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Effect of repeated electroconvulsive shocks on isoprenaline-induced changes of the endogenous inhibitor of cAMP-dependent protein kinase in rat brain
European Journal of Pharmacology, 126 (1986) 273-279 Elsevier
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E F F E C T O F REPEATED E L E C T R O C O N V U L S I V E S H O C K S O N I S O P R E N A L I N E - I N D U C E D C H A N G E S OF T H E E N D O G E N O U S I N H I B I T O R OF c A M P - D E P E N D E N T P R O T E I N KINASE IN RAT BRAIN JOLANTA ZAWILSKA* and ANDRZEJ SZMIGIELSKI Department of Pharmacodynamics, Division of Pharmacology, Medical Academy, Narutowicza 120a, 90-145 Lbclk, Poland Received 11 March 1986, accepted 29 April 1986
J. ZAWILSKA and A. SZMIGIELSKI, Effect of repeated electroconvulsive shocks on isoprenaline-induced changes of the endogenous inhibitor of cAMP-dependent protein kinase in rat brain, European J. Pharmacol. 126 (1986) 273-279. The effect of repeated electroconvulsive shocks (ECS) on the responsiveness to isoprenaline of an endogenous, specific inhibitor of cAMP-dependent protein kinase (type I inhibitor) was tested in rat hippocampus and brainstem. Isoprenaline produced a dose-dependent decrease in the type I inhibitor activity in these brain structures. Neither the basal activity nor the isoprenaline-induced changes of type I inhibitor activity were affected by a single ECS. However, a series of 11 ECS markedly reduced the response of the type I inhibitor activity to isoprenaline. The action of isoprenaline was completely blocked by propranolol and markedly enhanced by aminophylline. The results indicate that the subsensitivity of fl-adrenoceptors is accompanied by reduced responsiveness of type I inhibitor activity to isoprenaline. fl-Adrenoceptors
Electroconvulsive shocks
Type I inhibitor
1. Introduction Endogenous small molecular weight thermostable proteins that inhibit protein kinases are widely distributed in the tissues. These inhibitory proteins contain two different components: a selective inhibitor of cyclic AMP-dependent protein kinase, termed a type I inhibitor, and a type II inhibitor that inhibits phosphorylation catalyzed by cyclic AMP-dependent protein kinase, cyclic G M P - d e p e n d e n t protein kinase and cyclic nucleotide-independent protein kinases (Szmigielski et al., 1977). The type II inhibitor activity changes specifically in a reciprocal manner to alterations of cyclic G M P content in rat cerebellum (Szmigielski and Guidotti, 1979) and striatum (Szmigielski, 1984). It has been found that the type I inhibitor binds selectively to catalytic subunits of cyclic AMP-dependent protein kinase, yielding an inac* To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 ElsevierSciencePublishers B.V.
Isoprenaline
tive complex, and in this way blocks the activity of the enzyme (Ashby and Walsh, 1972; 1973; Demaille et al., 1977). An increase of cyclic AMP content generated by the stimulation of either fl-adrenoceptors in human lymphocytes and rat heart or D 1 dopamine receptors in rat striatum was followed by a simultaneous decrease in type I inhibitor activity (Szmigielski, 1981a,b; 1984). The response of the type I inhibitor activity to isoprenaline has been proposed as an index of fl-adrenoceptor reactivity in vivo (Szmigielski et al., 1984). Long-term administration of most antidepressants as well as repeated electroconvulsive shocks (ECS) is known to produce subsensitivity of fl-adrenoceptors in rat brain, as measured by the responsiveness of the noradrenergic cyclic AMP generating system or in radioligand binding studies (for reviews see: Sulser, 1978; Pandey and Davis, 1981; 1983; Sugrue, 1983). It has been demonstrated in our laboratory that prolonged treatment with some antidepressants (i.e. imipramine, nomifensin and mianserin)
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markedly reduced the response of the type I inhibitor activity to isoprenaline (Szmigielski et al., 1984). The purpose of the present study was to investigate the isoprenaline-induced changes of the type I inhibitor activity in rat brain after the fl-adrenoceptors had been made subsensitive by chronic ECS.
2. Materials and methods
2.1. Animals
Male albino Wistar rats, weighing 140-180 g, were used. The animals were kept under standard laboratory conditions (housed 10 to a cage, under a 12 h dark-light cycle, with free access to granulated food and tap water). Electroconvulsive shocks were produced by passing an electric current (170 mA, 50 Hz, 500 ms) through ear-clip electrodes; this invariably produced both clonic and tonic seizures. The rats received either a single shock or a series of 11 shocks once dally. Control animals were handled in the same way except that no current was passed through the electrodes. The rats were killed by decapitation 24 h after a single ECS and at specified time intervals after the cessation of the series of ECS. The animals were injected intraperitoneally with saline or various doses of isoprenaline (Novodrin, Germed) 10 rain before killing. 2.2. Measurement of the type I inhibitor activity
Type I inhibitor activity was measured according to Szmigielski (1981a,b) with some modifications. Briefly, the brains were removed quickly and the hippocampus and brainstem dissected out on an ice-cold glass plate. The tissues were homogenized in 0.6 and 1.5 ml of ice-cold potassium phosphate buffer (pH 7.2), respectively. The homogenate was centrifuged at 10000 × g for 15 rain at 0-4°C. The supernatant was heated for 10 min at 90-95°C. After cooling on ice the mixture was centrifuged at 10000 × g for 15 rain at 0-4°C. An aliquot of 200/~1 of the resulting supernatant was loaded onto Sephadex G-50 superfine (Pharmacia)
0.7 × 5 cm column previously equilibrated with the same buffer. The column was washed with 400 /~1 of the buffer and the type I inhibitor was eluted with the next 600 #1. The assay of type I inhibitor activity was based on its ability to inhibit the phosphorylation catalyzed by cyclic AMP-dependent protein kinase (Walsh et al., 1971; Ashby and Walsh, 1972). The standard assay system contained in a final volume of 85/~1:120 mM sodium acetate buffer (pH 6.0), 70 mM magnesium acetate, 0.2 ~tM cAMP, 20 /~g of calf thymus histone mixture (type II, Sigma), 20 units of partially purified cAMP-dependent protein kinase (prepared according to Kuo and Greengard, 1972), 0.1 mM ~,32p-ATP (specific activity 3.7 MBq//~mol; prepared according to the modified method of Schendel and Wells, 1973) and 30/~1 of the Sephadex eluate. The reaction was carried out at 30 o C for 10 rain and was terminated by pipetting a 25 /~1 aliquot of the incubation mixture onto phosphocellulose-paper (Whatman P-81). The amount of 32p incorporated into histone was measured according to Witt and Roskoski (1975). The assay was always run in tripficate, with a boiled incubation mixture as a blank. One unit (U) of cAMP-dependent protein kinase is the amount of the enzyme that catalyzes, in 1 rain at 30°C, the transfer of 1 pmol of phosphate from 32p-ATP tO calf histone mixture. Under these conditions one unit of type I inhibitor activity was defined as the amount of this inhibitor which inhibited the phosphorylation of histone by 20%. Since the type I inhibitor binds tightly to catalytic subunits of the cAMP-dependent protein kinase, giving an inactive complex (Ashby and Walsh, 1972; Demallle et al., 1977), the incubation mixture always contained a fixed amount of the enzyme (exactly 20 U) and an excess of histone (20/~g). Student's t-test was used for statistical analysis of the results. 3. Results
3.1. The effect of isoprenaline on the type I inhibitor activity in some brain structures of normal rats
Isoprenaline produced a dose-dependent decrease in the type I inhibitor activity in rat hippo-
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Fig. 1. The effect of various doses of isoprenaline on the type I inhibitor activity in hippocampus and brainstem of normal rats. Each point represents the mean + S.E.M. from 5-7 animals. • • Hippocampus, • . . . . . . • brainstem. * P < 0.05 when compared to the saline-treated group.
c a m p u s a n d b r a i n s t e m . A statistically significant decrease in t y p e I i n h i b i t o r activity was o b s e r v e d after a dose of 1 m g / k g o f the drug. I s o p r e n a l i n e in a dose o f 10 m g / k g r e d u c e d b y a b o u t 40% the t y p e I i n h i b i t o r activity in these two b r a i n structures (fig. 1). Similar results h a d b e e n o b t a i n e d in our l a b o r a t o r y for rat h e a r t a n d p i n e a l g l a n d (Szmigielski, 1981a; Szmigielski et al., 1984).
3.3. The effect of chronic ECS on the isoprenalineinduced decrease in type I inhibitor activity in rat brain ECS for 11 consecutive d a y s m a r k e d l y dim i n i s h e d the r e s p o n s e Of the t y p e I i n h i b i t o r
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Fig. 2. The effect of a single ECS on the isoprenaline-induced decrease in the type ] inhibitor activity in rat hippocampus (A) and brainstem (B). Each point represents the mean+_S.E.M. from 5-6 animals. • . . . . . , The actual basal type I inhibitor activity. * P < 0.05 when compared to the saline-treated group. activity to i s o p r e n a l i n e in b o t h the h i p p o c a m p u s a n d the b r a i n s t e m . Interestingly, the strongest red u c t i o n in the responsiveness of type I i n h i b i t o r activity to the i s o p r e n a l i n e a c t i o n was o b s e r v e d in the h i p p o c a m p u s a n d the b r a i n stem, respectively after 3, or 3 a n d 7 d a y s following the cessation of ECS (when no c h a n g e of the t y p e I i n h i b i t o r activity was seen after the d r u g a d m i n i s t r a t i o n ) . Sixteen d a y s after the last ECS the s a m e doses of i s o p r e n a l i n e (i.e. 1, 2, 5 m g / k g ) p r o d u c e d a significant decrease in t y p e I i n h i b i t o r activity in the h i p p o c a m p u s of b o t h c o n t r o l rats a n d those subj e c t e d to chronic ECS. I n contrast, 32 d a y s after the cessation of a
3.2. The effect of a single E C S on the isoprenalineinduced decrease in type I inhibitor activity in rat brain T h e decrease in t y p e I i n h i b i t o r activity p r o d u c e d b y i s o p r e n a l i n e was n o t affected b y a single ECS. A m a r k e d r e d u c t i o n in the t y p e I i n h i b i t o r activity was o b s e r v e d after the s a m e doses of the d r u g (i.e. 1, 2, 5 m g / k g ) b o t h in c o n t r o l a n d in E C S - t r e a t e d rats. A single E C S also h a d n o influence on the b a s a l activity of the t y p e I i n h i b i t o r in these b r a i n structures (fig. 2).
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Fig. 3. The effect of repeated ECS on the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus. Each diagram represents the mean+S.E.M, from 5-6 animals. . . . . . . , The actual basal type I inhibitor activity in rats killed 1, 3, 7 or 16 days after cessation of sham ECS. 'Control' represents the rats killed 3 days following termination of sham ECS. Isoprenaline (1 mg/kg i.p.) induced a statistically significant decrease of type I inhibitor activity when injected 1, 3, 7 or 16 days after cessation of sham ECS. * P < 0.05 when compared to the corresponding saline-treated group.
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Fig. 4. The effect of repeated ECS on the isoprenaline-induced decrease in type I inhibitor activity in rat brainstem. Each diagram represents the mean+S.E.M, from 5-6 animals. . . . . . . , The actual basal type I inhibitor activity in rats killed 1, 3, 7, 16 or 32 days after cessation of sham ECS~ 'Control' represents the rats killed 3 days following termination of sham ECS. Isoprenaline (1 mg/kg i.p.) induced a statistically significant decrease of the type I inhibitor activity when injected 1, 3, 7, 16 or 32 days after cessation of sham.ECS * P < 0.05 when compared to the corresponding saline~treated group. / series of ECSs the a d m i n i s t r a t i o n of 2 m g / k g of i s o p r e n a l i n e resulted in a m a r k e d r e d u c t i o n in t y p e I i n h i b i t o r activity in the b r a i n s t e m , whereas the 1 m g / k g dose (effective in the c o n t r o l group) was a l m o s t w i t h o u t effect. L o n g - t e r m ECS treatm e n t did n o t alter the b a s a l t y p e I i n h i b i t o r activity in the b r a i n structures a n a l y z e d (figs. 3 a n d 4). P r o p r a n o l o l (an anta.gonist of f l - a d r e n o c e p t o r s ) c o m p l e t e l y b l o c k e d the r e d u c t i o n in t y p e I i n h i b i tor activity p r o d u c e d b y i s o p r e n a l i n e in the hipp o c a m p u s of rats c h r o n i c a l l y treated with E C S (table 1). T h e a d m i n i s t r a t i o n of a m i n o p h y l l i n e ( a n
TABLE 1 The effect of propranolol on the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus after repeated ECS. The animals were killed 7 days following cessation of a series of ECS, 20 min after i.p. administration of propranolol and 10 rain after i.p. administration of isoprenaline. Each value represents the mean+S.E.M, from 5-6 animals, a p<0.05 when compared to the saline-treated group; b p < 0.05 when compared to the isoprenaline-treated group. Treatment Saline Isoprenaline Propranolol Propranolol + isoprenaline
Dose (mg/kg) 5 5 5 5
Type I inhibitor activity (U/g) 783 + 17 609 + 44 a 843 + 23 77 + 41 b
The effect of aminophylline on the isoprenaline-induced decrease in the type I inhibitor activity in rat hippocampus after repeated ECS. The animals were killed 7 days following cessation of a series of ECS, 20 min after i.p. administration of aminophylline and 10 min after i.p. administration of isoprenaline. Each value represents the mean+S.E.M, from 5-6 animals, a p < 0.05 when compared to the saline-treated group: b p < 0.05 when compared to the animals treated with isoprenaline in the dose of 1 mg/kg. Treatment Saline Isoprenaline Isoprenaline Aminophylline Aminophylline + isoprenaline
Dose (mg/kg) 1 5 80 80 1
Type I inhibitor activity (U/g) 783 + 17 760 + 11 609 -I-44 a 693 + 17 a 625 + 22 a.b
inhibitor of phosphodiesterase) significantly d e c r e a s e d the t y p e I i n h i b i t o r activity in the hipp o c a m p u s of the ECS group. Moreover, p r e t r e a t m e n t with a m i n o p h y l l i n e when c o m b i n e d with a d o s e of i s o p r e n a l i n e that was ineffective b y itself resulted in a further decrease in type I i n h i b i t o r activity (table 2).
4. Discussion It has b e e n r e p o r t e d b y several authors that chronic, b u t n o t acute ECS caused a decrease in n o r a d r e n a l i n e - s t i m u l a t e d cyclic A M P a c c u m u l a tion in rat b r a i n (Vetulani a n d Sulser, 1975; V e t u l a n i et al., 1976; Gillespie et al., 1979; D e a k i n et al., 1981) as well as a r e d u c t i o n in fl-adrenergic b i n d i n g sites (Bergstrom a n d Kellar, 1979; Gillespie et al., 1979; P a n d e y et al., 1979; D e a k i n et al., 1981; K e l l a r et al., 1981; Sugrue, 1982). Such effects have also b e e n f o u n d after a p r o l o n g e d a d m i n i s t r a t i o n o f m o s t a n t i d e p r e s s a n t s (for reviews see: Sulser, 1978; P a n d e y a n d Davis, 1981; 1983; Sugrue, 1983). It has been suggested that the d e c r e a s e d responsiveness (subsensitivity) of central B - a d r e n o c e p t o r s m a y be related to the t h e r a p e u t i c a c t i o n of several a n t i d e p r e s s a n t treatments. I s o p r e n a l i n e - i n d u c e d s t i m u l a t i o n of fl-adrenoc e p t o r s in h u m a n l y m p h o c y t e s , rat h e a r t a n d p i n e a l g l a n d was f o u n d to be followed b y a d o s e - d e p e n -
277
dent decrease in type I inhibitor activity (Szmigielski, 1981a,b; Szmigielski et al., 1984). It is known that isoprenaline penetrates the blood-brain barrier at least to some extent. Westerman (1973) has shown that intravenously injected isoprenaline produced a dose-dependent increase of cAMP content in mouse brain. Intraperitoneal administration of this drug induced a dose-related decrease of type I inhibitor activity in rat hypothalamus and brainstem (Szmigielski et al., 1984; Zawilska and Szmigielski, in press). Moreover, several studies have demonstrated that, after systemic injection, isoprenaline decreased spontaneous motor activity in mice. This action was completely blocked by t-receptor antagonists (Frances and Simon, 1978; Frances et al., 1978). We have previously shown that the subsensitivity of fl-adrenoceptors in rat pineal gland, hippocampus and brainstem produced by three weeks administration of some antidepressants (i.e. imipramine, nomifensin and mianserin) was accompanied by a marked reduction in the response of the type I inhibitor to isoprenaline. In contrast, acute treatment with these drugs did not affect the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus (Szmigielski et al., 1984). Similar data are now being reported to follow ECS. A series of 11 ECSs markedly diminished the response of the type I inhibitor activity to isoprenaline both in rat hippocampus and brainstem. The strongest effect was observed 3, or 3 and 7 days after the last shock for the hippocampus and the brainstem, respectively. The isoprenaline-induced changes in type I inhibitor activity in the hippocampus returned to the control values within 16 days following the cessation of ECS, while the isoprenaline effect in the brainstem was still different from that in the control animals as late as 32 days after the last shock. These results are in agreement with the findings of Vetulani et al. (1976) that the reduced responsiveness to noradrenaline of the cyclic AMP generating system in rat limbic forebrain slices, following a series of 8 ECS, was most pronounced 2 and 3 days after cessation of ECS and did not return to the normal values within 8 days after the last shock.
Diminished responsiveness of the type I inhibitor activity to isoprenaline after 1 series of 11 ECSs was also observed in vitro in rat hippocampus slices incubated with this compound (results not shown). The ability of the fl-adrenoceptor blocker, propranolol, to completely antagonize the isoprenaline-induced decrease in type I inhibitor activity in the hippocampus of long-term ECS-treated rats as well as the enhancement of the isoprenaline action by aminophylline indicates that the changes of the type I inhibitor activity following isoprenaline administration were related to both the stimulation of fl-adrenoceptors and the increase in the cyclic AMP content. Aminophylline inhibits phopshodiesterase and blocks adenosine receptors. Inhibition of phopshodiesterase should enhance the isoprenaline-induced increase in cyclic AMP content. On the other hand, blockade of adenosine receptors has been reported to decrease adenylate cyclase activity and cychc AMP content (Harris, 1978; Daly et al., 1980), At the dose used in our experiments aminophylline acts as a phosphodiesterase inhibitor and potentiates the action of isoprenaline. A single ECS did not affect the isoprenaline action tested thus indicating that the reduced effect of the drug on the type I inhibitor activity after repeated ECSs was probably due to the subsensitivity of fl-adrenoceptors in rat hippocampus and brainstem, and not to a direct action of ECS. We have also found that the supersensitivity of fl-adrenoceptors in rat brain produced by either subchronic reserpine administration or the degeneration of central noradrenergic neurons caused by 6-hydroxydopamine (given intraventricularly) was accompanied by an increase in responsiveness of type I inhibitor activity to isoprenaline (Zawilska and Szmigielski, 1986). The physiological role of type I inhibitor seems to be to prevent the spontaneous phosphorylation calatyzed by a cAMP-dependent system. Stimulation of dopamine D 1 receptors or/3-adrenoceptors induces an increase of cAMP content, liberation of free catalytic subunits of cAMP-dependent protein kinase (an active form of the enzyme) with a simulta-
278 n e o u s d e c r e a s e in t y p e I i n h i b i t o r activity. T h i s s e q u e n c e o f e v e n t s a l l o w s p h o p s h o r y l a t i o n to p r o c e e d (Szmigielski, 1981a; S z m i g i e l s k i a n d K o w n e r , 1984). R e d u c e d r e s p o n s i v e n e s s o f t h e t y p e I inh i b i t o r a c t i v i t y to i s o p r e n a l i n e a f t e r r e p e a t e d E C S s e e m s to b e of g r e a t i m p o r t a n c e b e c a u s e it s h o w s s u b s e n s i t i v i t y o f the c e l l u l a r c A M P - d e p e n d e n t p h o p s h o r y l a t i n g system.
Acknowledgement This research was supported by the Polish Academy of Sciences, Grant No. 763/VI.
References Ashby, C.D. and D.A. Walsh, 1972, Characterization of the interaction of a protein inhibitor with adenosine-3'5'-monophosphate dependent protein kinase. I. Interaction with the catalytic subunits of the protein kinase, J. Biol. Chem. 247, 6637. Ashby, C.D. and D.A. Walsh, 1973, Characterization of the interaction of a protein inhibitor with adenosine-3'5'-monophosphate dependent protein kinases. II. Mechanism of action with holoenzyme, J. Biol. Chem. 252, 1255. Bergstrom, D.A. and K.J. Kellar, 1979, Effect of electroconvulsive shock on monoaminergic binding sites in rat brain, Nature 278, 464. Daly, J.W., E. McNeal, C. Partington, M. Neuwirth and C.R. Creveling, 1980, Accumulation of cyclic AMP in adeninelabeled cell-free preparation from guinea pig cerebral cortex: Role of fl-adrenergic and H2-histaminergic receptors, J. Neurochem. 35, 326. Deakin, J.P.W., P. Owen, A.J. Cross and M.J. Dashwood, 1981, Studies on possible mechanisms of action of electroconvulsive therapy; effects of repeated electrically induced seizures on rat brain receptors for monoamines and other neurotransmitters, Psychopharmacology 73, 345. Demaille, J.G., K.A. Peters and E.H. Fisher, 1977, Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine-3'5'-monophosphate dependent protein kinase, Biochemistry 16, 3080. Frances, J., A.J. Puech, P. Simon, 1978, Profil psychopharmaoclogique de l'isoprenaline et du salbutamol, J. Pharmacol. (Pards) 9. 25. Frances, H., and P. Simon, 1978, isoproterenol and psychopharmacological tests: antagonism by beta-adrenergic antagonists, Pharmacol. Res. Commun. 10, 211. Harris, J.E., 1978, fl-Adrenergic receptor-sensitive adenosine cyclic 3'5'-monophosphate accumulation in homogenates of the rat corpus striatum, Biochem. Pharmacol. 27, 2919. Gillespie, D.D., D.H. Monier and S.H. Snyder, 1979, Elec-
troconvulsive treatment: Rapid subsensitivity of the norepinephrine receptor coupled adenylate cyclase system in brain linked to down-regulation of fl-adrenergic receptors, Commun. Psychopharmacol. 3, 191. Kellar, K.J., C.S. Cascio, D.A. Bergstrom, J.A. Butler and P. Iadarola, 1981, Electroconvulsive shock and reserpine: effects on fl-adrenergic receptors in rat brain, J. Neurochem. 37, 830. Kuo, J.F. and P. Greengard, 1972, An assay for cyclic AMP and cyclic GMP based upon their abilities to activate cyclic AMP-dependent and cyclic GMP-dependent protein kinase, in: Advances in Cyclic Nucleotides Research, Vol. 2, eds. P. Greengard, G.A. Robinson and R. Paoletti (Raven Press, New York) p. 41. Pandey, G.M. and J.M. Davis, 1981, Treatment with antidepressants sensitivity of fl-adrenergic receptors and affective illness, in: Neuroreceptors - Basic and Clinical Aspects, eds. E. Usdin, W.E. Bunney and J.M. Davis (John Wiley and Sons Ltd.) p. 99. Pandey, G.M. and J.M. Davis, 1983, Treatment with antidepressants and down regulation of beta-adrenergic receptors, Drug Develop. Res. 3, 393. Pandey, G.M., W.J. Heinze, B.D. Brown and J.M. Davis, 1979, Electroconvulsive shock treatment decrease fl-adrenergic receptor sensitivity in rat brain, Nature 280, 234. Schendel, P.P. and R.D. Wells, 1973, The synthesis and purification of (32p)-adenosine triphopshate with high specific activity, J. Biol. Chem. 248, 8319. Sugrue, M.P., 1982, A study of the sensitivity of rat brain alpha2-adrenoreceptors during chronic antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 320, 90. Sugrue, M.F., 1983, Do antidepressants a common mechanism of action, Biochem. Pharmacol. 32, 1811. Sulser, F., 1978, Functional aspects of the norepinephrine receptor coupled adenylate cyclase system in the limbic forebrain and its modification by drugs which precipitate or alleviate depression: Molecular approaches to an understanding of affective disorders, Pharmakopsychiatrie 11, 43. Szmigielski, A., 1981a, Modulation of the activity of endogenous protein kinase inhibitors in rat heart by the betaadrenergic receptor, Arch. Int. Pharmacodyn. 249, 64. Szmigielski, A., 1981b, The effect of beta-receptor stimulation on the activity of the inhibitor of cAMP dependent protein kinase, Acta Physiol. Pol. 32, 501. Szmigielski, A., 1984, Endogenous protein kinase inhibitors: Modulation by dopaminergic receptors, in: Dynamics of Neurotransmitter Function, ed. I. Hanin (Raven Press, New York) p. 339. Szmigielski, A. and A. Guidotti, 1979, Action of harmaline and diazepam on the cerebellar content of cyclic GMP and the activity of two endogenous inhibitors of protein kinase, Neurochem. Res. 4, 189. Szmigielski, A., A. Guidotti and E. Costa, 1977, Endogenous protein kinase inhibitors. Purification, characterization and distribution in different tissues, J. Biol. Chem. 252, 2848. Szmigielski, A. and A. Kowner, 1984, The effect of stimulation of pre- and postsynaptic dopamine receptors on the endog-
279 enous inhibitor of cAMP dependent protein kinase, Arch. Int. Pharmacodyn. 272, 205. Szmigielski, A., J. Zawilska and K. Kondracki, 1984, Isoprenaline-induced changes in the type I inhibitor activity as an index of beta-adrenergic receptor subsensitivity, Pol. J. Pharmacol. Pharm. 36, 281. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments: Reduction in the sensitivity of the noradrenergic cyclic AMP generating system in the rat limbic forebrain, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 19. Vetulani, J. and F. Sulser, 1975, Action of various antidepressant treatments reduces reactivity of noradrenergic cyclic AMP generating system in limbic forebrain, Nature 257, 495.
Walsh, D.A., C.D. Ashby, C. Golzales, D. Calkins, E.H. Fischer and F.G. Krebs, 1971. Purification and characterization of a protein inhibitor of adenosine-3'5'-monophosphate-dependent protein kinase, J. Biol. Chem. 246, 1977. Westerman, E., 1973, Effects of catecholamines and prostaglandins on cyclic AMP levels in brain in vivo, in: Frontiers of Catecholamine Research, eds. E. Usdin and S.H. Snyder (Pergamon Press) p. 339. Witt, J. and J.R. Roskoski, 1975, Rapid protein kinase assay using phosphocellulose-paper absorption, Anal. Biochem. 66, 253. Zawilska, J. and A. Szmigielski, Isoprenaline-induced changes in activity of the endogenous inhibitor of cAMP-dependent protein kinases under conditions of fl-adrenoceptor supersensitivity, J. Pharm. Pharmacol. 38 (in press).
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E F F E C T O F REPEATED E L E C T R O C O N V U L S I V E S H O C K S O N I S O P R E N A L I N E - I N D U C E D C H A N G E S OF T H E E N D O G E N O U S I N H I B I T O R OF c A M P - D E P E N D E N T P R O T E I N KINASE IN RAT BRAIN JOLANTA ZAWILSKA* and ANDRZEJ SZMIGIELSKI Department of Pharmacodynamics, Division of Pharmacology, Medical Academy, Narutowicza 120a, 90-145 Lbclk, Poland Received 11 March 1986, accepted 29 April 1986
J. ZAWILSKA and A. SZMIGIELSKI, Effect of repeated electroconvulsive shocks on isoprenaline-induced changes of the endogenous inhibitor of cAMP-dependent protein kinase in rat brain, European J. Pharmacol. 126 (1986) 273-279. The effect of repeated electroconvulsive shocks (ECS) on the responsiveness to isoprenaline of an endogenous, specific inhibitor of cAMP-dependent protein kinase (type I inhibitor) was tested in rat hippocampus and brainstem. Isoprenaline produced a dose-dependent decrease in the type I inhibitor activity in these brain structures. Neither the basal activity nor the isoprenaline-induced changes of type I inhibitor activity were affected by a single ECS. However, a series of 11 ECS markedly reduced the response of the type I inhibitor activity to isoprenaline. The action of isoprenaline was completely blocked by propranolol and markedly enhanced by aminophylline. The results indicate that the subsensitivity of fl-adrenoceptors is accompanied by reduced responsiveness of type I inhibitor activity to isoprenaline. fl-Adrenoceptors
Electroconvulsive shocks
Type I inhibitor
1. Introduction Endogenous small molecular weight thermostable proteins that inhibit protein kinases are widely distributed in the tissues. These inhibitory proteins contain two different components: a selective inhibitor of cyclic AMP-dependent protein kinase, termed a type I inhibitor, and a type II inhibitor that inhibits phosphorylation catalyzed by cyclic AMP-dependent protein kinase, cyclic G M P - d e p e n d e n t protein kinase and cyclic nucleotide-independent protein kinases (Szmigielski et al., 1977). The type II inhibitor activity changes specifically in a reciprocal manner to alterations of cyclic G M P content in rat cerebellum (Szmigielski and Guidotti, 1979) and striatum (Szmigielski, 1984). It has been found that the type I inhibitor binds selectively to catalytic subunits of cyclic AMP-dependent protein kinase, yielding an inac* To whom all correspondenceshould be addressed. 0014-2999/86/$03.50 © 1986 ElsevierSciencePublishers B.V.
Isoprenaline
tive complex, and in this way blocks the activity of the enzyme (Ashby and Walsh, 1972; 1973; Demaille et al., 1977). An increase of cyclic AMP content generated by the stimulation of either fl-adrenoceptors in human lymphocytes and rat heart or D 1 dopamine receptors in rat striatum was followed by a simultaneous decrease in type I inhibitor activity (Szmigielski, 1981a,b; 1984). The response of the type I inhibitor activity to isoprenaline has been proposed as an index of fl-adrenoceptor reactivity in vivo (Szmigielski et al., 1984). Long-term administration of most antidepressants as well as repeated electroconvulsive shocks (ECS) is known to produce subsensitivity of fl-adrenoceptors in rat brain, as measured by the responsiveness of the noradrenergic cyclic AMP generating system or in radioligand binding studies (for reviews see: Sulser, 1978; Pandey and Davis, 1981; 1983; Sugrue, 1983). It has been demonstrated in our laboratory that prolonged treatment with some antidepressants (i.e. imipramine, nomifensin and mianserin)
274
markedly reduced the response of the type I inhibitor activity to isoprenaline (Szmigielski et al., 1984). The purpose of the present study was to investigate the isoprenaline-induced changes of the type I inhibitor activity in rat brain after the fl-adrenoceptors had been made subsensitive by chronic ECS.
2. Materials and methods
2.1. Animals
Male albino Wistar rats, weighing 140-180 g, were used. The animals were kept under standard laboratory conditions (housed 10 to a cage, under a 12 h dark-light cycle, with free access to granulated food and tap water). Electroconvulsive shocks were produced by passing an electric current (170 mA, 50 Hz, 500 ms) through ear-clip electrodes; this invariably produced both clonic and tonic seizures. The rats received either a single shock or a series of 11 shocks once dally. Control animals were handled in the same way except that no current was passed through the electrodes. The rats were killed by decapitation 24 h after a single ECS and at specified time intervals after the cessation of the series of ECS. The animals were injected intraperitoneally with saline or various doses of isoprenaline (Novodrin, Germed) 10 rain before killing. 2.2. Measurement of the type I inhibitor activity
Type I inhibitor activity was measured according to Szmigielski (1981a,b) with some modifications. Briefly, the brains were removed quickly and the hippocampus and brainstem dissected out on an ice-cold glass plate. The tissues were homogenized in 0.6 and 1.5 ml of ice-cold potassium phosphate buffer (pH 7.2), respectively. The homogenate was centrifuged at 10000 × g for 15 rain at 0-4°C. The supernatant was heated for 10 min at 90-95°C. After cooling on ice the mixture was centrifuged at 10000 × g for 15 rain at 0-4°C. An aliquot of 200/~1 of the resulting supernatant was loaded onto Sephadex G-50 superfine (Pharmacia)
0.7 × 5 cm column previously equilibrated with the same buffer. The column was washed with 400 /~1 of the buffer and the type I inhibitor was eluted with the next 600 #1. The assay of type I inhibitor activity was based on its ability to inhibit the phosphorylation catalyzed by cyclic AMP-dependent protein kinase (Walsh et al., 1971; Ashby and Walsh, 1972). The standard assay system contained in a final volume of 85/~1:120 mM sodium acetate buffer (pH 6.0), 70 mM magnesium acetate, 0.2 ~tM cAMP, 20 /~g of calf thymus histone mixture (type II, Sigma), 20 units of partially purified cAMP-dependent protein kinase (prepared according to Kuo and Greengard, 1972), 0.1 mM ~,32p-ATP (specific activity 3.7 MBq//~mol; prepared according to the modified method of Schendel and Wells, 1973) and 30/~1 of the Sephadex eluate. The reaction was carried out at 30 o C for 10 rain and was terminated by pipetting a 25 /~1 aliquot of the incubation mixture onto phosphocellulose-paper (Whatman P-81). The amount of 32p incorporated into histone was measured according to Witt and Roskoski (1975). The assay was always run in tripficate, with a boiled incubation mixture as a blank. One unit (U) of cAMP-dependent protein kinase is the amount of the enzyme that catalyzes, in 1 rain at 30°C, the transfer of 1 pmol of phosphate from 32p-ATP tO calf histone mixture. Under these conditions one unit of type I inhibitor activity was defined as the amount of this inhibitor which inhibited the phosphorylation of histone by 20%. Since the type I inhibitor binds tightly to catalytic subunits of the cAMP-dependent protein kinase, giving an inactive complex (Ashby and Walsh, 1972; Demallle et al., 1977), the incubation mixture always contained a fixed amount of the enzyme (exactly 20 U) and an excess of histone (20/~g). Student's t-test was used for statistical analysis of the results. 3. Results
3.1. The effect of isoprenaline on the type I inhibitor activity in some brain structures of normal rats
Isoprenaline produced a dose-dependent decrease in the type I inhibitor activity in rat hippo-
275
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Fig. 1. The effect of various doses of isoprenaline on the type I inhibitor activity in hippocampus and brainstem of normal rats. Each point represents the mean + S.E.M. from 5-7 animals. • • Hippocampus, • . . . . . . • brainstem. * P < 0.05 when compared to the saline-treated group.
c a m p u s a n d b r a i n s t e m . A statistically significant decrease in t y p e I i n h i b i t o r activity was o b s e r v e d after a dose of 1 m g / k g o f the drug. I s o p r e n a l i n e in a dose o f 10 m g / k g r e d u c e d b y a b o u t 40% the t y p e I i n h i b i t o r activity in these two b r a i n structures (fig. 1). Similar results h a d b e e n o b t a i n e d in our l a b o r a t o r y for rat h e a r t a n d p i n e a l g l a n d (Szmigielski, 1981a; Szmigielski et al., 1984).
3.3. The effect of chronic ECS on the isoprenalineinduced decrease in type I inhibitor activity in rat brain ECS for 11 consecutive d a y s m a r k e d l y dim i n i s h e d the r e s p o n s e Of the t y p e I i n h i b i t o r
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Fig. 2. The effect of a single ECS on the isoprenaline-induced decrease in the type ] inhibitor activity in rat hippocampus (A) and brainstem (B). Each point represents the mean+_S.E.M. from 5-6 animals. • . . . . . , The actual basal type I inhibitor activity. * P < 0.05 when compared to the saline-treated group. activity to i s o p r e n a l i n e in b o t h the h i p p o c a m p u s a n d the b r a i n s t e m . Interestingly, the strongest red u c t i o n in the responsiveness of type I i n h i b i t o r activity to the i s o p r e n a l i n e a c t i o n was o b s e r v e d in the h i p p o c a m p u s a n d the b r a i n stem, respectively after 3, or 3 a n d 7 d a y s following the cessation of ECS (when no c h a n g e of the t y p e I i n h i b i t o r activity was seen after the d r u g a d m i n i s t r a t i o n ) . Sixteen d a y s after the last ECS the s a m e doses of i s o p r e n a l i n e (i.e. 1, 2, 5 m g / k g ) p r o d u c e d a significant decrease in t y p e I i n h i b i t o r activity in the h i p p o c a m p u s of b o t h c o n t r o l rats a n d those subj e c t e d to chronic ECS. I n contrast, 32 d a y s after the cessation of a
3.2. The effect of a single E C S on the isoprenalineinduced decrease in type I inhibitor activity in rat brain T h e decrease in t y p e I i n h i b i t o r activity p r o d u c e d b y i s o p r e n a l i n e was n o t affected b y a single ECS. A m a r k e d r e d u c t i o n in the t y p e I i n h i b i t o r activity was o b s e r v e d after the s a m e doses of the d r u g (i.e. 1, 2, 5 m g / k g ) b o t h in c o n t r o l a n d in E C S - t r e a t e d rats. A single E C S also h a d n o influence on the b a s a l activity of the t y p e I i n h i b i t o r in these b r a i n structures (fig. 2).
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Fig. 3. The effect of repeated ECS on the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus. Each diagram represents the mean+S.E.M, from 5-6 animals. . . . . . . , The actual basal type I inhibitor activity in rats killed 1, 3, 7 or 16 days after cessation of sham ECS. 'Control' represents the rats killed 3 days following termination of sham ECS. Isoprenaline (1 mg/kg i.p.) induced a statistically significant decrease of type I inhibitor activity when injected 1, 3, 7 or 16 days after cessation of sham ECS. * P < 0.05 when compared to the corresponding saline-treated group.
276 TABLE 2 ; 600
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Fig. 4. The effect of repeated ECS on the isoprenaline-induced decrease in type I inhibitor activity in rat brainstem. Each diagram represents the mean+S.E.M, from 5-6 animals. . . . . . . , The actual basal type I inhibitor activity in rats killed 1, 3, 7, 16 or 32 days after cessation of sham ECS~ 'Control' represents the rats killed 3 days following termination of sham ECS. Isoprenaline (1 mg/kg i.p.) induced a statistically significant decrease of the type I inhibitor activity when injected 1, 3, 7, 16 or 32 days after cessation of sham.ECS * P < 0.05 when compared to the corresponding saline~treated group. / series of ECSs the a d m i n i s t r a t i o n of 2 m g / k g of i s o p r e n a l i n e resulted in a m a r k e d r e d u c t i o n in t y p e I i n h i b i t o r activity in the b r a i n s t e m , whereas the 1 m g / k g dose (effective in the c o n t r o l group) was a l m o s t w i t h o u t effect. L o n g - t e r m ECS treatm e n t did n o t alter the b a s a l t y p e I i n h i b i t o r activity in the b r a i n structures a n a l y z e d (figs. 3 a n d 4). P r o p r a n o l o l (an anta.gonist of f l - a d r e n o c e p t o r s ) c o m p l e t e l y b l o c k e d the r e d u c t i o n in t y p e I i n h i b i tor activity p r o d u c e d b y i s o p r e n a l i n e in the hipp o c a m p u s of rats c h r o n i c a l l y treated with E C S (table 1). T h e a d m i n i s t r a t i o n of a m i n o p h y l l i n e ( a n
TABLE 1 The effect of propranolol on the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus after repeated ECS. The animals were killed 7 days following cessation of a series of ECS, 20 min after i.p. administration of propranolol and 10 rain after i.p. administration of isoprenaline. Each value represents the mean+S.E.M, from 5-6 animals, a p<0.05 when compared to the saline-treated group; b p < 0.05 when compared to the isoprenaline-treated group. Treatment Saline Isoprenaline Propranolol Propranolol + isoprenaline
Dose (mg/kg) 5 5 5 5
Type I inhibitor activity (U/g) 783 + 17 609 + 44 a 843 + 23 77 + 41 b
The effect of aminophylline on the isoprenaline-induced decrease in the type I inhibitor activity in rat hippocampus after repeated ECS. The animals were killed 7 days following cessation of a series of ECS, 20 min after i.p. administration of aminophylline and 10 min after i.p. administration of isoprenaline. Each value represents the mean+S.E.M, from 5-6 animals, a p < 0.05 when compared to the saline-treated group: b p < 0.05 when compared to the animals treated with isoprenaline in the dose of 1 mg/kg. Treatment Saline Isoprenaline Isoprenaline Aminophylline Aminophylline + isoprenaline
Dose (mg/kg) 1 5 80 80 1
Type I inhibitor activity (U/g) 783 + 17 760 + 11 609 -I-44 a 693 + 17 a 625 + 22 a.b
inhibitor of phosphodiesterase) significantly d e c r e a s e d the t y p e I i n h i b i t o r activity in the hipp o c a m p u s of the ECS group. Moreover, p r e t r e a t m e n t with a m i n o p h y l l i n e when c o m b i n e d with a d o s e of i s o p r e n a l i n e that was ineffective b y itself resulted in a further decrease in type I i n h i b i t o r activity (table 2).
4. Discussion It has b e e n r e p o r t e d b y several authors that chronic, b u t n o t acute ECS caused a decrease in n o r a d r e n a l i n e - s t i m u l a t e d cyclic A M P a c c u m u l a tion in rat b r a i n (Vetulani a n d Sulser, 1975; V e t u l a n i et al., 1976; Gillespie et al., 1979; D e a k i n et al., 1981) as well as a r e d u c t i o n in fl-adrenergic b i n d i n g sites (Bergstrom a n d Kellar, 1979; Gillespie et al., 1979; P a n d e y et al., 1979; D e a k i n et al., 1981; K e l l a r et al., 1981; Sugrue, 1982). Such effects have also b e e n f o u n d after a p r o l o n g e d a d m i n i s t r a t i o n o f m o s t a n t i d e p r e s s a n t s (for reviews see: Sulser, 1978; P a n d e y a n d Davis, 1981; 1983; Sugrue, 1983). It has been suggested that the d e c r e a s e d responsiveness (subsensitivity) of central B - a d r e n o c e p t o r s m a y be related to the t h e r a p e u t i c a c t i o n of several a n t i d e p r e s s a n t treatments. I s o p r e n a l i n e - i n d u c e d s t i m u l a t i o n of fl-adrenoc e p t o r s in h u m a n l y m p h o c y t e s , rat h e a r t a n d p i n e a l g l a n d was f o u n d to be followed b y a d o s e - d e p e n -
277
dent decrease in type I inhibitor activity (Szmigielski, 1981a,b; Szmigielski et al., 1984). It is known that isoprenaline penetrates the blood-brain barrier at least to some extent. Westerman (1973) has shown that intravenously injected isoprenaline produced a dose-dependent increase of cAMP content in mouse brain. Intraperitoneal administration of this drug induced a dose-related decrease of type I inhibitor activity in rat hypothalamus and brainstem (Szmigielski et al., 1984; Zawilska and Szmigielski, in press). Moreover, several studies have demonstrated that, after systemic injection, isoprenaline decreased spontaneous motor activity in mice. This action was completely blocked by t-receptor antagonists (Frances and Simon, 1978; Frances et al., 1978). We have previously shown that the subsensitivity of fl-adrenoceptors in rat pineal gland, hippocampus and brainstem produced by three weeks administration of some antidepressants (i.e. imipramine, nomifensin and mianserin) was accompanied by a marked reduction in the response of the type I inhibitor to isoprenaline. In contrast, acute treatment with these drugs did not affect the isoprenaline-induced decrease in type I inhibitor activity in rat hippocampus (Szmigielski et al., 1984). Similar data are now being reported to follow ECS. A series of 11 ECSs markedly diminished the response of the type I inhibitor activity to isoprenaline both in rat hippocampus and brainstem. The strongest effect was observed 3, or 3 and 7 days after the last shock for the hippocampus and the brainstem, respectively. The isoprenaline-induced changes in type I inhibitor activity in the hippocampus returned to the control values within 16 days following the cessation of ECS, while the isoprenaline effect in the brainstem was still different from that in the control animals as late as 32 days after the last shock. These results are in agreement with the findings of Vetulani et al. (1976) that the reduced responsiveness to noradrenaline of the cyclic AMP generating system in rat limbic forebrain slices, following a series of 8 ECS, was most pronounced 2 and 3 days after cessation of ECS and did not return to the normal values within 8 days after the last shock.
Diminished responsiveness of the type I inhibitor activity to isoprenaline after 1 series of 11 ECSs was also observed in vitro in rat hippocampus slices incubated with this compound (results not shown). The ability of the fl-adrenoceptor blocker, propranolol, to completely antagonize the isoprenaline-induced decrease in type I inhibitor activity in the hippocampus of long-term ECS-treated rats as well as the enhancement of the isoprenaline action by aminophylline indicates that the changes of the type I inhibitor activity following isoprenaline administration were related to both the stimulation of fl-adrenoceptors and the increase in the cyclic AMP content. Aminophylline inhibits phopshodiesterase and blocks adenosine receptors. Inhibition of phopshodiesterase should enhance the isoprenaline-induced increase in cyclic AMP content. On the other hand, blockade of adenosine receptors has been reported to decrease adenylate cyclase activity and cychc AMP content (Harris, 1978; Daly et al., 1980), At the dose used in our experiments aminophylline acts as a phosphodiesterase inhibitor and potentiates the action of isoprenaline. A single ECS did not affect the isoprenaline action tested thus indicating that the reduced effect of the drug on the type I inhibitor activity after repeated ECSs was probably due to the subsensitivity of fl-adrenoceptors in rat hippocampus and brainstem, and not to a direct action of ECS. We have also found that the supersensitivity of fl-adrenoceptors in rat brain produced by either subchronic reserpine administration or the degeneration of central noradrenergic neurons caused by 6-hydroxydopamine (given intraventricularly) was accompanied by an increase in responsiveness of type I inhibitor activity to isoprenaline (Zawilska and Szmigielski, 1986). The physiological role of type I inhibitor seems to be to prevent the spontaneous phosphorylation calatyzed by a cAMP-dependent system. Stimulation of dopamine D 1 receptors or/3-adrenoceptors induces an increase of cAMP content, liberation of free catalytic subunits of cAMP-dependent protein kinase (an active form of the enzyme) with a simulta-
278 n e o u s d e c r e a s e in t y p e I i n h i b i t o r activity. T h i s s e q u e n c e o f e v e n t s a l l o w s p h o p s h o r y l a t i o n to p r o c e e d (Szmigielski, 1981a; S z m i g i e l s k i a n d K o w n e r , 1984). R e d u c e d r e s p o n s i v e n e s s o f t h e t y p e I inh i b i t o r a c t i v i t y to i s o p r e n a l i n e a f t e r r e p e a t e d E C S s e e m s to b e of g r e a t i m p o r t a n c e b e c a u s e it s h o w s s u b s e n s i t i v i t y o f the c e l l u l a r c A M P - d e p e n d e n t p h o p s h o r y l a t i n g system.
Acknowledgement This research was supported by the Polish Academy of Sciences, Grant No. 763/VI.
References Ashby, C.D. and D.A. Walsh, 1972, Characterization of the interaction of a protein inhibitor with adenosine-3'5'-monophosphate dependent protein kinase. I. Interaction with the catalytic subunits of the protein kinase, J. Biol. Chem. 247, 6637. Ashby, C.D. and D.A. Walsh, 1973, Characterization of the interaction of a protein inhibitor with adenosine-3'5'-monophosphate dependent protein kinases. II. Mechanism of action with holoenzyme, J. Biol. Chem. 252, 1255. Bergstrom, D.A. and K.J. Kellar, 1979, Effect of electroconvulsive shock on monoaminergic binding sites in rat brain, Nature 278, 464. Daly, J.W., E. McNeal, C. Partington, M. Neuwirth and C.R. Creveling, 1980, Accumulation of cyclic AMP in adeninelabeled cell-free preparation from guinea pig cerebral cortex: Role of fl-adrenergic and H2-histaminergic receptors, J. Neurochem. 35, 326. Deakin, J.P.W., P. Owen, A.J. Cross and M.J. Dashwood, 1981, Studies on possible mechanisms of action of electroconvulsive therapy; effects of repeated electrically induced seizures on rat brain receptors for monoamines and other neurotransmitters, Psychopharmacology 73, 345. Demaille, J.G., K.A. Peters and E.H. Fisher, 1977, Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine-3'5'-monophosphate dependent protein kinase, Biochemistry 16, 3080. Frances, J., A.J. Puech, P. Simon, 1978, Profil psychopharmaoclogique de l'isoprenaline et du salbutamol, J. Pharmacol. (Pards) 9. 25. Frances, H., and P. Simon, 1978, isoproterenol and psychopharmacological tests: antagonism by beta-adrenergic antagonists, Pharmacol. Res. Commun. 10, 211. Harris, J.E., 1978, fl-Adrenergic receptor-sensitive adenosine cyclic 3'5'-monophosphate accumulation in homogenates of the rat corpus striatum, Biochem. Pharmacol. 27, 2919. Gillespie, D.D., D.H. Monier and S.H. Snyder, 1979, Elec-
troconvulsive treatment: Rapid subsensitivity of the norepinephrine receptor coupled adenylate cyclase system in brain linked to down-regulation of fl-adrenergic receptors, Commun. Psychopharmacol. 3, 191. Kellar, K.J., C.S. Cascio, D.A. Bergstrom, J.A. Butler and P. Iadarola, 1981, Electroconvulsive shock and reserpine: effects on fl-adrenergic receptors in rat brain, J. Neurochem. 37, 830. Kuo, J.F. and P. Greengard, 1972, An assay for cyclic AMP and cyclic GMP based upon their abilities to activate cyclic AMP-dependent and cyclic GMP-dependent protein kinase, in: Advances in Cyclic Nucleotides Research, Vol. 2, eds. P. Greengard, G.A. Robinson and R. Paoletti (Raven Press, New York) p. 41. Pandey, G.M. and J.M. Davis, 1981, Treatment with antidepressants sensitivity of fl-adrenergic receptors and affective illness, in: Neuroreceptors - Basic and Clinical Aspects, eds. E. Usdin, W.E. Bunney and J.M. Davis (John Wiley and Sons Ltd.) p. 99. Pandey, G.M. and J.M. Davis, 1983, Treatment with antidepressants and down regulation of beta-adrenergic receptors, Drug Develop. Res. 3, 393. Pandey, G.M., W.J. Heinze, B.D. Brown and J.M. Davis, 1979, Electroconvulsive shock treatment decrease fl-adrenergic receptor sensitivity in rat brain, Nature 280, 234. Schendel, P.P. and R.D. Wells, 1973, The synthesis and purification of (32p)-adenosine triphopshate with high specific activity, J. Biol. Chem. 248, 8319. Sugrue, M.P., 1982, A study of the sensitivity of rat brain alpha2-adrenoreceptors during chronic antidepressant treatments, Naunyn-Schmiedeb. Arch. Pharmacol. 320, 90. Sugrue, M.F., 1983, Do antidepressants a common mechanism of action, Biochem. Pharmacol. 32, 1811. Sulser, F., 1978, Functional aspects of the norepinephrine receptor coupled adenylate cyclase system in the limbic forebrain and its modification by drugs which precipitate or alleviate depression: Molecular approaches to an understanding of affective disorders, Pharmakopsychiatrie 11, 43. Szmigielski, A., 1981a, Modulation of the activity of endogenous protein kinase inhibitors in rat heart by the betaadrenergic receptor, Arch. Int. Pharmacodyn. 249, 64. Szmigielski, A., 1981b, The effect of beta-receptor stimulation on the activity of the inhibitor of cAMP dependent protein kinase, Acta Physiol. Pol. 32, 501. Szmigielski, A., 1984, Endogenous protein kinase inhibitors: Modulation by dopaminergic receptors, in: Dynamics of Neurotransmitter Function, ed. I. Hanin (Raven Press, New York) p. 339. Szmigielski, A. and A. Guidotti, 1979, Action of harmaline and diazepam on the cerebellar content of cyclic GMP and the activity of two endogenous inhibitors of protein kinase, Neurochem. Res. 4, 189. Szmigielski, A., A. Guidotti and E. Costa, 1977, Endogenous protein kinase inhibitors. Purification, characterization and distribution in different tissues, J. Biol. Chem. 252, 2848. Szmigielski, A. and A. Kowner, 1984, The effect of stimulation of pre- and postsynaptic dopamine receptors on the endog-
279 enous inhibitor of cAMP dependent protein kinase, Arch. Int. Pharmacodyn. 272, 205. Szmigielski, A., J. Zawilska and K. Kondracki, 1984, Isoprenaline-induced changes in the type I inhibitor activity as an index of beta-adrenergic receptor subsensitivity, Pol. J. Pharmacol. Pharm. 36, 281. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments: Reduction in the sensitivity of the noradrenergic cyclic AMP generating system in the rat limbic forebrain, Naunyn-Schmiedeb. Arch. Pharmacol. 293, 19. Vetulani, J. and F. Sulser, 1975, Action of various antidepressant treatments reduces reactivity of noradrenergic cyclic AMP generating system in limbic forebrain, Nature 257, 495.
Walsh, D.A., C.D. Ashby, C. Golzales, D. Calkins, E.H. Fischer and F.G. Krebs, 1971. Purification and characterization of a protein inhibitor of adenosine-3'5'-monophosphate-dependent protein kinase, J. Biol. Chem. 246, 1977. Westerman, E., 1973, Effects of catecholamines and prostaglandins on cyclic AMP levels in brain in vivo, in: Frontiers of Catecholamine Research, eds. E. Usdin and S.H. Snyder (Pergamon Press) p. 339. Witt, J. and J.R. Roskoski, 1975, Rapid protein kinase assay using phosphocellulose-paper absorption, Anal. Biochem. 66, 253. Zawilska, J. and A. Szmigielski, Isoprenaline-induced changes in activity of the endogenous inhibitor of cAMP-dependent protein kinases under conditions of fl-adrenoceptor supersensitivity, J. Pharm. Pharmacol. 38 (in press).