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II. Effect of aminooxyacetic acid and bicuculline on prolactin release in castrated male rats
Life Sciences, Vol. 27, pp. 2025-2029 Printed in the U.S.A.
Pergamon Press
CURRENT CONCEPTS:
II.
EFFECTOF AMINOOXYACETIC ACID AND BICUCULLINE ON PROLACTIN RELEASE IN CASTRATEDMALE RATS L. Debeljuk, S. Goijman, A. Seilicovich, M. C. Diaz and V. B. Rettori
Centro de Investigaciones en Reproducci6n, Facultad de Medicina, ll21 Buenos Aires, Argentina
Summary To investigate the role of gamma aminobutyric acid (GABA) on prolactin secretion, castrated male rats were infused with aminooxyacetic acid (AOAA) or bicuculline, two drugs that affect GABA metabolism or its binding to the receptors, respectively. The infusion of AOAAor bicuculline for 2 hr did not significantly modify serum prolactin levels. A quick iv injection of sulpiride, a drug that induces hyperprolactinemia, brought about a significantly lower release of prolactin in rats infused with AOAA than in control rats, infused with saline. The response to sulpiride in rats infused with bicuculline was significantly greater, in terms of prolactin release, than in control rats. These results suggest that GABAmay have an inhibitory role on the regulation of prolactin release. Evidence has been recently presented showing that gamma aminobutyric acid (GABA) may be involved in the regulation of prolactin secretion (1). Some reports have shown an apparent inhibitory influence of GABAon prolactin release (2, 3), whereas others supported a stimulatory influence (4). Some authors have recently suggested a dual action of GABA, being stimulatory through the central nervous system but inhibitory by acting directly on the pituitary gland (5). Several drugs have been used to affect GABA synthesis or metabolism or to interfere with its receptors. In the present investigation aminooxyacetic acid (AOAA), an inhibitor of GABA-transaminase, and bicuculline, a GABA receptor blocker (6), were used. They were administered as an infusion, to keep constant levels of the drug in blood in order to study their effect on prolactin levels. In addition,to study the effects of the alteration of GABA metabolism due to AOAA and the blockade of GABAreceptors due to bicuculline in a more dynamic way, the response of the pituitary gland to sulpiride, a drug known to induce prolactin release (7), was also investigated. Material and Methods Male rats of the Wistar strain (mean weight: lO0 g) were used throughout the experiment. They were maintained on a diet of Purina laboratory chow and tap water ad lib. The rats were castrated 6 days before starting the experiment. This investigation was carried out after 3:00 PM, since i t had previously been demonstrated that GABA levels in the hypothalamus fluctuate widely between 6:00 AM and 2:00 PM (8). 0024-3205/80/482025-05502.00/0 Copyright (c) 1980 Pergamon Press Ltd
2026
GABA and Prolactln Release
Vol. 27, No. 22, 1980
The day of the experiment, the rats were anesthetized with urethan@ (0.6 ml/lO0 g body wt, of a 25% solution). 90 min later blood was drawn and the materials to be tested were injected in a rapid pulse and then the administration was continued as a constant infusion, The administration of drugs was done by quick injection of I ml (containing 1/3 of the total dose) and then by constant infusion of 2 ml over a 2 hr period with a Harvard infusion pump. The rats were divided into 3 groups: the f i r s t being infused with saline, the second with AOAA hemi-hydrochloride (Sigma Chemical Co.) and the third with bicuculline (Sigma Chemical Co.). Sixteen rats per group were used. The total dose of AOAA administered was 6 mg/rat: 2 mg in the rapid pulse and 4 mg infused over 2 hr. The total dose of bicuculline administered was 0.21 mg/rat: 0.07 mg in the rapid pulse and 0.14 mg infused over 2 hr. AOAA was dissolved in saline and bicuculline was dissolved with 0.1N HCl and then neutralized in saline. 100 min after starting the infusion blood was drawn and saline or sulpiride sulphate (100pg/rat) were given iv as a quick injection. 20 min later the rats were killed by decapitation and blood was collected from the trunk. Prolactin was determined in all the serum samples by means of the double-antibody radioimmunoassay described by Niswender et al. (g) using reagents obtained through NIAMDDand Dr. A. F. Parlow. Purified rat prolactin was labeled with 1251 and NIAMDD-r Prol-RP1 was used as the standard preparation. The significance of the differences was investigated with the Student's t test and Duncan's new multiple range test (10). Results Basal levels of serum prolactin were similar in all the 3 groups studied (Table I ) . Neither AOAA nor bicuculline administration modified significantly serum prolactin levels 100 min after the beginning of the infusions, as compared with saline-treated controls. The rapid pulse of bicuculline induced convulsions in the rats. These convulsions were transient and subsided after a short period of time. The quick injection of sulpiride induced a significant increase of serum prolactin levels in all the 3 groups investigated. The magnitude of the increment of serum prolactin however, varied significantly according to the drugs infused. The prolactin increase after sulpiride in AOAA-infused rats was significantly lower than in control rats (p<0.05). On the other hand, serum prolactin levels after sulpiride injections were significantly higher in bicuculline-infused rats than in control or AOAAinfused rats (p<0.01). The quick injection of saline at 100 min of the beginning of infusions did not significantly modified serum prolactin levels in any of the 3 groups under study. Discussion The results here presented show that, under certain circumstances, when GABA catabolism or GABA receptors are modified, concomitant changes on prolactin release can be observed. Previous reports have demonstrated either a stimulatory or an inhibitory influence of GABA on prolactin secretion (1-5). The latest reports, however have shown that GABAmay have predominant role on prolactin secretion by acting d i r e c t l y on the pituitary gland (2, 11).
15.79 ± 1.27 17.84 ± 3.01
Bicuculline Bicuculline
saline salpiride
salpiride
saline
saline $u~piride
Acute injection
"
16.29 ± 1.72 792.61 ± 150.94 (a)(c)
99.44 ± 11.30 (a)(b)
19.57 ± 2.50
12.36 ± 1.26 286.28 i 52.8 (a)
120 minutes
*Eight rats were used in each group. Serum prolactin levels were expressed as NIAMDD-rProI-RP1, ng ± SE (a) Prolactin increase was significantly higher (p<0.01) than that of the respective saline injected control. (b) Prolactin increase was significantly lower (p<0.05) than that of saline-infused rats. (c) Prolactin increase was significantly higher (p< 0.01) than that of saline-infused rats.
14.46 ± 1.2g 19.16 ± 2.21
16.41 ± 2.09
21.40 ± 4.79
12.34 ± 1.07 14.74 ~ 1.23
AOAA
AOAA
10.65 ± 0.80 g.26 • 1.48
100 minutes
24.78 + 6.14 13.64 ~ 1.64
0
Saline Saline
Infusion*
Effect of the infusion with saline, AOAAor bicuculline on sulpiride-induced prolactin release in castrated male rats
Table I
0
rn
P~
fD
~o f} rt F~
0
Oo 0
Z 0
0
2028
GABA and Prolactin Release
Vol. 27, No. 22, 1980
Evidence has been also recently presented that GABA receptors exist in the p i t u i t a r y gland (12). In the present investigation, AOAAwas used as a mean of inhibiting GABAtransaminase, the enzyme involved in GABA catabolism (6). This drug induces therefore an accumulation of GABA in the brain. Rats treated with infusions of AOAA did not show significant modifications of basal levels of serum prolactin. I t is possible that in rats with normal or low serum prolactin levels the action of any inhibitor of prolactin release may be d i f f i c u l t to demonstrate. This may be the reason for the apparent lack of modification of basal prolactin levels in rats infused with AOAA. When p i t u i t a r y gland was strongly stimulated with sulpiride, however, prolactin release was considerably lower than in control rats. In this case, the hyperprolactinemia induced by sulpiride may have made apparent the inhibitory effect of GABA on prolactin release. Similarly, Libertun et al. (3) have also found that AOAA did not affect basal prolactin levels but did suppress the release of prolactin induced by suckling. The results indicate that GABA accumulation induced by AOAA inhibited prolactin release after a stimulation with sulpiride. On the other hand, the GABA receptor blocker bicuculline, while not affecting basal prolactin levels, induced a higher response to sulpiride in terms of prolactin release. These results, obtained with 2 drugs with d i f f e r e n t mechanisms of action, are consistent with the previous evidence supporting an inhibitory role of GABA on prolactin release. Acknowledgments The authors wish to express their gratitude to NIAMDD, NIH, and to Dr. A. F. Parlow for the g i f t of reagents used for rat prolactin radioimmunoassays. References 1. J. G. Ondo and K. A. Pass, Endocrinology 98 1248-1252 (1976). 2. A. V. Schally, T. W. Redding, A. Arimura, A. Dupont and G. L. Linthicum, Endocrinology 100 681-691 (1977). 3. C. Libertun, M. C. Arakelian, G. A. Larrea and V. G. Foglia, Proc. Soc. Exp. Biol. Med. 161 28-31 (1979). 4. R. Mioduszewski, L. Grandison and J. Meites, Proc. Soc. Exp. Biol. Med. 151 44-47 (1976). 5. V. Locatelli, D. Cocchi, C. Frigerio, R. Betti, P. Krogsgaard-Larsen, G. Racagni and E. E. Muller, Endocrinology 105 778-785 (1979). 6. T. N. Chase and J. R. Walters, in GABA in Nervous System Function, E. Roberts, T. N. Chase and D. B. Tower, Eds p. 497-513, Raven Press, New York (1976). 7. L. Debeljuk, V. Rettori, R. V. Rozados and C. Villegas V~lez, Proc. Soc. Exp. Biol. Med. 150 229-231 (1975). 8. F. Cattabeni, A. Maggi, M. Monduzzi, L. De Angelis and G. Racagni, J. Neurochem. 31 565-567 (1978). 9. G. D. Niswender, C. L. Chen, A. R. Midgley, J r . , J. Meites and S. E l l i s , Proc. Soc. Exp~ Biol. Med. 130 793-797 (1969).
Vol. 27, No. 22, 1980
GABA and Prolactin Release
2029
10. R. G. D. Steel and J. H. Torrie, Principles and Procedures of Statistics, Mc Graw-Hill, New York (1960). 11. A. Enjalbert, M. Ruberg, S. Arancibia, L. Fiore, M. Priam and C. Kordon, Endocrinology 105 823-826 (1979). 12. L. Grandison and A. Guidotti, Endocrinology 105 754-759 (1979).
Pergamon Press
CURRENT CONCEPTS:
II.
EFFECTOF AMINOOXYACETIC ACID AND BICUCULLINE ON PROLACTIN RELEASE IN CASTRATEDMALE RATS L. Debeljuk, S. Goijman, A. Seilicovich, M. C. Diaz and V. B. Rettori
Centro de Investigaciones en Reproducci6n, Facultad de Medicina, ll21 Buenos Aires, Argentina
Summary To investigate the role of gamma aminobutyric acid (GABA) on prolactin secretion, castrated male rats were infused with aminooxyacetic acid (AOAA) or bicuculline, two drugs that affect GABA metabolism or its binding to the receptors, respectively. The infusion of AOAAor bicuculline for 2 hr did not significantly modify serum prolactin levels. A quick iv injection of sulpiride, a drug that induces hyperprolactinemia, brought about a significantly lower release of prolactin in rats infused with AOAA than in control rats, infused with saline. The response to sulpiride in rats infused with bicuculline was significantly greater, in terms of prolactin release, than in control rats. These results suggest that GABAmay have an inhibitory role on the regulation of prolactin release. Evidence has been recently presented showing that gamma aminobutyric acid (GABA) may be involved in the regulation of prolactin secretion (1). Some reports have shown an apparent inhibitory influence of GABAon prolactin release (2, 3), whereas others supported a stimulatory influence (4). Some authors have recently suggested a dual action of GABA, being stimulatory through the central nervous system but inhibitory by acting directly on the pituitary gland (5). Several drugs have been used to affect GABA synthesis or metabolism or to interfere with its receptors. In the present investigation aminooxyacetic acid (AOAA), an inhibitor of GABA-transaminase, and bicuculline, a GABA receptor blocker (6), were used. They were administered as an infusion, to keep constant levels of the drug in blood in order to study their effect on prolactin levels. In addition,to study the effects of the alteration of GABA metabolism due to AOAA and the blockade of GABAreceptors due to bicuculline in a more dynamic way, the response of the pituitary gland to sulpiride, a drug known to induce prolactin release (7), was also investigated. Material and Methods Male rats of the Wistar strain (mean weight: lO0 g) were used throughout the experiment. They were maintained on a diet of Purina laboratory chow and tap water ad lib. The rats were castrated 6 days before starting the experiment. This investigation was carried out after 3:00 PM, since i t had previously been demonstrated that GABA levels in the hypothalamus fluctuate widely between 6:00 AM and 2:00 PM (8). 0024-3205/80/482025-05502.00/0 Copyright (c) 1980 Pergamon Press Ltd
2026
GABA and Prolactln Release
Vol. 27, No. 22, 1980
The day of the experiment, the rats were anesthetized with urethan@ (0.6 ml/lO0 g body wt, of a 25% solution). 90 min later blood was drawn and the materials to be tested were injected in a rapid pulse and then the administration was continued as a constant infusion, The administration of drugs was done by quick injection of I ml (containing 1/3 of the total dose) and then by constant infusion of 2 ml over a 2 hr period with a Harvard infusion pump. The rats were divided into 3 groups: the f i r s t being infused with saline, the second with AOAA hemi-hydrochloride (Sigma Chemical Co.) and the third with bicuculline (Sigma Chemical Co.). Sixteen rats per group were used. The total dose of AOAA administered was 6 mg/rat: 2 mg in the rapid pulse and 4 mg infused over 2 hr. The total dose of bicuculline administered was 0.21 mg/rat: 0.07 mg in the rapid pulse and 0.14 mg infused over 2 hr. AOAA was dissolved in saline and bicuculline was dissolved with 0.1N HCl and then neutralized in saline. 100 min after starting the infusion blood was drawn and saline or sulpiride sulphate (100pg/rat) were given iv as a quick injection. 20 min later the rats were killed by decapitation and blood was collected from the trunk. Prolactin was determined in all the serum samples by means of the double-antibody radioimmunoassay described by Niswender et al. (g) using reagents obtained through NIAMDDand Dr. A. F. Parlow. Purified rat prolactin was labeled with 1251 and NIAMDD-r Prol-RP1 was used as the standard preparation. The significance of the differences was investigated with the Student's t test and Duncan's new multiple range test (10). Results Basal levels of serum prolactin were similar in all the 3 groups studied (Table I ) . Neither AOAA nor bicuculline administration modified significantly serum prolactin levels 100 min after the beginning of the infusions, as compared with saline-treated controls. The rapid pulse of bicuculline induced convulsions in the rats. These convulsions were transient and subsided after a short period of time. The quick injection of sulpiride induced a significant increase of serum prolactin levels in all the 3 groups investigated. The magnitude of the increment of serum prolactin however, varied significantly according to the drugs infused. The prolactin increase after sulpiride in AOAA-infused rats was significantly lower than in control rats (p<0.05). On the other hand, serum prolactin levels after sulpiride injections were significantly higher in bicuculline-infused rats than in control or AOAAinfused rats (p<0.01). The quick injection of saline at 100 min of the beginning of infusions did not significantly modified serum prolactin levels in any of the 3 groups under study. Discussion The results here presented show that, under certain circumstances, when GABA catabolism or GABA receptors are modified, concomitant changes on prolactin release can be observed. Previous reports have demonstrated either a stimulatory or an inhibitory influence of GABA on prolactin secretion (1-5). The latest reports, however have shown that GABAmay have predominant role on prolactin secretion by acting d i r e c t l y on the pituitary gland (2, 11).
15.79 ± 1.27 17.84 ± 3.01
Bicuculline Bicuculline
saline salpiride
salpiride
saline
saline $u~piride
Acute injection
"
16.29 ± 1.72 792.61 ± 150.94 (a)(c)
99.44 ± 11.30 (a)(b)
19.57 ± 2.50
12.36 ± 1.26 286.28 i 52.8 (a)
120 minutes
*Eight rats were used in each group. Serum prolactin levels were expressed as NIAMDD-rProI-RP1, ng ± SE (a) Prolactin increase was significantly higher (p<0.01) than that of the respective saline injected control. (b) Prolactin increase was significantly lower (p<0.05) than that of saline-infused rats. (c) Prolactin increase was significantly higher (p< 0.01) than that of saline-infused rats.
14.46 ± 1.2g 19.16 ± 2.21
16.41 ± 2.09
21.40 ± 4.79
12.34 ± 1.07 14.74 ~ 1.23
AOAA
AOAA
10.65 ± 0.80 g.26 • 1.48
100 minutes
24.78 + 6.14 13.64 ~ 1.64
0
Saline Saline
Infusion*
Effect of the infusion with saline, AOAAor bicuculline on sulpiride-induced prolactin release in castrated male rats
Table I
0
rn
P~
fD
~o f} rt F~
0
Oo 0
Z 0
0
2028
GABA and Prolactin Release
Vol. 27, No. 22, 1980
Evidence has been also recently presented that GABA receptors exist in the p i t u i t a r y gland (12). In the present investigation, AOAAwas used as a mean of inhibiting GABAtransaminase, the enzyme involved in GABA catabolism (6). This drug induces therefore an accumulation of GABA in the brain. Rats treated with infusions of AOAA did not show significant modifications of basal levels of serum prolactin. I t is possible that in rats with normal or low serum prolactin levels the action of any inhibitor of prolactin release may be d i f f i c u l t to demonstrate. This may be the reason for the apparent lack of modification of basal prolactin levels in rats infused with AOAA. When p i t u i t a r y gland was strongly stimulated with sulpiride, however, prolactin release was considerably lower than in control rats. In this case, the hyperprolactinemia induced by sulpiride may have made apparent the inhibitory effect of GABA on prolactin release. Similarly, Libertun et al. (3) have also found that AOAA did not affect basal prolactin levels but did suppress the release of prolactin induced by suckling. The results indicate that GABA accumulation induced by AOAA inhibited prolactin release after a stimulation with sulpiride. On the other hand, the GABA receptor blocker bicuculline, while not affecting basal prolactin levels, induced a higher response to sulpiride in terms of prolactin release. These results, obtained with 2 drugs with d i f f e r e n t mechanisms of action, are consistent with the previous evidence supporting an inhibitory role of GABA on prolactin release. Acknowledgments The authors wish to express their gratitude to NIAMDD, NIH, and to Dr. A. F. Parlow for the g i f t of reagents used for rat prolactin radioimmunoassays. References 1. J. G. Ondo and K. A. Pass, Endocrinology 98 1248-1252 (1976). 2. A. V. Schally, T. W. Redding, A. Arimura, A. Dupont and G. L. Linthicum, Endocrinology 100 681-691 (1977). 3. C. Libertun, M. C. Arakelian, G. A. Larrea and V. G. Foglia, Proc. Soc. Exp. Biol. Med. 161 28-31 (1979). 4. R. Mioduszewski, L. Grandison and J. Meites, Proc. Soc. Exp. Biol. Med. 151 44-47 (1976). 5. V. Locatelli, D. Cocchi, C. Frigerio, R. Betti, P. Krogsgaard-Larsen, G. Racagni and E. E. Muller, Endocrinology 105 778-785 (1979). 6. T. N. Chase and J. R. Walters, in GABA in Nervous System Function, E. Roberts, T. N. Chase and D. B. Tower, Eds p. 497-513, Raven Press, New York (1976). 7. L. Debeljuk, V. Rettori, R. V. Rozados and C. Villegas V~lez, Proc. Soc. Exp. Biol. Med. 150 229-231 (1975). 8. F. Cattabeni, A. Maggi, M. Monduzzi, L. De Angelis and G. Racagni, J. Neurochem. 31 565-567 (1978). 9. G. D. Niswender, C. L. Chen, A. R. Midgley, J r . , J. Meites and S. E l l i s , Proc. Soc. Exp~ Biol. Med. 130 793-797 (1969).
Vol. 27, No. 22, 1980
GABA and Prolactin Release
2029
10. R. G. D. Steel and J. H. Torrie, Principles and Procedures of Statistics, Mc Graw-Hill, New York (1960). 11. A. Enjalbert, M. Ruberg, S. Arancibia, L. Fiore, M. Priam and C. Kordon, Endocrinology 105 823-826 (1979). 12. L. Grandison and A. Guidotti, Endocrinology 105 754-759 (1979).