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Chronic pentobarbital diminishes GABA- and pentobarbital-enhanced [3H]diazepam binding to benzodiazepine receptors
European Journal of Pharmacology, 87 (1983) 169-170 Elsevier Biomedical Press
169
Rapid communication CHRONIC PENTOBARBITAL DIMINISHES GABA- AND PENTOBARBITAL-ENHANCED ]3H]DIAZEPAM BINDING TO BENZODIAZEPINE RECEPTORS B A R B A R A F. R O T H - S C H E C H T E R i,,, C O R I N N E EBEL
I
and PIERRE M A L L O R G A 2.,,
I Laboratotre de Pharmacodynamie, Universit~ Louis Pasteur, B.P. 10, 67048 Strasbourg Cedex. France, and 2 Centre de Recherche Merrell International, 16, rue d'Ankara, 67084 Strasbourg Cedex, France Received 13 December 1982, accepted 16 December 1982
One of the recently identified high-affinity benzodiazepine binding sites present in the mammalian CNS forms part of a macromolecular complex consisting of a GABA receptor, a benzodiazepine receptor and a chloride ionophore with binding sites for dihydroxypicrotoxinin and some barbiturates. The complex possesses drug receptor sites which have been studied by radioactive ligand binding, and their relevance to the in vivo actions of the drugs that bind to them in vitro has been established (Skolnick and Paul, 1981; Tallman et al., 1980). Modulation of the postsynaptic response to GABA can be considered as one of the direct actions of both the benzodiazepines and the barbiturates (Tallman et al., 1980; Skolnick and Paul, 1981). Changes in receptor density and affinity in response to chronic drug administration is a wellrecognized phenomenon of cellular adaptation. Little is known about adaptive responses of the GABA-benzodiazepine receptor-ionophore complex after chronic administration of drugs affecting one or all of its components (Crawley et al., 1982). Recently, we have been able to identify specific neuronal [3 H]diazepam (DZ) binding sites in isolated neurons grown in primary culture. Their biochemical characterization will be published elsewhere. Briefly, these neurons contain DZ binding sites which in the presence of chloride can be activated in a dose-dependent manner by the addition of GABA and pentobarbital. They appear, * To whom all correspondence should be addressed. ** Present address; Centre de Recherche Merck Sharp and Dohme-Cbibret, B.P. 88, 63203 Riom Cedex, France. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
therefore, to be identical to the binding sites described for the pharmacologically relevant benzodiazepines in the mammalian CNS. We now report that chronic exposure of these neurons to pentobarbital significantly reduces GABA- and pentobarbital-enhanced [3H]DZ binding to neuronal sites. Primary cultures of isolated neurons were prepared from the cerebral hemispheres of 8-day old chick embryos and maintained for 48 h in a medium consisting of DEM fortified by 20% fetal calf serum (Pettmann et al., 1979). Thereafter, this medium was replaced by serum-free medium (Bottenstein and Sato, 1980). All cultures were maintained for an additional 5 days, half of them in drug-free medium, the other half containing 5 × 1 0 - 4 M pentobarbital. No morphological modifications could be detected in the barbiturate-treated neurons (Phase contrast, 200 × ). For the binding studies the medium was carefully aspirated from the culture plates and the cells were rinsed twice with 5 ml of phosphate-buffered saline (PBS) (pH 7.4). The cells were then removed from the dish with a rubber spatula, transferred to a centrifuge tube containing PBS, centrifuged at 900 X g and the pellet kept at - 2 5 ° C until use. Prior to the binding experiment, the pellet was homogenized for 15 s in Tris-HCl 50 mM (pH 7.0 at 25°C) using a Polytron (Kinematica, Luzern, Switzerland). The homogenate was centrifuged at 40000 x g for 10 min and the supernatant discarded. The resuspension-centrifugation cycle was repeated twice and the final pellet resuspended in Tris-HC1 to a protein concentration of 0.5-0.9 mg/ml. The [3H]DZ binding incubations were carried o u t in triplicate at 4°C for 30 min in a
170
140
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Fig. 1. Effect of G A B A and pentobarbital on [3H]diazepam
binding to membranes of neuronal cultures. Neurons were prepared from the hemispheres of 8-day old chick embryosand maintained in serum-free medium in the absence ( ) and presence (. . . . . . ) of 5 × 10-4 M pentobarbital as described in the text. The specific binding of [3HIDZ (0.5 nM) was measured in the presence of Tris-chloride buffer. This experiment was replicated twice. Within each experiment the differences between the control and treated cultures were highlysignificant (P < 0.01, Student's t-test), but variation of absolute values between experiments did not permit pooling of the data.
final volume of 1 ml containing [3H]DZ (0.5 nM), 50 mM Tris-HC1 (pH 7.0 at 25°C), 600 p.l of the membrane preparation, in the presence or absence of the drug tested (sodium pentobarbital (Abbott), GABA (Sigma), or clonazepam (gift from Hoffmann-La-Roche)). The specific binding was defined as the difference between [3H]DZ bound in the absence and in the presence of 1 0 - 6 M clonazepam. In the presence of chloride ions the specific [3H]DZ binding was 4.887 f m o l / m g protein (34% of total binding) in control cultures and 5.776 f m o l / m g protein (34% of total binding) in cultures grown in the presence of pentobarbitai. GABA was found to enhance, in a dose-dependent fashion (10 -7 to 10-5 M), [3H]DZ binding to membranes from neurons grown in the absence or presence of pentobarbital, but the magnitude of the enhancement was reduced in the pentobarbital-treated neurons (fig. 1). When pentobarbital
(10 s to 10 3 M) was added to the membrane preparations, it stimulated [3H]DZ binding only in control neurons. It had no significant effect on [3H]DZ binding to membranes from pentobarbital-treated neurons. It might be argued that the lack of response to the direct action of pentobarbital is due to the presence of some residual pentobarbital resulting in maximal enhancement of [3H]DZ binding. However, this appears unlikely since the proportion of specifically bound [3H]DZ is identical in both membrane preparations. Therefore, chronic exposure (5 days) of neurons to pentobarbital resulted in decreased sensitivity to the enhancement by pentobarbitai and GABA of the interactions between the neuronal binding sites of diazepam and pentobarbital within the GABA-benzodiazepine receptor-ionophore complex. At this time we do not know whether this phenomenon is due to a change in number of binding sites or their affinities. Scatchard analysis data obtained following chronic exposure to various doses of pentobarbital and study of the GABA-pentobarbital synergism will be needed to clarify this point. Our observations confirm the functional coupling between the GABA- and barbiturate-binding sites of the GABA-benzodiazepine receptor complex. Chronic pentobarbital treatment not only affects the sensitivity of the receptor complex to the barbiturate but to GABA as well. Thus, altered sensitivity to barbiturates after its chronic administration may be due to functional modulation of this neuronal receptor complex.
References Bottenstein, J.E. and G.H. Sato, 1980, Fibronectin and polylysine requirement for proliferation of neuroblastoma in defined medium, Exp. Cell Res. 129, 361. Crawley, J.N., P.J. Marangos, J. Stivers and K.J. Goodwin, 1982, Chronic clonazepam administration induces benzodiazepine subsensitivity, Neuropharmacol. 21, 85. Pettmann, B., J.C. Louis and M. Sensenbrenner, 1979, Morphological and biochemical maturation of neurons cultured in the absence of glial cells, Nature 281, 378. Skolnick, P. and S. Paul, 1981, The mechanism(s) of action of benzodiazepines, Med. Res. Rev. I, 3. Tallman, J., S. Paul, P. Skolnick and R.D. Gallager, 1980, Receptors for the age of anxiety: Pharmacology of the benzodiazepines, Science 207, 274.
169
Rapid communication CHRONIC PENTOBARBITAL DIMINISHES GABA- AND PENTOBARBITAL-ENHANCED ]3H]DIAZEPAM BINDING TO BENZODIAZEPINE RECEPTORS B A R B A R A F. R O T H - S C H E C H T E R i,,, C O R I N N E EBEL
I
and PIERRE M A L L O R G A 2.,,
I Laboratotre de Pharmacodynamie, Universit~ Louis Pasteur, B.P. 10, 67048 Strasbourg Cedex. France, and 2 Centre de Recherche Merrell International, 16, rue d'Ankara, 67084 Strasbourg Cedex, France Received 13 December 1982, accepted 16 December 1982
One of the recently identified high-affinity benzodiazepine binding sites present in the mammalian CNS forms part of a macromolecular complex consisting of a GABA receptor, a benzodiazepine receptor and a chloride ionophore with binding sites for dihydroxypicrotoxinin and some barbiturates. The complex possesses drug receptor sites which have been studied by radioactive ligand binding, and their relevance to the in vivo actions of the drugs that bind to them in vitro has been established (Skolnick and Paul, 1981; Tallman et al., 1980). Modulation of the postsynaptic response to GABA can be considered as one of the direct actions of both the benzodiazepines and the barbiturates (Tallman et al., 1980; Skolnick and Paul, 1981). Changes in receptor density and affinity in response to chronic drug administration is a wellrecognized phenomenon of cellular adaptation. Little is known about adaptive responses of the GABA-benzodiazepine receptor-ionophore complex after chronic administration of drugs affecting one or all of its components (Crawley et al., 1982). Recently, we have been able to identify specific neuronal [3 H]diazepam (DZ) binding sites in isolated neurons grown in primary culture. Their biochemical characterization will be published elsewhere. Briefly, these neurons contain DZ binding sites which in the presence of chloride can be activated in a dose-dependent manner by the addition of GABA and pentobarbital. They appear, * To whom all correspondence should be addressed. ** Present address; Centre de Recherche Merck Sharp and Dohme-Cbibret, B.P. 88, 63203 Riom Cedex, France. 0014-2999/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
therefore, to be identical to the binding sites described for the pharmacologically relevant benzodiazepines in the mammalian CNS. We now report that chronic exposure of these neurons to pentobarbital significantly reduces GABA- and pentobarbital-enhanced [3H]DZ binding to neuronal sites. Primary cultures of isolated neurons were prepared from the cerebral hemispheres of 8-day old chick embryos and maintained for 48 h in a medium consisting of DEM fortified by 20% fetal calf serum (Pettmann et al., 1979). Thereafter, this medium was replaced by serum-free medium (Bottenstein and Sato, 1980). All cultures were maintained for an additional 5 days, half of them in drug-free medium, the other half containing 5 × 1 0 - 4 M pentobarbital. No morphological modifications could be detected in the barbiturate-treated neurons (Phase contrast, 200 × ). For the binding studies the medium was carefully aspirated from the culture plates and the cells were rinsed twice with 5 ml of phosphate-buffered saline (PBS) (pH 7.4). The cells were then removed from the dish with a rubber spatula, transferred to a centrifuge tube containing PBS, centrifuged at 900 X g and the pellet kept at - 2 5 ° C until use. Prior to the binding experiment, the pellet was homogenized for 15 s in Tris-HCl 50 mM (pH 7.0 at 25°C) using a Polytron (Kinematica, Luzern, Switzerland). The homogenate was centrifuged at 40000 x g for 10 min and the supernatant discarded. The resuspension-centrifugation cycle was repeated twice and the final pellet resuspended in Tris-HC1 to a protein concentration of 0.5-0.9 mg/ml. The [3H]DZ binding incubations were carried o u t in triplicate at 4°C for 30 min in a
170
140
° Z :3 0
/
120
I00
80 ~' 60
/
,
40
t
BA
/0 t/
/
°
Z
o=
=o
io
F~ NTOeAI~ITAL
Ioo
~)00 [ , , M 3
Fig. 1. Effect of G A B A and pentobarbital on [3H]diazepam
binding to membranes of neuronal cultures. Neurons were prepared from the hemispheres of 8-day old chick embryosand maintained in serum-free medium in the absence ( ) and presence (. . . . . . ) of 5 × 10-4 M pentobarbital as described in the text. The specific binding of [3HIDZ (0.5 nM) was measured in the presence of Tris-chloride buffer. This experiment was replicated twice. Within each experiment the differences between the control and treated cultures were highlysignificant (P < 0.01, Student's t-test), but variation of absolute values between experiments did not permit pooling of the data.
final volume of 1 ml containing [3H]DZ (0.5 nM), 50 mM Tris-HC1 (pH 7.0 at 25°C), 600 p.l of the membrane preparation, in the presence or absence of the drug tested (sodium pentobarbital (Abbott), GABA (Sigma), or clonazepam (gift from Hoffmann-La-Roche)). The specific binding was defined as the difference between [3H]DZ bound in the absence and in the presence of 1 0 - 6 M clonazepam. In the presence of chloride ions the specific [3H]DZ binding was 4.887 f m o l / m g protein (34% of total binding) in control cultures and 5.776 f m o l / m g protein (34% of total binding) in cultures grown in the presence of pentobarbitai. GABA was found to enhance, in a dose-dependent fashion (10 -7 to 10-5 M), [3H]DZ binding to membranes from neurons grown in the absence or presence of pentobarbital, but the magnitude of the enhancement was reduced in the pentobarbital-treated neurons (fig. 1). When pentobarbital
(10 s to 10 3 M) was added to the membrane preparations, it stimulated [3H]DZ binding only in control neurons. It had no significant effect on [3H]DZ binding to membranes from pentobarbital-treated neurons. It might be argued that the lack of response to the direct action of pentobarbital is due to the presence of some residual pentobarbital resulting in maximal enhancement of [3H]DZ binding. However, this appears unlikely since the proportion of specifically bound [3H]DZ is identical in both membrane preparations. Therefore, chronic exposure (5 days) of neurons to pentobarbital resulted in decreased sensitivity to the enhancement by pentobarbitai and GABA of the interactions between the neuronal binding sites of diazepam and pentobarbital within the GABA-benzodiazepine receptor-ionophore complex. At this time we do not know whether this phenomenon is due to a change in number of binding sites or their affinities. Scatchard analysis data obtained following chronic exposure to various doses of pentobarbital and study of the GABA-pentobarbital synergism will be needed to clarify this point. Our observations confirm the functional coupling between the GABA- and barbiturate-binding sites of the GABA-benzodiazepine receptor complex. Chronic pentobarbital treatment not only affects the sensitivity of the receptor complex to the barbiturate but to GABA as well. Thus, altered sensitivity to barbiturates after its chronic administration may be due to functional modulation of this neuronal receptor complex.
References Bottenstein, J.E. and G.H. Sato, 1980, Fibronectin and polylysine requirement for proliferation of neuroblastoma in defined medium, Exp. Cell Res. 129, 361. Crawley, J.N., P.J. Marangos, J. Stivers and K.J. Goodwin, 1982, Chronic clonazepam administration induces benzodiazepine subsensitivity, Neuropharmacol. 21, 85. Pettmann, B., J.C. Louis and M. Sensenbrenner, 1979, Morphological and biochemical maturation of neurons cultured in the absence of glial cells, Nature 281, 378. Skolnick, P. and S. Paul, 1981, The mechanism(s) of action of benzodiazepines, Med. Res. Rev. I, 3. Tallman, J., S. Paul, P. Skolnick and R.D. Gallager, 1980, Receptors for the age of anxiety: Pharmacology of the benzodiazepines, Science 207, 274.