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Effect of swim stress on neurosteroid binding sites of the GABA-A receptor complex labelled with [35S]-TBPS: An autoradiographic study in rat brain
EFFECT OF SWIM STRESS ON NEUROSTEROID BINDING OF THE GABA-A RECEPTOR COMPLEX LABELLED [35S]-TBPS:AN AUTORADIOGRAPHIC STUDY IN BRAIN.
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Unsaturated Free Fatty Acids Increase Agonist Benzodiazepine Receptor Binding Depending On The Suhanit Composition M.R.Witt. S.E. Westh-Hansen and M.Nielsen Dep. of Biochemistry, Research Institute of Biological Psychiatry, Set. Hans Hospital, Roskilde, Denmark It has been previously shown that unsaturated free fatty acids (FFA) strongly enhance the binding of agonist benzodiazepine receptor ligands in the CNS invitro (Witt and Nielsen, 1994). To investigate the selectivity of this interactions, recombinant human GABAA/Benzodiazepine receptor complexes assembled by
M.Vincens, S. Behar, M.Valli. Pharmacologie Endocrinienne, Hopital Lariboisi~re, 2 rue Ambroise Par6, 75010 Paris, France. Stress has been associated with alterations of monoaminergic systems in the brain and particularly of Gabaergic function. It has been demonstrated that stress alters the number and/or functional properties of GABA-A receptors in the CNS. In addition, an increase of the level of neurosteroids such as allopregnanolone (Sa3aP) and allotetrahydrodoc (THDOC) were detected in the brain of rats after exposure to swim stress. These two neurosteroids have been reported to interact with the [35S]-TBPS binding sites of the GABA-A receptor complex and to enhance GABA-A activity in the CNS . In order to further adress the role of these neurosteroids in stress , we studied the interaction of 5a3aP with [35S]-TBPS binding sites and their modulation by GABA in different structures of the brain using quantitative autoradiographic technique.. After exposure to a brief(10 rain) swim stress, rats were sacrificed either immediately (group A) or 10 min after stress (groupB).Results: The apparent number of [35S.]-TBPS binding sites was increased in group B and decreased in group A as compared to control group. In the absence of GABA the IC50 values of 5a3aP for displacement of [35S-TBPS] binding were 2 to 8 xlO-6 M as well as in control and in stressed groups in all brain regions studied. In the presence of GABA 5x10-6M, the IC50 values of 5a3aP for [35S-TBPS] binding inhibition was decreased by a factor of 10 in control group(ICS0=2 to 5x10-7M) .In contrast, IC50 values for 5a3aP were not significantly altered in stressed group (:1C50 =2 to 6xl0-6M).Conclusion:These data show that swim stress induces modification of the interaction of neurosteroids with [35S TBPS] binding sites of the GABA-A receptor complex.
different subunit compositions (~1~272, 0~2~2~t2, (1~3~2"~2,0~5[~2~2)were expressed using the baculovirus-transfected Sf9 insect cell sytem. At 10 M, unsaturated FFA, in particular, arachidonic (20:4) and docosahexaenoic (22:6) strongly stimulated (> 200 % of control) the binding of 3H-Flunitrazepam to the ~3~272 receptor combination in whole cell preparations. No effect of unsaturated FFA on 3H-Funitrazepam binding to t~i~2"/2and tx2~23'2 receptor combinations was observed, and only effects (30 % of contrnl) where observed using the %~272 receptor combination. The saturated FFAs, stearic and palmitie acid were without effect. The hydroxylated unsaturated FFAs, Ricinoleic and Ricinelaidic acid were shown to decrease the binding of 3H-Flunitrazepamto only if an ctll3£t2receptor combination was used, while being inactive in all other receptor combinations.The binding of the benzodiazepine antagonist Ro15-1788 was not influenced or weakly enhanced by unsaturated FFA in any receptor combination. Seatehard plot analysis of the stimulation of 3H-Flunitrazepam binding to an a3[~2Y: combination shows that the enhancement is due to an increase in binding affinity (decrease of the dissociation constant KD). Our data indicate the effect of FFA on agonist benzodiazepine receptor ligands is dependent on the receptor subunit combination used and that the t~ subunit is essential to mediate these effects. Given the heterogeneity of the GABAA/Benzodiazepine receptor subunit distribution in the CNS, the overall effect of FFA on the benzodiazepine receptor can be assumed to vary at both cellular and regional level. References M-R. Witt & M. Nielsen (1994) Journal of Neuroehemistry 62, No.4, 1432-1439
THE ANTICONVULSANT PROFILE OF GYKI 52 466
P e n t y l e n e t e t r a z o l and s t r y c h n i n e s e n s i t i v i t y i n b r a i n w e i g h t s e l e c t e d mice N.N.Boqdano~, ~ I . I . P o l e t a e v a , ~ N.V.Popova Institute of H i g h e r Nervous A c t i v i t y and N e u r o p h y s i o l o g y , B u t l e r o v 5a~ Moscow, R u s s i a ; ~Lomonosov S t a t e U n i v e r s i t y , Moscow, Russia The heterogenous mice p o p u l a t i o n - t h e h y b r i d s o f s i x i n b r e d s t r a i n s (CBA/Lac/Sto, DBA/20/Sto, C57Bl/60/Sto, CS?Br/cd/Sto, A/Sn, BALB/c/Sto) served for brain weight selection. The s e l e c t i o n f o r h i g h (HB) and low (LB) b r a i n w e i g h t s was performed on the basis of b o d y - b r a i n weights relationships so t h a t t h e r e g r e s s i o n l i n e and c o n f i d e n c e intervals were used for each g e n e r a t i o n data. HB and LB s t o c k s e x i s t now as t h e r e s u l t of i l generations of t h i s s e l e c t i o n . The b r a i n w e i g h t d i f f e r e n c e s i s a p p r o x i m a t e l y 75 m g . P e n t y l e n e t e t r a z o l ( from 25 t o I00 mg/kg, s.c.) and strychnine ( 2 mg/kg ) i n j e c t i o n s t o HB and LB male mice were p e r formed. Number o f a n i m a l s a f f e c t e d , s e i z u r e latencies and mortality were used as i n d i c e s . L B m i c e p r o v e d to be m o r e p r o n e to c o n v u l s a n a c t i o n than HB. A l t h o u g h the r e l a tive brain weights of two s a m p l e s u s e d in this study demousted partial overlap. This probably means t h a t LB l o w e r t h r e s h o l d s of seizure susceptibility c o u l d be d e t e r m i n e d by s u b t l e n e u r o n a l o r g a n i z a t i o n differences induced as the result of brain weight selection.
F. Andrasi, P. Berzsenyi, I Tarnawa Institute for Drug Research, Berlini 0t 47-49, H-1045 Budapest, Hungary The selective non-competitive AMPA-antagonist GYKI52466 has neuroprotective skeletal muscle relaxant and anticonvulsive effects. We investigated this feature compared to that of established myorelaxants. Anticonvulsant activity in CFLP mice ED50 mg/kg p.o. Method GYKI 52 466 Baclofen Chlorzoxazone Electroshock 38 155 >400 (20mA, 1.5s,0.4ms) Pentetrazole 130 i.p. 115 >200 >400 Strychnine 3 i.p. 87 >200 >400 Bemegride 50 i.p. 73 >200 >400 Picrotoxin 17 i.p. >200 >200 >400 Bicuculline 1 i.v. 35 >200 440 Nicotine 12 i.v. 100 >200 >400 3-SH-propionic acid 47 >200 >400 90 i.v. (3-MPA) 4-aminopyridine 43 >200 >400 12.5 i.p. The broad anticonvulsant spectrum of GYKI 52 466 suggests a potential role in the therapy of epilepsy.
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SITES WITH RA T
Unsaturated Free Fatty Acids Increase Agonist Benzodiazepine Receptor Binding Depending On The Suhanit Composition M.R.Witt. S.E. Westh-Hansen and M.Nielsen Dep. of Biochemistry, Research Institute of Biological Psychiatry, Set. Hans Hospital, Roskilde, Denmark It has been previously shown that unsaturated free fatty acids (FFA) strongly enhance the binding of agonist benzodiazepine receptor ligands in the CNS invitro (Witt and Nielsen, 1994). To investigate the selectivity of this interactions, recombinant human GABAA/Benzodiazepine receptor complexes assembled by
M.Vincens, S. Behar, M.Valli. Pharmacologie Endocrinienne, Hopital Lariboisi~re, 2 rue Ambroise Par6, 75010 Paris, France. Stress has been associated with alterations of monoaminergic systems in the brain and particularly of Gabaergic function. It has been demonstrated that stress alters the number and/or functional properties of GABA-A receptors in the CNS. In addition, an increase of the level of neurosteroids such as allopregnanolone (Sa3aP) and allotetrahydrodoc (THDOC) were detected in the brain of rats after exposure to swim stress. These two neurosteroids have been reported to interact with the [35S]-TBPS binding sites of the GABA-A receptor complex and to enhance GABA-A activity in the CNS . In order to further adress the role of these neurosteroids in stress , we studied the interaction of 5a3aP with [35S]-TBPS binding sites and their modulation by GABA in different structures of the brain using quantitative autoradiographic technique.. After exposure to a brief(10 rain) swim stress, rats were sacrificed either immediately (group A) or 10 min after stress (groupB).Results: The apparent number of [35S.]-TBPS binding sites was increased in group B and decreased in group A as compared to control group. In the absence of GABA the IC50 values of 5a3aP for displacement of [35S-TBPS] binding were 2 to 8 xlO-6 M as well as in control and in stressed groups in all brain regions studied. In the presence of GABA 5x10-6M, the IC50 values of 5a3aP for [35S-TBPS] binding inhibition was decreased by a factor of 10 in control group(ICS0=2 to 5x10-7M) .In contrast, IC50 values for 5a3aP were not significantly altered in stressed group (:1C50 =2 to 6xl0-6M).Conclusion:These data show that swim stress induces modification of the interaction of neurosteroids with [35S TBPS] binding sites of the GABA-A receptor complex.
different subunit compositions (~1~272, 0~2~2~t2, (1~3~2"~2,0~5[~2~2)were expressed using the baculovirus-transfected Sf9 insect cell sytem. At 10 M, unsaturated FFA, in particular, arachidonic (20:4) and docosahexaenoic (22:6) strongly stimulated (> 200 % of control) the binding of 3H-Flunitrazepam to the ~3~272 receptor combination in whole cell preparations. No effect of unsaturated FFA on 3H-Funitrazepam binding to t~i~2"/2and tx2~23'2 receptor combinations was observed, and only effects (30 % of contrnl) where observed using the %~272 receptor combination. The saturated FFAs, stearic and palmitie acid were without effect. The hydroxylated unsaturated FFAs, Ricinoleic and Ricinelaidic acid were shown to decrease the binding of 3H-Flunitrazepamto only if an ctll3£t2receptor combination was used, while being inactive in all other receptor combinations.The binding of the benzodiazepine antagonist Ro15-1788 was not influenced or weakly enhanced by unsaturated FFA in any receptor combination. Seatehard plot analysis of the stimulation of 3H-Flunitrazepam binding to an a3[~2Y: combination shows that the enhancement is due to an increase in binding affinity (decrease of the dissociation constant KD). Our data indicate the effect of FFA on agonist benzodiazepine receptor ligands is dependent on the receptor subunit combination used and that the t~ subunit is essential to mediate these effects. Given the heterogeneity of the GABAA/Benzodiazepine receptor subunit distribution in the CNS, the overall effect of FFA on the benzodiazepine receptor can be assumed to vary at both cellular and regional level. References M-R. Witt & M. Nielsen (1994) Journal of Neuroehemistry 62, No.4, 1432-1439
THE ANTICONVULSANT PROFILE OF GYKI 52 466
P e n t y l e n e t e t r a z o l and s t r y c h n i n e s e n s i t i v i t y i n b r a i n w e i g h t s e l e c t e d mice N.N.Boqdano~, ~ I . I . P o l e t a e v a , ~ N.V.Popova Institute of H i g h e r Nervous A c t i v i t y and N e u r o p h y s i o l o g y , B u t l e r o v 5a~ Moscow, R u s s i a ; ~Lomonosov S t a t e U n i v e r s i t y , Moscow, Russia The heterogenous mice p o p u l a t i o n - t h e h y b r i d s o f s i x i n b r e d s t r a i n s (CBA/Lac/Sto, DBA/20/Sto, C57Bl/60/Sto, CS?Br/cd/Sto, A/Sn, BALB/c/Sto) served for brain weight selection. The s e l e c t i o n f o r h i g h (HB) and low (LB) b r a i n w e i g h t s was performed on the basis of b o d y - b r a i n weights relationships so t h a t t h e r e g r e s s i o n l i n e and c o n f i d e n c e intervals were used for each g e n e r a t i o n data. HB and LB s t o c k s e x i s t now as t h e r e s u l t of i l generations of t h i s s e l e c t i o n . The b r a i n w e i g h t d i f f e r e n c e s i s a p p r o x i m a t e l y 75 m g . P e n t y l e n e t e t r a z o l ( from 25 t o I00 mg/kg, s.c.) and strychnine ( 2 mg/kg ) i n j e c t i o n s t o HB and LB male mice were p e r formed. Number o f a n i m a l s a f f e c t e d , s e i z u r e latencies and mortality were used as i n d i c e s . L B m i c e p r o v e d to be m o r e p r o n e to c o n v u l s a n a c t i o n than HB. A l t h o u g h the r e l a tive brain weights of two s a m p l e s u s e d in this study demousted partial overlap. This probably means t h a t LB l o w e r t h r e s h o l d s of seizure susceptibility c o u l d be d e t e r m i n e d by s u b t l e n e u r o n a l o r g a n i z a t i o n differences induced as the result of brain weight selection.
F. Andrasi, P. Berzsenyi, I Tarnawa Institute for Drug Research, Berlini 0t 47-49, H-1045 Budapest, Hungary The selective non-competitive AMPA-antagonist GYKI52466 has neuroprotective skeletal muscle relaxant and anticonvulsive effects. We investigated this feature compared to that of established myorelaxants. Anticonvulsant activity in CFLP mice ED50 mg/kg p.o. Method GYKI 52 466 Baclofen Chlorzoxazone Electroshock 38 155 >400 (20mA, 1.5s,0.4ms) Pentetrazole 130 i.p. 115 >200 >400 Strychnine 3 i.p. 87 >200 >400 Bemegride 50 i.p. 73 >200 >400 Picrotoxin 17 i.p. >200 >200 >400 Bicuculline 1 i.v. 35 >200 440 Nicotine 12 i.v. 100 >200 >400 3-SH-propionic acid 47 >200 >400 90 i.v. (3-MPA) 4-aminopyridine 43 >200 >400 12.5 i.p. The broad anticonvulsant spectrum of GYKI 52 466 suggests a potential role in the therapy of epilepsy.
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