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Carbamazepine and electroconvulsive shock attenuate β-adrenoceptor and muscarinic cholinoceptor coupling to G protein in rat cortex
European Journal of Pharraacolog~. - Molecular Pharmacology. Section. 189 (1990) 99-),03
99
Elsevier EJPMOL 80028
Short communication
Carbamazepine and electroconvulsive shock attenuate/2-adrenoceptor and muscarinic cholinoceptor coupling to G proteins in rat cortex Sofia Avissar ~, Gabriel Schreiber k Charanjit S. Aulakh ~. Kr3"styna M. Wozniak 2 and Dennis L. Murphy 1 I Laboratory of Clinical Science, National Institute of Mental Health and : Lt~boratory of Clmi~ff Studies, National hlstitute on Alcohol Abtme and Alcoholism. Bethesd~ Maryland. t~S.A.
Received 28 February 1990, accepted 28 March 199,3
We have recently found that lithium attenuates the function of G proteins, suggesting these proteins as the common site for both the antimanic and antidepressant therapeutic effects of lithium. Perturbation of G protein function may thus be a general characteristic of treatments effective in bipolar affective disorder. In the present study, we demonstrate that both chronic carbamazepine and repeated electroconvulsive shock treatment inhibit the coupling of both muscarinic cholinoceptors and fl-adrenoceptors to pertussis toxin-sensitive and cholera toxin-sensitive G proteins, respectively. Carbamazepine: Eiectrocon,~a!siveshock: G proteins: fl-Adrenoceptors: Muscarinic chotinoceptors
1. Introduction Lithium is now well established as the drug of choice for treating manic-depressive illness. Carbamazepine also represents an alternative treatment for bipolar affective disorder. Clinical evidence suggests that carbamazepine has acute antimanic and long-term prophylactic effects in the treatment of bipolar affective disorder. Preliminary evidence suggests that carbanmzepine may also have acute antidepressant properties (for review, see Post, 1987). Electroconvulsive shock (ECS) therapy also resembles lithium, as it is effective in the acute treatment of mania and in the prevention of affcctive recurrences (for review, see Lerer, 1987). The most striking aspect of these 'bipolar' treatments is their ability to prevent a n d / o r treat both mania and depression.
Correspondence to: Sofia Avissar. Ph.D.. Beer-Sheva Mental Health Coaled. P,O. Box 4600, Beer-Sbeva. 84170. Israel
In recent years, research addressing the mechanism of action of treatments effective in bipolar affective disorder has focused on the perturbation of signal transduction phenomena, beyond receptors (for review, see Baraban et al., 1989). The family of guanine nuc!eotide-binding proteins (G proteins) plays a pivotal rote in post-receptor information transduction by mediating extraceltular signals from membrane receptors to intracellular second messenger systems, such as the adenytate cyclase and phospbatidylinositol (PI) cycle (for review, see Gilman, 1987), Recently, we found that therapeutic concentrations of lithium attenuate both /Ladrenoceptor and muscarinic eholinoceptor coupling to cholera toxin-sensitive and pe~ussis toxin-~;ensitive G proteins, respectively. Therefore we proposed attenuation of G protein function as the common mechanism for both the antimanic and antidepressant therapeutic effects of tith.;um (Avissar et al., 1988; Avissar and Schreiber. 1989). In these studies, we assessed lithium aheration of G pro-
0922-4106/90/$03.50 ~J t990 Elsevier Science Publishers B.V. (3iomedica! Divisio.)
100 tein function by producing agonist-induced increases in GTP binding to these proteins, which enabled us to directly measure G protein function distal to receptors and independent of second messenger activity. In the present study, we used the saree technique to assess the possible interaction of carbamazepine and ECS with G proteins and thus determine whether altered G protein function is a general characteristic of successful treatments for bipolar affective disorder.
generalized tonic-clonic seizure. Control animals had the electrodes placed in their ears but had no current applied (sham ECS). Animals were killed 24 h after the last ECS treatment. 2.3. Membrane preparation
Rats were decapitated, and the cerebral cortex was removed rapidly. The tissue was homogenized in buffer A (25 mM "Iris, pH 7.4, 1 mM dithiothreitol (DTT)) containing 1 mM EGTA. Membranes were obtained by centrifuging the preparations twice for 10 min, 10000 × g, at 4°C.
2. Materials and methods
Male Wistar rats weighing approximately 250 g at the be~uning of the study were used. Animals were housed in a temperature-controlled (24 + I°C) room with a 12-~, light-dark cycle (lights on at 7:00 a.m.). 2.L Chronic carbamazepine treatment
Animals were divided into a control group and a carbamazepine-treated group. In the carbamazepine-treated group, animals were given food (Purina food pallets) containing carbamazepine (5.0 g/kg rat chow food) for 14 days. All animals had free access to food and water. Plasma levels of carbamazepine in rats maintained on this diet were 1.0 + 0.15 meq/l, at the time of sacrifice. Plasma levels of the carbamazepine active metabolite, 10,11-epoxide, were 5.4 + 0.5 meq/1. It should be noted that carbamazepine-10,11epoxide is a major metabolite of carbamazepine in humans, but only represents approximately 25% of the parent compound. In contrast, this ratio is reversed in the rat, and levels of the epoxide are about four-fold higher than carbamazepine itself following chronic administration (Marangos et al., 1985).
2.4. [3H] GTP binding to rat cerebral cortical membranes
A 200 #1 sample of membranes (0.5-1.5 mg protein), suspended in (mM): 25 "Iris (pH 7.4), 1 ATP, 2.2 Mg 2+, 1 EGTA, and 1 DTT, was pipetted into plastic microfuge tubes containing varying concentrations of [3H]GTP (0.5-4.0 #M). The incubation was performed at room temperature for 5 min (equilibrium conditions) and the reaction was stopped by adding five volumes of ice-cold buffer A, followed by centrifugation at 10000 × g for 1 rain. The pellets were washed rapidly three times with ice-cold buffer A by repeating centrifugation and were then resuspended in 200/tl of the same buffer. Bound radioactivity was measured by liquid scintillation spectrometry by adding a 150-/d aliquot dissolved in scintillation liquid. All assays were carried out in triplicate, together with triplicate control samples containing 100 #M unlabelled guanyl-5'-yl imidodiphosphate [Gpp (NH)p], to determine nonspecific binding. Isoproterenol or carbamylcholine was added at a final concentration of 50/tM and 100 #M, respectively, which are maximally effective doses established previously (Avissar et al., 1988). 2.5. A D P ribosylation
2.2. Electroconvulsive shock administration Rats were shocked through ear clip electrodes once daily in the morning for 11 days. Current (30 mA, 0.5 s) was delivered by a Dvostat electroshock generator and all shocked rats experienced a
Membranes were suspended in 1 ml buffer containing (mM): 25 Tris, (pH 7.4), 10 NAD, 1 ATP, 1 EGTA, 2 DTT, 5 MgC12, 10 thymidine, 20 creatine phosphate; 100/tM GTP, and 40/tg mlcreatine phosphokinase. ADP ribesylation was
t01 carried out for 25 min at 3 0 ° C by adding cholera toxin (50 #g ml-~), preactivated for ~6 min at 3 7 ° C with 20 mM DTT, or pertussis toxin (25 pg m1-1) preactivated for 10 min at 30~C with 20 mM Tris. The reaction was stopped by adding 25 ml ice-cold 25 mM Tris (pH 7.4) and 5 mM MgCI 2 immediately followed by centrifugation at 10000 × g for 10 min. [3H]GTP binding was then carried out as described.
tively (Avissar et al., i988; fig. 1A). The fl-adrenergic agonist effect was exerted through G~, as ribosylation by -holera toxin totally abolished the isoproterenol effect, and ribosylation with pertussis toxin had no effec:. Ribosylation of G proteins other than G,, catalyzed by pertussls toxin, inhibited carbamylchohne-induced increases in [3H]GTP binding capacity, but ribosylation catalyzed by cholera toxin did not affect carbamyicholine action, thus supporting a muscarinic effect through G proteins other than G~ (i.e., Gi,
3. Results
G J.
In the present study, G protein function was assessed directly by measuring isoproterenol- and carbamylcholine-induced increases in [3H]GTP binding to membranes prepared from rat cerebral cortex, lsoproterenol- and carbamylcholine-induced increases in the binding capacity for GTP were blocked by propranolol and atropine, respec-
Chronic 2-week treatment with carbamazepine, which yielded therapeutic blood levels, totally abolished the ability of both lsoproterenol mad cafoamylcholine to induce increases in GTP bindin~ capacity (fig. IB). The drug d;d not affect G'fP basal bind;ng characteristics. As with carbamazepine treatmenL repeated administration of ECS on 1 i consecutive days tota'.ly abolished the
C
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40
t
20 O
\
E CL "/!
I 40
}: 50
,~-50
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40
50
=~30
4O
Bound (pmoi x mg protein-~ ) Fig. 1. Chronic carbamazepine and repeated ECA administration: attenuation of fl-adrenoecptor- and musea~finicreceptor-induced increases in GTP binding capacity. Representative Scatchard plots of basal and agonist-induced [3H]GTP binding to membranes prepared fromcerebral cortexof(A) untreated rats (rats givensham ECS bad similarresul:.s);(B) rats treated with carbamazepinefor 14 days; (C) rats given ECS treatment for 11 days. (®) Basal |3H]GTP binding,~r2}in the presenceof 50 .aM i.~o~r,~erenol and (o) in the presenceof 100 ,aM of carbamylchohne. Each groupconsistedof 6-8 rats. t~'~protereaol-and carbamylehoiine-inducedincreases in [3H]GFP specific binding were assessed in membranes of cerebral cortex prepared from each anir,~at and compared to isoproterenol and carbamylchohne effects in membranes prepared ?rein untreated rata given sham ECS ~soproterenot and earbamylcbohneinduced, respectively, 15.1± 1.2 and 16.5-+1.6% incre,.ase~in GTP bindingcapacity in contrc~!al~ii~mls;in sham-ECS animals, 15.4_+1.4 and 19.2+2.4% respectiveincreaseswereobsers'ed.In the carbarnazepineand ECS groups,both agonistsfailed ~.o induce significant increasesin GTP binding capacity abovebasal.
102 effects of both isoproterenol and carbamylcholine on GTP binding (fig. 1C), and had no effect on GTP basal binding.
4. Discussion Carbamazepine and lithium share a common clinical profile of acute and prophylactic therapeutic effects in manic-depressive illness (for review, see Post, 1987). Likewise, the clinical spectrum of ECS treatment differs substantially from that of chemical antidepressants and resembles more closely the clinical profile of lithium (for review, see Lerer, 1987). Two main theories attempting to explain the lithium mechanism of action have focused on the perturbation of two second messenger effector systems: adenylate cyclase and PI turnover (for review, see Baraban et al., 1989). Recently, we reported (Avissar et al., 1988; Avissar and Schreiber, 1989) that therapeutic concentrations of lithium can alter the function of a core feature of both these signalling systems, namely G proteins. We showed that in rat cerebral cortex, lithium inhibited the coupling of both /8adrenoceptors and muscarinic cholinoceptors to cholera toxin- and pertussis toxin-sensitive G proteins, respectively (Avissar et al., 1988; Avissar and Schreiber, 1989). In the present study, we used the same method to determine G protein function independent of the subsequent activities of second messenger effector systems, and found that chronic earbamazepine and ECS alter receptor-coupled cholera toxin- and pertussis toxin-sensitive G protein function. Both chronic treatment with carbamazepine and repeated administration of ECS attenuated isoproterenol- and carbamylcholine-induced increases in GTP binding to rat cerebral cortical membranes. Although a diversity of neurochemical effects produced by carbamazepine and ECS have been described (for reviews, see Post, 1987; Lerer, 1987), both treatments have been reported to reduce cyclic AMP (cAMP) production in response to norepinephrine in rat cortical slices (Newman et al., 1986; Palmer et al., 1979). As carbamazepine does not down-regulate fl-adrenoceptor density
(Marangos et al., 1985), its effects are likely to be distal to the fl-adrenoceptor. ECS is known to down-regulate fl-adrenoceptor density (Lerer, 1987); however, its effects on cAMP accumulation may be located distal to the receptor because it also attenuates the accumulation of cAMP induced by forskolin (Newman et al., 1986), which bypasses the fl-adrenoceptor. These postreceptor effects are similar to those reported for lithium (for review, see Bunney and Gadand-Bunney, 1987) and they suggest that both carbamazepine and ECS act at a post-receptor site, which might be similar to that of lithium. G~ plays a crucial role in transducing fl-adrenoceptor-induced stimulation of adenylate cyclase. After chronic treatment with either lithium, carbamazepine, or ECS, reduced cAMP production, in response to fl-adrenoceptor stimulation, can result from the attenuated ,8-adrenoceptorcoupled G~ protein function caused by these treatments. Muscarinic cholinoceptors in the central nervous system are involved both in the inhibition of adenylate cyclase activity and in the activation of PI turnover. These second messenger systems are thought to be associated with muscarinic cholinoceptors via G proteins: G~ or G O in the case of adenylate cyclase, and the recently designated G protein, Gp, in the case of PI turnover (for review, see Gilman, 1987). Perturbation of muscarinic cholinoceptor-coupied, pertussis toxin-sensitive G proteins by chronic treatment with lithium (Avissar et al., 1988; Avissar and Schreiber, 1989), carbamazepine and ECS (described in the present study) predicts alteration by these treatments of muscarinic cholinoceptor-coupled inhibition of adenylate cyclase or activation of the PI cycle. Recent studies confirm this prediction. Chronic ECS was found to reduce the degree of inhibition by serotonin and carbachol of forskolin-sti'nutated adenylate cyclase in rat hippocampal membranes (Newman and Lerer, 1988). Lithium, given chronically, inhibits rather than enhances inositol triphosphate response to muscarinic and other receptor agonists in the rat cortex (Batty and Nahorski, 1985; Godfrey et al., 1989). This inhibition could not be explained by the classical site (inositol-l-phosphatase) of the effects of lithium
103 o n PI m e t a b o l i s m . P r e l i m i n a r y reports suggest p e r t u r b a t i o n o f inositol p h o s p h a t e a c c u m u l a t i o n in rat b r a i n slices b y b o t h ECS a n d c a r b a m a z e p i n e ( V a d n a l a n d Bazan, 1988). W e suggest that altered G p r o t e i n function is a general corm,n o n m e c h a n i s m for b o t h the antim a n i c a n d the a n t i d e p r e s s a n t effects of a v a r i e t y o f t r e a t m e n t s used in b i p o l a r affective disorder.
References Avissar, S. and G. Schreiber, 1989, Muscarinic receptor subclassification and G proteins: significance for lithium action in affective disorders and for the treatment of the extrapyramidal side effects of neurolepties, Biol. Psychiatry 26, 113. Avissar, S., G. Schreiber, A. Danon. and R.H. Belmaker, 1988, Lithium inhibits adrenergic and cholinergic increases in GTP binding in rat cortex, Nature 331, 440. 13araban, J.M., P.F. Worley, and S.H. Snyder, 1989. Second messenger systems and psychoactive drug action: focus on the phosphoinositide system and lithium, Am. J. Psychiatry 146, 1251. Batty, I. and S.R. Nahorski, 1985, Differential effects of lithium on musearinic receptor stimulation of inositol phosphates in rat cerebral cortex slices, J. Neurochem. 45, 1514. Bunney, Jr., W.E., and B.L. Garlan,t-Bunney, 1987, Mechanisms of action of lithium in affec6ve illness: basic and clinical implications, in Psyehopharmacology: The Tlfird Generation of Progress, ed. H.Y. Mehzer (Raven Press. New York). Gilman, A.G., 1987, G proteins: transduce~-sof receptor-generated signals, Annu. Rev. Biocherr- 36. 6i5.
Godfrey, P.P., SJ. McClue. A.M. White, A.J. Wood, and D.G. Grahame-Smith, 1989. Subacute and chronic in vivo lithium treatment inhibits agonist- and sodium fluoride-stimulated inositol phosphate ,~roduction in, rat cortex, J. Neurochem. 52, 498. Lerer. B., 1987. Neurochemieal and other neurobiological consequences of ECT: implications for the pathogenesis and treatment of affective disorders, in Psychopharmacology: The Third Generation of Progress, ed. H.Y. Mehzer (Raven Press, New York) p. 577. Marangos. P.J.. S.R. Weiss. P. Montgomery, J. Patel, P.K. Narang, A.M. Cappabianca and R.M. Post, t985, Chronic carbamazepine treatment increases brain adenosine receptors. Epilepsia 26. 493. Newman, M.E. and B. Lerer, 1988, Chronic electroconvulsive shock and desimipramine reduce the degree of inhibition by 5-HT and carbachol of forskolin-stimulated adenylate eyclase in rat hippocampal membranes. European J. Pharmacol. 148, 257. Newman, M.E., H. Solomon and 13. Lerer, 1986, Electroconvulsive shock and cyclic AMP signal transduction: effects distal to the receptor, J. Neurochem. 46, 1667. Palmer, G.C.. D.J. Jones, M,A. Medina and W.B. Stavinoha. 1979, Anticonvulsant drug actions on in vitro and in vivo levels of cychc AMP in the mouse brain, Epilepsia 20, 95. Post. R.M., 1987. Mechanisms of action of earbamazepine and related anliconvulsants in affective illness, in Psychopharmacology: The Third Generation of Progress. ed. H.Y. Mehzer (Raven Press, New York) p. 567. Vadnal, R.E. and N.G. 13azan, lO88, Carbamaz~epineinhibits electroconvulsive shock-induced inositol triphosphate (IP3) accumulation in rat cerebral cortex and hippocampus, Binchem. Biophys. Res. Commun. 153, 128.
99
Elsevier EJPMOL 80028
Short communication
Carbamazepine and electroconvulsive shock attenuate/2-adrenoceptor and muscarinic cholinoceptor coupling to G proteins in rat cortex Sofia Avissar ~, Gabriel Schreiber k Charanjit S. Aulakh ~. Kr3"styna M. Wozniak 2 and Dennis L. Murphy 1 I Laboratory of Clinical Science, National Institute of Mental Health and : Lt~boratory of Clmi~ff Studies, National hlstitute on Alcohol Abtme and Alcoholism. Bethesd~ Maryland. t~S.A.
Received 28 February 1990, accepted 28 March 199,3
We have recently found that lithium attenuates the function of G proteins, suggesting these proteins as the common site for both the antimanic and antidepressant therapeutic effects of lithium. Perturbation of G protein function may thus be a general characteristic of treatments effective in bipolar affective disorder. In the present study, we demonstrate that both chronic carbamazepine and repeated electroconvulsive shock treatment inhibit the coupling of both muscarinic cholinoceptors and fl-adrenoceptors to pertussis toxin-sensitive and cholera toxin-sensitive G proteins, respectively. Carbamazepine: Eiectrocon,~a!siveshock: G proteins: fl-Adrenoceptors: Muscarinic chotinoceptors
1. Introduction Lithium is now well established as the drug of choice for treating manic-depressive illness. Carbamazepine also represents an alternative treatment for bipolar affective disorder. Clinical evidence suggests that carbamazepine has acute antimanic and long-term prophylactic effects in the treatment of bipolar affective disorder. Preliminary evidence suggests that carbanmzepine may also have acute antidepressant properties (for review, see Post, 1987). Electroconvulsive shock (ECS) therapy also resembles lithium, as it is effective in the acute treatment of mania and in the prevention of affcctive recurrences (for review, see Lerer, 1987). The most striking aspect of these 'bipolar' treatments is their ability to prevent a n d / o r treat both mania and depression.
Correspondence to: Sofia Avissar. Ph.D.. Beer-Sheva Mental Health Coaled. P,O. Box 4600, Beer-Sbeva. 84170. Israel
In recent years, research addressing the mechanism of action of treatments effective in bipolar affective disorder has focused on the perturbation of signal transduction phenomena, beyond receptors (for review, see Baraban et al., 1989). The family of guanine nuc!eotide-binding proteins (G proteins) plays a pivotal rote in post-receptor information transduction by mediating extraceltular signals from membrane receptors to intracellular second messenger systems, such as the adenytate cyclase and phospbatidylinositol (PI) cycle (for review, see Gilman, 1987), Recently, we found that therapeutic concentrations of lithium attenuate both /Ladrenoceptor and muscarinic eholinoceptor coupling to cholera toxin-sensitive and pe~ussis toxin-~;ensitive G proteins, respectively. Therefore we proposed attenuation of G protein function as the common mechanism for both the antimanic and antidepressant therapeutic effects of tith.;um (Avissar et al., 1988; Avissar and Schreiber. 1989). In these studies, we assessed lithium aheration of G pro-
0922-4106/90/$03.50 ~J t990 Elsevier Science Publishers B.V. (3iomedica! Divisio.)
100 tein function by producing agonist-induced increases in GTP binding to these proteins, which enabled us to directly measure G protein function distal to receptors and independent of second messenger activity. In the present study, we used the saree technique to assess the possible interaction of carbamazepine and ECS with G proteins and thus determine whether altered G protein function is a general characteristic of successful treatments for bipolar affective disorder.
generalized tonic-clonic seizure. Control animals had the electrodes placed in their ears but had no current applied (sham ECS). Animals were killed 24 h after the last ECS treatment. 2.3. Membrane preparation
Rats were decapitated, and the cerebral cortex was removed rapidly. The tissue was homogenized in buffer A (25 mM "Iris, pH 7.4, 1 mM dithiothreitol (DTT)) containing 1 mM EGTA. Membranes were obtained by centrifuging the preparations twice for 10 min, 10000 × g, at 4°C.
2. Materials and methods
Male Wistar rats weighing approximately 250 g at the be~uning of the study were used. Animals were housed in a temperature-controlled (24 + I°C) room with a 12-~, light-dark cycle (lights on at 7:00 a.m.). 2.L Chronic carbamazepine treatment
Animals were divided into a control group and a carbamazepine-treated group. In the carbamazepine-treated group, animals were given food (Purina food pallets) containing carbamazepine (5.0 g/kg rat chow food) for 14 days. All animals had free access to food and water. Plasma levels of carbamazepine in rats maintained on this diet were 1.0 + 0.15 meq/l, at the time of sacrifice. Plasma levels of the carbamazepine active metabolite, 10,11-epoxide, were 5.4 + 0.5 meq/1. It should be noted that carbamazepine-10,11epoxide is a major metabolite of carbamazepine in humans, but only represents approximately 25% of the parent compound. In contrast, this ratio is reversed in the rat, and levels of the epoxide are about four-fold higher than carbamazepine itself following chronic administration (Marangos et al., 1985).
2.4. [3H] GTP binding to rat cerebral cortical membranes
A 200 #1 sample of membranes (0.5-1.5 mg protein), suspended in (mM): 25 "Iris (pH 7.4), 1 ATP, 2.2 Mg 2+, 1 EGTA, and 1 DTT, was pipetted into plastic microfuge tubes containing varying concentrations of [3H]GTP (0.5-4.0 #M). The incubation was performed at room temperature for 5 min (equilibrium conditions) and the reaction was stopped by adding five volumes of ice-cold buffer A, followed by centrifugation at 10000 × g for 1 rain. The pellets were washed rapidly three times with ice-cold buffer A by repeating centrifugation and were then resuspended in 200/tl of the same buffer. Bound radioactivity was measured by liquid scintillation spectrometry by adding a 150-/d aliquot dissolved in scintillation liquid. All assays were carried out in triplicate, together with triplicate control samples containing 100 #M unlabelled guanyl-5'-yl imidodiphosphate [Gpp (NH)p], to determine nonspecific binding. Isoproterenol or carbamylcholine was added at a final concentration of 50/tM and 100 #M, respectively, which are maximally effective doses established previously (Avissar et al., 1988). 2.5. A D P ribosylation
2.2. Electroconvulsive shock administration Rats were shocked through ear clip electrodes once daily in the morning for 11 days. Current (30 mA, 0.5 s) was delivered by a Dvostat electroshock generator and all shocked rats experienced a
Membranes were suspended in 1 ml buffer containing (mM): 25 Tris, (pH 7.4), 10 NAD, 1 ATP, 1 EGTA, 2 DTT, 5 MgC12, 10 thymidine, 20 creatine phosphate; 100/tM GTP, and 40/tg mlcreatine phosphokinase. ADP ribesylation was
t01 carried out for 25 min at 3 0 ° C by adding cholera toxin (50 #g ml-~), preactivated for ~6 min at 3 7 ° C with 20 mM DTT, or pertussis toxin (25 pg m1-1) preactivated for 10 min at 30~C with 20 mM Tris. The reaction was stopped by adding 25 ml ice-cold 25 mM Tris (pH 7.4) and 5 mM MgCI 2 immediately followed by centrifugation at 10000 × g for 10 min. [3H]GTP binding was then carried out as described.
tively (Avissar et al., i988; fig. 1A). The fl-adrenergic agonist effect was exerted through G~, as ribosylation by -holera toxin totally abolished the isoproterenol effect, and ribosylation with pertussis toxin had no effec:. Ribosylation of G proteins other than G,, catalyzed by pertussls toxin, inhibited carbamylchohne-induced increases in [3H]GTP binding capacity, but ribosylation catalyzed by cholera toxin did not affect carbamyicholine action, thus supporting a muscarinic effect through G proteins other than G~ (i.e., Gi,
3. Results
G J.
In the present study, G protein function was assessed directly by measuring isoproterenol- and carbamylcholine-induced increases in [3H]GTP binding to membranes prepared from rat cerebral cortex, lsoproterenol- and carbamylcholine-induced increases in the binding capacity for GTP were blocked by propranolol and atropine, respec-
Chronic 2-week treatment with carbamazepine, which yielded therapeutic blood levels, totally abolished the ability of both lsoproterenol mad cafoamylcholine to induce increases in GTP bindin~ capacity (fig. IB). The drug d;d not affect G'fP basal bind;ng characteristics. As with carbamazepine treatmenL repeated administration of ECS on 1 i consecutive days tota'.ly abolished the
C
A :=. X
tT..~
40
t
20 O
\
E CL "/!
I 40
}: 50
,~-50
"
40
50
=~30
4O
Bound (pmoi x mg protein-~ ) Fig. 1. Chronic carbamazepine and repeated ECA administration: attenuation of fl-adrenoecptor- and musea~finicreceptor-induced increases in GTP binding capacity. Representative Scatchard plots of basal and agonist-induced [3H]GTP binding to membranes prepared fromcerebral cortexof(A) untreated rats (rats givensham ECS bad similarresul:.s);(B) rats treated with carbamazepinefor 14 days; (C) rats given ECS treatment for 11 days. (®) Basal |3H]GTP binding,~r2}in the presenceof 50 .aM i.~o~r,~erenol and (o) in the presenceof 100 ,aM of carbamylchohne. Each groupconsistedof 6-8 rats. t~'~protereaol-and carbamylehoiine-inducedincreases in [3H]GFP specific binding were assessed in membranes of cerebral cortex prepared from each anir,~at and compared to isoproterenol and carbamylchohne effects in membranes prepared ?rein untreated rata given sham ECS ~soproterenot and earbamylcbohneinduced, respectively, 15.1± 1.2 and 16.5-+1.6% incre,.ase~in GTP bindingcapacity in contrc~!al~ii~mls;in sham-ECS animals, 15.4_+1.4 and 19.2+2.4% respectiveincreaseswereobsers'ed.In the carbarnazepineand ECS groups,both agonistsfailed ~.o induce significant increasesin GTP binding capacity abovebasal.
102 effects of both isoproterenol and carbamylcholine on GTP binding (fig. 1C), and had no effect on GTP basal binding.
4. Discussion Carbamazepine and lithium share a common clinical profile of acute and prophylactic therapeutic effects in manic-depressive illness (for review, see Post, 1987). Likewise, the clinical spectrum of ECS treatment differs substantially from that of chemical antidepressants and resembles more closely the clinical profile of lithium (for review, see Lerer, 1987). Two main theories attempting to explain the lithium mechanism of action have focused on the perturbation of two second messenger effector systems: adenylate cyclase and PI turnover (for review, see Baraban et al., 1989). Recently, we reported (Avissar et al., 1988; Avissar and Schreiber, 1989) that therapeutic concentrations of lithium can alter the function of a core feature of both these signalling systems, namely G proteins. We showed that in rat cerebral cortex, lithium inhibited the coupling of both /8adrenoceptors and muscarinic cholinoceptors to cholera toxin- and pertussis toxin-sensitive G proteins, respectively (Avissar et al., 1988; Avissar and Schreiber, 1989). In the present study, we used the same method to determine G protein function independent of the subsequent activities of second messenger effector systems, and found that chronic earbamazepine and ECS alter receptor-coupled cholera toxin- and pertussis toxin-sensitive G protein function. Both chronic treatment with carbamazepine and repeated administration of ECS attenuated isoproterenol- and carbamylcholine-induced increases in GTP binding to rat cerebral cortical membranes. Although a diversity of neurochemical effects produced by carbamazepine and ECS have been described (for reviews, see Post, 1987; Lerer, 1987), both treatments have been reported to reduce cyclic AMP (cAMP) production in response to norepinephrine in rat cortical slices (Newman et al., 1986; Palmer et al., 1979). As carbamazepine does not down-regulate fl-adrenoceptor density
(Marangos et al., 1985), its effects are likely to be distal to the fl-adrenoceptor. ECS is known to down-regulate fl-adrenoceptor density (Lerer, 1987); however, its effects on cAMP accumulation may be located distal to the receptor because it also attenuates the accumulation of cAMP induced by forskolin (Newman et al., 1986), which bypasses the fl-adrenoceptor. These postreceptor effects are similar to those reported for lithium (for review, see Bunney and Gadand-Bunney, 1987) and they suggest that both carbamazepine and ECS act at a post-receptor site, which might be similar to that of lithium. G~ plays a crucial role in transducing fl-adrenoceptor-induced stimulation of adenylate cyclase. After chronic treatment with either lithium, carbamazepine, or ECS, reduced cAMP production, in response to fl-adrenoceptor stimulation, can result from the attenuated ,8-adrenoceptorcoupled G~ protein function caused by these treatments. Muscarinic cholinoceptors in the central nervous system are involved both in the inhibition of adenylate cyclase activity and in the activation of PI turnover. These second messenger systems are thought to be associated with muscarinic cholinoceptors via G proteins: G~ or G O in the case of adenylate cyclase, and the recently designated G protein, Gp, in the case of PI turnover (for review, see Gilman, 1987). Perturbation of muscarinic cholinoceptor-coupied, pertussis toxin-sensitive G proteins by chronic treatment with lithium (Avissar et al., 1988; Avissar and Schreiber, 1989), carbamazepine and ECS (described in the present study) predicts alteration by these treatments of muscarinic cholinoceptor-coupled inhibition of adenylate cyclase or activation of the PI cycle. Recent studies confirm this prediction. Chronic ECS was found to reduce the degree of inhibition by serotonin and carbachol of forskolin-sti'nutated adenylate cyclase in rat hippocampal membranes (Newman and Lerer, 1988). Lithium, given chronically, inhibits rather than enhances inositol triphosphate response to muscarinic and other receptor agonists in the rat cortex (Batty and Nahorski, 1985; Godfrey et al., 1989). This inhibition could not be explained by the classical site (inositol-l-phosphatase) of the effects of lithium
103 o n PI m e t a b o l i s m . P r e l i m i n a r y reports suggest p e r t u r b a t i o n o f inositol p h o s p h a t e a c c u m u l a t i o n in rat b r a i n slices b y b o t h ECS a n d c a r b a m a z e p i n e ( V a d n a l a n d Bazan, 1988). W e suggest that altered G p r o t e i n function is a general corm,n o n m e c h a n i s m for b o t h the antim a n i c a n d the a n t i d e p r e s s a n t effects of a v a r i e t y o f t r e a t m e n t s used in b i p o l a r affective disorder.
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