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Effects of aminooxyacetic acid on in vivo γ-aminobutyric acid system of rat oviduct
(;c~I. IHlarmac. Vol. 14. No. 2. pp. 281 283, 1983 Printed in Great Briuml. All rights reserved
0306-3623 83 020281-03503.00/0 Copyright © 1983 Pergamon Press Ltd
EFFECTS OF A M I N O O X Y A C E T I C ACID ON IN V I V O ;c-AMINOBUTYRIC ACID SYSTEM OF RAT O V I D U C T RAFAEL MARTI'N DEL RIO and MARGARITA SIERRA Lf)PEZ Departamento de Investigaci6n, Centro "Ram6n y Cajal", Ctra. de Colmenar Km 9, Madrid 34, Spain (Reeeiced 4 May 19821
Abstract 1. Effects of aminooxyacetic acid (AOAA) on in cico 7-aminobutyric acid system of rat oviduct have been studied. 2. After i.p. injection of the drug the oviduct G A B A level becomes 50°, of control values, GAD activity remains apparently unchanged while GABA-T activity decreases. 3. The radioactive G A B A found in oviducts incubated in the presence of b[U->~C]glutamate, decreases with increasing concentrations of A O A A through the inhibition of L-glutamate dccarboxylase. 4. GABA decrease seen in rito after AOAA administration is probably due to GAD inhibition.
INTRODt CTION
E{A'cts o/" AOAA in vivo Animals in the estrous phase of the ovarian cycle were injected i.p. with AOAA dissolved in Ringer solution (10mg/mll adjusted to pH 7.0 with NaOH in a 50mg/kg dose. Four hr later the rats were decapitated and the dissected oviducts were used both for GABA content evaluation and enzymatic assay (Martin & Latorre, 19801. A similar group of animals was injected with saline and used as control. The owlrian phase cycle was assessed by vaginal frotis.
It is well known that mammalian brain contains high levels of 7-aminobutyric acid (Roberts & Frankel, 1950) and the enzymes that control its synthesis L-glutamate decarboxylase (GAD) (EC 4.1.1.15) and degradation 4-aminobutyrate 2-oxoglutarate amino transferase (GABA-T) (EC 2.6.1.19) have been thoroughly studied (Wu, 1978). The existence of a high concentration of GABA, higher than in brain, and the presence of G A D and GABA-T activities have recently been described in crude homogenates of rat oviduct {Martin & Latorre 1980; Martin del Rio, 1981). In both organs the two enzymes are inhibited by aminooxyacetic acid (AOAA) in citro (Martin, 1981): Sytinsky & Vasilijev, 1969). The in riro administration of AOAA has been shown to increase the brain GABA content through the inhibition of GABA-T while G A D activity remained unchanged (Baxter & Roberts, 1961). An opposite effect of AOAA on the GABA levels in ovary (Martin & Latorre 1980) and in oviduct (Erda et al., 1982) was shown after its in I,ivo administration. Here the effect of AOAA on the G A D and GABA-T activities of oviduct both in vivo and on the incubated organ is presented. It is proposed that the decrease of oviduct GABA level seen before, is due to inhibition of the synthesising enzyme L-glutamate decarboxylase.
E[l~'ets O[ AOAA on ineuhated ociduets Dissected oviducts were incubated for different periods of time at 41 C in 0.3 ml of Ringer solution pH 7.4 containing l ltCi of L-[U-~°C]glutamatc. AOAA was added at concentrations shown in the text. After incubation and washing with distilled water, a fraction containing the oviduct free amino acids was extracted. These samples were chromatographed using a Beckman 121MB automatic amino acid analyzer and 2 min fractions collected from the outlet column were counted for radioactivity. Fractions containing glutamate and GABA were assessed in a previous run using "'cold'" amino acids and assaying the fractions with ninhydrin reagent. The GAD and GABA-T enzyme activities of oviducts, incubated with AOAA as above, were assayed both in the presence and absence of pyridoxal-5'-phosphatc. Controls were done in the same conditions with no inhibitor in the incubation medium.
RESULTS E~l~,cts q [ ' A O A A in vivo
MATERIALS AND M E T H O D S Adult female Sprague Dawley rats weighing 170 200g were used. GABA. AOAA, glutamate, succinic semialdchyde. pyridoxal-5'-phosphatc and Tris were from Sigma Co. Ninhydrin reagent and buffers from Beckman Instruments were used in the analysis of amino acids. l.-[U-~'*C]glutamic acid (specific activity 285 mCi,,'mmol) was from Radiochcmical Centre Amersham, while other chemicals were reagent grade. L-glutamate decarboxylasc and ?-aminobutyrate transaminase were assayed by measuring the GABA formation as previously described. (Martin & Latorre, 1980).
A group of female rats was injected i.p. with an AOAA solution as described in Methods and the GABA content was measured in both oviduct and brain. At the same time. G A D and GABA-T activities were assayed in crude homogenates prepared from oviduct. Table 1 shows the GABA values obtained in both groups of animals for whole brain and oviduct tissue. In the treated animals there was a 380°41 increase in brain GABA and a 500, decrease in oviduct. When compared with controls, these changes were statistically highly significant (P < 0.001). It was evident then that the inhibitor produced opposite effects in oviduct and brain GABA concentrations.
281
_8.':' ~
RAtAI:I MARIi:-, I}H RIO and MAR(i,\I~.IIA SIIP,RA L()PIZ
Table 1. GABA levels in brain and oxiduct in treated IAOAA} and untreated {control} animals
Brain Oviduct
Control
AOA,~
1.98 ± 0.0216} 4.67 + 0.03 (91
7.80 ± O.l~{6P** 2.40 + 0.221121***
13o j GLU
,o!
GABA values {imlol g of frcsh tissue} arc the mean SEM. Numbers in brackets indicate animals used for each determination. A nonpaired Student's Dtest was used to compare the treated and control rats. *** P < 0.001.
T h e AOAA effect on the two enzymatic activities in oviduct is shown in Table 2. L-glutamate decarboxylase activity was always the same in b o t h control and treated animals, although the activity with no coenzyme in the incubation medium was 87°, of that found in the presence of a high pyridoxal-5'-phosphate concentration (0.33 mM). Results with GABA-T were very different. In animals injected with the inhibitor, the enzyme activity without pyridoxal-5'phosphate in the incubation medium was vcry low (only 18'!o of controls). This inhibition was abolished by a high concentration of coenzyme 10.14 raM} in the assay mixture.
E~l('cts Of A OAA (m incubated oriducts Dissected oviducts when incubated in Ringer solution containing L[US~'C]ghmtmate incorporate the amino acid from the medium. Figure I shows the radioactive proiile of an ion-exchange chromatography of the free amino acid fraction obtained from oviducts after 60 min incubation as in M e t h o d s in the presence of L [ U - t 4 q g h m t m a t e {I t £ i } at a 1 × 10-s M concentration. The c h r o m a t o g r a m shows only two peaks, the first corresponding to glutamate and the second to GABA. It is clear the GABA radioactivity comes from the glutamate uptake, probably through the action of L-glutamate decarboxylase. The radioactivity in the G A B A peak position {absolute and percent values) increases with incubation time as shown in Table 3. both in the absence (control) and presence of AOAA 15 × 10- 4 M}. However the a m o u n t of radioactive GABA found with the inhibitor in the incubation medium is very small compared with that o f the control.
Table 2. Homogcnatcs GAD and GABA-T activities after AOAA in iiro administratiem PLP {mMI
Control
AOAA
GA D
0.00 0.33
87 1{}{}
82 100
GABA-T
{1.0(1 (}.14
82
lS
100
100
Enzymatic activity values are expressed as percentage of control activity 110{)) in thc prescncc of pyridoxal-5'-phosphatc (PLP).
o_ x Q. o
Gtt~,A
5-
- -
-
- 7 :
tO
-
, - -
~'0
-
.
.
.
.
.
30
•
40 TIME
~0 (rain)
Fig. I. Radioactive Ion-exchange chromatography profile of frcc amino acid fraction obtained from I[U-laC]gluta mate incubated oviducts. Total radioactivity {cpm} in 2 min haction is represented.
The effects of AOAA concentrations on GABA formation in oviducts incubated for 60rain with the same a m o u n t of radioactive glutamate {1 tLCi} arc shown in Fig. 2. The radioactive GABA content decreases progressively with augmented concentration of the inhibitor. as shown by the almost linear increase of the G L U {cpm)/GABA (cpm} ratio. The G A D and G A B A - T activities in homogenates obtained from oviducts incubated for 60 min the presence of AOAA 15 × 10 5M} were assayed m the presence and absence of pyridoxal-5'-phosphate in the assay mixture. With no coenzyme, the activity was 14". of the control, being almost fully active {80".) when the coenzyme {0.33mM) was added to the assay. G A B A - T activity was 25",, in the presence of coenzyme {0.14mM) and 14",, m its absence compared with controls. I)lS('l SSION
Aminooxyacetic acid, a carbonyl trapping agent, is an i~z citro inhibitor of brain G A D and GABA-T enzymes through the production of oximes with pyridoxal-5'-phosphate {Sytinsky & Vasilijev. 1969). A similar inhibition by AOAA has r e c e n t b been
Table 3. Time course of radioactive GABA content in LI [ - l aC~glutamate incubated oviducts Time Imin) A O A A {M}
0
5 × 10 "*
20
41)
60
A 2474
B 4.4
k 3090
B 7.0
a 8616
B 7.4
246
0.3
342
0.5
708
0.9
Values are expresscd (AI as [~a(.] radioactMty lepta} and {B) as percentage of I [t!-~a(]glutamate uptake transformed into GABA.
283
AOA,A and oviduct GABA system
I00 £
,3
50-
,K
~6
~o
~o
[aOaA] x 16s, (M)
Fig. 2. Effect of aminooxyacetic acid on radioactive GABA formation from L[-U-~'*C]glutamate. The ratio of total radioactivity collected in the glutamate and GABA fractions obtained from amino acid analyzer is represented.
reported in oviduct G A D and GABA-T enzymes (Martin, 1981). After i.p. injection of AOAA, G A D brain enzyme activity remains unchanged wile GABA-T is greatly inhibited and the GABA level consequently rises sharply (Baxter & Roberts, 1961). We have apparently observed a similar in vivo inhibition pattern caused by AOAA, but after 4 h r of drug action, GABA levels were only 50°/,, of control values. A 30% decrease on oviduct GABA level caused by AOAA has recently been reported (Erd6 et al., 1982) but they used half the dose and kill the animals 2 hr after administration. A possible explanation for this decrease is that the oviduct enzymes may be located in two different compartments, the one containing G A D being more accessible to the inhibitor causing greater G A D inhibition and therefore GABA decrease in the in t;ivo situation. When homogenates are obtained, this separate enzyme storage may be lost. Pyridoxal-5'-phosphate present in the homogenates may now be
enough to restore G A D activity competing with the AOAA while GABA-T becomes accessible and because in homogenates is more susceptible to the inhibition by AOAA than G A D (Martin, 1981) is inhibited by the available drug. This explanation is in agreement with the results obtained in incubation experiments. The presence of AOAA in the Ringer medium produces a decrease in the radioactive GABA content formed from the uptake of radioactive glutamate which is proportional to the increasing inhibitor concentration, indicating a decrease in the de novo synthesis as a consequence of G A D inhibition. This preparation resembles the results obtained in crude homogenates (Martin, 1981) in that the GABA-T is also inhibited by AOAA. The results presented here clearly indicate that oviduct GABA enzymes have a different behavior with AOAA in the in vivo and in the in Eitro situations and also that the in t'ivo response to AOAA is different in the brain and oviduct GABA system. Acknowledgements The authors wish to thank Dr J. M. R. Delgado for research facilities, Dr E. Herrera for commenting on the manuscript, Miss A. Latorre for technical assistance and Mrs C. S. Delgado for editorial help.
REFERENCES BAXTER C. F. • ROBERTS E. (1961) Elevation of ?-aminobutyric acid in brain: Selective inhibition of 7-aminobutyric acid :~-ketoglutaric acid transaminase. J. biol. Chem. 236,
3287 3294. ERDO S. L., ROSD¥ B. & SZPORNY L. (1982) Higher GABA concentrations in Fallopian tube than in brain of rat. J. Neurochem. 38, I174-1176. MARTIN DEL RIO R. & LATORRIiA. C. (1980) Presence of 7-aminobutyric acid in rat ovary. J. Neurochem. 34, 1584 1586. MARTIN DEL RIo R. (1981) 7-Aminobutyric acid system in rat oviduct. J. hiol Chem. 256, 9816~9819. RORERTS E. & FRANKELS. (1950) 7-Aminobutyric acid in brain. Fedn. Proc. Fedn Am. Sots. exp. Biol. 9, 219. SVTINSKY J. A. & VASIL1JEVV. Y. (1969) Some catalytic properties of purified ?-aminobutyrate-:c-oxoglutarate transaminase from the rat brain. Enzymologia 39, 1 11. Wu J. Y. (1978) GABA in Nervous System Function. pp. 7 55. Raven Press, New York.
0306-3623 83 020281-03503.00/0 Copyright © 1983 Pergamon Press Ltd
EFFECTS OF A M I N O O X Y A C E T I C ACID ON IN V I V O ;c-AMINOBUTYRIC ACID SYSTEM OF RAT O V I D U C T RAFAEL MARTI'N DEL RIO and MARGARITA SIERRA Lf)PEZ Departamento de Investigaci6n, Centro "Ram6n y Cajal", Ctra. de Colmenar Km 9, Madrid 34, Spain (Reeeiced 4 May 19821
Abstract 1. Effects of aminooxyacetic acid (AOAA) on in cico 7-aminobutyric acid system of rat oviduct have been studied. 2. After i.p. injection of the drug the oviduct G A B A level becomes 50°, of control values, GAD activity remains apparently unchanged while GABA-T activity decreases. 3. The radioactive G A B A found in oviducts incubated in the presence of b[U->~C]glutamate, decreases with increasing concentrations of A O A A through the inhibition of L-glutamate dccarboxylase. 4. GABA decrease seen in rito after AOAA administration is probably due to GAD inhibition.
INTRODt CTION
E{A'cts o/" AOAA in vivo Animals in the estrous phase of the ovarian cycle were injected i.p. with AOAA dissolved in Ringer solution (10mg/mll adjusted to pH 7.0 with NaOH in a 50mg/kg dose. Four hr later the rats were decapitated and the dissected oviducts were used both for GABA content evaluation and enzymatic assay (Martin & Latorre, 19801. A similar group of animals was injected with saline and used as control. The owlrian phase cycle was assessed by vaginal frotis.
It is well known that mammalian brain contains high levels of 7-aminobutyric acid (Roberts & Frankel, 1950) and the enzymes that control its synthesis L-glutamate decarboxylase (GAD) (EC 4.1.1.15) and degradation 4-aminobutyrate 2-oxoglutarate amino transferase (GABA-T) (EC 2.6.1.19) have been thoroughly studied (Wu, 1978). The existence of a high concentration of GABA, higher than in brain, and the presence of G A D and GABA-T activities have recently been described in crude homogenates of rat oviduct {Martin & Latorre 1980; Martin del Rio, 1981). In both organs the two enzymes are inhibited by aminooxyacetic acid (AOAA) in citro (Martin, 1981): Sytinsky & Vasilijev, 1969). The in riro administration of AOAA has been shown to increase the brain GABA content through the inhibition of GABA-T while G A D activity remained unchanged (Baxter & Roberts, 1961). An opposite effect of AOAA on the GABA levels in ovary (Martin & Latorre 1980) and in oviduct (Erda et al., 1982) was shown after its in I,ivo administration. Here the effect of AOAA on the G A D and GABA-T activities of oviduct both in vivo and on the incubated organ is presented. It is proposed that the decrease of oviduct GABA level seen before, is due to inhibition of the synthesising enzyme L-glutamate decarboxylase.
E[l~'ets O[ AOAA on ineuhated ociduets Dissected oviducts were incubated for different periods of time at 41 C in 0.3 ml of Ringer solution pH 7.4 containing l ltCi of L-[U-~°C]glutamatc. AOAA was added at concentrations shown in the text. After incubation and washing with distilled water, a fraction containing the oviduct free amino acids was extracted. These samples were chromatographed using a Beckman 121MB automatic amino acid analyzer and 2 min fractions collected from the outlet column were counted for radioactivity. Fractions containing glutamate and GABA were assessed in a previous run using "'cold'" amino acids and assaying the fractions with ninhydrin reagent. The GAD and GABA-T enzyme activities of oviducts, incubated with AOAA as above, were assayed both in the presence and absence of pyridoxal-5'-phosphatc. Controls were done in the same conditions with no inhibitor in the incubation medium.
RESULTS E~l~,cts q [ ' A O A A in vivo
MATERIALS AND M E T H O D S Adult female Sprague Dawley rats weighing 170 200g were used. GABA. AOAA, glutamate, succinic semialdchyde. pyridoxal-5'-phosphatc and Tris were from Sigma Co. Ninhydrin reagent and buffers from Beckman Instruments were used in the analysis of amino acids. l.-[U-~'*C]glutamic acid (specific activity 285 mCi,,'mmol) was from Radiochcmical Centre Amersham, while other chemicals were reagent grade. L-glutamate decarboxylasc and ?-aminobutyrate transaminase were assayed by measuring the GABA formation as previously described. (Martin & Latorre, 1980).
A group of female rats was injected i.p. with an AOAA solution as described in Methods and the GABA content was measured in both oviduct and brain. At the same time. G A D and GABA-T activities were assayed in crude homogenates prepared from oviduct. Table 1 shows the GABA values obtained in both groups of animals for whole brain and oviduct tissue. In the treated animals there was a 380°41 increase in brain GABA and a 500, decrease in oviduct. When compared with controls, these changes were statistically highly significant (P < 0.001). It was evident then that the inhibitor produced opposite effects in oviduct and brain GABA concentrations.
281
_8.':' ~
RAtAI:I MARIi:-, I}H RIO and MAR(i,\I~.IIA SIIP,RA L()PIZ
Table 1. GABA levels in brain and oxiduct in treated IAOAA} and untreated {control} animals
Brain Oviduct
Control
AOA,~
1.98 ± 0.0216} 4.67 + 0.03 (91
7.80 ± O.l~{6P** 2.40 + 0.221121***
13o j GLU
,o!
GABA values {imlol g of frcsh tissue} arc the mean SEM. Numbers in brackets indicate animals used for each determination. A nonpaired Student's Dtest was used to compare the treated and control rats. *** P < 0.001.
T h e AOAA effect on the two enzymatic activities in oviduct is shown in Table 2. L-glutamate decarboxylase activity was always the same in b o t h control and treated animals, although the activity with no coenzyme in the incubation medium was 87°, of that found in the presence of a high pyridoxal-5'-phosphate concentration (0.33 mM). Results with GABA-T were very different. In animals injected with the inhibitor, the enzyme activity without pyridoxal-5'phosphate in the incubation medium was vcry low (only 18'!o of controls). This inhibition was abolished by a high concentration of coenzyme 10.14 raM} in the assay mixture.
E~l('cts Of A OAA (m incubated oriducts Dissected oviducts when incubated in Ringer solution containing L[US~'C]ghmtmate incorporate the amino acid from the medium. Figure I shows the radioactive proiile of an ion-exchange chromatography of the free amino acid fraction obtained from oviducts after 60 min incubation as in M e t h o d s in the presence of L [ U - t 4 q g h m t m a t e {I t £ i } at a 1 × 10-s M concentration. The c h r o m a t o g r a m shows only two peaks, the first corresponding to glutamate and the second to GABA. It is clear the GABA radioactivity comes from the glutamate uptake, probably through the action of L-glutamate decarboxylase. The radioactivity in the G A B A peak position {absolute and percent values) increases with incubation time as shown in Table 3. both in the absence (control) and presence of AOAA 15 × 10- 4 M}. However the a m o u n t of radioactive GABA found with the inhibitor in the incubation medium is very small compared with that o f the control.
Table 2. Homogcnatcs GAD and GABA-T activities after AOAA in iiro administratiem PLP {mMI
Control
AOAA
GA D
0.00 0.33
87 1{}{}
82 100
GABA-T
{1.0(1 (}.14
82
lS
100
100
Enzymatic activity values are expressed as percentage of control activity 110{)) in thc prescncc of pyridoxal-5'-phosphatc (PLP).
o_ x Q. o
Gtt~,A
5-
- -
-
- 7 :
tO
-
, - -
~'0
-
.
.
.
.
.
30
•
40 TIME
~0 (rain)
Fig. I. Radioactive Ion-exchange chromatography profile of frcc amino acid fraction obtained from I[U-laC]gluta mate incubated oviducts. Total radioactivity {cpm} in 2 min haction is represented.
The effects of AOAA concentrations on GABA formation in oviducts incubated for 60rain with the same a m o u n t of radioactive glutamate {1 tLCi} arc shown in Fig. 2. The radioactive GABA content decreases progressively with augmented concentration of the inhibitor. as shown by the almost linear increase of the G L U {cpm)/GABA (cpm} ratio. The G A D and G A B A - T activities in homogenates obtained from oviducts incubated for 60 min the presence of AOAA 15 × 10 5M} were assayed m the presence and absence of pyridoxal-5'-phosphate in the assay mixture. With no coenzyme, the activity was 14". of the control, being almost fully active {80".) when the coenzyme {0.33mM) was added to the assay. G A B A - T activity was 25",, in the presence of coenzyme {0.14mM) and 14",, m its absence compared with controls. I)lS('l SSION
Aminooxyacetic acid, a carbonyl trapping agent, is an i~z citro inhibitor of brain G A D and GABA-T enzymes through the production of oximes with pyridoxal-5'-phosphate {Sytinsky & Vasilijev. 1969). A similar inhibition by AOAA has r e c e n t b been
Table 3. Time course of radioactive GABA content in LI [ - l aC~glutamate incubated oviducts Time Imin) A O A A {M}
0
5 × 10 "*
20
41)
60
A 2474
B 4.4
k 3090
B 7.0
a 8616
B 7.4
246
0.3
342
0.5
708
0.9
Values are expresscd (AI as [~a(.] radioactMty lepta} and {B) as percentage of I [t!-~a(]glutamate uptake transformed into GABA.
283
AOA,A and oviduct GABA system
I00 £
,3
50-
,K
~6
~o
~o
[aOaA] x 16s, (M)
Fig. 2. Effect of aminooxyacetic acid on radioactive GABA formation from L[-U-~'*C]glutamate. The ratio of total radioactivity collected in the glutamate and GABA fractions obtained from amino acid analyzer is represented.
reported in oviduct G A D and GABA-T enzymes (Martin, 1981). After i.p. injection of AOAA, G A D brain enzyme activity remains unchanged wile GABA-T is greatly inhibited and the GABA level consequently rises sharply (Baxter & Roberts, 1961). We have apparently observed a similar in vivo inhibition pattern caused by AOAA, but after 4 h r of drug action, GABA levels were only 50°/,, of control values. A 30% decrease on oviduct GABA level caused by AOAA has recently been reported (Erd6 et al., 1982) but they used half the dose and kill the animals 2 hr after administration. A possible explanation for this decrease is that the oviduct enzymes may be located in two different compartments, the one containing G A D being more accessible to the inhibitor causing greater G A D inhibition and therefore GABA decrease in the in t;ivo situation. When homogenates are obtained, this separate enzyme storage may be lost. Pyridoxal-5'-phosphate present in the homogenates may now be
enough to restore G A D activity competing with the AOAA while GABA-T becomes accessible and because in homogenates is more susceptible to the inhibition by AOAA than G A D (Martin, 1981) is inhibited by the available drug. This explanation is in agreement with the results obtained in incubation experiments. The presence of AOAA in the Ringer medium produces a decrease in the radioactive GABA content formed from the uptake of radioactive glutamate which is proportional to the increasing inhibitor concentration, indicating a decrease in the de novo synthesis as a consequence of G A D inhibition. This preparation resembles the results obtained in crude homogenates (Martin, 1981) in that the GABA-T is also inhibited by AOAA. The results presented here clearly indicate that oviduct GABA enzymes have a different behavior with AOAA in the in vivo and in the in Eitro situations and also that the in t'ivo response to AOAA is different in the brain and oviduct GABA system. Acknowledgements The authors wish to thank Dr J. M. R. Delgado for research facilities, Dr E. Herrera for commenting on the manuscript, Miss A. Latorre for technical assistance and Mrs C. S. Delgado for editorial help.
REFERENCES BAXTER C. F. • ROBERTS E. (1961) Elevation of ?-aminobutyric acid in brain: Selective inhibition of 7-aminobutyric acid :~-ketoglutaric acid transaminase. J. biol. Chem. 236,
3287 3294. ERDO S. L., ROSD¥ B. & SZPORNY L. (1982) Higher GABA concentrations in Fallopian tube than in brain of rat. J. Neurochem. 38, I174-1176. MARTIN DEL RIO R. & LATORRIiA. C. (1980) Presence of 7-aminobutyric acid in rat ovary. J. Neurochem. 34, 1584 1586. MARTIN DEL RIo R. (1981) 7-Aminobutyric acid system in rat oviduct. J. hiol Chem. 256, 9816~9819. RORERTS E. & FRANKELS. (1950) 7-Aminobutyric acid in brain. Fedn. Proc. Fedn Am. Sots. exp. Biol. 9, 219. SVTINSKY J. A. & VASIL1JEVV. Y. (1969) Some catalytic properties of purified ?-aminobutyrate-:c-oxoglutarate transaminase from the rat brain. Enzymologia 39, 1 11. Wu J. Y. (1978) GABA in Nervous System Function. pp. 7 55. Raven Press, New York.