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Chronic ethanol administration increases the binding of the benzodiazepine inverse agonist and alcohol antagonist [3H]RO15-4513 in rat brain
European Journal of Pharmacology, 153 (1988) 141-145
141
Elsevier EJP 20185 Short communication
Chronic ethanol administration increases the binding of the benzodi~epine inverse agonist and alcohol antagonist [3HlRO15-4513 in rat brain Molina Mhatre, Ashok K. Mehta and Maharaj
K. Ticku *
Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
Received 6 June 1988, accepted 14 June 1988
Chronic ethanol treatment which produced intoxication and physical dependence in rats, produced an increase in the specific binding of ethanol antagonist [3H]RO15-4513 in rat brain cerebral cortex and cerebellum, but not in hippocampus and striatum. The increase in both the regions was due to an increase in the number (Bmax) of receptor sites. These results suggest that the RO15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of RO15-4513 as an ethanol antagonist. Ethanol; GABA-benzodiazepine receptor complex; Ro 15-4513
1. Introduction Involvement of GABA-benzodiazepine receptor system in the actions of ethanol has been speculated for some time now (e.g. Ticku et al., 1983; Hunt, 1983). Recent studies have supported this contention, since ethanol potentiated GABA-induced 36C1- fluxes in cultured spinal cord neurons (Ticku et al., 1986), neurosynaptosomes (Suzdak et al., 1986) and microsacs (Allan and Harris, 1986). Furthermore, an azido analogue of the benzodiazepine receptor antagonist RO15-1788, RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6oxo-4H-imidazo[1,5a],[1,4]benzodiazepine-3-carboxylate was reported to be a selective ethanol antagonist using biochemical and behavioral paradigms in rats (Suzdak et al., 1986). We and others have previously reported that chronic ethanol treatment does not alter the bind-
* To whom all correspondence should be addressed: Dept. of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
ing parameters of benzodiazepine agonists to the central type of receptors (e.g. Rastogi et al., 1986). However, since RO15-4513 is a partial inverse agonist and it reverses ethanol's behavioral and biochemical effects, and its binding was shown to be slightly different from that of diazepam and RO15-1788 (Sieghart et al., 1987; Sadzot et al., 1987), we have investigated the effect of chronic ethanol treatment on the binding of [3H]RO154513 in brain regions of the rat.
2. Materials and methods Male Sprague-Dawley rats (150-180 g) were purchased from Harlan (Indianapolis, IN), [3H] RO15-4513 was purchased from New England Nuclear (Boston, MA), RO15-4513 was a gift from Dr. W. Haefely (Hoffmann-La-Roche, Basal, Switzerland) and RO15-1788 and diazepam were a gift from Hoffmann-La-Roche (Nutley, N J). BCA protein assay reagent was purchased from Pierce Chemical Co. (Rockford, IL).
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
142
2.1. Chronic treatment with ethanol The animals were intoxicated by a modification of the intragastric intubation method (Majchrowicz, 1975), as described earlier (Rastogi et al., 1986). Briefly, at the beginning of the experiment, a priming dose of ethanol of 5 g / k g was administered to all animals. Following this, doses were adjusted individually for each animal according to the presence or absence of ataxia, loss of righting reflex and motor activity. Animals were given routinely 20% v / v ethanol in normal saline three times a day for six days. On the day of withdrawal from ethanol, the animals were allowed to detoxify and the degree of intoxication was assessed as described elsewhere (Rastogi et al., 1986).
~l aliquots were filtered on W h a t m a n G F / B filter and washed rapidly thrice with 3.5 ml of buffer. The filters were dried and the radioactivity was determined as described (Rastogi et al., 1986). Nonspecific binding was determined under identical conditions using 2 × 10 6 M nonradioactive RO15-4513, and was subtracted from the total binding. Nonspecific binding ranged from 11 _+ 0.5% (0.25 nM) to 42 _+ 10% (20 nM) of the total binding. Protein was estimated using BCA protein assay reagent. The counting efficiency was determined by the external standard method and was 38%. The K D and Bmax values were obtained by the linear regression of the Scatchard data. The statistical significance was calculated by using the Student's t-test.
2.2. Evaluation of development of audiogenic seizures and changes in body temperature
3. Results
Audiogenic seizures were recorded by exposing individual rats to the sound by 98 db electric bell for 1 min as described earlier (Frye et al., 1983). Rectal temperature of rats was recorded in different groups with the help of a NBS traceable digital rectal thermometer (Markson, USA).
2.3. Preparation of tissue Following chronic treatment with ethanol, rats were decapitated and regions of the brain were dissected, pooled in 0.32 M sucrose and kept frozen at - 7 0 ° C till the day of the assay. The mitochondrial plus microsomal (P2 + P3) fraction was prepared as described previously (Rastogi et al., 1986). On the day of the assay the tissue was thawed, centrifuged, washed twice using KrebsTris buffer (116 m M NaC1, 4.8 m M KC1, 1.2 m M CaC12, 1.2 m M MgC12, 15 m M Tris HC1, p H 7.4) and resuspended in the same buffer for the binding studies.
2.4. Binding studies Aliquots (0.15-0.4 mg p r o t e i n / m l ) of the membrane fraction in Krebs-Tris buffer were incubated with [3H]RO15-4513 (0.25-20 nM) for 60 min at 0-4 ° C. Following incubation, triplicate 250
3.1. Behavioral studies All three groups of rats were monitored for weight changes, locomotor activity, audiogenic seizures and body temperature. Following six days of treatment, control group of rats gained 4.4 + 0.8% (n = 6), chronic ethanol maintained group lost 21.6 + 3.2% (n = 8; P < 0.001 as compared to control group) and alcohol withdrawn (24 h) group lost 24.8 _+ 3.2% (n = 8; P < 0,001 as compared to control group) of their body weight. Alcohol maintained group exhibited different stages of intoxication including decreased locomotor activity, sedation, ataxia and coma, while as alcohol withdrawn group exhibited increased locomotor activity, rigid posture and high irritability. Alcohol withdrawn (24 h) group of rats, but not the control or alcohol maintained rats, showed susceptibility to audiogenic seizures including wild running and clonic and tonic seizures. Alcohol maintained group also showed a decrease in body temperature (36.6 _ 0.2°C, n = 10; P < 0.001 as compared to control or withdrawn groups) versus control group (39.5 _+ 0.08 ° C, n = 5) and alcohol withdrawn group (38.8 + 0.15°C, n = 6). The experimental protocol used in this study for chronic ethanol treatment has been previously reported to produce blood ethanol concentration of 475 _+ 65
143
m g / d l , with no ethanol being detected in the 24 h withdrawal group (Rastogi et al., 1986).
3.2. Binding studies Initial preliminary studies indicated that [3H] RO15-4513 bound in a specific and saturable manner to rat brain cerebral cortex and cerebellar membranes. The binding was also displaced in a concentration-dependent manner by RO15-4513, RO15-1788 and diazepam. While RO15-1788 and RO15-4513 gave same level of maximal displace-
ment, diazepam gave 10-15% less maximal displacement (data not shown). In vitro addition of ethanol (5-100 mM) did not alter the specific binding of [3H]RO15-4513 binding to cerebral cortex or cerebellar membranes. Figure 1 shows typical Scatchard plots of the binding of [3H]RO15-4513 in the cerebral cortex (fig. 1A) and cerebellum (fig. 1B) of control, chronic and the withdrawn group of rats. The effect of chronic treatment with ethanol and during withdrawal (24 h) and K D and Bm,x values of [3H]RO15-4513 in regions of the brain of the rat
A. Cerebral Cortex
6Conlrol 0 Chronic Z~ Withdrawal •
B. Cerebellum
5• Control o Chronic Withdrawal
4~
o
4-"
v a)
'_o 3-
3-
v ¢--
x (1)
0
03 t-
A
0=
2-
•
~o
o
•
o
2-
03
•
o
o
o I.o 2.0 30 [3H] R015-4513 Bound (pmol/mg protein)
0
0
IIb
Z~0
~0
[3H] R015-4513 Bound (pmol/mg protein)
Fig. 1. Typical Scatchard plots of the binding of [ 3H]RO15-4513 in the cerebral cortex (A) and cerebellum (B) of control, chronic and withdrawn (24 h) groups of rats. The binding of [3H]RO15-4513 was measured as described in Methods. The K D and Bm~ values obtained by linear regression are summarized in table 1.
144 TABLE 1 Effect of chronic administration of ethanol on the binding of [3H]RO15-4513 in rats (Scatchard analysis). Rats were chronically treated with ethanol by the intubation method as described in Methods. The withdrawn group was sacrificed 24 h after the last intubation of ethanol. Saturation isotherms were analyzed by Scatchard analysis to determine the K o and Bmax values. The results are the means_+ S.D. of number of experiments indicated in parentheses. Region
Control Ko (nM)
Cerebral cortex Cerebellum Hippocampus Striatum
Specific binding of [ 3H]RO15-4513 Bm~x ( p m o l / m g protein)
Chronic KD
Bmax
Withdrawn KD
(nM)
( p m o l / m g protein)
(nM)
( p m o l / m g protein)
Bmax
0.72__+0.40
2.116_+0.40 (4)
0.61_+0.09
3.404_+0.43 (5) ~
0.83_+0.34
2.557+0.27 (6)
0.57_+0.18
1.452_+0.22 (4)
0.84_+0.25
2.576_+0.33 (6) ~
0.70-+0.27
2.446__+0.23 (5) a
0.62_+0.11 1.37_+0.47
1.511_+0.28(2) 1.362-+0.27(2)
1.03_+0.11 0.83_+0.10
1.696_+0.04(2) b 1.210_+0.10(2)
1.05_+0.07 b 0.83-+0.04
1.696 +__0.04 (2) 1.167__+0.13 (2)
a p < 0.001, as compared to control group, b p < 0.05, as compared to control group.
is summarized in table 1. Specific [3H]RO15-4513 binding was increased selectively following chronic ethanol treatment in cerebral cortex (61%) and cerebellum (77%), but not in hippocampus and striatum. This increase was due to an increase in the Bmax value in both the regions, without any significant effect on the K o value. At 24 h withdrawal, the number of receptor sites returned to the control value in cerebral cortex, however, they were still elevated in the cerebellum (table 1).
4. Discussion Based on several lines of biochemical (Ticku et al., 1983; 1986; Hunt, 1983; Allan and Harris, 1986; Suzdak et al., 1986) and behavioral (Liljequist and Engel, 1982; Rastogi and Ticku, 1986) observations, it is becoming apparent that ethanol may mediate most if not all of its effects via G A B A benzodiazepine receptor-ionophore complex. Recent findings that an inverse agonist of the central benzodiazepine receptor, RO15-4513 could selectively block ethanol's behavioral effects and ethanol's effect on GABA-induced 3 6 C 1 - flux (Suzdak et al., 1986), have produced a great deal of interest in this area of investigation. Chronic ethanol treatment produced the expected behavioral changes indicating that the rats were intoxicated (e.g. Majchrowicz, 1975). This
was also confirmed by the behavioral changes observed during withdrawal, which were consistent with the development of physical dependence in the rats. Our results demonstrate that chronic ethanol treatment produces a selective increase in the number of binding sites for RO154513 in cerebral cortex and cerebellum. This effect cannot be a nonspecific effect, since no changes were observed in hippocampus and striatum of the same rats. The decrease in body weight produced by chronic ethanol treatment cannot account for the observed changes, since similar treatment does not alter the binding of benzodiazepine agonists and picrotoxin to their receptor sites (Rastogi et al., 1986). Since ethanol does not interact with the [3H]RO15-4513 binding sites in vitro using membrane homogenates, it is possible that the integrity of the membranes and the necessary physiochemical environment may be crucial for the observed in vivo effect. This would also be consistent with the notion that ethanol per se does not have a specific receptor site, but most likely it exerts its modulatory effect on GABAergic transmission by interacting with the coupling mechanism which could involve a unique lipoprotein ' d o m a i n ' in close proximity to the C1- channels coupled to G A B A receptors. The significance of an elevated number of receptor sites for RO15-4513 following chronic ethanol treatment is not clear at present. It is possible that the chronic ethanol treatment
145 e x p o s e s s o m e less a c c e s s i b l e sites o r r e m o v e s s o m e e n d o g e n o u s l i g a n d b o u n d to t h e i n v e r s e a g o n i s t b i n d i n g sites. H o w e v e r , at p r e s e n t the p r e s e n c e o f s u c h a n e n d o g e n o u s l i g a n d a n d its n a t u r e is still a matter of debate. F u r t h e r , since b e n z o d i a z e p i n e a g o n i s t b i n d i n g is n o t a l t e r e d f o l l o w i n g c h r o n i c e t h a n o l t r e a t m e n t o r d u r i n g w i t h d r a w a l f r o m e t h a n o l ( R a s t o g i et al., 1986), o u r results s u g g e s t t h a t t h e b i n d i n g p r o p e r ties o f i n v e r s e a g o n i s t s like R O 1 5 - 4 5 1 3 m a y b e s l i g h t l y d i f f e r e n t f r o m t h e a g o n i s t b i n d i n g . A rec e n t s t u d y h a s also d e m o n s t r a t e d t h a t the b i n d i n g distribution of RO15-1788 and RO15-4513 was n o t i d e n t i c a l ( S a d z o t et al., 1987). In s u m m a r y , the a b i l i t y of c h r o n i c e t h a n o l t r e a t m e n t to s e l e c t i v e l y enhance [3H]RO15-4513 binding further supports t h e r o l e o f G A B A e r g i c s y s t e m in t h e a c t i o n s o f e t h a n o l , a n d t h e p o t e n t i a l role o f R O 1 5 - 4 5 1 3 as a n a l c o h o l r e c e p t o r a n t a g o n i s t a n d its r e c e p t o r sites in a l c o h o l d e p e n d e n c e .
Acknowledgements We thank Ms. Diana Reese for excellent secretarial help. This work was supported in part by funds from NIAAA Grant AA04090.
References Allan, A.M. and A.R. Harris, 1986, Gamma-aminobutyric acid and alcohol actions: Neurochemical studies of long sleep and short sleep mice, Life Sci. 39, 2005.
Frye, G.D., T.J. McCown and G.R. Breese, 1983, Differential sensitivity of ethanol withdrawal signs in the rat to ~,aminobutyric acid (GABA) mimetics: Blockade of audiogenic but not forelimb tremors, J. Pharmacol. Exp. Ther. 226, 720. Hunt, W.A., 1983, The effect of ethanol on GABAergic transmission, Neurosci. Biobehav. Rev. 7, 87. Liljequist, S. and J. Engel, 1982, Effect of GABAergic agonists and antagonists on ethanol-induced behavioral changes, Psychopharmacology 78, 71. Majchrowicz, E., 1975, Induction of physical dependence upon ethanol and the associated behavioral changes in rats, Psychopharmacology 43, 245. Rastogi, S.K., R. Thyagarajan, J. Clothier and M.K. Ticku, 1986, Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat, Neuropharmacology 25, 1179. Rastogi, S.K. and M.K. Ticku, 1986, Anticonvulsant profile of drugs which facilitate GABAergic transmission on convulsions mediated by a GABAergic mechanism, Neuropharmacology 25, 175. Sadzot, B., J.J. Frost and H.N. Wagner, 1987, In vivo labeling of central benzodiazepine receptors with [3H]RO15-4513, Neurosci. Abstracts 13, 960 (2165.1). Sieghart, W., A. Eichinger, J.G. Richards and H. MiShler, 1987, Photoaffinity labeling of benzodiazepine receptor proteins with the partial inverse agonist [3H]RO15-4513: A biochemical and autoradiography study, J. Neurochem. 48, 46. Suzdak, P., J.R. Glowa, J.N. Crawley, R.D. Schwartz, P. Skolnick and S.M. Paul, 1986, A selective imidazodiazepine antagonist of ethanol in the rat, Science 234, 1243. Ticku, M.K., T.P. Burch and W.C. Davis, 1983, The interactions of ethanol with the benzodiazepine-GABA receptor ionophore complex, Pharmacol. Biochem. Behav. 18 (Suppl. 1), 15. Ticku, M.K., P. Lowrimore and P. Lehoullier, 1986, Ethanol enhances GABA-induced 36C1- influx in primary spinal cord cultured neurons, Brain Res. Bull. 17, 23.
141
Elsevier EJP 20185 Short communication
Chronic ethanol administration increases the binding of the benzodi~epine inverse agonist and alcohol antagonist [3HlRO15-4513 in rat brain Molina Mhatre, Ashok K. Mehta and Maharaj
K. Ticku *
Department of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
Received 6 June 1988, accepted 14 June 1988
Chronic ethanol treatment which produced intoxication and physical dependence in rats, produced an increase in the specific binding of ethanol antagonist [3H]RO15-4513 in rat brain cerebral cortex and cerebellum, but not in hippocampus and striatum. The increase in both the regions was due to an increase in the number (Bmax) of receptor sites. These results suggest that the RO15-4513 binding sites on the oligomeric GABA receptor complex are altered following chronic ethanol administration, and support the notion of a unique role of RO15-4513 as an ethanol antagonist. Ethanol; GABA-benzodiazepine receptor complex; Ro 15-4513
1. Introduction Involvement of GABA-benzodiazepine receptor system in the actions of ethanol has been speculated for some time now (e.g. Ticku et al., 1983; Hunt, 1983). Recent studies have supported this contention, since ethanol potentiated GABA-induced 36C1- fluxes in cultured spinal cord neurons (Ticku et al., 1986), neurosynaptosomes (Suzdak et al., 1986) and microsacs (Allan and Harris, 1986). Furthermore, an azido analogue of the benzodiazepine receptor antagonist RO15-1788, RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6oxo-4H-imidazo[1,5a],[1,4]benzodiazepine-3-carboxylate was reported to be a selective ethanol antagonist using biochemical and behavioral paradigms in rats (Suzdak et al., 1986). We and others have previously reported that chronic ethanol treatment does not alter the bind-
* To whom all correspondence should be addressed: Dept. of Pharmacology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284-7764, U.S.A.
ing parameters of benzodiazepine agonists to the central type of receptors (e.g. Rastogi et al., 1986). However, since RO15-4513 is a partial inverse agonist and it reverses ethanol's behavioral and biochemical effects, and its binding was shown to be slightly different from that of diazepam and RO15-1788 (Sieghart et al., 1987; Sadzot et al., 1987), we have investigated the effect of chronic ethanol treatment on the binding of [3H]RO154513 in brain regions of the rat.
2. Materials and methods Male Sprague-Dawley rats (150-180 g) were purchased from Harlan (Indianapolis, IN), [3H] RO15-4513 was purchased from New England Nuclear (Boston, MA), RO15-4513 was a gift from Dr. W. Haefely (Hoffmann-La-Roche, Basal, Switzerland) and RO15-1788 and diazepam were a gift from Hoffmann-La-Roche (Nutley, N J). BCA protein assay reagent was purchased from Pierce Chemical Co. (Rockford, IL).
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
142
2.1. Chronic treatment with ethanol The animals were intoxicated by a modification of the intragastric intubation method (Majchrowicz, 1975), as described earlier (Rastogi et al., 1986). Briefly, at the beginning of the experiment, a priming dose of ethanol of 5 g / k g was administered to all animals. Following this, doses were adjusted individually for each animal according to the presence or absence of ataxia, loss of righting reflex and motor activity. Animals were given routinely 20% v / v ethanol in normal saline three times a day for six days. On the day of withdrawal from ethanol, the animals were allowed to detoxify and the degree of intoxication was assessed as described elsewhere (Rastogi et al., 1986).
~l aliquots were filtered on W h a t m a n G F / B filter and washed rapidly thrice with 3.5 ml of buffer. The filters were dried and the radioactivity was determined as described (Rastogi et al., 1986). Nonspecific binding was determined under identical conditions using 2 × 10 6 M nonradioactive RO15-4513, and was subtracted from the total binding. Nonspecific binding ranged from 11 _+ 0.5% (0.25 nM) to 42 _+ 10% (20 nM) of the total binding. Protein was estimated using BCA protein assay reagent. The counting efficiency was determined by the external standard method and was 38%. The K D and Bmax values were obtained by the linear regression of the Scatchard data. The statistical significance was calculated by using the Student's t-test.
2.2. Evaluation of development of audiogenic seizures and changes in body temperature
3. Results
Audiogenic seizures were recorded by exposing individual rats to the sound by 98 db electric bell for 1 min as described earlier (Frye et al., 1983). Rectal temperature of rats was recorded in different groups with the help of a NBS traceable digital rectal thermometer (Markson, USA).
2.3. Preparation of tissue Following chronic treatment with ethanol, rats were decapitated and regions of the brain were dissected, pooled in 0.32 M sucrose and kept frozen at - 7 0 ° C till the day of the assay. The mitochondrial plus microsomal (P2 + P3) fraction was prepared as described previously (Rastogi et al., 1986). On the day of the assay the tissue was thawed, centrifuged, washed twice using KrebsTris buffer (116 m M NaC1, 4.8 m M KC1, 1.2 m M CaC12, 1.2 m M MgC12, 15 m M Tris HC1, p H 7.4) and resuspended in the same buffer for the binding studies.
2.4. Binding studies Aliquots (0.15-0.4 mg p r o t e i n / m l ) of the membrane fraction in Krebs-Tris buffer were incubated with [3H]RO15-4513 (0.25-20 nM) for 60 min at 0-4 ° C. Following incubation, triplicate 250
3.1. Behavioral studies All three groups of rats were monitored for weight changes, locomotor activity, audiogenic seizures and body temperature. Following six days of treatment, control group of rats gained 4.4 + 0.8% (n = 6), chronic ethanol maintained group lost 21.6 + 3.2% (n = 8; P < 0.001 as compared to control group) and alcohol withdrawn (24 h) group lost 24.8 _+ 3.2% (n = 8; P < 0,001 as compared to control group) of their body weight. Alcohol maintained group exhibited different stages of intoxication including decreased locomotor activity, sedation, ataxia and coma, while as alcohol withdrawn group exhibited increased locomotor activity, rigid posture and high irritability. Alcohol withdrawn (24 h) group of rats, but not the control or alcohol maintained rats, showed susceptibility to audiogenic seizures including wild running and clonic and tonic seizures. Alcohol maintained group also showed a decrease in body temperature (36.6 _ 0.2°C, n = 10; P < 0.001 as compared to control or withdrawn groups) versus control group (39.5 _+ 0.08 ° C, n = 5) and alcohol withdrawn group (38.8 + 0.15°C, n = 6). The experimental protocol used in this study for chronic ethanol treatment has been previously reported to produce blood ethanol concentration of 475 _+ 65
143
m g / d l , with no ethanol being detected in the 24 h withdrawal group (Rastogi et al., 1986).
3.2. Binding studies Initial preliminary studies indicated that [3H] RO15-4513 bound in a specific and saturable manner to rat brain cerebral cortex and cerebellar membranes. The binding was also displaced in a concentration-dependent manner by RO15-4513, RO15-1788 and diazepam. While RO15-1788 and RO15-4513 gave same level of maximal displace-
ment, diazepam gave 10-15% less maximal displacement (data not shown). In vitro addition of ethanol (5-100 mM) did not alter the specific binding of [3H]RO15-4513 binding to cerebral cortex or cerebellar membranes. Figure 1 shows typical Scatchard plots of the binding of [3H]RO15-4513 in the cerebral cortex (fig. 1A) and cerebellum (fig. 1B) of control, chronic and the withdrawn group of rats. The effect of chronic treatment with ethanol and during withdrawal (24 h) and K D and Bm,x values of [3H]RO15-4513 in regions of the brain of the rat
A. Cerebral Cortex
6Conlrol 0 Chronic Z~ Withdrawal •
B. Cerebellum
5• Control o Chronic Withdrawal
4~
o
4-"
v a)
'_o 3-
3-
v ¢--
x (1)
0
03 t-
A
0=
2-
•
~o
o
•
o
2-
03
•
o
o
o I.o 2.0 30 [3H] R015-4513 Bound (pmol/mg protein)
0
0
IIb
Z~0
~0
[3H] R015-4513 Bound (pmol/mg protein)
Fig. 1. Typical Scatchard plots of the binding of [ 3H]RO15-4513 in the cerebral cortex (A) and cerebellum (B) of control, chronic and withdrawn (24 h) groups of rats. The binding of [3H]RO15-4513 was measured as described in Methods. The K D and Bm~ values obtained by linear regression are summarized in table 1.
144 TABLE 1 Effect of chronic administration of ethanol on the binding of [3H]RO15-4513 in rats (Scatchard analysis). Rats were chronically treated with ethanol by the intubation method as described in Methods. The withdrawn group was sacrificed 24 h after the last intubation of ethanol. Saturation isotherms were analyzed by Scatchard analysis to determine the K o and Bmax values. The results are the means_+ S.D. of number of experiments indicated in parentheses. Region
Control Ko (nM)
Cerebral cortex Cerebellum Hippocampus Striatum
Specific binding of [ 3H]RO15-4513 Bm~x ( p m o l / m g protein)
Chronic KD
Bmax
Withdrawn KD
(nM)
( p m o l / m g protein)
(nM)
( p m o l / m g protein)
Bmax
0.72__+0.40
2.116_+0.40 (4)
0.61_+0.09
3.404_+0.43 (5) ~
0.83_+0.34
2.557+0.27 (6)
0.57_+0.18
1.452_+0.22 (4)
0.84_+0.25
2.576_+0.33 (6) ~
0.70-+0.27
2.446__+0.23 (5) a
0.62_+0.11 1.37_+0.47
1.511_+0.28(2) 1.362-+0.27(2)
1.03_+0.11 0.83_+0.10
1.696_+0.04(2) b 1.210_+0.10(2)
1.05_+0.07 b 0.83-+0.04
1.696 +__0.04 (2) 1.167__+0.13 (2)
a p < 0.001, as compared to control group, b p < 0.05, as compared to control group.
is summarized in table 1. Specific [3H]RO15-4513 binding was increased selectively following chronic ethanol treatment in cerebral cortex (61%) and cerebellum (77%), but not in hippocampus and striatum. This increase was due to an increase in the Bmax value in both the regions, without any significant effect on the K o value. At 24 h withdrawal, the number of receptor sites returned to the control value in cerebral cortex, however, they were still elevated in the cerebellum (table 1).
4. Discussion Based on several lines of biochemical (Ticku et al., 1983; 1986; Hunt, 1983; Allan and Harris, 1986; Suzdak et al., 1986) and behavioral (Liljequist and Engel, 1982; Rastogi and Ticku, 1986) observations, it is becoming apparent that ethanol may mediate most if not all of its effects via G A B A benzodiazepine receptor-ionophore complex. Recent findings that an inverse agonist of the central benzodiazepine receptor, RO15-4513 could selectively block ethanol's behavioral effects and ethanol's effect on GABA-induced 3 6 C 1 - flux (Suzdak et al., 1986), have produced a great deal of interest in this area of investigation. Chronic ethanol treatment produced the expected behavioral changes indicating that the rats were intoxicated (e.g. Majchrowicz, 1975). This
was also confirmed by the behavioral changes observed during withdrawal, which were consistent with the development of physical dependence in the rats. Our results demonstrate that chronic ethanol treatment produces a selective increase in the number of binding sites for RO154513 in cerebral cortex and cerebellum. This effect cannot be a nonspecific effect, since no changes were observed in hippocampus and striatum of the same rats. The decrease in body weight produced by chronic ethanol treatment cannot account for the observed changes, since similar treatment does not alter the binding of benzodiazepine agonists and picrotoxin to their receptor sites (Rastogi et al., 1986). Since ethanol does not interact with the [3H]RO15-4513 binding sites in vitro using membrane homogenates, it is possible that the integrity of the membranes and the necessary physiochemical environment may be crucial for the observed in vivo effect. This would also be consistent with the notion that ethanol per se does not have a specific receptor site, but most likely it exerts its modulatory effect on GABAergic transmission by interacting with the coupling mechanism which could involve a unique lipoprotein ' d o m a i n ' in close proximity to the C1- channels coupled to G A B A receptors. The significance of an elevated number of receptor sites for RO15-4513 following chronic ethanol treatment is not clear at present. It is possible that the chronic ethanol treatment
145 e x p o s e s s o m e less a c c e s s i b l e sites o r r e m o v e s s o m e e n d o g e n o u s l i g a n d b o u n d to t h e i n v e r s e a g o n i s t b i n d i n g sites. H o w e v e r , at p r e s e n t the p r e s e n c e o f s u c h a n e n d o g e n o u s l i g a n d a n d its n a t u r e is still a matter of debate. F u r t h e r , since b e n z o d i a z e p i n e a g o n i s t b i n d i n g is n o t a l t e r e d f o l l o w i n g c h r o n i c e t h a n o l t r e a t m e n t o r d u r i n g w i t h d r a w a l f r o m e t h a n o l ( R a s t o g i et al., 1986), o u r results s u g g e s t t h a t t h e b i n d i n g p r o p e r ties o f i n v e r s e a g o n i s t s like R O 1 5 - 4 5 1 3 m a y b e s l i g h t l y d i f f e r e n t f r o m t h e a g o n i s t b i n d i n g . A rec e n t s t u d y h a s also d e m o n s t r a t e d t h a t the b i n d i n g distribution of RO15-1788 and RO15-4513 was n o t i d e n t i c a l ( S a d z o t et al., 1987). In s u m m a r y , the a b i l i t y of c h r o n i c e t h a n o l t r e a t m e n t to s e l e c t i v e l y enhance [3H]RO15-4513 binding further supports t h e r o l e o f G A B A e r g i c s y s t e m in t h e a c t i o n s o f e t h a n o l , a n d t h e p o t e n t i a l role o f R O 1 5 - 4 5 1 3 as a n a l c o h o l r e c e p t o r a n t a g o n i s t a n d its r e c e p t o r sites in a l c o h o l d e p e n d e n c e .
Acknowledgements We thank Ms. Diana Reese for excellent secretarial help. This work was supported in part by funds from NIAAA Grant AA04090.
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