Reinctkatrr Oowrr 1 x 8 Ac0.05N H A c
1
Choline, Choline erltrr k p r chrnatograpby, BuOH, EthOH. A c A , Watrr
I
Srparatrd quricrnrry rmincr Elution A b s o l u t r Methanol
1
Propunol
1
0 1 N HC1 Reinecke 1011 (4Oc. 3 0 r n i n l
Prctioitrtrd Kcinrtkrttr
Acetone
-
\
Devtlopin(
rilb Drrqrndorll or Ilinhydrinr rcr~mtr
P u r n t i t r t i w detrrminrtion o f GABACh
FIG.1. Flow sheet for separation of y-aminobutyrylcholine from trichloroacetic acid brain extracts.
y-Aminobutyrylcholine in cat cerebral cortex
1463
TABLE I. DESCENDING CHROMATOGRAPHY OF QUATERNARY AMMONIUM BASES, SEPARATED FROM BRAIN TCA EXTRACTS. RF of spots found
No. of Developing reagent analyses Dragendorff Ninhydrine
4* 4*
Control
Stimulated
0.293; 0.40§; 0.5411 0.437
0.18t; 0.29$; 0.408;0.541) 0.18t ; 0.431
* Each analysis was made on combined TCA extracts obtained from 6 cats. TCA was removed and Reinecke precipitation was performed. The bases were separated from the Reinecke and spotted on the chromatograms; Whatman No. 1. The moving phase: BuOH; EtOH; acetic acid; water. t Corresponds to y-aminobutyrylcholine standard R,. 1Corresponds to choline standard R,. 5 Corresponds to acetylcholine standard R,. I( Corresponds to GABA standard RF. Unidentified spot. return to control values 5 s following cessation of stimulation as was found for GABA (LICHTSHTEIN & DOBKIN,1976). Table 4 shows that no reversibility was apparent. Unfortunately, our preparation using the freezing technique does not permit us to examine this over longer periods of time.
DISCUSSION
Our results clearly demonstrate the presence of GABACh in the cat cerebral cortex (Table 1) and that unilateral AES of 30s duration produces a 4.6-fold increase in the ester content of the contralateral stimulated cortex (Table 2). The question raised by this observation concerns the physiological context in which this may occur. The relationship of GABACh to electrophysiological phenomena in the CNS is unclear. A few authors have demonstrated a significant inhibitory action showing that local application of the ester on the cerebral cortex surface TABLE2. EFFECT OF
of a cat causes a decrease of the evoked potentials recorded from this area after afferent electrical stimulation or direct stimulation of the brain (TAKAHASHI et al., 1969; ASANO1960; ASHIDAet al., 1965). Based on these observations and the structural similarity of GABACh to Bicuculline, it has been suggested that GABACh functions as a neurotransmitter (HOWELIS, 1971). Other electrophysiological investigations (HONOUR& MCLENNAN,1960; CURTIS& WATKINS, & CURTIS,1964, 1960; CURTISet al., 1961; CRANFORD KRNJEVIC,1964) show that GABACh has no specific inhibitory action. Injection of GABACh into the circulatory system of the cat produces a decrease in arterial blood pressure and causes inhibition at the neuromuscular junction (HOLMSTED& SJOQVIST, 1960). Application of the ester to cat sympathetic ganglion has shown a weak ‘acetylcholine-like’ and a strong ‘GABA-like’ activity (BOWERY& BROWN, 1972). The authors correlate these reactions with the hydrolysis of GABACh by the choline esterase present in the tissue. According to these investiga-
AFFERENT ELECTRICAL STIMULATION ON
GABACh
LEVEL IN THE CEREBRAL CORTEX
Direct determination Product determination-after hydrolysis GABACh pmo1/100g GABA pmo1/100g Choline pmo1/100 g Difference Ipsilateral Contralateral contralateral minus Ipsilateral Contralateral Ipsilateral Contralateral ipsilateral 2.11 1.14 1.05 0.96 1.31 1.09 -
kS.E.M.
6.80 5.84 5.31 5.07 5.82 6.21
4.69 4.70 4.26 4.11 4.51 5.12
1.91 0.74 0.87
5.90 4.17 5.05
-
-
-
-
-
-
-
-
1.32 1.50
5.12 7.03
-
-
-
1.27 k 0.17
5.84 k 0.25
4.56 f 0.14t
_ _ _ _ ~ ~ ~
-~
1.26
-
5.45 k 0.48
0.22 ~~~
~
-
-
-
1.16 1.15 0.80 1.73 2.30
4.65 5.25 5.82 6.21 8.20
1.43 k 0.26
6.03 f 0.60
~~
* The control cerebral cortex, ipsilateral to the electrically stimulated brachial plexus, was frozen before commencement of stimulation. After 30 s of stimulation the stimulated contralateral cortex was frozen while stimulation was still under way. Each value for ipsilateral and contralateral was obtained from 6 cats. GABACh levels were measured after elution of the ester from paper chromatograms (Direct determination).GABA or choline were measured after hydrolysis (2 N-HCI 5 h 100°C) of the ester following its determination (product determination). t Significantly greater than zero. P < 0.001 N.C. 3 0 / 6 4
D. LICHTSHTEIN and J. DOBKIN
I464
TABLE3. GABACh No. of
analyses
LEVELS IN THE CEREBRAL CORTEX OF NON-STIMULATED CATS
GABACh pmo1/100 g Hemisphere Difference frozen second first minus second
Hemisphere frozen first 1.02
4
+ 0.88
1.06
0.09
0.04 & 0.12*
A 30s interval was allowed to elapse between the application of the freezing containers to each hemisphere. The chemical procedure was as described in Table 2. Each analysis was made on combined extracts obtained from 6 cats. Data are means f S.E.M. * Not significantly different from zero.
TABLE 4. DIFFERENCE IN GABACh LEVELS IN THE CEREBRAL CORTEX 5 S
AFTER CESSATION
OF STlMlJLATlON
GABACh prno1/100g No. of
analyses 4
Ipsilateral 1.10
0.09
Contralateral 6.39
+ 0.42
Difference ipsilateral minus contralateral 5.29 f 0.35*
Experimental conditions were the same as in Table 2 except that after 30 s of stimulation the current was switched off and freezing of the contralateral (stimulated)cortex was initiated 5 s later. Each analysis was made on combined extracts obtained from 6 cats. Data are means f S.E.M. * Significantly greater than zero. P < 0.001. tions, studying the ester as a neurotransmitter would not seem to be a fruitful approach. The fact that GABACh concentration rises during electrical stimulation does not support the concept of its action as a neurotransmitter. If it were a neurotransmitter, one would expect a decrease in its concentration (if it is degraded as acetylcholine), or at least that it should remain constant (if it is recycled) during stimulation. Another approach might be that GABACh participates in the process of GABA inactivation. One possibility would be that following its action on the postsynaptic membrane, GABA combines with choline to form GABACh, which is inactive at the synapse. Such an inactivation mechanism, involvind an anabolic reaction, was proposed (WERMAN,1966) and it may occur along with other mechanisms of GABA inactivation. In this mechanism of inactivation, knowledge about the subcellular distribution of a GABACh synthesizing enzyme about which nothing is known, is of major importance. A second possibility is that the formation of GABACh is a second stage in the inactivation process. The process of high affinity uptake of GABA into the nerve or glial cells is generally considered to be the one responsible for GABA inactivation (for review see MARTIN,1976). GABA uptake cannot by itself explain the decrease found upon stimulation, unless it is related to other mechanisms such as degradation with GABA-T or an anabolic proccss such as GABACh synthesis. The aim of this study as presented in the Introduction was to try and elucidate the mechanism responsible for the reduction of GABA in the cerebral cortex by AES. Is the increase of GABACh concentration related to the GABA decrease? It must be emphasized
that although we found an increase in GABACh in our search for the missing GABA, we have no proof that it is the same GABA which is utilized in the synthesis of GABACh. The fact that following cessation of AES a return t o control GABACh levels does not take place (Table 4), in spite of the observed return of GABA concentrations to normal, suggests that there is not a simple buffer-like relationship between these two compounds. Even if we consider the increase in GABACh levels by AES to be due to a process which binds GABA and causes its decrease, this mechanism can account for only about 10% of the amount of GABA which disappears (4.6 pmol GABACh/100 g brain produced during 30s AES as compared to a decrease of 43 pmol GABA/100 g brain during 30 s AES). Since the levels of GABA peptide, Homocarnosine and Homoanserine d o not change during AES (LICHTSHTEIN & DOBKIN, unpublished data) it would be worthwhile to focus further studies on other GABA products in the brain such as y-butyrobetaine and Carnitine, y-Guanidinobutyric acid, and y-amino 8-hydroxybutyric acid, which have not yet been determined in our experiments. Finally, it might be suggested as an alternative hypothesis that GABACh functions as a metabolic bridge between the GABA-ergic and cholinergic systems. Although there is no evidence in the literature supporting such a role, this possibility is too important to be ignored. Unfortunately the paucity of information available concerning the enzymatic systems involved in GABACh metabolism prevents our describing a more factually based hypothesis for the physiological role of this compound.
y-Aminobutyrylcholine in cat cerebral cortex
1465
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