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The insensitivity of developing benzodiazepine receptors to chronic treatment with diazepam, GABA and muscimol in brain cell cultures
Brain Research, 210 (1981) 4 7 1 4 7 4 © Elsevier/North-Holland Biomedical Press
471
The insensitivity of developing benzodiazepine receptors to chronic treatment with diazepam, GABA and rnuscimol in brain cell cultures
D E B O R A H B. S H I B L A , M A R Y A N N G A R D E L L a n d J O S E P H H. N E A L E
The Department of Biology, Georgetown University, Washington, D.C. 20057 and The Laboratory of Neurophysiology, NINCDS, National Institute of Health, Bethesda, Md. 20205 (U.S.A.) (Accepted N o v e m b e r 20th, 1980)
Key words: benzodiazepine receptor - - diazepam - - G A B A - - m u s c i m o l - - d e v e l o p m e n t
The benzodiazepines (BZ) are a widely prescribed class of drugs which act as hypnotics, muscle relaxants, anti-anxiety agents and anti-convulsants. The demonstration that nervous system membranes bind BZ in a saturable and stereospecific manner suggests that this class of drugs may act through cell surface receptors1,~. The interaction of BZ both pharmacologically and physiologically with 7-aminobutyric acid (GABA) indicates that the membrane binding sites for BZ and GABA may, in some cases, be functionally related. BZ/GABA interaction is not completely understood and the possible existence of an endogenous ligand for the BZ receptor has resulted in uncel~ainty as to the mode of action of this class of drugs on a cellular level (for review see ref. 6). We have followed the course of appearance of the BZ receptor in the mouse central nervous system, both in vivo and in cell culture. We have also examined the effect of chronic treatment with BZ, GABA and the GABA agonist, muscimol, on the development of the BZ receptor in vitro in an attempt to determine the role of receptor ligands in the regulation of BZ receptor development. Dissociated cell cultures were prepared from total brain tissue which had been removed from C57B1/6 mouse fetuses between 13 and 15 days of gestation. The cell cultures were prepared by a method which has been described for murine spinal cord tissue 4. Cells were inoculated at a density of one brain per 60 mm culture dish and treated with a mitotic inhibitor after 3 days and again after 10 days in culture. The major modification of the published method involved the use of HEPES buffered minimal essential medium (20-30 mM HEPES, pH 7.4) rather than the traditional COz-bicarbonate buffer system. The HEPES buffer was utilized in order to avoid the pH fluctuations which result from the failure to maintain adequate CO2 concentrations in cell culture incubators. (Murine brain, spinal cord and dorsal sensory ganglia which develop in this HEPES buffered medium appear morphologically similar to those which develop in the CO2 system.) Membrane homogenates were prepared from mouse brain tissue which was
472 grown in culture, from brains removed directly f r o m fetuses which were 14,16 or t8 days in gestation and f r o m adult mouse brains. The membranes were washed extensively and assayed for [aH]diazepam (New England Nuclear, 64 and 83 Ci/mmol) binding as determined by a v a c u u m filtration assay 1,3. Specific binding was determined as the difference between the m e m b r a n e - b o u n d radioactivity determined in reactions containing 1000-fold excess unlabeled diazepam (non-specific binding) and those reactions performed in the absence o f unlabeled competitor (total binding). The protein concentration was determined from aliquots o f the membrane homogenate obtained at the time of assay and solubilized in N a O H . The binding o f [ZH]diazepam to brain cell membranes from adult mice was determined over a concentration range o f 0.1-20 nM o f radiolabeled ligand. Scatchard analysis o f this data yields an apparent dissociation constant (Ka) o f 8.4 n M ( ± 0.9 nM, S.D.) and the total n u m b e r of binding sites (Bmax) was estimated as 2.3 j : 0.3 fmol/#g protein. Brain cells obtained from 13.5 days in term fetal mice and permitted to develop for 3 weeks in dissociated cell cultures were similarly assayed for the presence o f diazepam binding sites over a radiolabeled ligand range o f 1-12 nM. The cultured cell membranes had an apparent K d of 8.7 ~ 1.7 n M and a Bmax of 3.1 :~ 0.1 fmol//tg protein. The cell cultures consistently developed a greater density o f binding sites than the adult brain when expressed in terms o f the total culture or brain protein. The Bmax value for [3H]DZP binding to cultured brain is influenced by the relative number o f neuronal and non-neuronal cells in the culture, a ratio which may be controlled to some extent by the timing and duration of the chemical inhibition o f non-neuronal cell division. TABLE 1 Development of brain B Z binding sites in vivo and in primary culture
Membrane homogenates were prepared from mouse brain cells after the indicated intervals in dissociated cell culture or development in vivo. Membrane preparations were incubated with 1, 2 or 4 nM [3H]diazepam and the specific binding to saturable BZ receptors was determined at each concentration. Cell cultures were prepared from dissociated 13.5 day fetal brains and the receptor binding in this fetal tissue is presented as the zero time reference for the cell cultures. Each value represents the data from triplicate determinations of both total and non-specific binding. The range of these values for each data point was less than 15 ~. Tissue
Specific 3H-diazepam binding (fmol/pg protein) 1 nM
2 nM
4 nM
0.04 0.12 0.25 0.29
0.06 0.25 0.48 0.51
0.10 0.43 0.72 0.88
0.04 0.05 0.15 0.21
0.06 0.08 0.27 0.35
0.10 0.15 0.45 0.61
(A) Brain in culture
0 Time (13.5 days in utero) 1 (weeks) 2 (weeks) 3 (weeks) ( B) Brain in vivo
13.5 days in utero 16.5 days in utero 18.5 days in utero Adult
473 The time course of the BZ receptor development in fetal mouse brain and in cultured brain is presented in Table I. The progressive increase in diazepam binding during 3 weeks in culture parallels the development of these membrane receptors in vivo. The cell culture inoculum was prepared from fetal brain obtained at 13.5 days of of gestation and thus represents the day O time point for the cultured cells. During the first week in culture there was increase in diazepam binding which very closely matches the increased binding of [3H]DZP to fetal brain between day 13.5 and day 18.5 of development in utero. As has been demonstrated for a variety of other biochemical and morphologic characteristics, the dissociated neuronal cell cultures reproduce, within the limits of this assay system, events associated with the development of the intact central nervous system. Both neuronal and non-neuronal brain cells may bind BZ. The receptor ligands clonazepam and R05-4864 were used to discriminate [SH]diazepam binding using membranes from these two classes of cells. Clonazepam is reported to bind neuronal BZ receptors with high affinity while binding to the non-neuronal and peripheral tissue receptor less effectively. When 10-11-10 -5 M clonazepam was used to compete 5 nM labeled diazepam binding to mouse brain membranes, we observed an IC50 value of 2.5 nM for the competitor and a calculated K~ of 1.6 nM. Little additional inhibition of diazepam binding was obtained at concentrations of clonazepam below 10-8 M. The 10-7 clonazepam decreased the binding of 5 nM [3H]diazepam to adult mouse brain membranes by 89 ~ while blocking by 78 ~ the binding of the radioligand to TABLE II Development of 3H-diazepam binding in mouse brain cell cultures treated with diazepam, GABA and muscimol
Cells were from 3 separate inocula of dissociated fetal brain differentiated for 3 weeks in the presence of 1 #M diazepam (part A). Homogenates from cultured cell membranes were prepared for receptor assay and the binding was determined in the indicated concentrations of [3H]diazepam. The apparent receptor affinity(Ka) and receptor density(Binax)were estimated from a Scatchard analysisof the specific binding data. In a separate experiment (part B) cells were maintained for 3 weeks in culture in the presence of 1/~M GABA or muscimoland the membranes similarlyassayed for saturable [SH]diazepam binding sites. Culture conditions
Specific [3HJdiazepam binding (fmol/l~g protein) 1 nM
2 nM
4 nM
8 nM
12 n M
0.35 0.32
0.55 0.50
0.96 0.92
1.38
1.92 1.89
0.26 0.27
0.49 0.54
0.98 0.96
----
0.74 0.72 0.78
1.08 1.04 1.22
Ka n M
Bmax(fmol/ I~g protein)
(A) Expt. 1
Control Diazepam-treated
1.43
8.7 10.7
3.1 3.4
6.3
3.0
6.8 6.2
3.0 3.1
Expt. 2
Control Diazepam-treated
m
(B) Expt. 3
Control GABA-treated Muscimol-treated
1.65 1.58
1.97 1.98
1.88
1.98
474 membranes from cells which had differentiated for 3 weeks in culture. The nonneuronal BZ receptor ligand, R05-4864 (10 -5 M), decreased labeled diazepam binding to adult brain membranes by less than 5 o/,, and to the cultured brain cells by 16 ~ . When applied together, the effects of these two competitors were additive. These data suggest that the non-neuronal elements in the cell cultures may contribute more to the total culture BZ receptors than they do to the adult brain in vivo. The factors which regulate the presence of BZ receptors in nervous system cell membranes have not yet been established. Yet the widespread clinical application of BZ provides considerable opportunity for inadvertent exposure of the developing nervous system to this class of drugs. A study of the effect of chronic BZ treatment suggests that the presence of the ligand itself might influence the density of brain receptors 5 while another report indicates that repeated seizure activity may increase the quantity but not the affinity of BZ receptors in the hippocampus 2. In an attempt to determine the influence of a BZ on the development of the BZ receptor, cell cultures were treated with 1 # M diazepam for 3 weeks. In each of the experiments presented in Table IIA, cells from a single inoculum of dissociated fetal brains were cultured in the presence or absence of the BZ. Similar binding of [3H]diazepam was observed in the control and drug-treated cells in both experiments. Because of the considerable data suggesting that the BZ and G A B A receptors may be structurally and functionally coupled in some neuronal systems, we further tested the effect of chronic exposure of the cultured brain cells to 1 /~M GABA or the GABA agonist, muscimol (Table liB). Again, the binding of [aH]diazepam to the membrane homogenates prepared from these cells after 3 weeks in culture was very similar to the binding to the non-drugtreated control cells. Neither the binding affinity nor the density of receptors appeared to be influenced by these chronic treatments. We conclude that at the concentration of ligands tested, the synthesis and catabolism of the BZ receptor do not appear to be regulated through long term receptor occupancy. This work was supported by grants from the Marshall B. Coyne Foundation and the National Institute on Drug Abuse, DA02297, and N I H training Grant G M 07443.
1 Braestrup, C. and Squires, R. F., Specific benzodiazepine receptors in rat brain characterized by high-affinity [aH]diazepam binding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805-3809. 2 McNamara, J. O., Peper, A. M. and Patrone, V., Repeated seisures induced long-term increase in hippocampal benzodiazepine receptors, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 3029-3032. 3 MShler, H. and Okada, T., Benzodiazepine receptor: demonstration in the central nervous system, Science, 198 (1977) 849-851. 4 Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N. and Nelson, P. G., The mouse spinal cord in cell culture. I. Morphology and intrinsic electrophysiologic properties, J. Neurophysiol., 40 (1977) 1132-1150. 5 Rosenberg, H. C. and Chiu, T. H., Decreased aH-diazepam binding is a specific response to chronic benzodiazepine treatment, Life Sci., 24 (1979) 803-808. 8 Taltman, J. F., Paul, S. M., Skolnick, P. and Gallager, D. W., Receptors for the age of anxiety : pharmacology of the benzodiazepines, Science, 207 (1980) 274-281.
471
The insensitivity of developing benzodiazepine receptors to chronic treatment with diazepam, GABA and rnuscimol in brain cell cultures
D E B O R A H B. S H I B L A , M A R Y A N N G A R D E L L a n d J O S E P H H. N E A L E
The Department of Biology, Georgetown University, Washington, D.C. 20057 and The Laboratory of Neurophysiology, NINCDS, National Institute of Health, Bethesda, Md. 20205 (U.S.A.) (Accepted N o v e m b e r 20th, 1980)
Key words: benzodiazepine receptor - - diazepam - - G A B A - - m u s c i m o l - - d e v e l o p m e n t
The benzodiazepines (BZ) are a widely prescribed class of drugs which act as hypnotics, muscle relaxants, anti-anxiety agents and anti-convulsants. The demonstration that nervous system membranes bind BZ in a saturable and stereospecific manner suggests that this class of drugs may act through cell surface receptors1,~. The interaction of BZ both pharmacologically and physiologically with 7-aminobutyric acid (GABA) indicates that the membrane binding sites for BZ and GABA may, in some cases, be functionally related. BZ/GABA interaction is not completely understood and the possible existence of an endogenous ligand for the BZ receptor has resulted in uncel~ainty as to the mode of action of this class of drugs on a cellular level (for review see ref. 6). We have followed the course of appearance of the BZ receptor in the mouse central nervous system, both in vivo and in cell culture. We have also examined the effect of chronic treatment with BZ, GABA and the GABA agonist, muscimol, on the development of the BZ receptor in vitro in an attempt to determine the role of receptor ligands in the regulation of BZ receptor development. Dissociated cell cultures were prepared from total brain tissue which had been removed from C57B1/6 mouse fetuses between 13 and 15 days of gestation. The cell cultures were prepared by a method which has been described for murine spinal cord tissue 4. Cells were inoculated at a density of one brain per 60 mm culture dish and treated with a mitotic inhibitor after 3 days and again after 10 days in culture. The major modification of the published method involved the use of HEPES buffered minimal essential medium (20-30 mM HEPES, pH 7.4) rather than the traditional COz-bicarbonate buffer system. The HEPES buffer was utilized in order to avoid the pH fluctuations which result from the failure to maintain adequate CO2 concentrations in cell culture incubators. (Murine brain, spinal cord and dorsal sensory ganglia which develop in this HEPES buffered medium appear morphologically similar to those which develop in the CO2 system.) Membrane homogenates were prepared from mouse brain tissue which was
472 grown in culture, from brains removed directly f r o m fetuses which were 14,16 or t8 days in gestation and f r o m adult mouse brains. The membranes were washed extensively and assayed for [aH]diazepam (New England Nuclear, 64 and 83 Ci/mmol) binding as determined by a v a c u u m filtration assay 1,3. Specific binding was determined as the difference between the m e m b r a n e - b o u n d radioactivity determined in reactions containing 1000-fold excess unlabeled diazepam (non-specific binding) and those reactions performed in the absence o f unlabeled competitor (total binding). The protein concentration was determined from aliquots o f the membrane homogenate obtained at the time of assay and solubilized in N a O H . The binding o f [ZH]diazepam to brain cell membranes from adult mice was determined over a concentration range o f 0.1-20 nM o f radiolabeled ligand. Scatchard analysis o f this data yields an apparent dissociation constant (Ka) o f 8.4 n M ( ± 0.9 nM, S.D.) and the total n u m b e r of binding sites (Bmax) was estimated as 2.3 j : 0.3 fmol/#g protein. Brain cells obtained from 13.5 days in term fetal mice and permitted to develop for 3 weeks in dissociated cell cultures were similarly assayed for the presence o f diazepam binding sites over a radiolabeled ligand range o f 1-12 nM. The cultured cell membranes had an apparent K d of 8.7 ~ 1.7 n M and a Bmax of 3.1 :~ 0.1 fmol//tg protein. The cell cultures consistently developed a greater density o f binding sites than the adult brain when expressed in terms o f the total culture or brain protein. The Bmax value for [3H]DZP binding to cultured brain is influenced by the relative number o f neuronal and non-neuronal cells in the culture, a ratio which may be controlled to some extent by the timing and duration of the chemical inhibition o f non-neuronal cell division. TABLE 1 Development of brain B Z binding sites in vivo and in primary culture
Membrane homogenates were prepared from mouse brain cells after the indicated intervals in dissociated cell culture or development in vivo. Membrane preparations were incubated with 1, 2 or 4 nM [3H]diazepam and the specific binding to saturable BZ receptors was determined at each concentration. Cell cultures were prepared from dissociated 13.5 day fetal brains and the receptor binding in this fetal tissue is presented as the zero time reference for the cell cultures. Each value represents the data from triplicate determinations of both total and non-specific binding. The range of these values for each data point was less than 15 ~. Tissue
Specific 3H-diazepam binding (fmol/pg protein) 1 nM
2 nM
4 nM
0.04 0.12 0.25 0.29
0.06 0.25 0.48 0.51
0.10 0.43 0.72 0.88
0.04 0.05 0.15 0.21
0.06 0.08 0.27 0.35
0.10 0.15 0.45 0.61
(A) Brain in culture
0 Time (13.5 days in utero) 1 (weeks) 2 (weeks) 3 (weeks) ( B) Brain in vivo
13.5 days in utero 16.5 days in utero 18.5 days in utero Adult
473 The time course of the BZ receptor development in fetal mouse brain and in cultured brain is presented in Table I. The progressive increase in diazepam binding during 3 weeks in culture parallels the development of these membrane receptors in vivo. The cell culture inoculum was prepared from fetal brain obtained at 13.5 days of of gestation and thus represents the day O time point for the cultured cells. During the first week in culture there was increase in diazepam binding which very closely matches the increased binding of [3H]DZP to fetal brain between day 13.5 and day 18.5 of development in utero. As has been demonstrated for a variety of other biochemical and morphologic characteristics, the dissociated neuronal cell cultures reproduce, within the limits of this assay system, events associated with the development of the intact central nervous system. Both neuronal and non-neuronal brain cells may bind BZ. The receptor ligands clonazepam and R05-4864 were used to discriminate [SH]diazepam binding using membranes from these two classes of cells. Clonazepam is reported to bind neuronal BZ receptors with high affinity while binding to the non-neuronal and peripheral tissue receptor less effectively. When 10-11-10 -5 M clonazepam was used to compete 5 nM labeled diazepam binding to mouse brain membranes, we observed an IC50 value of 2.5 nM for the competitor and a calculated K~ of 1.6 nM. Little additional inhibition of diazepam binding was obtained at concentrations of clonazepam below 10-8 M. The 10-7 clonazepam decreased the binding of 5 nM [3H]diazepam to adult mouse brain membranes by 89 ~ while blocking by 78 ~ the binding of the radioligand to TABLE II Development of 3H-diazepam binding in mouse brain cell cultures treated with diazepam, GABA and muscimol
Cells were from 3 separate inocula of dissociated fetal brain differentiated for 3 weeks in the presence of 1 #M diazepam (part A). Homogenates from cultured cell membranes were prepared for receptor assay and the binding was determined in the indicated concentrations of [3H]diazepam. The apparent receptor affinity(Ka) and receptor density(Binax)were estimated from a Scatchard analysisof the specific binding data. In a separate experiment (part B) cells were maintained for 3 weeks in culture in the presence of 1/~M GABA or muscimoland the membranes similarlyassayed for saturable [SH]diazepam binding sites. Culture conditions
Specific [3HJdiazepam binding (fmol/l~g protein) 1 nM
2 nM
4 nM
8 nM
12 n M
0.35 0.32
0.55 0.50
0.96 0.92
1.38
1.92 1.89
0.26 0.27
0.49 0.54
0.98 0.96
----
0.74 0.72 0.78
1.08 1.04 1.22
Ka n M
Bmax(fmol/ I~g protein)
(A) Expt. 1
Control Diazepam-treated
1.43
8.7 10.7
3.1 3.4
6.3
3.0
6.8 6.2
3.0 3.1
Expt. 2
Control Diazepam-treated
m
(B) Expt. 3
Control GABA-treated Muscimol-treated
1.65 1.58
1.97 1.98
1.88
1.98
474 membranes from cells which had differentiated for 3 weeks in culture. The nonneuronal BZ receptor ligand, R05-4864 (10 -5 M), decreased labeled diazepam binding to adult brain membranes by less than 5 o/,, and to the cultured brain cells by 16 ~ . When applied together, the effects of these two competitors were additive. These data suggest that the non-neuronal elements in the cell cultures may contribute more to the total culture BZ receptors than they do to the adult brain in vivo. The factors which regulate the presence of BZ receptors in nervous system cell membranes have not yet been established. Yet the widespread clinical application of BZ provides considerable opportunity for inadvertent exposure of the developing nervous system to this class of drugs. A study of the effect of chronic BZ treatment suggests that the presence of the ligand itself might influence the density of brain receptors 5 while another report indicates that repeated seizure activity may increase the quantity but not the affinity of BZ receptors in the hippocampus 2. In an attempt to determine the influence of a BZ on the development of the BZ receptor, cell cultures were treated with 1 # M diazepam for 3 weeks. In each of the experiments presented in Table IIA, cells from a single inoculum of dissociated fetal brains were cultured in the presence or absence of the BZ. Similar binding of [3H]diazepam was observed in the control and drug-treated cells in both experiments. Because of the considerable data suggesting that the BZ and G A B A receptors may be structurally and functionally coupled in some neuronal systems, we further tested the effect of chronic exposure of the cultured brain cells to 1 /~M GABA or the GABA agonist, muscimol (Table liB). Again, the binding of [aH]diazepam to the membrane homogenates prepared from these cells after 3 weeks in culture was very similar to the binding to the non-drugtreated control cells. Neither the binding affinity nor the density of receptors appeared to be influenced by these chronic treatments. We conclude that at the concentration of ligands tested, the synthesis and catabolism of the BZ receptor do not appear to be regulated through long term receptor occupancy. This work was supported by grants from the Marshall B. Coyne Foundation and the National Institute on Drug Abuse, DA02297, and N I H training Grant G M 07443.
1 Braestrup, C. and Squires, R. F., Specific benzodiazepine receptors in rat brain characterized by high-affinity [aH]diazepam binding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805-3809. 2 McNamara, J. O., Peper, A. M. and Patrone, V., Repeated seisures induced long-term increase in hippocampal benzodiazepine receptors, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 3029-3032. 3 MShler, H. and Okada, T., Benzodiazepine receptor: demonstration in the central nervous system, Science, 198 (1977) 849-851. 4 Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N. and Nelson, P. G., The mouse spinal cord in cell culture. I. Morphology and intrinsic electrophysiologic properties, J. Neurophysiol., 40 (1977) 1132-1150. 5 Rosenberg, H. C. and Chiu, T. H., Decreased aH-diazepam binding is a specific response to chronic benzodiazepine treatment, Life Sci., 24 (1979) 803-808. 8 Taltman, J. F., Paul, S. M., Skolnick, P. and Gallager, D. W., Receptors for the age of anxiety : pharmacology of the benzodiazepines, Science, 207 (1980) 274-281.