Brain Research, 88 (1975) 115-119 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
G A B A and amino acids in the electric organ of
N. N. OSBORN*, H. Z I M M E R M A N N * * ,
115
Torpedo
M. J. D O W D A L L * * AND N. SELLER***
*Max-Planck-lnstitut fiir experimentelle Medizin, Forschungsstelh, Neurochemie, GSttingen, **MaxPlanck-lnstitut fiir biophysikalische Chemie, A bteilung Neurochemie, GSttingen, and *** Max-Plancklnstitut fiir Hirnforschung, Arbeitsgruppe Neurochemie, Frankfurt (G.F.R.)
(Accepted January 9th, 1975)
While carrying out studies with a micro-dansyl chromatographic procedure to analyze the contents in the electric tissue of Torpedo with the aim of quantitatively determining the content of choline, it was noticed that the tissue contained a substance which had the same chromatographic mobility as dansyl-GABA (see Fig. l a). Since GABA is considered to be a transmitter substance in certain situations 1,2, we decided t o see whether it was indeed a constituent of electric tissue and whether it, together with other amino acids, could be detected in the isolated synaptic vesicles even though all the evidence available shows acetylcholine to be the transmitter substance in this tissuea, 13, For the unequivocal identification of GABA in the electric tissue, tissue was homogenized immediately after dissection in 10 vot. 0.2 N perchloric acid and about 0.1 ml aliquots were reacted with excess dansyl chloride s,l°. The dansylated products were then extracted with toluene, evaporated, applied to a chromatographic plate (Silica Gel G layer, 200/~m thick) and subjected to two-dimensional chromatography. In the first dimension we used two chromatographic runs with the solvent system benzene-cyclohexane-methanol (85:15:2; by vol.) and for the second dimension two chromatographic runs of diethylether-cyclohexane (3 : 1 ; by vol.). The spots which cochromatographed with authentic dansyl-2-oxopyrrolidine, the reaction product of GABA with excessive dansyl chloride H, were extracted with ethyl acetate 12 and subjected to mass spectrometry. Spectra, though showing the peaks of dansyl-2-oxopyrrolidine, were, however, not uniform, thus necessitating a re-chromatography of the zone containing the GABA-derivatives using the solvents described above. On these chromatograms the characteristic orange fluorescent colour of the GABA derivative was clearly visible. Further mass-spectrometric analysis of these extracted spots showed the characteristic peaks of dansyl-2-oxopyrrolidine (M + at m / e 318 and M - at m / e 254)9. Moreover, the content of GABA in the electric organ as estimated from fluorescence intensity and mass spectrometry was in the order 10 nmoles/g fresh tissue. This amount of GABA is very low compared with the tissue's acetylcholine content, viz. 924 nmoles/g fresh tissue, as determined by bioassay 15.
116 b
Cl
d
•
c
e
01~ 0 6
'2
1
( ~ Chofinel
,Po2o i W
2ND
T
-~ 1~.'~
kOansyl OB
Fig. I. Autoradiograms of microchromatograms showing substances occurring in the electric organ (a), nerves innervating the electric organ (b), bh)od (c), muscle (d) and synaptic vesicles from Ihe electric organ (e). Also present is a map to help identification (f). The direction of chromatography is indicated by arrows: (t) water formic acid (1()():3, v/v) and (2) benzene-acetic acid (9:1, vv). S starting poinl. Unmarked spots belong to excess dansyl chloride (clearly identified in d as a group of about 6 spots on the upper right hand side of the chromatogram) and unknown dansyl substa~ces.
In other experiments the amino acid distribution in the electric tissue, muscle tissue, nerves innervating the electric organ and blood was analyzed by the microdansyl procedure 6,7. Briefly, the procedure involves extracting amino acids and related substances from the tissues by homogenizing in 0.01 M sodium bicarbonate solution (pH 10) and then treating the substances with [~4C]dansyl chloride (specific activity 49 mCi/mmole). The 14C-dansylated substances are then c h r o m a t o g r a p h e d on 3 cm , 3 cm polyamide layers (see Fig. 1), autoradiograms prepared, and the individual spots removed from the c h r o m a t o g r a m s for assessment by liquid scintillation spectrometry. Fig. la, b, c and d show the separation o f dansyl derivatives in the 4 tissues analyzed and each substance can be identified by comparing the c h r o m a t o g r a m s with the map (Fig. lf) and Table 1. Moreover, the radioactivity associated with the same 20 dansyl spots on c h r o m a t o g r a m s from each sample in terms o f percentage is shown in Table I. It can be seen that while the major single amino acid in all 4 tissues is taurine, a number o f differences in the amino acid content occur in the various tissues. For example, the a m o u n t o f proline, glycine and u n k n o w n 4 in relation to the other substances is specifically high in the muscle compared with the other 3 samples. Interesting, too, is to note that c o m p a r e d to other tissues, the electric organ contains more lysine, ornithine, leucine, isoleucine and unknown 1. It is also clear from theresults that the blood,
117 TABLE I PERCENTAGE COMPOSITION OF SUBSTANCES ( £
S , E , M . FOR
3
EXPERIMENTS) SEPARATED IN THEIR DANSYL
FORMS BY MICROCHROMATOGRAPHY
Spot
Substance
Blood
Muscle
Nerve
Electric organ
Tryptophan Lysine Ornithine Taurine Methionine Phenylalanine Unknown 1 Leucine lsoleucine Unknown 2 Unknown 3 Proline Valine Unknown 4 GABA Alanine + --NH2 + unknown 5 Ethanolamine Glycine Glutamic and aspartic acids Glutamine, arginine, cysteine, serine, threonine, asparagine, hydroxyproline, e-lysine and 6-amino-histidine
0.71 ± 0.08 0.36 7_ 0.07 0.43 ~ 0.01 50.62 ± 7 . 1 2 0.08 ± 0.02 0.15 _i: 0.01 0.17 ± 0.01 0.71 ~ 0.07 0.42 ± 0.03 0.58 ± 0.02 0.65 ± 0.04 0.92 ± 0.05 0.95 ± 0.02 4.03 ± 0.15 1.21 ± 0.08
0.36 ± 0.06 0.16 ± 0.03 0.07 ± 0.01 43.23 ± 6.01 0.02 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.25 ± 0.03 0.18 ± 0.01 0.14 ± 0.02 0.34 -- 0.03 10.46 ± 0.09 0.23 ± 0.03 9.83 ~: 0.31 0.03 ± 0.01
0.63 ± 0.06 0.35 _- 0.04 0.31 _~ 0.0l 35.96 _ 5.32 0.04 _: 0.01 0.30 ± 0.11 0.18 ± 0.03 0.86 ± 0.08 0.52 ± 0.02 0.15 -- 0.01 0.23 :~ 0.03 0.75 ± 0.02 0.85 ± 0.21 1.36 -: 0.21 0.04 £ 0.01
0.84 - 0.08 5.87 - 0.09 3.56 - 0.10 12.22 _t 1.21 0.16 :! 0.01 0.46 ~q 0.12 1.00 ± 0.06 1.58 ± 0.09 1.03 ± 0.09 0.25 ± 0.01 0.49 ~ 0.03 4.76 ± 0.41 1.70 ~ 0.24 7.65 :~ 0.64 1.03 ~ 0.07
14.16 i 3.21 2.94 k_0.98 9.43 :t 0.91
2.12 ± 0 . 1 1 0.65 -- 0.06 26.64 ± 1.21
33.44±4.1 1.61 i 0.07 9.91 ± 0.91
9.65± 1.10 0.69 ± 0.07 2.01 ~ 0.81
3.03 ± 0.99
0.82 ± 0.07
6.24 ± 0.71
4.98 ± 0.61
8.00 ± 2.96
3.89 _R 0.41
5.38 :! 0.81
39.56 ± 5.12
llO,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
in a d d i t i o n to the electric organ, c o n t a i n s substantial a m o u n t s o f G A B A , while only trace quantities are present in the muscle and electric nerve trunks. It may at first be argued that the G A B A in the electric o r g a n originates f r o m b l o o d c o n t a m i n a t i o n . H o w e v e r , the muscle and nerves also contain traces o f blood, yet the G A B A c o n c e n t r a tion is barely detectable. M o r e o v e r , the r e l a t i o n s h i p between the various a m i n o acids in the b l o o d and electric organs is different. F o r example, the ratio o f g l y c i n e to G A B A in the b l o o d is a p p r o x i m a t e l y 8:1, whilst in the electric o r g a n it is 2:1. The question thus arises as to the origin o f G A B A in the electric organ, especially since the nerves i n n e r v a t i n g this organ reveal the barest a m o u n t o f the substance. In o r d e r to investigate this further, synaptic vesicles f r o m the tissue were p r e p a r e d as described elsewhere a,la and the amine and a m i n o acid c o n t e n t e x a m i n e d by the m i c r o - d a n s y l procedure. As can be seen f r o m Fig. le, very few substances can be identified in the vesicles. Only lysine, leucine, t r y p t o p h a n , e t h a n o l a m i n e and choline can be identified as o c c u r r i n g in the synaptic vesicles. T h e huge glycine spot is e x o g e n o u s in origin, belonging to the T r i s - g l y c i n e isolation m e d i u m used in the p r e p a r a t i o n o f the vesicles.
118 In no instance could G A B A be identilied in the vesicles in c o n f i r m a t i o n o f its ~bscucc in the electric nerves. O f interest is to note the presence o f a dansyl derivative c~tc:s p o n d i n g to the position o f dansyl choline in the vesicle lYaction. Since acetylchoiinc w o u l d be r a p i d l y h y d r o l y z e d to choline under the alkaline c o n d i t i o n s used for dansytation this s p o t could reflect the k n o w n acetylcholine content 5. T a k e n as a whole, these results clearly illustrate t h a t G A B A is present in the electric o r g a n in r e a s o n a b l y high a m o u n t s , t h o u g h not in a s s o c i a t i o n with isolated p a r t s (nerve t r u n k s a n d vesicles) o f the k n o w n nervous i nnervation. The role o f G A BA in the electric organ is difficult to e x p l a i n ; firstly because it is barely present in the nerves i n n e r v a t i n g the o r g a n a n d o r i g i n a t i n g from the brain, and secondly because all the evidence shows acetylcholine to be the t r a n s m i t t e r substance in this situation. It m a y well be that the a m i n o acid has a general m e t a b o l i c function, as it has been found in n o n - n e u r o n a l tissues ~2,~4 and it also occurs in Torpedo blood. The absence o f G A B A in s y n a p t i c vesicles c a n n o t be t a k e n as a b s o l u t e p r o o f t h a t it does not act as a t r a n s m i t t e r in the electric organ. However, if this is true then it m u s t be present in hitherto u n d i s c o v e r e d nerves, t h o u g h this seems unlikely in view o f the extensive m o r p h o l o g i c a l studies on this system (for review see ref. 4). W h a t e v e r the case, the exact localization o f G A B A within the electric organ should be investigated, p e r h a p s by a u t o r a d i o g r a p h y , before a clue to its functional role can be found.
1 BAXTER,C., The nature of T-aminobutyric acid. In A. LAJTHA(Ed.), Handbook of Neuroehemistry, Vol. 3, Plenum Press, New York, N.Y., 1970, pp. 289-353. 2 CURTIS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Rev. Physiol., 69 (1974) 97-188. 3 FELDBERG,W., AND FESSARD,m., The cholinergic nature of the nerves to the electric organ of Torpedo (T. marmorata), J. Physiol. (Lond.), 101 (1942) 200-216. 4 FESSARD,A., Les organes electriques. In P. P. GRASS~(Ed.), Traitd de Zoologie, Vol. XIII, Masson, Paris, 1958, pp. 1143-1238. 5 ISRAi~L,M., GAUTRON, J., ANt) LESBATS, B., Subcellular fractionation of the electric organ of Torpedo marmorata, J. Neurochem., 17 (1970) 1441-1450. 6 NEUHOFE, V., Micro-determination of amino acids and related compounds. In V. NEUHOF~ (Ed.), Micromethods in Molecular Biology, Vol. 14, Springer, Berlin, 1973, pp. 85-147. 7 OSBORNE,N. N., The analysis of amines and amino acids in microquantities of tissues. In G. A. KERKUT AND J. PHILLIS(Eds.), Progress in Neurobiology, Vol. 1, Pergamon Press, Oxford, 1973, pp. 301-332. 8 SEILER,N., Use of the dansyl reaction in biochemical analysis, Meth. Biochem. Anal., 18 (t970) 259-337. 9 SEILER,N., SCHNEIDER,H., UND SONNENBERG,K.-D., Die massenspektrometrische Identifizierung yon biogenen Aminen in Form ihrer 1-Dimethylamino-naphthalin-5-sulfonyl-Derivate, Z. analyt. Chem., 252 (1970) 127-136. 10 SEILER, N., AND WIECHMANN, M., TLC analysis of amines as their DANS-derivatives. In A. NIEDERWIESERAND G. PATAKI(Eds.), Progr. Thin-Layer Chromatography and Related Methods, Vol. 1, Humphrey Science Publ., Ann Arbor, Mich., 1970, pp. 94-144. 11 SEILER, N., UND WIECHMANN, M., Die Bestimmung der ~-Amino-butters0.ure im 10 a~ MolBereich als 1-Dimethylamino-naphthalin-5-sulfonyl Derivat, Hoppe-Seylers Z. Physiol. Chem., 349 (1968) 588-594. 12 SELLER,N., UND WIECHtMANN,M., Zum Vorkommen der 6-Amino-buttersiiure und der 0-Amino-~hydroxy-buttersfiure im tierischen Gewebe, Hoppe-Seylers Z. Physiol. Chem., 350 (1969) 14931500.
119 13 WHITTAKER, V. P., DOWDALL, M. J., AND BOYNE, A. F., The storage and release of acetylcholine by cholinergic nerve terminals: recent results with non-mammalian preparations, Biochem. Sot'. Syrup., 36 (1972) 49-68. 14 ZACHMANN, M., Tocci, P., AND NYHAN, W. L., The occurrence of 7-aminobutyric acid in human tissues other than brain, J. biol. Chem., 241 (1966) 1355-1358. 15 ZIMMERMANN,H., AND WHITTAKER, V. P., Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo : a combined biochemical, electrophysiological and morphological study, J. Neurochem., 22 (1974) 435-450.
G A B A and amino acids in the electric organ of
N. N. OSBORN*, H. Z I M M E R M A N N * * ,
115
Torpedo
M. J. D O W D A L L * * AND N. SELLER***
*Max-Planck-lnstitut fiir experimentelle Medizin, Forschungsstelh, Neurochemie, GSttingen, **MaxPlanck-lnstitut fiir biophysikalische Chemie, A bteilung Neurochemie, GSttingen, and *** Max-Plancklnstitut fiir Hirnforschung, Arbeitsgruppe Neurochemie, Frankfurt (G.F.R.)
(Accepted January 9th, 1975)
While carrying out studies with a micro-dansyl chromatographic procedure to analyze the contents in the electric tissue of Torpedo with the aim of quantitatively determining the content of choline, it was noticed that the tissue contained a substance which had the same chromatographic mobility as dansyl-GABA (see Fig. l a). Since GABA is considered to be a transmitter substance in certain situations 1,2, we decided t o see whether it was indeed a constituent of electric tissue and whether it, together with other amino acids, could be detected in the isolated synaptic vesicles even though all the evidence available shows acetylcholine to be the transmitter substance in this tissuea, 13, For the unequivocal identification of GABA in the electric tissue, tissue was homogenized immediately after dissection in 10 vot. 0.2 N perchloric acid and about 0.1 ml aliquots were reacted with excess dansyl chloride s,l°. The dansylated products were then extracted with toluene, evaporated, applied to a chromatographic plate (Silica Gel G layer, 200/~m thick) and subjected to two-dimensional chromatography. In the first dimension we used two chromatographic runs with the solvent system benzene-cyclohexane-methanol (85:15:2; by vol.) and for the second dimension two chromatographic runs of diethylether-cyclohexane (3 : 1 ; by vol.). The spots which cochromatographed with authentic dansyl-2-oxopyrrolidine, the reaction product of GABA with excessive dansyl chloride H, were extracted with ethyl acetate 12 and subjected to mass spectrometry. Spectra, though showing the peaks of dansyl-2-oxopyrrolidine, were, however, not uniform, thus necessitating a re-chromatography of the zone containing the GABA-derivatives using the solvents described above. On these chromatograms the characteristic orange fluorescent colour of the GABA derivative was clearly visible. Further mass-spectrometric analysis of these extracted spots showed the characteristic peaks of dansyl-2-oxopyrrolidine (M + at m / e 318 and M - at m / e 254)9. Moreover, the content of GABA in the electric organ as estimated from fluorescence intensity and mass spectrometry was in the order 10 nmoles/g fresh tissue. This amount of GABA is very low compared with the tissue's acetylcholine content, viz. 924 nmoles/g fresh tissue, as determined by bioassay 15.
116 b
Cl
d
•
c
e
01~ 0 6
'2
1
( ~ Chofinel
,Po2o i W
2ND
T
-~ 1~.'~
kOansyl OB
Fig. I. Autoradiograms of microchromatograms showing substances occurring in the electric organ (a), nerves innervating the electric organ (b), bh)od (c), muscle (d) and synaptic vesicles from Ihe electric organ (e). Also present is a map to help identification (f). The direction of chromatography is indicated by arrows: (t) water formic acid (1()():3, v/v) and (2) benzene-acetic acid (9:1, vv). S starting poinl. Unmarked spots belong to excess dansyl chloride (clearly identified in d as a group of about 6 spots on the upper right hand side of the chromatogram) and unknown dansyl substa~ces.
In other experiments the amino acid distribution in the electric tissue, muscle tissue, nerves innervating the electric organ and blood was analyzed by the microdansyl procedure 6,7. Briefly, the procedure involves extracting amino acids and related substances from the tissues by homogenizing in 0.01 M sodium bicarbonate solution (pH 10) and then treating the substances with [~4C]dansyl chloride (specific activity 49 mCi/mmole). The 14C-dansylated substances are then c h r o m a t o g r a p h e d on 3 cm , 3 cm polyamide layers (see Fig. 1), autoradiograms prepared, and the individual spots removed from the c h r o m a t o g r a m s for assessment by liquid scintillation spectrometry. Fig. la, b, c and d show the separation o f dansyl derivatives in the 4 tissues analyzed and each substance can be identified by comparing the c h r o m a t o g r a m s with the map (Fig. lf) and Table 1. Moreover, the radioactivity associated with the same 20 dansyl spots on c h r o m a t o g r a m s from each sample in terms o f percentage is shown in Table I. It can be seen that while the major single amino acid in all 4 tissues is taurine, a number o f differences in the amino acid content occur in the various tissues. For example, the a m o u n t o f proline, glycine and u n k n o w n 4 in relation to the other substances is specifically high in the muscle compared with the other 3 samples. Interesting, too, is to note that c o m p a r e d to other tissues, the electric organ contains more lysine, ornithine, leucine, isoleucine and unknown 1. It is also clear from theresults that the blood,
117 TABLE I PERCENTAGE COMPOSITION OF SUBSTANCES ( £
S , E , M . FOR
3
EXPERIMENTS) SEPARATED IN THEIR DANSYL
FORMS BY MICROCHROMATOGRAPHY
Spot
Substance
Blood
Muscle
Nerve
Electric organ
Tryptophan Lysine Ornithine Taurine Methionine Phenylalanine Unknown 1 Leucine lsoleucine Unknown 2 Unknown 3 Proline Valine Unknown 4 GABA Alanine + --NH2 + unknown 5 Ethanolamine Glycine Glutamic and aspartic acids Glutamine, arginine, cysteine, serine, threonine, asparagine, hydroxyproline, e-lysine and 6-amino-histidine
0.71 ± 0.08 0.36 7_ 0.07 0.43 ~ 0.01 50.62 ± 7 . 1 2 0.08 ± 0.02 0.15 _i: 0.01 0.17 ± 0.01 0.71 ~ 0.07 0.42 ± 0.03 0.58 ± 0.02 0.65 ± 0.04 0.92 ± 0.05 0.95 ± 0.02 4.03 ± 0.15 1.21 ± 0.08
0.36 ± 0.06 0.16 ± 0.03 0.07 ± 0.01 43.23 ± 6.01 0.02 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 0.25 ± 0.03 0.18 ± 0.01 0.14 ± 0.02 0.34 -- 0.03 10.46 ± 0.09 0.23 ± 0.03 9.83 ~: 0.31 0.03 ± 0.01
0.63 ± 0.06 0.35 _- 0.04 0.31 _~ 0.0l 35.96 _ 5.32 0.04 _: 0.01 0.30 ± 0.11 0.18 ± 0.03 0.86 ± 0.08 0.52 ± 0.02 0.15 -- 0.01 0.23 :~ 0.03 0.75 ± 0.02 0.85 ± 0.21 1.36 -: 0.21 0.04 £ 0.01
0.84 - 0.08 5.87 - 0.09 3.56 - 0.10 12.22 _t 1.21 0.16 :! 0.01 0.46 ~q 0.12 1.00 ± 0.06 1.58 ± 0.09 1.03 ± 0.09 0.25 ± 0.01 0.49 ~ 0.03 4.76 ± 0.41 1.70 ~ 0.24 7.65 :~ 0.64 1.03 ~ 0.07
14.16 i 3.21 2.94 k_0.98 9.43 :t 0.91
2.12 ± 0 . 1 1 0.65 -- 0.06 26.64 ± 1.21
33.44±4.1 1.61 i 0.07 9.91 ± 0.91
9.65± 1.10 0.69 ± 0.07 2.01 ~ 0.81
3.03 ± 0.99
0.82 ± 0.07
6.24 ± 0.71
4.98 ± 0.61
8.00 ± 2.96
3.89 _R 0.41
5.38 :! 0.81
39.56 ± 5.12
llO,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
in a d d i t i o n to the electric organ, c o n t a i n s substantial a m o u n t s o f G A B A , while only trace quantities are present in the muscle and electric nerve trunks. It may at first be argued that the G A B A in the electric o r g a n originates f r o m b l o o d c o n t a m i n a t i o n . H o w e v e r , the muscle and nerves also contain traces o f blood, yet the G A B A c o n c e n t r a tion is barely detectable. M o r e o v e r , the r e l a t i o n s h i p between the various a m i n o acids in the b l o o d and electric organs is different. F o r example, the ratio o f g l y c i n e to G A B A in the b l o o d is a p p r o x i m a t e l y 8:1, whilst in the electric o r g a n it is 2:1. The question thus arises as to the origin o f G A B A in the electric organ, especially since the nerves i n n e r v a t i n g this organ reveal the barest a m o u n t o f the substance. In o r d e r to investigate this further, synaptic vesicles f r o m the tissue were p r e p a r e d as described elsewhere a,la and the amine and a m i n o acid c o n t e n t e x a m i n e d by the m i c r o - d a n s y l procedure. As can be seen f r o m Fig. le, very few substances can be identified in the vesicles. Only lysine, leucine, t r y p t o p h a n , e t h a n o l a m i n e and choline can be identified as o c c u r r i n g in the synaptic vesicles. T h e huge glycine spot is e x o g e n o u s in origin, belonging to the T r i s - g l y c i n e isolation m e d i u m used in the p r e p a r a t i o n o f the vesicles.
118 In no instance could G A B A be identilied in the vesicles in c o n f i r m a t i o n o f its ~bscucc in the electric nerves. O f interest is to note the presence o f a dansyl derivative c~tc:s p o n d i n g to the position o f dansyl choline in the vesicle lYaction. Since acetylchoiinc w o u l d be r a p i d l y h y d r o l y z e d to choline under the alkaline c o n d i t i o n s used for dansytation this s p o t could reflect the k n o w n acetylcholine content 5. T a k e n as a whole, these results clearly illustrate t h a t G A B A is present in the electric o r g a n in r e a s o n a b l y high a m o u n t s , t h o u g h not in a s s o c i a t i o n with isolated p a r t s (nerve t r u n k s a n d vesicles) o f the k n o w n nervous i nnervation. The role o f G A BA in the electric organ is difficult to e x p l a i n ; firstly because it is barely present in the nerves i n n e r v a t i n g the o r g a n a n d o r i g i n a t i n g from the brain, and secondly because all the evidence shows acetylcholine to be the t r a n s m i t t e r substance in this situation. It m a y well be that the a m i n o acid has a general m e t a b o l i c function, as it has been found in n o n - n e u r o n a l tissues ~2,~4 and it also occurs in Torpedo blood. The absence o f G A B A in s y n a p t i c vesicles c a n n o t be t a k e n as a b s o l u t e p r o o f t h a t it does not act as a t r a n s m i t t e r in the electric organ. However, if this is true then it m u s t be present in hitherto u n d i s c o v e r e d nerves, t h o u g h this seems unlikely in view o f the extensive m o r p h o l o g i c a l studies on this system (for review see ref. 4). W h a t e v e r the case, the exact localization o f G A B A within the electric organ should be investigated, p e r h a p s by a u t o r a d i o g r a p h y , before a clue to its functional role can be found.
1 BAXTER,C., The nature of T-aminobutyric acid. In A. LAJTHA(Ed.), Handbook of Neuroehemistry, Vol. 3, Plenum Press, New York, N.Y., 1970, pp. 289-353. 2 CURTIS, D. R., AND JOHNSTON, G. A. R., Amino acid transmitters in the mammalian central nervous system, Rev. Physiol., 69 (1974) 97-188. 3 FELDBERG,W., AND FESSARD,m., The cholinergic nature of the nerves to the electric organ of Torpedo (T. marmorata), J. Physiol. (Lond.), 101 (1942) 200-216. 4 FESSARD,A., Les organes electriques. In P. P. GRASS~(Ed.), Traitd de Zoologie, Vol. XIII, Masson, Paris, 1958, pp. 1143-1238. 5 ISRAi~L,M., GAUTRON, J., ANt) LESBATS, B., Subcellular fractionation of the electric organ of Torpedo marmorata, J. Neurochem., 17 (1970) 1441-1450. 6 NEUHOFE, V., Micro-determination of amino acids and related compounds. In V. NEUHOF~ (Ed.), Micromethods in Molecular Biology, Vol. 14, Springer, Berlin, 1973, pp. 85-147. 7 OSBORNE,N. N., The analysis of amines and amino acids in microquantities of tissues. In G. A. KERKUT AND J. PHILLIS(Eds.), Progress in Neurobiology, Vol. 1, Pergamon Press, Oxford, 1973, pp. 301-332. 8 SEILER,N., Use of the dansyl reaction in biochemical analysis, Meth. Biochem. Anal., 18 (t970) 259-337. 9 SEILER,N., SCHNEIDER,H., UND SONNENBERG,K.-D., Die massenspektrometrische Identifizierung yon biogenen Aminen in Form ihrer 1-Dimethylamino-naphthalin-5-sulfonyl-Derivate, Z. analyt. Chem., 252 (1970) 127-136. 10 SEILER, N., AND WIECHMANN, M., TLC analysis of amines as their DANS-derivatives. In A. NIEDERWIESERAND G. PATAKI(Eds.), Progr. Thin-Layer Chromatography and Related Methods, Vol. 1, Humphrey Science Publ., Ann Arbor, Mich., 1970, pp. 94-144. 11 SEILER, N., UND WIECHMANN, M., Die Bestimmung der ~-Amino-butters0.ure im 10 a~ MolBereich als 1-Dimethylamino-naphthalin-5-sulfonyl Derivat, Hoppe-Seylers Z. Physiol. Chem., 349 (1968) 588-594. 12 SELLER,N., UND WIECHtMANN,M., Zum Vorkommen der 6-Amino-buttersiiure und der 0-Amino-~hydroxy-buttersfiure im tierischen Gewebe, Hoppe-Seylers Z. Physiol. Chem., 350 (1969) 14931500.
119 13 WHITTAKER, V. P., DOWDALL, M. J., AND BOYNE, A. F., The storage and release of acetylcholine by cholinergic nerve terminals: recent results with non-mammalian preparations, Biochem. Sot'. Syrup., 36 (1972) 49-68. 14 ZACHMANN, M., Tocci, P., AND NYHAN, W. L., The occurrence of 7-aminobutyric acid in human tissues other than brain, J. biol. Chem., 241 (1966) 1355-1358. 15 ZIMMERMANN,H., AND WHITTAKER, V. P., Effect of electrical stimulation on the yield and composition of synaptic vesicles from the cholinergic synapses of the electric organ of Torpedo : a combined biochemical, electrophysiological and morphological study, J. Neurochem., 22 (1974) 435-450.
