Alcohol, Vol. 9, pp. 261-269, 1992

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Functional Properties of GABAA Receptors in Two Rat Lines Selected for High and Low Alcohol Sensitivity M . U U S I - O U K A R I * A N D E. R. K O R P I t l

*Tampere Brain Research Center, Department o f Biomedical Sciences, University o f Tampere, Tampere, Finland tResearch Laboratories o f the Finnish State Alcohol Company, Alko Ltd., Helsinki, Finland Received 28 A u g u s t 1991; A c c e p t e d 6 D e c e m b e r 1991 UUSI-OUKARI, M. AND E. R. KORPI. Functionalproperties of GABAA receptorsin two rat linesselectedfor high and low alcoholsensitivity. ALCOHOL 9(3) 261-269, 1992.-The effects of lorazepam and sodium barbital on GABA^ receptor function were evaluated in rat lines selected for differential sensitivity to the motor-impairing effects of ethanol [alcoholinsensitive (AT) and alcohol-sensitive (ANT) lines]. The effect of GABA on [3Hlflunitrazepam and [3H]Ro 15-4513 binding and the effects of lorazepam and sodium barbital on [3H]muscimol binding were measured in cerebellar, cerebrocortical, and hippocampal membrane preparations. The effects of lorazepam and sodium barbital on muscimol-stimulated 36C1- influx were measured using membrane vesicle suspensions from the same brain areas. No differences were found between the rat lines in the GABA-induced stimulation of [3H]flunitrazepam binding or in the lorazepam and sodium barbital-induced enhancement of either [3]muscimol binding or muscimol-stimulated ~CI- flux, Neither was desensitization of the ~CI- flux affected differently by ethanol, lorazepam, and barbital in vitro between the lines. The affinity of cerebellar diazepaminsensitive (DZ-IS) [3H]Ro 15-4513-binding sites for benzodiazepine agonists has been shown to be much greater in the ANT than the AT rats. In the present study, at 0°C, GABA decreased [3H]Ro 15-4513 binding in the presence of diazepam only in ANT rats. Similarly, GABA decreased this binding at 37°C in ANT rats having a high affinity for diazepam, whereas it enhanced the binding in all AT samples and in those ANT samples where diazepam had a poor AT-like affinity. The decrease in binding in ANT samples is apparently caused by the enhancing effect of GABA on diazepam binding to DZ-IS [3H]Ro 15-4513-binding sites. This enhanced interaction between GABA and a benzodiazepine agonist suggests that the altered DZ-IS binding sites might be involved in the augmented sensitivity of the ANT rats to sedative drugs. GABA^ Benzodiazepine receptors Ethanol sensitivity Cerebellum Diazepam-insensitive binding

BENZODIAZEPINES and barbiturates have been shown to modulate the action of -r-aminobutyric acid A-type (GABAA) receptors. Both classes of drugs allosterically enhance the binding o f GABA, an effect which can be blocked by hicuculline, a GABA^ antagonist (42). Both drugs also stimulate receptor-mediated chloride ion flux (1,8,38,41,44). Electrophysiological studies have indicated that henzodiazepines increase the probability of the chloride channel opening in response to GABA, while barbiturates prolong the mean open time of the GABA-activated chloride channel (13). Several lines o f evidence also suggest that ethanol enhances the effects of GABAergic synaptic transmission. Drugs that enhance GABA^ receptor function enhance most of the behavioral effects of ethanol, whereas drugs that decrease the receptor function reduce these effects of ethanol (10,19, 21,30,36). Electrophysiological studies using cerehrocortical, hippocampal, or spinal cord neurons have demonstrated that

Selected rat lines

[3H]Ro15-4513

ethanol potentiates GABA-activated chloride currents (6,9, 40). In biochemical 36C1- flux assay, ethanol stimulates GABAA receptor-mediated ~Cl- flux into cerebrocortical and cerebellar membrane vesicles and to cultured spinal cord neurons (2,34,35,46,48). Selective breeding of rodents for differential sensitivity to ethanol or benzodiazepines have produced lines with differences in GABAA receptor function. Long-sleep (LS) and short-sleep (SS) mice, high alcohol sensitive (HAS), and low alcohol sensitive (LAS) rats, and diazepam-sensitive (DS) and diazepam-resistant (DR) mice differ in ethanol, barbiturate, and benzodiazepine-induced enhancement of 36C1- flux into membrane vesicles, the enhancement being more pronounced in the sensitive rodent lines (2-5,15). There are also differences between the lines in some ligand-binding interactions of GABA^ receptors, a n d / o r in the effect o f ethanol on the binding (2,4,28,29,32,37). The sensitive mice lines (LS and DS) are

1 Requests for reprints should be addressed to E. R. Korpi, M.D., Research Laboratories, Alko Ltd., P.O.B. 350, SF-00101 Heisinki, Finland. 261

262

UUSI-OUKARI AND KORPI

also more sensitive to the behavioral effects of the abovementioned agents, which have not been used in the selection of the lines (4,7,12,26,27,31). These findings lend further support to the notion of GABAA receptors playing a role in the intoxicating effects of ethanol. Two rat lines, the AT (alcohol-insensitive, "alcohol-tolerant") and ANT (alcohol-sensitive, "alcohol-nontolerant") rats, have been selectively outbred for differential sensitivity to the motor-impairing effects of ethanol at the Research Laboratories of the Finnish State Alcohol Company (Alko, Ltd.) (11). The ANT rats are also more sensitive to the motorimpairing effects of lorazepam and sodium barbital, suggesting that differences in GABAergic mechanisms might be involved in these behavioral differences of the rat lines (17,18). Studies concerning GABAA receptors in AT and ANT rats have mainly focused on ligand binding and muscimol stimulation of 36C1- flux, but no consistent differences have been found between the rat lines in receptor density or muscimolstimulated 36C1- flux (20,24,49-51). No comprehensive study of the function of GABAA receptors in these rat lines has been carried out. In the present study, GABAA receptor functions in brain homogenates of AT and ANT rats were compared by studying allosteric interactions using ligand-binding assays and by studying drug actions on the stimulation and desensitization of G A B A A receptor function using 36Ci- flux assays. METHOD

Materials [3H]Flunitrazepam (specific activity 77 Ci/mmol) and Imethylamine-~H]muscimol (21.2 Ci/mmol) were purchased from Amersham (UK); [3H]Ro 15-4513 (29.0 Ci/mmol) from Du Pont de Nemours Gmbh, NEN Division (Germany); ethyl-/3-carboline-3-carboxylate (~-CCE) from Research Biochemicals (Natick, MA); diazepam from Orion Pharmaceutica (Espoo, Finland); and lorazepam from Wyeth/Huhtam/iki Pharmaceuticals (Turku, Finland). Ro 15-1788 was a gift from Hoffmann-La Roche, Ltd. (Basel, Switzerland). Sodium barbital was purchased from Sigma Chemical Co. (St. Louis, MO).

Animals The animals were maintained in groups of four to six rats in stainless-steel wire cages at 22 _+ 2°C under artificial lighting with a 12-h light-dark cycle starting with lights on at 6:00 a.m. The rats had free access to tap water and R3 rodent pellet feed (Ewos Ab, S6dert/ilje, Sweden). A total of 53 AT and 61 ANT rats of the F37 and F40 generations were used.

Preparation of Brain Membranes The rats were killed by decapitation, their brains were removed, and the cerebral cortices, cerebelli, and hippocampi were frozen on dry ice and stored at - 800C. The tissues were weighed, thawed, and homogenized in 50 volumes of 50 mmol/l Tris-citrate buffer, pH 7.1, with a Kinematica Polytron PT 10/35 for 15 s at the setting of 6. The homogenates were centrifuged at 48,000 g and at + 4 ° C for 10 rain, and the resulting pellets washed four times by resuspension and centrifugation. The suspensions were stored at - 800C for at least 24 h, thawed, centrifuged at 48,000 g for 10 min, washed three times by resuspension and centrifugation in Tris-citrate, and the resulting suspensions stored at - 8 0 0 C . On the day of binding experiment, portions of the

suspensions were thawed, centrifuged at 48,000 g for 10 min, and washed three times by resuspension and centrifugation in a solution of 50 mmol/1 Tris and 150 mmol/1 KCI buffer, pH 7.4, adjusted with HC1 (assay buffer).

Ligand-Binding Studies The membrane preparations were suspended in assay buffer together with 1 nmol/1 [3H]flunitrazepam, 2 nmol/1 [3H]Ro 15-4513 or 5 nmol/1 [3H]muscimol, with or without compounds acting at GABAA receptors, in a total volume of 0.5 ml. For saturation analyses of [aH]Ro 15-4513 binding to cerebellar membranes from ANT rats, eight radioligand concentrations (0.5-50 nmol/l) were used. Displacement of 1 nmol/l [3H]Ro 15-4513 by diazepam was determined using 14 different diazepam concentrations (1 nmol/1-100 #mol/1). Nonspecific binding was determined in the presence of 10 /~mol/l Ro 15-1788 for [3H]flunitrazepam and [9H]Ro 15-4513 binding, and with I mmol/l GABA for [3H]muscimol binding. In [3H]flunitrazepam and [3H]Ro 15-4513-binding studies, the suspensions were incubated in the dark at 0°C for 60 min or at 37°C for 30 min. The incubation period for [3H]muscimol binding at 370C was 15 min. The incubations were terminated by filtrating the samples under vacuum onto Whatman GF/B filters using a Brandel cell harvester (model M-48 R). The filters were washed twice with 5 ml of ice-cold assay buffer and immersed in 10 ml of Optiphase "Hisafe" II (LKB Wallac). The radioactivity was measured with an LKB Wallac Ultrobeta 1210 scintillation counter at about 45o70 efficiency using the external standard channels ratio method.

Chloride Flux The radioactive chloride (36C1-) influx assays were performed essentially as described in refs. 20 and 49. The assay measures GABAA receptor-mediated chloride influx, which can be blocked by picrotoxin (14,20). One animal of each line at a time was decapitated, the brains were rinsed in ice-cold saline, and cerebral cortices, cerebelli, and hippocampi were removed. The brain parts were weighed, homogenized manually in 7 volumes (mg = ~1) of ice-cold assay buffer (NaC1 145 mmol/l, KC1 5 mmol/l, MgC12 1 mmol/l, CaC12 1 mmol/ 1, D-glucose 10 mmol/l, Hepes 10 mmol/l, pH 7.5, adjusted with Tris-base) using Kontes glass-glass homogenizers (Duall 23). The homogenates were diluted to 30 vols, and allowed to pass by gravity through a single layer of nylon cloth (mesh size 6I #m). Then the homogenates were centrifuged at I000 g and + 2 " C for 15 min. The pellets were resuspended and centrifuged once and the final pellets suspended in 10 vols of assay buffer. Aliquots of 125 ~1 were incubated at 30°C for 10 min, and the flux assay was started by adding 125 /zl of assay buffer containing 0.4 #Ci 3~C1- with or without 3 ~mol/ 1 muscimol, which produces a half-maximal stimulation (20), and various concentrations of lorazepam (10 nmol/l-10/~mol/ 1) or sodium barbital (0.3-10 mmol/l). The flux was stopped after 3 s by adding 4 ml of ice-cold assay buffer containing 100 ~mol/1 picrotoxin, and by filtrating the suspension onto Whatman GF/C filters using a Hoefer filtration unit at a vacuum pressure of 25 cm of Hg. The filters were quickly washed twice with 4 ml of ice-cold assay buffer containing 100 ~tmol/l picrotoxin, with the funnels removed during the last wash. The filters were dried, and the radioactivity measured as described above for the ligand-binding studies. In the desensitization studies, the cerebrocortical and cerebellar homogenates, prepared as described above were incu-

A L C O H O L S E N S I T I V I T Y A N D GABAA R E C E P T O R F U N C T I O N TABLE 1 GABA STIMULATION OF [3H]FLUNITRAZEPAM BINDING TO BRAIN MEMBRANES OF AT AND ANT RATS AT 0°C IN THE ABSENCE OR PRESENCE OF /~-CCE

Brain Area Cerebral cortex ATrat line ANT rat line Cerebellum ATrat line ANTratline Hippocampus AT rat line ANT rat line

~-CCE (100 nmol/I)

ECso ~mol/l)

+ +

1.27 0.94 1.09 2.63

_+ 0.15 + 0.27 ± 0.09 -+ 0.88

+ +

0.72 1.69 0.68 3.51

+ ± ± +

0.08 0.39 0.14 3.16

+ +

3.28 2.00 1.65 2.34

± + ± ±

1.70 0.52 0.17 0.21

Maximal Stimulation (~/e)

87.2 381 77.9 435

± ± ± ±

9.2t" 92 8.9 160

118.9 + 9.7 NC 124.5 ± 8.9 NC 99.3 246 88.7 195

+ + ± ±

7.7~ 13" 7.0~ 16

The membranes were incubated at 0°C with 1 nmol/i [3H]flunitrazepam with or without various concentrations of GABA (0.1-100 ~tmol/I) in the presence or absence of 100 nmol/1/~-CCE. Nonspecific binding was determined in the presence of 10 #moi/l Ro 15-1788. The values represent the mean ± SEM of four rats from each rat line. Students t-test: *p < 0.05, significance of difference between the lines; t"P < 0.05, ~p < 0.001, significantly different from maximal stimulation in the presence of ~-CCE. NC = Due to negligible baseline binding in the presence of /~-CCE, the percentage of maximal stimulation could not be calculated. bated at 3 0 ° C for 10 min in a total volume o f 400 #1 with or without 5-50 # m o l / l G A B A + 50 mmol/1 ethanol, 2 #mol/1 lorazepam, or 1 m m o l / l sodium barbital. After incubation, the ability o f 50 ~ m o l / l muscimol to stimulate GABAA receptor-mediated chloride flux was measured by adding 100 #1 o f assay buffer containing 0.4 #Ci 36C1- with or without muscimol. The flux was stopped as described above. Protein concentrations in all experiments were determined using a modification o f the Lowry method (22,43).

Analysis of Binding Data The Pearson correlation coefficients between cerebellar [3H]Ro 15-4513 binding and the effect o f G A B A on the binding were calculated using c o m p u t e r programs by the SAS Institute, Inc. (see SAS User's Guide: Statistics, version 5 edition). The displacement o f [3H]flunitrazepam binding by ~-CCE, displacement o f [3H]Ro 15-4513 binding by diazepam, and saturation analyses o f [3H]Ro 15-4513 binding were analyzed using the E B D A / L 1 G A N D computer program (33,39). The K d values for [3H]flunitrazepam binding from ref. 24 were used to obtain the K~ values for ~ - C C E in both rat lines. Maximal G A B A stimulation (percent over baseline binding without G A B A ) and t h e G A B A concentrations producing half-maximal stimulation (ECso) o f [3H]flunitrazepam binding were estimated assuming a hyperbolic relationship between concentration and stimulation, using the L O W R Y program o f G. A. M c P b e r s o n (Elsevier, Biosoft). Significance o f differences between two groups was assessed using Student's t-test.

263 TABLE 2 EFFECT OF SODIUM BARBITAL AND LORAZEPAM ON [3HIMUSCIMOL BINDING TO BRAIN MEMBRANES OF AT AND ANT RATS

Drug/Brain Area Sodium barbital (mmol/l) Cerebral cortex AT rat line ANT rat line Cerebellum AT rat line ANT rat line Hippocampus ATratline ANTratline Lorazepam (#tool/l) Cerebral cortex AT rat line ANTratline Cerebellum AT rat line ANT rat line Hippocampus ATrat line ANT rat line

EC~0

Maximal Stimulation (%)

1.33 ± 0.24 1.79 + 0.91

54.8 ± 5.0 62.7 + 13.3

0.91 + 0.45 1.57 + 0.98

77.9 + 14.0 54.8 ± 4.5

2.1 + 0.60* 3.8 + 0.40

86.4 ± 3.8 118.1 ± 25.6

0.44 ± 0.07* 0.15 ± 0.01

112.9 + 33.7 72.8 + 18.8

0.57 + 0.38 0.78 ± 0.31

12.5 ± 2.1 23.0 ± 4.3

0.15 ± 0.04 0.13 ± 0.03

12.0 + 2.8 27.0 ± 6.4

The membranes were incubated at 37 °C with 5 nmol/l [~H]muscimol with or without various concentrations of sodium barbital (30 #mol/l to 10 mmol/l) or lorazepam (30 nmol/l to 10 #tool/l). Nonspecific binding was determined in the presence of 1 mmol/I GABA. The values represent the mean + SEM of four rats from each rat line. Student's t-test: *p < 0.05, significance of difference between rat lines.

RESULTS

GABA-Induced Stimulation of [~H]Flunitrazepam Binding KI values for [3H]flunitrazepam displacement by /~-CCE were lowest in the cerebellum and highest in the hippocampus, which is consistent with the predominance o f BZ1 sites in the former and the high proportion o f BZ2 sites in the latter. No difference was found between the rat lines in the K~ values or slope factors. Five micromoles per liter fl-CCE displaced all specific binding from all the tissues studied./3-CCE, at a concentration o f 100 n m o l / l , displaced 95°70 o f the cerebellar binding in both rat lines, and this concentration was thus used to study the stimulation by G A B A o f [3H]flunitrazepam binding at BZ2-binding sites. G A B A stimulated [3H]flunitrazepam binding in a dosedependent manner in all the brain areas tested. A t 0 ° C , the stimulation was strongest in cerebellar membranes (Table 1), and at 3 7 n c in hippocampal membranes. There were no differences between the rat lines in the ECs0 values or in the maximal stimulation o f binding, with the exception o f binding to hippocampal membranes at 0 ° C in the presence o f 100 n m o l / l / ~ - C C E , where the percentage o f maximal stimulation was higher in the A T rats (p < 0.05). Since the baseline binding without G A B A was 27.5 and 31.5 f m o l / m g protein and the maximal stimulated binding 94.2 and 94.3 f m o l / m g protein for the A T and A N T rats, respectively, the greater maximal percent stimulation by G A B A in the A T rats was thus

264

UUSI-OUKARI AND KORPI

TABLE 3 EFFECT OF GABA ON [~H]Ro 15-4513 BINDING TO CEREBELLAR MEMBRANES OF AT AND ANT RATS IN THE ABSENCE OR PRESENCE OF DIAZEPAM AT Rats GABA (100 ~zmol/l) 0oC Total binding + 10 #mol/l diazepam 37oC Total binding + 10/~mol/1 diazepam

ANT Rats

o70of Control

+ +

567 484 265 277

_+ 22 ± 22* ± 11 ± 13

+

240 + 16 251 ± 19

-

62 ± 4

+

87 ± 5-~

o70of Control

104.5 + 1.8

778 706 156 136

104.0 ± 2.5

227 + 12 234 ± 14

85.3 ± 1.1

± ± ± ±

31§ 31§ 21§ 23§

90.8 ± 1.0, 80.7 ± 7.3,

103.4 ± 2.4

35 ± 2§

139.8 ± 4.6

41 ± 5§

118.9 + 9.6

The cerebellar membranes were incubated with 2 nmol/1 [3H]Ro 15-4513 with or without 100 t~mol/1 GABA in the presence or absence of 10 t~mol/1 diazepam. Nonspecific binding was determined in the presence of 10 t~mol/l Ro 15-1788. The values are expressed in fmol/mg protein (mean ± SEM of eight and 14 rats from both rat lines at 0°C and 370C, respectively). Student's t-test: *p < 0.05 and ?p < 0.01, significantly different from the control value; ~tp < 0.01, §p < 0.001, significance of difference between rat lines.

due to the lower baseline binding w i t h o u t G A B A . A t 37°C, there was a tendency t o w a r d higher maximal stimulation in A N T rats in all three brain areas. The maximal stimulation o f binding to cerebellar m e m b r a n e s at 0 ° C in the presence o f 100 n m o l / l B-CCE was impossible to determine, since in m o s t samples, no specific binding occurred w i t h o u t G A B A (Table

Barbital and Lorazepam-Induced Stimulation of [3H]Muscimol Binding The stimulating effect o f sodium barbital o n the binding was highest in h i p p o c a m p a l m e m b r a n e h o m o g e n a t e s , in which the ECso value was lower ( p < 0.05) in the A T rats (Table 2).

1).

100

m~

100 D •

+

80

~ EN

[]

ANT r a l s AT rats

[]

85

[]

10

60

t3

O

o

~5c0

[] []

cP

~

40

-~- £3

O

[]

.~_ ~

[]

[]

N

20 D

C~

D

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£3

D

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1

o

•

60

,

.

80

r

100

-

=

120

•

l

140

'

l

160

•

,

180

•

200

Effect of GABA (% of control)

FIG. 1. Relationship between the affinity of diazepam for cerebeUar [3H]Ro 15-4513-binding sites and the effect of GABA on DZ-IS binding in individual ANT rats. The effect of GABA was studied on the cerebellar membranes of AT and ANT rats with 2 nmol/l [3H]Ro 15-4513 at 37°C for 30 min in a solution of 50 mmol/l Tris and 150 mmol/I KCI buffer with 10 ~,mol/l diazepam in the presence or absence of 1130 t~mol/l GABA. Diazepam affinity was estimated in the same samples by incubating the membranes at 0°C for 60 min with 1 nmol/l [3H]Ro 15-4513 in the presence of 30 t~mol/l diazepam. The AT point depicts the mean + SEM of 14 rats. Nonspecific binding was determined in the presence of 10 t~mol/1 Ro 15-1788.

50

i

i

i

i

f

i

6O

70

80

90

100

110

120

Effect of GABA (% of control) FIG. 2. Association between high diazeparn affinity of the ANT cerebellar [3H]Ro 15-4513-binding sites and GABA-induced inhibition of binding in the presence of 10 lzmol/1 diazcpam. The cerebellar membranes were incubated with 2 nmol/l [3HIRo 15-4513 at 0oc for 60 rain in a solution of 50 mmol/l Tris and 150 mmol/l KC! buffer containing 10 t~mol/l diazepam in the absence or presence of 100 /~mol/l GABA. The Kt values for displacement of [3I-l]Ro 15-4513 binding by diazepam from DZ-IS-binding sites was determined using a radioligand concentration of 1 nmol/l and diazepam concentrations ranging from 1 nmol/l to 100/zmol/l. Nonspecific binding was determined in the presence of 10 ~mol/I Ro 15-1788.

ALCOHOL SENSITIVITY AND GABAA RECEPTOR FUNCTION There were no other statistically significant differences between the lines (p > 0.05). The stimulation by lorazepam was highest in the cerebrocortical membranes and very low in the cerebellar and hippocampal membranes (Table 2). The ECs0 value in the cerebral cortex was lower in the ANT rats (p < 0.05). No differences between the lines were found in the cerebellum and hippocampus.

Effects of GABA and Temperature on Cerebellar ['H]Ro 15-4513 Binding.u The effect of GABA on [3H]Ro 15-4513 binding to cerebellar membranes of AT and ANT rats was studied in the absence and presence of 10/~mol/1 diazepam (Table 3). At 0°C, the total binding of 2 nmol/l [3H]Ro 15-4513 was significantly higher in ANT rats (p < 0.001). One hundred micromoles per liter GABA decreased the total binding more in AT rats when the values were expressed in percent of control binding (p < 0.01). In the presence of 10 #mol/l diazepam, diazepam-insensitive (DZ-IS) binding was lower in ANT rats (p < 0.001), and 100 #mol/l GABA further decreased the binding only in ANT rats (percentage of the binding without GABA in the presence of 10 #mol/l diazepam: p < 0.01, significance of the difference between rat lines). At 37°C, the total binding of 2 nmol/l [3H]Ro 15-4513 was similar in both rat lines, and 100 #M GABA had no effect on it (Table 3). In the presence of 10 #mol/l diazepam, GABA enhanced the binding in all samples of AT rats. In samples of ANT rats, GABA stimulated the binding only if there was a high amount of binding in the presence of 30 #mol/l diazepam, whereas it inhibited the binding in samples with low

265

[3H]Ro 15-4513 binding in the presence of diazepam (Fig. 1). There was a significant correlation in ANT rats between the binding in the presence of diazepam and the percent effect of GABA on the binding. The Pearson correlation coefficient was 0.87 (p < 0.003). In the presence of 10/tmol/l diazepam, GABA inhibited cerebellar [3H]Ro 15-4513 binding at both temperatures in ANT rats exhibiting low amount of DZ-IS binding (Table 3). This was further confirmed by a finding that the logarithm of the K~ values for diazepam displacement of the DZ-IS binding at 0°C (1.3-84.8 #mol/1, n = 8) correlated significantly with the GABA effect on the binding in the presence of 10 #mol/l diazepam (Fig. 2). The Pearson correlation coefficients were 0.87 (p = 0.005) and 0.91 (p < 0.002) for absolute (data not shown) and percentage values of binding (Fig. 2), respectively. The total binding of 2 nmol/l [3H]Ro 15-4513 at 0°C was also higher in rats with low DZ-IS binding. These results prompted us to test whether the ANT samples with high affinity to diazepam at their DZ-IS binding sites might also possess higher affinity to [3HIRo 15-4513. The Kd values for [3H]Ro 15-4513 binding at 0°C were between 3.4-8.6 nmol/l (n = 8) [total binding was measured because it was not possible to determine the affinity of the DZ-IS binding separately, due to inhibition of the DZ-IS binding by diazepam at micromolar concentrations in ANT rats (50)]. There was, however, no significant correlation between the K~ and the K~ values.

Enhancement of Muscimol-Stimulated 36C1- Flux by Barbital and Lorazepam Sodium barbital and lorazepam enhanced muscimolinduced stimulation of 36C1- flux in brain homogenates of AT

TABLE 4 DESENSITIZINGEFFECT OF GABA, LORAZEPAM, AND SODIUM BARBITALON GABA^RECEPTOR-MEDIATED 36C1- FLUX IN BRAIN HOMOGENATESOF AT AND ANT RATS BrainArea

20-sPreincubation

Cerebral cortex AT rat line

GABA GABA + lorazepam GABA + barbital

20.5 19.3 17.6 20.8 20.0 17.4 16.5 21.1

± ± ± + ± ± ± ±

0.72 0.69 0.66* 0.25 0.48 0.84* 0.39* 1.03

-GABA GABA GABA GABA GABA GABA

11.9 + 11.4 + 9.71 ± 12.0 + 11.5 ± 10.8 ± 9.04 ± 11.6 ±

0.79 0.59 0.58 0.86 0.52 0.67 0.39* 0.67

GABA GABA + lorazepam GABA + barbital ANT rat line

Cerebellum AT rat line

ANT rat line

nmoiCF/mg Proteinin 3 s

+ lorazepam + barbital

+ lorazepam + barbital

etaof Control

94.3 ± 0.9 85.9 ± 1.5 102.0 ± 2.7 87.1 ± 3.1 82.6 + 0.6 105.4 ± 3.5

95.8 + 1.6 81.6 + 2.3 100.9 ± 2.7 93.7 ± 2.1 78.9 ± 2.3 104.3 ± 4.2

The membrane homogenates were preincubated with or without 10 t~mol/l GABA with or without 2 #mol/l lorazepam or 1 mmol/l sodium barbital for 20 s. The homogenates were then incubated for 3 s with radioactive chloride with or without 50 t~mol/l muscimol as described in METHODS. The values represent the mean + SEM of five rats from each rat line. Student's t-test: *p < 0.05, significantly different from the control value.

266

UUSI-OUKARI AND KORPI

a n d A N T rats. T h e effect o f b o t h drugs was greatest in the cerebral cortex. T h e r e were n o differences between the rat lines in the effect o f the drugs w h e n expressed in absolute values, percent e n h a n c e m e n t s , or ECso values o f the potentiation.

flUX in h o m o g e n a t e s o f b o t h b r a i n areas f r o m b o t h rat lines, a n d 50 m m o l / l e t h a n o l h a d no effect o n this reduced stimulation. T h e r e were n o differences between the rat lines in either b r a i n area. DISCUSSION

Desensitization of Muscimol-Stimulated 36Cl- Flux A 20-s p r e i n c u b a t i o n in the presence o f 1 0 / ~ m o l / l G A B A decreased significantly the s u b s e q u e n t chloride flux induced by 50 t t m o l / l m u s c i m o l only in t h e cerebrocortical samples o f A N T rats Go < 0.05). W h e n p r e i n c u b a t i o n was p e r f o r m e d with G A B A a n d 2/~mol/1 l o r a z e p a m , the s t i m u l a t i o n by muscimol was significantly reduced in b o t h rat lines (Table 4). P r e i n c u b a t i o n with G A B A a n d 1 m m o l / 1 s o d i u m b a r b i t a l h a d n o effect o n the s u b s e q u e n t s t i m u l a t i o n by muscimol. N o differences were f o u n d between the r a t lines. A 20-s p r e i n c u b a t i o n with 50 m m o l / l e t h a n o l , 5 /zmol/l G A B A , or 5 ttmol/1 G A B A + 50 mol/1 e t h a n o l h a d n o effect o n the s u b s e q u e n t s t i m u l a t i o n by m u s c i m o l in cerebrocortical a n d cerebellar h o m o g e n a t e s o f A T a n d A N T rats (Table 5). P r e i n c u b a t i o n with 50 # m o l / l G A B A decreased the chloride

No consistent differences were f o u n d between the A T a n d A N T rat lines in the m o d u l a t i o n o f GABAA receptor f u n c t i o n by l o r a z e p a m , a benzodiazepine, or s o d i u m b a r b i t a l , a b a r b i turate. In the three b r a i n areas examined, the effect o f these drugs o n [3H]muscimol binding a n d r e c e p t o r - m e d i a t e d 36Clflux was quite similar in the two rat lines. T h e effect o f e t h a n o l a n d p e n t o b a r b i t a l o n m u s c i m o l - s t i m u l a t e d 36C1- flux was recently studied in cerebrocortical a n d cerebellar h o m o g e n a t e s o f A T a n d A N T rats, b u t n o differences were f o u n d between the rat lines in the p o t e n t i a t i o n o f the s t i m u l a t i o n by these drugs (49). A c u t e a d m i n i s t r a t i o n o f ethanol or l o r a z e p a m was s h o w n to reduce the m u s c i m o l - s t i m u l a t e d 36C1- flux in c o m b i n e d cerebral cortical a n d cerebellar h o m o g e n a t e s in A N T rats, whereas these t r e a t m e n t s h a d n o effect o n the s t i m u l a t i o n in

TABLE 5 DESENSITIZING EFFECT OF GABA,AND ETHANOL ON GABA^/ RECEPTOR-MEDIATED ~CI FLUX IN BRAIN HOMOGENATES OF AT AND ANT RATS Brain Area Cerebral cortex AT rat line

ANT rat line

nmol CI-/mg Protein in 3 s

20-s Preincubation

EtOH 5 ttmol/1GABA 5 gmol/1GABA + EtOH 50/~mol/1GABA 50 #mol/l GABA + EtOH EtOH 5 #mol/1 5 #mol/l GABA + EtOH 50 #mol/1 GABA 50#mol/1GABA + EtOH

Cerebellum AT rat line

-EtOH 5/~mol/1 GABA 5 t,mol/IGABA + EtOH 50/zmol/l GABA 50/~mol/l GABA + EtOH

ANT rat line

-

EtOH 5 gmol/l 5 gmol/l 50#mol/I 50 gmol/l

GABA GABA + EtOH GABA GABA + EtOH

16.1 16.3 15.9 15.5 9.2 9.5 15.3 15.6 15.9 15.3 9.9 9.1

+_ 0.66 ± 0.59 ± 0.53 ± 0.54 ± 0.31" ± 0.26 ± 0.57 _+ 0.47 ± 0.52 ± 0.46 ± 0.46* ± 0.18

10.8 11.1 10.4 10.7 7.2 7.3 10.9 10.9 10.6 10.8 7.5 7.2

± 0.65 + 0.75 ± 0.75 ± 0.56 ± 0.47t + 0.37 ± 0.49 ± 0.88 ± 0.72 +_ 0.69 ± 0.52t ± 0.43

% of Control

101.8 99.1 96.7 57.6 59.0

± + ± + ±

2.3 1.3 1.2 2.0 1.4

101.6 103.7 100.2 64.8 59.4

:t: ± ± ± ±

2.2 3.6 3.4 2.7 2.4

102.7 98.5 99.1 66.6 67.5

± ± ± ± ±

3.0 4.4 4.7 2.7 3.0

99.3 97.2 99.0 68.2 66.4

± ± ± ± ±

9.5 3.2 4.1 2.3 1.7

The membrane homogenates were preincubated with or without GABA in the absence or presence of 50 mmol/l ethanol. T h e h o m o g e n a t e s w e r e t h e n incubated for 3 s with radioactive chloride with 50 ~mol/1 muscimol as described in METHODS. The values represent the mean ± SEM of six rats from each rat line. Student's t-test: */7 < 0.001, tP < 0.01, significantly different from the corresponding control value.

A L C O H O L SENSITIVITY AND GABAA RECEPTOR FUNCTION AT rats (20). This difference suggests that the reduced stimulation in ANT rats might be due to desensitization of the GABAA receptors following a greater enhancement of the receptor function by ethanol and lorazepam, or to differences in the desensitization mechanism itself. In the present study, preincubation of cerebrocortical or cerebellar homogenates with 10 or 50 #mol/l GABA resulted in a decrease of the subsequent muscimol-stimulated 36C1- flux. Only lorazepam, in combination with GABA, was able to further decrease the subsequent muscimol-induced stimulation, whereas ethanol had no effect on it, and sodium barbital abolished the decrease induced by GABA alone. The effects were similar in both rat lines, giving no indication of greater desensitization of GABAA receptor activity by these drugs in ANT rats in vitro. There were no clear interline differences in the GABAinduced stimulation of [3H]flunitrazepam binding in the absence or presence of /3-CCE, which displaces [3H]flunitrazepam predominantly from BZl-binding sites. The previously found greater potentiation of [3H]flunitrazepam binding to the cerebrocortical membranes of AT rats (24) was no confirmed in the present study. These results suggest that there is no such general functional difference in the GABAA receptors of the AT and ANT rat lines, which would explain the differential sensitivity of these rat lines to ethanol and sedative drugs. In contrast, flunitrazepam-induced enhancement of muscimol-stimulated 360- flux in the brain vesicles of LS, DS, and HAS rats is higher than to those of the corresponding insensitive rodent lines (2,4,5). The lines differ in a similar way regarding phenobarbital-enhanced 36C1- flux (4,5,16). Furthermore, in a recent study, there was no difference between the AT and ANT rats in the ethanol-induced enhancement of muscimolstimulated 36C1- flux (49), whereas this enhancing effect of ethanol is greater in the three sensitive lines mentioned above (2-5). These functional differences in GABAA receptors in LS versus SS mice, HAS versus LAS rats, and DS versus DR mice, suggest that GABAA receptors play a role in the behavioral differences of these line pairs in response to ethanol and sedative drugs, whereas the present results on 36C1- flux, [3H]flunitrazepam binding, and [3H]muscimol binding in AT and ANT rats do not indicate any general difference between the rat lines in the function of GABAA receptors. [3HIRo 15-4513, a partial inverse agonist of benzodiazepine receptors, binds to a pharmacologically novel type of GABAA/ benzodiazepine receptor in the cerebellum. The binding is insensitive to benzodiazepine agonists (diazepam-insensitive, DZ-IS), stimulated by micromolar concentrations of GABA at 37°C, and is dependent on the presence of a6 subunit in recombinant receptors (23,25). We have recently demonstrated differences between the AT and ANT rat lines in the displacement of [3H]Ro 15-4513 binding to these sites. Benzodiazepine agonists displace the binding more efficiently in the membranes of ANT rats (50,51). This difference was not seen in the membranes of several other rodent line pairs (including LS/SS mice and HAS/LAS rats) selected for differences in ethanol sensitivity (51). The cerebellar [3H]Ro 15-4513 binding shows temperature-dependent modulation by GABA (25). At 0°C, GABA reduces [3H]Ro 15-4513 binding only at classical (diazepamsensitive, DZ-S) benzodiazepine-binding sites (25). In the present study, GABA reduced the total binding by 70-80 fmol/ mg protein in both AT and ANT rat lines, the reduction being smaller in ANT rats when expressed in percent values. However, the total binding of 2 nmol/l [3H]Ro 15-4513 was higher in the membranes of ANT rats due to the higher affinity of

267

cerebellar [3H]Ro 15-4513 binding in these rats (50). Thus, the modulation of GABA at 0°C is fairly similar in both rat lines. In the presence of 10/~mol/1 diazepam, the cerebellar binding of [3H]Ro 15-4513 was lower in the ANT rats at both temperatures. This is consistent with the previous finding of higher affinity of DZ-IS binding sites for benzodiazepine agonists in the ANT rats (50,51). At 0°C, GABA had no effect on the binding in AT rats in the presence of diazepam, whereas it decreased the binding in ANT rats. The decrease was seen only in the membranes of those four animals, out of the totai of eight, which had highest amounts of the binding sensitive to diazepam. In a previous study with cultured granule cells, GABA had no effect on total [3H]Ro 15-4513 binding at 37°C, while it stimulated the binding in the presence of 10/~mol/l diazepam (25). At that temperature, the rat lines did not differ in the amount of total cerebellar [3H]Ro 15-4513 binding. GABA had no effect on the total binding, while in the presence of 10 #mol/1 diazepam, GABA increased the binding in all AT rats. In ANT rats, the increase was seen only in rats with high, AT-like binding in the presence of diazepam, whereas GABA decreased the binding in rats with low [3H]Ro 15-4513 binding. Since GABA had no effect on the total binding at 37°C, and decreased the binding at both temperatures in the presence of diazepam only in ANT samples with the highest affinity for diazepam, the latter decrease is apparently caused by an enhancing effect of GABA on diazepam binding to DZ-IS sites. Thus, diazepam would displace [3H]Ro 15-4513 binding from these sites, indicating that diazepam acts as an agonist of DZ-IS-binding sites in these rats. Although benzodiazepine agonists bind with higher affinity to the DZ-IS sites of ANT rats, and the coupling of GABA and benzodiazepine agonists is stronger at these sites at least in some ANT rats, no difference was found between the rat lines in lorazepam-induced enhancement of muscimolstimulated 36C1- flux in the cerebellum. The DZ-IS [3H]Ro 15-4513 binding accounts for about 25°70 of the total binding sites in the cerebellum (45,47), and the predominant site of lorazepam action at GABAA receptors is at the DZ-S-binding sites. Therefore, it is not surprising that there was no difference in lorazepam-induced enhancement of the chloride flux between the rat lines. Selective ligands, either acting as agonists at DZ-IS sites or as antagonists at DZ-S sites, are needed for further assessment of the possible role of the DZ-IS-binding sites in the ANT rat cerebelli. In the present study, the only difference in GABAA receptors between the AT and ANT rats was found in the cerebellar DZ-IS-binding sites of [3H]Ro 15-4513. Diazepam displaces [3H]Ro 15-4513 binding more efficiently from the binding sites of ANT rats. The higher affinity of these sites in ANT rats was also recently demonstrated in the binding of [3H]Ro 15-4513 in vivo after acute administration of lorazepam at a dose causing greater impairment of motor performance in the ANT rats (Korpi et al., submitted). The present findings in vitro, showing that the interaction between benzodiazepine agonists and GABA at DZ-IS-binding sites is stronger in the ANT rats, also indicates that the differences between the rat lines in their DZ-IS-binding sites might at least partly explain the differential sensitivity of the rat lines to lorazepam. ACKNOWLEDGEMENTS We thank Ms. Pirkko Johansson and Ms. Arja Mikkeiinen for expert technical assistance. This study was supported by a grant from the Finnish Foundation for Alcohol Studies.

268

UUSI-OUKARI AND KORPI

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