Naunyn-Schmiedeberg'sArch Pharmacol (1992) 346:432- 436
Naunyn-Schmiedeberg's
Archivesof
Pharmacology © Springer-Verlag 1992
The effect of chronic treatment with peripheral benzodiazepine receptor ligands on behavior and GABAA/benzodiazepine receptors in rat Lembit R/igo 1, Veijo Saano 1, Timo Auvinen 1, Aleksander Adojaan 2, Risto Holma 1, and Mauno M. Airaksinen I 1Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland 2Department of Pharmacology, Tartu University, ~likooli 18, EE-2400 Tartu, Estonia Received December 2, 1991/Accepted May 4, 1992
Summary. Rats were twice daily (2x 10 mg/kg, i.p.) treated for three weeks with the peripheral benzodiazepine (BZ) receptor ligands Ro 5-4864 (4'-chlorodiazepam) and PK 11 195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide). After the first injection there were no differences between the drug-treated and control animals in behavioral tests. After 10 days treatment, the number of sniffings was increased in Ro 5-4864-treated rats. After the last injection, sniffings and ambulations were decreased in PK ll195-treated animals. The number of rearings and groomings remained unchanged throughout the treatment, and there were no changes in the results in the elevated plus-maze test. Apparently these compounds are devoid of anxiolytic and anxiogenic effects at moderate doses. The effect of 72 a h withdrawal from the above mentioned chronic treatment on peripheral and central BZ receptors as well as on GABAA receptors was studied with receptor binding techniques using 3H-Ro 5-4864, 3Hflumazenil and 3H-muscimol, respectively, as ligands. The number of GABAA and central BZ receptors was lower after Ro 5-4864 treatment, as was the effect of progesterone-induced stimulation of 3H-muscimol binding. The number of peripheral BZ receptors was decreased after Ro 5-4864 and PK 11 195 treatments in the olfactory bulb but not in the cerebral cortex. The chronic treatment with peripheral BZ receptor ligands Ro 5-4864 and PK 11 195 produced only little behavioral effects. Ro 5-4864, often presented as an agonist of peripheral BZ receptors, was behaviorally inactive. PK 11 195, often considered to be an antagonist of Ro 5-4864 developed a small sedative action during chronic treatment. The withdrawal from chronic treatment with these ligands similarly affected peripheral BZ receptors but only Ro 5-4864 affected GABAA/BZ receptor complex in the CNS. The present data support the idea that Ro 5-4864 has independent of peripheral BZ receptors effects on GABA,
Correspondence to V. Saano at the above address
receptors while PK 11 195 is rather a partial agonist than antagonist of peripheral BZ receptors.
Key words: Ro 5-4864 - PK 11 195 - Chronic treatment - Behavior withdrawal - GABAA and benzodiazepine receptors
Introduction The existence of at least two clearly different classes of benzodiazepine (BZ) receptors has been well documented. The central type BZ receptors are present mainly in the CNS (M6hler and Okada 1977) and a close correlation between the clinical potencies of BZs and their affinities for these binding sites indicates that they mediate the therapeutic actions of these drugs (Tallman et al. 1980). The peripheral type BZ receptors are present both in peripheral tissues (adrenals, testis, kidney, heart, liver, spleen etc.) and in the CNS (for review see Saano et al. 1989). In spite of numerous studies relatively little is known about the regulation and physiological role of the peripheral BZ receptors. 4'-Chlorodiazepam (Ro 5-4864) and the isoquinoline carboxamide, PK 11 195, bind with high affinities to peripheral, but not to central BZ receptors (Marangos et al. 1982). Ro 5-4864 induces convulsions and anxiety in rodents (File and Lister 1983), displaces 35S-TBPS (t-butylbicyclophosphorothionate) from picrotoxinin binding sites on the GABAA receptors (Weissman et al. 1984) and antagonizes the depolarizing effect of muscimol on rat cuneate nucleus slices (Simmonds 1984). Hence, Ro 5-4864 appears to bind also to a modulatory site on the GABAA receptor chloride channel complex. Molecular cloning of subunit cDNAs has revealed a structural heterogenity of GABAA receptors, compromising a repertoire of several different a, fl, 7 and fi subunits (M6hler et al. 1990; Olsen and Tobin 1990). It is possible that Ro 5-4864 can stimulate at least some GABAA receptor subunit compositions (Costa and
433 G u i d o t t i 1991). Moreover, recent studies provide evidence t h a t peripheral BZ b i n d i n g sites m a y be involved in cholesterol t r a n s p o r t into m i t o c h o n d r i a a n d steroidogenesis ( P a p a d o p o u l o s et al. 1990, 1991). P K 11 195 has b e e n reported to a n t a g o n i z e some, b u t n o t all, o f the effects o f Ro 5-4864 (Saano et al. 1989). Thus, Ro 5-4864 a n d P K 11 195 have b e e n p r o p o s e d to be a n agonist a n d a n t a g o n i s t o f peripheral BZ b i n d i n g sites, respectively. T h e effects o f chronic t r e a t m e n t with central BZ lig a n d s have b e e n extensively studied. However, very little is k n o w n a b o u t the effects o f chronic t r e a t m e n t with specific peripheral BZ receptor ligands o n behavior as well as o n GABAA a n d b e n z o d i a z e p i n e receptors. O n l y recently it has b e e n reported that two weeks t r e a t m e n t with P K 11 195 reduces anxiety in h u m a n s (Ansseau et al. 1990). T h e a i m o f the current study was to examine c o m p a r atively the effect o f a three weeks t r e a t m e n t with Ro 5-4864 a n d P K 11 195 o n behavior. I n parallel the effect o f withdrawal from chronic t r e a t m e n t o n G A B A a a n d b o t h types of BZ receptors in the rat b r a i n was studied.
In vitro binding studies. Binding studies were carried out 72 h after the last injection. ~H-Flumazenil(Ro 15-1788, spec. act. 79 Ci/mmol, New England Nuclear, Boston, Mass., USA), 3H-muscimol (spec. act. 22 Ci/mmol, Amersham Radiochemicals, England) and 3I-I-Ro 5-4864 (spec. act. 89 Ci/mmol, New England Nuclear) binding was carried out as described earlier (Rggo et al. 1989, 1990). Steroid hormone-stimulate d 3H-muscimol binding was carried out as described by L6pezColom6 et al. (1990) with minor modifications. The cerebral cortex tissues were homogenized in 30 vol. 25 retool/1 KzHPO4/KHzPO4 containing 50 mmol/1 KC1 (pH 7.4) with an Ultraturrax homogenizer (5 s, setting 6) and washed twice in the same buffer by centrifugation (48,000×g for 20rain). The final pellets were stored overnight at -20 °C. After thawing they were rehomogenized and washed additionally four times in the same buffer for basal and steroid-stimulated 3Hmuscimol binding. The reaction was initiated by the addition of tissue and terminated after incubation (30 min on ice) by rapid filtration over Whatman GF/B filters. GABA (Sigma, St. Louis, Mo., USA), flunitrazepam (Hoffmann-La Roche, Basle, Switzerland)and PK 11195 (Pharmuka Laboratories, Gennevilliers, France) were used to determine the specific binding to GABA, central- and peripheral BZ receptors, respectively.Progesterone and 4-androstene-3,17-dione(androstenedione; both Sigma, St. Louis, Mo., USA) were used to stimulate 3H-muscimolbinding. Specific binding was calculated by subtracting the nonspecific from total binding. Protein content was measured by the Lowry et al. (1951) method.
Calculations and statistics. Maximum binding (Bmax)and affinity conMaterials and methods
Animals and drugs. Male Kuo:Wistar rats (National Laboratory Animal Center, Kuopio, Finland, 220-250 g) were housed five animals per cage in a room with a lighting cycle 20.00-07.00 h dark period and allowed free access to food and water. 4'-Chlorodiazepam (Ro 5-4864, Fluka AG, Basle, Switzerland) and 1-(2-chlorophenyl)-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide (PK 11195, Pharmuka Laboratories, Gennevilliers, France) were suspended in saline with a drop of Tween-80. The drugs were injected twice a day (at 9.00 and 20.00 h) intraperitoneally in concentrations to give an injection volume of 5 ml/kg and a dose 10 mg/kg (doses refer to drug bases). A total dose of 20 mg/kg/day was administered during 21 days. The last injection was given in the morning of the 22nd day. Behavioral experiments were carried out after the morning injections and binding studies 72 h after withdrawal. The drugs were administered 60 and 120 min before testing in the open field and elevated plus-maze, respectively.Saline containing the same amount of Tween-80 was used for control animals.
Apparatuses and registration procedure. A white circular test arena (diameter 83 cm, divided into 19 equal areas, walls 40 cm) with 65 lux light was used as an open-field. On the test day, the rats were transferred into the laboratory in the morning and allowed to habituate for at least 30 min. The behavior of rats was observed for 5 rain directly by an experienced experimentator. The number of rearings, sniffings, self-groomings, ambulations (stepping across the lines on the floor) and the time of non-locomotor exploration (rats sniffing and looking around while remaining in one place) were recorded. The elevatedplus-maze method described by Pellowand File (Pellow and File 1986) was used. In brief, the plus-maze consisted of two open arms, 50x 10, and two enclosed arms, 50x 10x25, with an open roof, arranged such that the two arms of the same kind were opposite to each other. The central compartment of the plus-maze was an open square, 10x 10 cm. During a 4-rain test period the following observations were made: (a) number of open arm entries, (b) time spent in open and closed arms and (c) total number of all arm entries. To begin the experiments, the animals were placed at the center of the plus-maze. The exploratory activity of rats treated with Ro 5-4864 and PK 11195 was studied in an elevated plus-maze on the 2nd and 22nd day of chronic treatment with these drugs. The data of behavioral studies were manually transferred to a computer for automatic data handling (program by PC Soft Co, Joensuu, Finland).
stants (KD) were calculated using Scatchard analysis. The Scatchard plots were computed with the curve-fitting program Enzfitter. StatView 512+ program was used for statistical calculations. The significance of the differencesbetween the binding data was calculated using two-tailed Student's t-test. The differences between control and treated animals in the open-field and elevated plus-maze were assessed by ANOVAwith repeated measures on the factor treatment day followedby Fischers PLSD test. P
Naunyn-Schmiedeberg's
Archivesof
Pharmacology © Springer-Verlag 1992
The effect of chronic treatment with peripheral benzodiazepine receptor ligands on behavior and GABAA/benzodiazepine receptors in rat Lembit R/igo 1, Veijo Saano 1, Timo Auvinen 1, Aleksander Adojaan 2, Risto Holma 1, and Mauno M. Airaksinen I 1Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland 2Department of Pharmacology, Tartu University, ~likooli 18, EE-2400 Tartu, Estonia Received December 2, 1991/Accepted May 4, 1992
Summary. Rats were twice daily (2x 10 mg/kg, i.p.) treated for three weeks with the peripheral benzodiazepine (BZ) receptor ligands Ro 5-4864 (4'-chlorodiazepam) and PK 11 195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide). After the first injection there were no differences between the drug-treated and control animals in behavioral tests. After 10 days treatment, the number of sniffings was increased in Ro 5-4864-treated rats. After the last injection, sniffings and ambulations were decreased in PK ll195-treated animals. The number of rearings and groomings remained unchanged throughout the treatment, and there were no changes in the results in the elevated plus-maze test. Apparently these compounds are devoid of anxiolytic and anxiogenic effects at moderate doses. The effect of 72 a h withdrawal from the above mentioned chronic treatment on peripheral and central BZ receptors as well as on GABAA receptors was studied with receptor binding techniques using 3H-Ro 5-4864, 3Hflumazenil and 3H-muscimol, respectively, as ligands. The number of GABAA and central BZ receptors was lower after Ro 5-4864 treatment, as was the effect of progesterone-induced stimulation of 3H-muscimol binding. The number of peripheral BZ receptors was decreased after Ro 5-4864 and PK 11 195 treatments in the olfactory bulb but not in the cerebral cortex. The chronic treatment with peripheral BZ receptor ligands Ro 5-4864 and PK 11 195 produced only little behavioral effects. Ro 5-4864, often presented as an agonist of peripheral BZ receptors, was behaviorally inactive. PK 11 195, often considered to be an antagonist of Ro 5-4864 developed a small sedative action during chronic treatment. The withdrawal from chronic treatment with these ligands similarly affected peripheral BZ receptors but only Ro 5-4864 affected GABAA/BZ receptor complex in the CNS. The present data support the idea that Ro 5-4864 has independent of peripheral BZ receptors effects on GABA,
Correspondence to V. Saano at the above address
receptors while PK 11 195 is rather a partial agonist than antagonist of peripheral BZ receptors.
Key words: Ro 5-4864 - PK 11 195 - Chronic treatment - Behavior withdrawal - GABAA and benzodiazepine receptors
Introduction The existence of at least two clearly different classes of benzodiazepine (BZ) receptors has been well documented. The central type BZ receptors are present mainly in the CNS (M6hler and Okada 1977) and a close correlation between the clinical potencies of BZs and their affinities for these binding sites indicates that they mediate the therapeutic actions of these drugs (Tallman et al. 1980). The peripheral type BZ receptors are present both in peripheral tissues (adrenals, testis, kidney, heart, liver, spleen etc.) and in the CNS (for review see Saano et al. 1989). In spite of numerous studies relatively little is known about the regulation and physiological role of the peripheral BZ receptors. 4'-Chlorodiazepam (Ro 5-4864) and the isoquinoline carboxamide, PK 11 195, bind with high affinities to peripheral, but not to central BZ receptors (Marangos et al. 1982). Ro 5-4864 induces convulsions and anxiety in rodents (File and Lister 1983), displaces 35S-TBPS (t-butylbicyclophosphorothionate) from picrotoxinin binding sites on the GABAA receptors (Weissman et al. 1984) and antagonizes the depolarizing effect of muscimol on rat cuneate nucleus slices (Simmonds 1984). Hence, Ro 5-4864 appears to bind also to a modulatory site on the GABAA receptor chloride channel complex. Molecular cloning of subunit cDNAs has revealed a structural heterogenity of GABAA receptors, compromising a repertoire of several different a, fl, 7 and fi subunits (M6hler et al. 1990; Olsen and Tobin 1990). It is possible that Ro 5-4864 can stimulate at least some GABAA receptor subunit compositions (Costa and
433 G u i d o t t i 1991). Moreover, recent studies provide evidence t h a t peripheral BZ b i n d i n g sites m a y be involved in cholesterol t r a n s p o r t into m i t o c h o n d r i a a n d steroidogenesis ( P a p a d o p o u l o s et al. 1990, 1991). P K 11 195 has b e e n reported to a n t a g o n i z e some, b u t n o t all, o f the effects o f Ro 5-4864 (Saano et al. 1989). Thus, Ro 5-4864 a n d P K 11 195 have b e e n p r o p o s e d to be a n agonist a n d a n t a g o n i s t o f peripheral BZ b i n d i n g sites, respectively. T h e effects o f chronic t r e a t m e n t with central BZ lig a n d s have b e e n extensively studied. However, very little is k n o w n a b o u t the effects o f chronic t r e a t m e n t with specific peripheral BZ receptor ligands o n behavior as well as o n GABAA a n d b e n z o d i a z e p i n e receptors. O n l y recently it has b e e n reported that two weeks t r e a t m e n t with P K 11 195 reduces anxiety in h u m a n s (Ansseau et al. 1990). T h e a i m o f the current study was to examine c o m p a r atively the effect o f a three weeks t r e a t m e n t with Ro 5-4864 a n d P K 11 195 o n behavior. I n parallel the effect o f withdrawal from chronic t r e a t m e n t o n G A B A a a n d b o t h types of BZ receptors in the rat b r a i n was studied.
In vitro binding studies. Binding studies were carried out 72 h after the last injection. ~H-Flumazenil(Ro 15-1788, spec. act. 79 Ci/mmol, New England Nuclear, Boston, Mass., USA), 3H-muscimol (spec. act. 22 Ci/mmol, Amersham Radiochemicals, England) and 3I-I-Ro 5-4864 (spec. act. 89 Ci/mmol, New England Nuclear) binding was carried out as described earlier (Rggo et al. 1989, 1990). Steroid hormone-stimulate d 3H-muscimol binding was carried out as described by L6pezColom6 et al. (1990) with minor modifications. The cerebral cortex tissues were homogenized in 30 vol. 25 retool/1 KzHPO4/KHzPO4 containing 50 mmol/1 KC1 (pH 7.4) with an Ultraturrax homogenizer (5 s, setting 6) and washed twice in the same buffer by centrifugation (48,000×g for 20rain). The final pellets were stored overnight at -20 °C. After thawing they were rehomogenized and washed additionally four times in the same buffer for basal and steroid-stimulated 3Hmuscimol binding. The reaction was initiated by the addition of tissue and terminated after incubation (30 min on ice) by rapid filtration over Whatman GF/B filters. GABA (Sigma, St. Louis, Mo., USA), flunitrazepam (Hoffmann-La Roche, Basle, Switzerland)and PK 11195 (Pharmuka Laboratories, Gennevilliers, France) were used to determine the specific binding to GABA, central- and peripheral BZ receptors, respectively.Progesterone and 4-androstene-3,17-dione(androstenedione; both Sigma, St. Louis, Mo., USA) were used to stimulate 3H-muscimolbinding. Specific binding was calculated by subtracting the nonspecific from total binding. Protein content was measured by the Lowry et al. (1951) method.
Calculations and statistics. Maximum binding (Bmax)and affinity conMaterials and methods
Animals and drugs. Male Kuo:Wistar rats (National Laboratory Animal Center, Kuopio, Finland, 220-250 g) were housed five animals per cage in a room with a lighting cycle 20.00-07.00 h dark period and allowed free access to food and water. 4'-Chlorodiazepam (Ro 5-4864, Fluka AG, Basle, Switzerland) and 1-(2-chlorophenyl)-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide (PK 11195, Pharmuka Laboratories, Gennevilliers, France) were suspended in saline with a drop of Tween-80. The drugs were injected twice a day (at 9.00 and 20.00 h) intraperitoneally in concentrations to give an injection volume of 5 ml/kg and a dose 10 mg/kg (doses refer to drug bases). A total dose of 20 mg/kg/day was administered during 21 days. The last injection was given in the morning of the 22nd day. Behavioral experiments were carried out after the morning injections and binding studies 72 h after withdrawal. The drugs were administered 60 and 120 min before testing in the open field and elevated plus-maze, respectively.Saline containing the same amount of Tween-80 was used for control animals.
Apparatuses and registration procedure. A white circular test arena (diameter 83 cm, divided into 19 equal areas, walls 40 cm) with 65 lux light was used as an open-field. On the test day, the rats were transferred into the laboratory in the morning and allowed to habituate for at least 30 min. The behavior of rats was observed for 5 rain directly by an experienced experimentator. The number of rearings, sniffings, self-groomings, ambulations (stepping across the lines on the floor) and the time of non-locomotor exploration (rats sniffing and looking around while remaining in one place) were recorded. The elevatedplus-maze method described by Pellowand File (Pellow and File 1986) was used. In brief, the plus-maze consisted of two open arms, 50x 10, and two enclosed arms, 50x 10x25, with an open roof, arranged such that the two arms of the same kind were opposite to each other. The central compartment of the plus-maze was an open square, 10x 10 cm. During a 4-rain test period the following observations were made: (a) number of open arm entries, (b) time spent in open and closed arms and (c) total number of all arm entries. To begin the experiments, the animals were placed at the center of the plus-maze. The exploratory activity of rats treated with Ro 5-4864 and PK 11195 was studied in an elevated plus-maze on the 2nd and 22nd day of chronic treatment with these drugs. The data of behavioral studies were manually transferred to a computer for automatic data handling (program by PC Soft Co, Joensuu, Finland).
stants (KD) were calculated using Scatchard analysis. The Scatchard plots were computed with the curve-fitting program Enzfitter. StatView 512+ program was used for statistical calculations. The significance of the differencesbetween the binding data was calculated using two-tailed Student's t-test. The differences between control and treated animals in the open-field and elevated plus-maze were assessed by ANOVAwith repeated measures on the factor treatment day followedby Fischers PLSD test. P
