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Pharmacology of (+)- and (−)-baclofen: GABA-dependant rotational behaviour and [3H]GABA receptor binding studies
Neuroscience Letters, 14 (1979) 123--127
123
© Elsevier/North-Holland Scientific Publishers Ltd.
PHARMACOLOGY OF ( + ) - A N D ( - ) - B A C L O F E N : GABA.DEPENDANT ROTATIONAL BEHAVIOUR AND [aH]GABA RECEPTOR BINDING STUDIES
J.L. WADDINGTON and A.L. CROSS Division o f Psychiatry, M.R.C. Clinical Research Centre, Harrow, H A I 3UJ (U.K.)
(Received March 3rd, 1979) (Accepted May 16th, 1979)
SUMMARY (+)- and (--)-baclofen were equiactive in inducing contralateral rotational behaviour following their unilateral injection into the zona reticulata of the rat substantia nigra, a proposed GABA-dependent rotational behaviour model, and in displacing the specific binding of [3H]GABA to frozen and thawed membrane preparations of rat brain. These non-stereospecific actions of baclofen at a population of cerebral GABA receptors contrast with stereospecific neuronal depressant effects probably mediated via presynaptic inhibition of the release of excitatory amino acid neurotransmitters. The present results indicate that baclofen may have t w o distinct mechanisms of action and this may account for the debate on the pharmacology of this compound. The development of clinically active agents which stimulate cerebral 7aminobutyric acid (GABA) receptors has generated considerable interest as it has been proposed that such compounds may be of theropeutic potential in neuropsychiatric conditions, including Huntington's chorea and schizophrenia [2]. Baclofen, the ~-(p-chlorophenyl) derivative of GABA, provides a means of conveying a GABA moiety into the brain [5]. However, confusion over the status of baclofen as a GABA agonist has led to difficulty in interpreting animal [7,19] and clinical [10] studies using this compound. While many studies have concluded that baclofen is devoid of GABA agonist activity [3,13], others have clearly indicated a GABA mimetic action [6,15]. This study describes~the effects of the resolved enantiomers of baclofen [ 14] in a GABA-dependant, dopamine (DA)-independent rotational behaviour model [20,21] and in a [3H]GABA receptor binding assay [4]. We have recently reported that racemic baclofen is active in both models [23], though in a manner not entirely consistent with a pure GABA agonist
124
action, and here propose how studies with it's stereoisomers may help resolve the diverse pharmacological properties of this compound. Rotational behaviour studies were performed on male Sprague--Dawley rats, 150--200 g, as previously described [20,21,23]. Briefly, rats received unilateral intracerebral injections of 500 ng (+)- or (-)-baclofen (as the hydrochloride, Ciba-Geigy, Basel) into the zona reticulata of the substantia nigra (SNR), in 1 ul 0.9% physiological saline. The intracerebral injections were made by slow motion infusion, under light ether anaesthesia, using stereotaxic coordinates [12] A2.2, V-2.8, L2.0. Immediately after the intracerebral injection procedure animals were placed in rotameters [ 22] for recording of rotational behaviour over a 45-min period. The specific binding of [3H] GABA to crude synaptic membrane preparations of whole rat brain was assessed in the presence and absence of (+)- or (-)-baclofen as previously described [4,23]. Membranes were frozen and thawed before use, and binding assays performed with 10 nM [SH]GABA {30--60 Ci/mmol, Radiochemical Centre, Amersham, U.K.). Specific binding was defined as the difference between binding occurring in the presence and in the absence of an excess (1 mM) of unlabelled GABA. Rotational responses to histologically-confirmed unilateral injections of 500 ng (+)- and (-}-baclofen are shown in Fig. 1. Both stereoisomers induced contralateral circling and the magnitudes and time-courses of these rotational responses were indistinguishable both from each other and from those produced by 500 ng racemic baclofen in an identical behavioural assay in which injections of saline or/3-phenylethylamine failed to induce contralateral turning [23]. These rotational responses to unilateral injections of 500 ng baclofen into SNR were, however, considerably less intense than those produced by smaller quantities of the potent and specific GABA agonist muscimol administered under identical conditions [ 23]. 0 m
-100
-200
-S -3OO E -400~
I
0
I
15
I
30
I
45
Time post injection (rain) Fig. 1. Rotational responses of rats to unilateral intranigral injections (500 ng) of (+)- or (--)-baclofen in 1 ~I ~llne. Rotations are plotted cumulatively against time after injection, and are the means + S.E.M. of 6 animals/group, o, (+)-baclofen; e, (--)-baclofen.
125
Both (+)- and (-)-baclofen were equiactive in displacing [3H]GABA binding. Log-probit plots indicated that the ICs0 for each isomer was 38/~M. This is essentially identical with the potency of racemic baclofen, though considerably less than the potency of muscimol and GABA itself, in inhil> iting [3H]GABA binding in an identical assay [23] (Table I). We have proposed that the contralateral rotational response widely shown to follow unilateral injections of GABA and GABAergic drugs into SNR may constitute a GABA
ICs0 (pM) b
(+)-baclofen (--)-baclofen (+)/(--)-baclofen GABA Muscimol
38 38 40 c 0.4 c 0.04 c
aThe receptor-binding procedure was performed essentially to the m e t h o d o f Enna and S n y d e r [4] with [ 3 H ] G A B A at a c o n c e n t r a t i o n o f 10 nM. b . . . ICs0 d e t e r m i n e d f r o m log-problt plots o f d u p h c a t e determinations. ~Data f r o m Waddington and Cross [23] for comparison.
126
characterised by the removal of an endogenous protein inhibitor of GABA binding which reveals a class of low density, high affinity GABA binding sites [8,18]. Both low and high affinity GABA binding sites were inhibited by receptor-specific drugs in proportion with their pharmacological potency [ 8] and any functional dissociation between them remains to be determined. However, we (unpublished data) and others [17] have found that baclofen does not displace [3H]GABA specifically bound to high affinity binding sites in Triton-treated membrane preparations. Thus, the seemingly preferential action of baclofen on low affinity GABA binding sites would support our contention, based on behavioural studies [23], that this compound does not possess pure GABA agonist activity. It may act on a particular population of GABA receptors, and we find that this GABAergic action, like that in the GABA-dependent rotational model, does not show stereospecificity. Whilst the present study indicates that the previously described GABA mimetic action of baclofen [6,15,23] is a non-stereospecific effect, there is clear evidence that baclofen has potent neuronal depressant properties that are unrelated to effects on GABAergic mechanisms [3,6] and which are stereospecific for the (-)-isomer [1,14]. These properties seem to be mediated by a presynaptic inhibition of the release of excitatory amino acid neurotransmitters [1,3,6,16]. From our data we suggest that the debate as to whether baclofen does or does not show GABA mimetic properties may reflect it's ability to induce depression of neuronal activity by two distinct mechanisms. Thus, while (-)-baclofen may potently and stereospecifically inhibit the release of excitatory aminoacid neurotransmitters, high concentrations of baclofen can also stimulate a population of cerebral GABA receptors in a non-stereospecific manner. This duplicity of action would indicate that baclofen is a poor pharmacological tool for either animal or clinical studies of GABAergic mechanisms. ACKNOWLEDGEMENT
J.L.W. and A.J.C. are M.R.C Scholars. A preliminary account of these results has been communicated to the British Pharmacological Society, University of Nottingham, U.K., September 1978. We are grateful to CibaGeigy, Basel, Switzerland, for gifts of drugs. REFERENCES 1 Ault, B. and Evans, H., Central depressant actions of baclofen, J. Physiol., 284 (1978) 131P. 2 Chase, T.N. and Walters, J.R., Pharmacologic approaches to the manipulation of GABA-mediated synaptic function in man. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven Press, New York, 1976, pp. 497--513.
127 3 Davidoff, R.A., Pharmacology of spasticity, Neurology, 28 (1978) 46--51. 4 Enna, S.J. and Snyder, S.H., Properties of ~-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Res., 100 (1975) 81--97. 5 Fehr, H.U. and Bein, H.J., Sites of action of a new muscle relaxant (baclofen, Lioresal ®, Ciba 34 647-Ba), J. Int. Med. Res., 2 (1974) 36--37. 6 Fox, S., Krnjevic, K., Morris, M.E., Pull, E. and Werman, R., Action of baclofen on mammalian synaptic transmission, Neuroscience, 3 (1978) 495--515. 7 Fuxe, K., HSkfelt, T., Ljungdahl, A., Agnati, L., Johansson, O. and Perez de la Mora, M., Evidence for an inhibitory GABAergic control of the mesolimbic dopamine neurons: possibility of improving treatment of schizophrenia by combined treatment with neuroleptic8 and GABAergic drugs, Med. Biol., 53 (1975) 177--183. 8 Greenlee, D., Van Ness, P.C. and Olsen, R.W., Endogenous inhibitor of GABA binding in mammalian brain, Life Sci., 22 (1978) 1653--1662. 9 Greenlee, D., Van Ness, P.C. and Olsen, R.W., Gamma-aminobutyric acid binding in mammalian brain: receptor-like specificity of sodium-independent sites, J. Neurochem. 31 (1978) 933--938. 10 Gulman, N.C., Bahr, B., Anderson, B. and Eliassen, H.M., A double blind trial of baclofen against placebo in the treatment of schizophrenia, Acta psychiat, scand., 54 (1976) 287--29. 11 Iversen, L.L., Biochemical pharmacology of GABA. In M.A. Lipton, A. Di Mascio and K.F. Killam (Eds.), Psychopharmacology: A Generation of Progress, Raven Press, New York, 1978, pp. 25--38. 12 Konig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotoxic Atlas, Williams and Wilkins, Baltimore, 1963, 162pp. 13 Naik, S.R., Guidotti, A. and Costa, E. Central GABA receptor agonists: comparison of muscimol and baclofen, Neuropharmacology, 15 (1976) 479--484. 14 Olpe, H.R., Demieville, H., Baitzer, V., Bencze, W.L., Koella, W.P., Wolf, P. and Ha~, H.L., The biological activity of d- and l-baclofen (LIORESAL®) Europ. J. Pharmacol., 52 (1978) 133--136. 15 Olsen, R.W., Ticku, M.K., Van Ness, P.C. and Greenlee, D. Effects of drugs on ~aminobutyric acid receptors, uptake, release and synthesis in vitro, Brain Res., 139 (1978) 277--294. 16 Potashner, S.J., Baclofen: Effects on amino acid release and metabolism in slices of Guinea Pig cerebral cortex, J. Neurochem., 32 (1979) 103--109. 17 Roberts, P.J., Gupta, fl.K. and Shargill, N.S. The interaction of baclofen (~-(4-chlorphenyl) GABA) systems in rat brain: evidence for a releasing action, Brain Res., 155 (1978) 209--212. 18 Toffano, G., Guidotti, A. and Costa, E. Purification of an endogenous protein inhibitor of the high affinity binding of ~,-aminobutyric acid to synaptic membranes of rat brain, Proc. Nat. Acad. Sci. USA, 75 (1978) 4024--4028. 19 Waddington, J.L., Induction of rotational behaviour by intranigral baclofen suggests possible GABA agonist activity, Experientia, 33 (1977) 1345--1346. 20 Waddington, J.L., Behavioural evidence for GABAergic activity of the benzodiazepine flurazepam, Europ. J. Pharmacol., 51 (1978) 417--422. 21 Waddington, J.L. and Cross, A.J. Denervation supersensitivity in the striatonigral GABA pathway, Nature, 276 (1978) 618--620. 22 Waddington, J.L. and Crow, T.J. Methodological problems in the measurement of drug-induced rotational behaviour, Psychopharmacology, 58 (1978) 153--155. 23 Waddington, J.L. and Cross, A.J. Baclofen and muscimol: Behavioural and neurochemical sequelae of unilateral intranigral administration and effects on 3H-GABA receptor binding, Naunyn-Schmiedeberg's Arch. Pharmacol., 306 (1979) 275--280.
123
© Elsevier/North-Holland Scientific Publishers Ltd.
PHARMACOLOGY OF ( + ) - A N D ( - ) - B A C L O F E N : GABA.DEPENDANT ROTATIONAL BEHAVIOUR AND [aH]GABA RECEPTOR BINDING STUDIES
J.L. WADDINGTON and A.L. CROSS Division o f Psychiatry, M.R.C. Clinical Research Centre, Harrow, H A I 3UJ (U.K.)
(Received March 3rd, 1979) (Accepted May 16th, 1979)
SUMMARY (+)- and (--)-baclofen were equiactive in inducing contralateral rotational behaviour following their unilateral injection into the zona reticulata of the rat substantia nigra, a proposed GABA-dependent rotational behaviour model, and in displacing the specific binding of [3H]GABA to frozen and thawed membrane preparations of rat brain. These non-stereospecific actions of baclofen at a population of cerebral GABA receptors contrast with stereospecific neuronal depressant effects probably mediated via presynaptic inhibition of the release of excitatory amino acid neurotransmitters. The present results indicate that baclofen may have t w o distinct mechanisms of action and this may account for the debate on the pharmacology of this compound. The development of clinically active agents which stimulate cerebral 7aminobutyric acid (GABA) receptors has generated considerable interest as it has been proposed that such compounds may be of theropeutic potential in neuropsychiatric conditions, including Huntington's chorea and schizophrenia [2]. Baclofen, the ~-(p-chlorophenyl) derivative of GABA, provides a means of conveying a GABA moiety into the brain [5]. However, confusion over the status of baclofen as a GABA agonist has led to difficulty in interpreting animal [7,19] and clinical [10] studies using this compound. While many studies have concluded that baclofen is devoid of GABA agonist activity [3,13], others have clearly indicated a GABA mimetic action [6,15]. This study describes~the effects of the resolved enantiomers of baclofen [ 14] in a GABA-dependant, dopamine (DA)-independent rotational behaviour model [20,21] and in a [3H]GABA receptor binding assay [4]. We have recently reported that racemic baclofen is active in both models [23], though in a manner not entirely consistent with a pure GABA agonist
124
action, and here propose how studies with it's stereoisomers may help resolve the diverse pharmacological properties of this compound. Rotational behaviour studies were performed on male Sprague--Dawley rats, 150--200 g, as previously described [20,21,23]. Briefly, rats received unilateral intracerebral injections of 500 ng (+)- or (-)-baclofen (as the hydrochloride, Ciba-Geigy, Basel) into the zona reticulata of the substantia nigra (SNR), in 1 ul 0.9% physiological saline. The intracerebral injections were made by slow motion infusion, under light ether anaesthesia, using stereotaxic coordinates [12] A2.2, V-2.8, L2.0. Immediately after the intracerebral injection procedure animals were placed in rotameters [ 22] for recording of rotational behaviour over a 45-min period. The specific binding of [3H] GABA to crude synaptic membrane preparations of whole rat brain was assessed in the presence and absence of (+)- or (-)-baclofen as previously described [4,23]. Membranes were frozen and thawed before use, and binding assays performed with 10 nM [SH]GABA {30--60 Ci/mmol, Radiochemical Centre, Amersham, U.K.). Specific binding was defined as the difference between binding occurring in the presence and in the absence of an excess (1 mM) of unlabelled GABA. Rotational responses to histologically-confirmed unilateral injections of 500 ng (+)- and (-}-baclofen are shown in Fig. 1. Both stereoisomers induced contralateral circling and the magnitudes and time-courses of these rotational responses were indistinguishable both from each other and from those produced by 500 ng racemic baclofen in an identical behavioural assay in which injections of saline or/3-phenylethylamine failed to induce contralateral turning [23]. These rotational responses to unilateral injections of 500 ng baclofen into SNR were, however, considerably less intense than those produced by smaller quantities of the potent and specific GABA agonist muscimol administered under identical conditions [ 23]. 0 m
-100
-200
-S -3OO E -400~
I
0
I
15
I
30
I
45
Time post injection (rain) Fig. 1. Rotational responses of rats to unilateral intranigral injections (500 ng) of (+)- or (--)-baclofen in 1 ~I ~llne. Rotations are plotted cumulatively against time after injection, and are the means + S.E.M. of 6 animals/group, o, (+)-baclofen; e, (--)-baclofen.
125
Both (+)- and (-)-baclofen were equiactive in displacing [3H]GABA binding. Log-probit plots indicated that the ICs0 for each isomer was 38/~M. This is essentially identical with the potency of racemic baclofen, though considerably less than the potency of muscimol and GABA itself, in inhil> iting [3H]GABA binding in an identical assay [23] (Table I). We have proposed that the contralateral rotational response widely shown to follow unilateral injections of GABA and GABAergic drugs into SNR may constitute a GABA
ICs0 (pM) b
(+)-baclofen (--)-baclofen (+)/(--)-baclofen GABA Muscimol
38 38 40 c 0.4 c 0.04 c
aThe receptor-binding procedure was performed essentially to the m e t h o d o f Enna and S n y d e r [4] with [ 3 H ] G A B A at a c o n c e n t r a t i o n o f 10 nM. b . . . ICs0 d e t e r m i n e d f r o m log-problt plots o f d u p h c a t e determinations. ~Data f r o m Waddington and Cross [23] for comparison.
126
characterised by the removal of an endogenous protein inhibitor of GABA binding which reveals a class of low density, high affinity GABA binding sites [8,18]. Both low and high affinity GABA binding sites were inhibited by receptor-specific drugs in proportion with their pharmacological potency [ 8] and any functional dissociation between them remains to be determined. However, we (unpublished data) and others [17] have found that baclofen does not displace [3H]GABA specifically bound to high affinity binding sites in Triton-treated membrane preparations. Thus, the seemingly preferential action of baclofen on low affinity GABA binding sites would support our contention, based on behavioural studies [23], that this compound does not possess pure GABA agonist activity. It may act on a particular population of GABA receptors, and we find that this GABAergic action, like that in the GABA-dependent rotational model, does not show stereospecificity. Whilst the present study indicates that the previously described GABA mimetic action of baclofen [6,15,23] is a non-stereospecific effect, there is clear evidence that baclofen has potent neuronal depressant properties that are unrelated to effects on GABAergic mechanisms [3,6] and which are stereospecific for the (-)-isomer [1,14]. These properties seem to be mediated by a presynaptic inhibition of the release of excitatory amino acid neurotransmitters [1,3,6,16]. From our data we suggest that the debate as to whether baclofen does or does not show GABA mimetic properties may reflect it's ability to induce depression of neuronal activity by two distinct mechanisms. Thus, while (-)-baclofen may potently and stereospecifically inhibit the release of excitatory aminoacid neurotransmitters, high concentrations of baclofen can also stimulate a population of cerebral GABA receptors in a non-stereospecific manner. This duplicity of action would indicate that baclofen is a poor pharmacological tool for either animal or clinical studies of GABAergic mechanisms. ACKNOWLEDGEMENT
J.L.W. and A.J.C. are M.R.C Scholars. A preliminary account of these results has been communicated to the British Pharmacological Society, University of Nottingham, U.K., September 1978. We are grateful to CibaGeigy, Basel, Switzerland, for gifts of drugs. REFERENCES 1 Ault, B. and Evans, H., Central depressant actions of baclofen, J. Physiol., 284 (1978) 131P. 2 Chase, T.N. and Walters, J.R., Pharmacologic approaches to the manipulation of GABA-mediated synaptic function in man. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven Press, New York, 1976, pp. 497--513.
127 3 Davidoff, R.A., Pharmacology of spasticity, Neurology, 28 (1978) 46--51. 4 Enna, S.J. and Snyder, S.H., Properties of ~-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions, Brain Res., 100 (1975) 81--97. 5 Fehr, H.U. and Bein, H.J., Sites of action of a new muscle relaxant (baclofen, Lioresal ®, Ciba 34 647-Ba), J. Int. Med. Res., 2 (1974) 36--37. 6 Fox, S., Krnjevic, K., Morris, M.E., Pull, E. and Werman, R., Action of baclofen on mammalian synaptic transmission, Neuroscience, 3 (1978) 495--515. 7 Fuxe, K., HSkfelt, T., Ljungdahl, A., Agnati, L., Johansson, O. and Perez de la Mora, M., Evidence for an inhibitory GABAergic control of the mesolimbic dopamine neurons: possibility of improving treatment of schizophrenia by combined treatment with neuroleptic8 and GABAergic drugs, Med. Biol., 53 (1975) 177--183. 8 Greenlee, D., Van Ness, P.C. and Olsen, R.W., Endogenous inhibitor of GABA binding in mammalian brain, Life Sci., 22 (1978) 1653--1662. 9 Greenlee, D., Van Ness, P.C. and Olsen, R.W., Gamma-aminobutyric acid binding in mammalian brain: receptor-like specificity of sodium-independent sites, J. Neurochem. 31 (1978) 933--938. 10 Gulman, N.C., Bahr, B., Anderson, B. and Eliassen, H.M., A double blind trial of baclofen against placebo in the treatment of schizophrenia, Acta psychiat, scand., 54 (1976) 287--29. 11 Iversen, L.L., Biochemical pharmacology of GABA. In M.A. Lipton, A. Di Mascio and K.F. Killam (Eds.), Psychopharmacology: A Generation of Progress, Raven Press, New York, 1978, pp. 25--38. 12 Konig, J.F.R. and Klippel, R.A., The Rat Brain: A Stereotoxic Atlas, Williams and Wilkins, Baltimore, 1963, 162pp. 13 Naik, S.R., Guidotti, A. and Costa, E. Central GABA receptor agonists: comparison of muscimol and baclofen, Neuropharmacology, 15 (1976) 479--484. 14 Olpe, H.R., Demieville, H., Baitzer, V., Bencze, W.L., Koella, W.P., Wolf, P. and Ha~, H.L., The biological activity of d- and l-baclofen (LIORESAL®) Europ. J. Pharmacol., 52 (1978) 133--136. 15 Olsen, R.W., Ticku, M.K., Van Ness, P.C. and Greenlee, D. Effects of drugs on ~aminobutyric acid receptors, uptake, release and synthesis in vitro, Brain Res., 139 (1978) 277--294. 16 Potashner, S.J., Baclofen: Effects on amino acid release and metabolism in slices of Guinea Pig cerebral cortex, J. Neurochem., 32 (1979) 103--109. 17 Roberts, P.J., Gupta, fl.K. and Shargill, N.S. The interaction of baclofen (~-(4-chlorphenyl) GABA) systems in rat brain: evidence for a releasing action, Brain Res., 155 (1978) 209--212. 18 Toffano, G., Guidotti, A. and Costa, E. Purification of an endogenous protein inhibitor of the high affinity binding of ~,-aminobutyric acid to synaptic membranes of rat brain, Proc. Nat. Acad. Sci. USA, 75 (1978) 4024--4028. 19 Waddington, J.L., Induction of rotational behaviour by intranigral baclofen suggests possible GABA agonist activity, Experientia, 33 (1977) 1345--1346. 20 Waddington, J.L., Behavioural evidence for GABAergic activity of the benzodiazepine flurazepam, Europ. J. Pharmacol., 51 (1978) 417--422. 21 Waddington, J.L. and Cross, A.J. Denervation supersensitivity in the striatonigral GABA pathway, Nature, 276 (1978) 618--620. 22 Waddington, J.L. and Crow, T.J. Methodological problems in the measurement of drug-induced rotational behaviour, Psychopharmacology, 58 (1978) 153--155. 23 Waddington, J.L. and Cross, A.J. Baclofen and muscimol: Behavioural and neurochemical sequelae of unilateral intranigral administration and effects on 3H-GABA receptor binding, Naunyn-Schmiedeberg's Arch. Pharmacol., 306 (1979) 275--280.