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Influence of dopamine receptor agonists and antagonists on calmodulin translocation in different brain regions
European Journal of Pharmacology - Molecular Pharmacology Section, 172 (1989) 205-210
205
Elsevier EJPMOL 90019
Influence of dopamine receptor agonists and antagonists on calmodulin translocation in different brain regions Nikolai Popov * and Hansjiirgen Matthies Institute of Pharmacology and Toxicology, Medical Academy, 3090 Magdeburg, G.D.R.
Received 21 February 1989, accepted 14 March 1989
Acute and chronic experiments were performed to study the effects of intraperitoneally administered dopamine receptor agonists and antagonists on the translocation of cytosolic and membrane-bound calmodulin in the striatum and hippocampus of the rat brain. Single doses of apomorphine and a low-dose amphetamine (1.25 mg/kg) resulted in a translocation of calmodulin, as measured by a decrease in membrane-bound and increase in cytosolic calmodulin in the striatum, whereas bromocryptine was ineffective. Amphetamine exerted a similar effect in the hippocampus and striatum. However, a high-dose amphetamine (5 mg/kg) had an opposite effect on translocation, in that there was an increase in membrane-bound calmodulin. Chronically applied amphetamine (5 mg/kg) and haloperidol (1 mg/kg), i.e. under conditions of dopamine receptor supersensitivity, tended to decrease cytosolic calmodulin in the striatum and hippocampus, and to increase membrane-bound calmodulin. The findings and, especially, the chronic effects of haloperidol and the high dose of amphetamine are interpreted in the fight of the current concept which suggests that transmitters and intraneuronal signalling systems converge, thereby influencing the more complex processes of neuronal plasticity, rather than receptor sensitivity only. Amphetamine; Haloperidol; Dopamine; Striatum; Hippocampus; Calmodulin translocation
1. Introduction
Calmodulin, a calcium-binding protein, has been found to play an essential role in various systems of intracellular signalling by mediating transmitter-induced changes in Ca 2+ concentrations (for review see Stoclet et al., 1987). The translocation of calmodulin from the cytosol to membranes and vice versa seems to be of crucial significance in these processes (Hanbauer et al., 1980; Costa, 1981). This involvement of transmitter signals in intraneuronal transduction suggests that the action of specific neurotropic drugs could also induce characteristic changes in the distribution a n d / o r content of calmodulin. Eluci-
dation of these events would contribute to a better understanding of the interplay between messenger systems in the intracellular control of neuronal functions and the mechanisms of action of pharmacologically active substances. It was our aim to study whether calmodulin translocates from the cytosolic c o m p a r t m e n t to membrane and vice versa in brain structures after acute or repeated intraperitoneal (i.p.) administration of dopamine receptor agonists and antagonists. The chronic treatment was applied under conditions chosen to attain dopamine receptor supersensitivity.
2. Materials and methods 2.1. A n i m a l s a n d t r e a t m e n t
* To whom all correspondence should be addressed: Institute of Pharmacology and Toxicology, Medical Academy, Leipzigerstrasse 44, 3090 Magdeburg, G.D.R.
Male rats of Wistar origin from our own breeding stock were used. Eight-week-old animals were
0922-4106/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 used for the acute experiments, while 5-week-old rats were used for the chronic treatment. The animals were provided with food and water ad libitum. All drugs used were injected i.p.: apomorphine hydrochloride (SPOFA, Prague, Czechoslovakia), bromocryptine mesylate (Sigma, Deisenhofen, F.R.G.), amphetamine racemate (VEB FahlbergList, Magdeburg, G.D.R.), haloperidol (Gedeon Richter, Budapest, Hungary) and clozapine (Leponex*, Sandoz, Basel, Switzerland). The animals in the acute experiments were killed 30-120 min after drug administration as detailed below (the number of animals per group is indicated in parentheses): 1 mg/kg apomorphine, 30 min (5) and 60 min (5); 10 mg/kg bromocryptine, 40 min (5) and 120 min (5); 1.25 mg/kg amphetamine, 40 min (10); 5 mg/kg amphetamine, 40 min (5); 1 mg/kg haloperidol, 40 min (10); 20 mg/kg clozapine, 40 min (5). The control animals (at least 5) received saline; these animals were killed after the corresponding periods following drug administration. The chronic series involved 3 groups of 8 rats each; the animals were treated daily with 1 mg/kg haloperidol, 5 mg/kg amphetamine or saline for 3 weeks. The rats were killed 2 days after withdrawal and the brains were dissected out as described elsewhere (Popov et al., 1973). The latter procedure was also used for the acute experiments, and the caudate putamen, n, accumbens and substantia nigra were dissected additionally in the chronic experiments (Horn et al., 1974). The brain regions were frozen on dry ice and stored at - 20 ° C until use.
2.2. Biochemical analysis Brain tissue from the individual regions was homogenized with a microhomogenizer in a solution containing 0.32 M sucrose and 1 mM disodium EDTA and, using a microtube adaptor, was centrifuged in a refrigerated ultracentrifuge at 100000 × g for 60 min. The resulting supernatant was heated (90 ° C, 1 min) and re-centrifuged at 100000 x g for 30 min. The supernatant obtained was designated as the water-soluble (cytosolic) fraction. The water-insoluble pellet from the first
centrifugation step was homogenized in 0.05 M Tris-HC1 buffer, pH 7.4, containing 0.1% Lubrol, heated (90°C, 1 min), sonicated (3 times for 15 s each) and kept at 4°C for 16 h. After centrifugation at 100 000 x g for 30 min, the resulting supernatant was designated the Lubrol-soluble fraction and contained membrane-bound calmodulin. The protein content was determined in aliquots of each fraction by the method of Lowry et al. (1951). The content of calmodulin was measured in triplicate by a modified, competitive, solid-phase radioimmunoassay (Schulzeck et al., 1983; Struy, 1983) with antiserum raised against performic acid-oxidized calmodulin from bovine brain (Van Eldik and Watterson, 1981). Briefly, the samples were pretreated with 20 mM chloramine T (to increase the sensitivity of the antigen-antibody reaction), and after 2 min the reaction was stopped by the addition of 40 mM potassium metabisulphite (Biber et al., 1984). Thereafter, each sample was incubated in the presence of a defined amount of rabbit antiserum to calmodulin. The amount of non-bound antibody was then detected by the addition of a25I-labelled anti-rabbit sheep globulin to the complex which consisted of PVC blister as solid phase covered by adsorbed calmodulin (antigen) on the inner surface, and calmodulin antibodies (unbound during the preceding step). This approach of labelling a third component (detector) in addition to the unlabelled antigen and antibody was adopted to preclude iodination-induced alterations of calmodulin and the calmodulin antibodies. The content was calculated with the aid of associated calibration curves, and is reported as big protein. The two-tailed Student's t-test in groups was used to compute the statistical significance of differences between means obtained for the control and drug-treated groups.
3. Results
3.1. Acute effects of dopaminergic drugs Single doses of the substances used did not influence the calmodulin content of the cytosolic and membrane-bound fractions in the frontal
207
51
cytosolic
4 3 2 1
DQ
striatum L
5 t membrane-t
hippocamp.
4
J
3 2 1
apo
mg/kg 5 rain 30 60
brcr
amph
5
1.2s 5
40 120
40
halo cloz 1 20 40
amph
1.2s 5 40
Fig. l. Influence of dopamine receptor agonists and antagonists on the translocation of calmodulin from cytosolic to membrane-bound forms, and vice versa, in the rat striatum and hippocampus. The ordinate indicates the calmodulin values ( n g / # g protein). M e a n s + S . E . M . of 10 saline controls are represented by horizontal lines: cytosolic calmodulin 2.92 + 0.38 and 2.52 for the striatum and hippocampus, respectively; membrane-bound calmodulin 3.20+0.21 and 2.90+0.35, respectively, l.p. doses and time of treatment are shown in the bottom part of the figure; a p o = apomorphine, brcr = bromocryptine, a m p h = amphetamine, halo = haloperidol, cloz = clozapine. The means of drug-treated animal determinations + S.E.M. are represented as bars. Empty circle, one filled circle and two filled circles stand for P < 0.05, P < 0.02 and P < 0.01, respectively, revealing statistical significance of differences between drug-treated animals and saline controls.
cortex and cerebellum. Figure 1 depicts the results obtained (a) in the dopamine-rich striatum, which is frequently used to study biochemical alterations related to the action of dopaminergic drugs and to changes in dopamine receptor responsiveness, and (b) in the hippocampus, which contains little dopamine and which is assumed to play a role in learning and memory formation (for review see Matthies, 1989) and which exhibits phenomena of neuronal plasticity, such as post-tetanic long-term
potentiation. As demonstrated in fig. 1, the D1/D-2 dopamine receptor agonist, apomorphine, and the preferential D-2 receptor agonist, bromocryptine, had different effects: apomorphine caused a significant decrease in membrane-bound calmodulin accompanied by a tendency to an increased content in the cytosolic fraction in the striatum, while bromocryptine did not influence the calmodulin content in either of the fractions from this region. After application of a low dose (1.25 mg/kg) of the indirect agonist, amphetamine, the distribution of calmodulin changed in a manner similar to that noted after apomorphine: a significant increase in the cytosolic fraction was accompanied by a significant decrease in membrane-bound calmodulin. In contrast, a relatively high dose of amphetamine (5.0 mg/kg) resulted in an opposite effect: a significant increase in membrane-bound calmodulin occurred with a simultaneous distinct, even though insignificant, decrease in cytosolic calmodulin. The dopamine receptor antagonists, haloperidol (1.0 mg/kg) and clozapine (20.0 mg/kg), had no effect on the distribution of calmodulin in either striatal fraction. In the hippocampus, only the low dose of amphetamine caused detectable changes: the cytosolic fraction of calmodulin was significantly enhanced and the membrane-bound fraction tended to decrease, thus exhibiting qualitative alterations identical to those occurring after the low-dose amphetamine in the striatum.
3.2. Effects of chronic treatment with haloperidol and amphetamine Chronic treatment with haloperidol and a high dose of amphetamine was given in the manner used in our laboratories to produce supersensitivity in rats as determined by the behavioural effects of single doses of apomorphine (Tarsy and Baldessarini, 1974; Honza, 1983) and amphetamine (Toru, 1982; Haselhorst, 1984). Table 1 shows the results obtained in the rat striatum, nucleus accumbens, substantia nigra and hippocampus. Two days after a 3 week treatment with a daily dose of 1.0 mg/kg haloperidol or 5.0 mg/kg amphetamine, cytosolic calmodulin was found to be diminished or not altered, while membrane-
208 TABLE 1 Alterations in the cytosolic and membrane-bound content of caimodulin in rat brain regions by chronically applied drugs. The animals received daily i.p. injections of saline, 1 m g / k g haloperidol or 5 m g / k g amphetamine for 21 days. Two days after withdrawal, the rats were killed and tissue fractions were prepared for biochemical analysis. In all cases, n = 8 per group. Drugs
Calmodulin content (ng//~g protein ± S.E.M.) Cytosolic
Membranebound
Striatum (caudate putaraen) Saline Haloperidol Amphetamine
2.66 ± 0.42 2.89 ± 0.22 2.12 ± 0.46
3.14±0.26 6.03±0.87 c 4.85±0.60 a
2.46 ± 0.25 2.10 + 0.35 2.33 5:0.60
3.11 5:0.22 4.32+0.98 3.73 + 0.57
2.58 ± 0.23 2.50 ± 0.33 3.39 ± 0.40
3.04 + 0.21 3.62 + 0.39 4.71 + 0.50 b
2.69 ± 0.22 1.71 ± 0.37 a 1.66 ± 0.20 a
2.94 + 0.27 3.43 + 0.29 3.95+0.32 a
Nucleus accumbens Saline controls Haloperidol Amphetamine
Substantia nigra Saline controls Haloperidol Amphetamine
H ippocampus Saline controls Haloperidol Amphetamine
a p < 0.05, b p < 0.02, c p < 0.01, controls vs. drug-treated rats.
bound calmodulin increased. The haloperidol-induced increase in membrane-bound calmodulin was highly significant in the striatum. A significant increase in membrane-bound calmodulin after amphetamine occurred in the striatum, substantia nigra and hippocampus. The changes in calmodulin in the cytosolic fractions were less pronounced; the decrease of calmodulin was significant in the hippocampus after both haloperidol and amphetamine treatment.
4. Discussion
The present findings suggest that calmodulin is involved in the dopamine receptor-mediated actions of drugs. Occupation of dopamine receptors
by a dopamine receptor agonist, given as a single dose, and activation of dopamine release seem to induce the translocation of calmodulin from the membrane-bound to the cytosolic fraction. In view of the significance of the different receptor subtypes for dopamine (for review see Clark and White, 1987; Chiodo, 1988), the ineffectiveness of bromocryptine suggests that D-1 receptors are predominantly involved in calmodulin-related intracellular events. The low amphetamine dose induced the same extent of translocation as apomorphine did and the changes were similar in the nigrostriatal system and the hippocampus. The results obtained after apomorphine and the low dose of amphetamine were consistent with previous data reporting an increase in cytosolic and a decrease in membrane-bound calmodulin (Hanbauer and Phyall, 1980; Hanbauer et al., 1980). However, an opposite effect was observed in the striatum after a high dose of amphetamine (5.0 mg/kg) which increased membrane-bound calmodulin considerably and reduced cytosolic calmodulin. Even though such an inverse effect would be expected to occur after application of dopamine receptor antagonists, single doses of haloperidol or clozapine, which do not cause noticeable behavioural changes, did not influence the calmodulin content in either fraction of the striatum and hippocampus. Membrane-bound calmodulin, however, did increase and cytosolic calmodulin decreased after a 3 week treatment with the antagonist, haloperidol, just as it did after repeated high doses of amphetamine. Under the present experimental conditions, dopamine receptor supersensitivity (Honza, 1983), a particular form of neuronal plasticity, developed after the long-lasting blockade of dopamine receptors or the repeated strong activation of dopaminergic transmission. The occurrence of this supersensitivity is well documented for the caudate putamen which is amply provided with dopaminergic terminals originating from the cell bodies located in distinct structures of substantia nigra (Domesick, 1981; Di Chiara et al., 1981). Supersensitivity to the actions of dopamine can also be obtained in the hippocampus, as demonstrated by the effects of local microinjection of dopaminergic drugs into the hippocampal structure on the retention of a
209
learned behaviour. Furthermore, dopaminergic drugs induce an increased fucosylation of hippocampal glycoproteins (Honza, 1983; Jork and Matthies, 1985). The existence of a particular mesohippocampal dopamine system has been evidenced (for review see Bischoff, 1986), and recent studies have revealed the relevance of dopaminergic influences for the longer-term maintenance of hippocampal post-tetanic potentiation (Frey et al., 1989). In view of these results, dopaminergic activation, when coinciding with other specific transmitter-mediated signals, is very likely to produce the effects outlined above in the hippocampus. Such an assumption could explain the opposing effects of low and high doses of amphetamine: a low dose of amphetamine only stimulates dopamine release and causes the same translocation of calmodulin from membranes to the cytosol as that observed after direct activation of the dopamine D-1 receptor by apomorphine. Inversely, high doses of amphetamine also stimulate other transmitter systems and functional structures which, by postsynaptic convergence, induce other intraneuronal signalling systems related to the more complex processes of neuronal plasticity, such as those that occur in the course of chronic treatment and the development of receptor supersensitivity. This suggestion is consistent with the initial translocation of calmodulin from the cytosolic compartment to membranes observed after the induction of hippocampal long-term potentiation (Popov et al., 1988). Therefore, high doses of amphetamine result in similar increases in membrane-bound calmodulin to those caused by repeated administration of either haloperidol or amphetamine, thus producing changes in receptor responsiveness. Further investigations are needed to elucidate which subtypes of dopamine receptors are involved in the different effects of low and high doses of amphetamine.
Acknowledgements The skilful technical assistance of Mrs. Dora Ntiss and Mrs. Helga Tischrneyer is gratefully acknowledged.
References Biber, A., G. Schmid and K. Hempel, 1984, Calmodulin content in specific brain areas, Exp. Brain Res. 56, 323. Bischoff, S., 1986, Mesohippocampal dopamine system. Characterization, functional' and clinical implications, in: The Hippocampus, Vol. 3, eds. R.L. Isaacson and K.H. Pribram (Plenum Press, New York) p. 1. Chiodo, L.A., 1988, Dopamine-containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neurosci. Biobehav. Rev. 12, 49. Clark, D. and F.J. White, 1987, The D1 dopamine receptors: the search for a function. A critical evaluation of the D 1 / D 2 dopamine receptor classification and its functional implications, Synapse 1, 347. Costa, E., 198l, Intraneuronal translocation of modulators: a regulatory mechanism for interneuronal communication, in: Apomorphine and Other Dopaminomimetics, Vol. 1, Basic Pharmacology, eds. G.L. Gessa and G.U. Corsini (Raven Press, New York) p. 143. Di Chiara, G., M. Morelli, A. Imperato and M.L Porceddu, 198l, Substantia nigra as an efferent station for dopaminergic behavioural syndromes arising in the striaturn, in: Apomorphine and Other Doparninomimetics, Vol. 1, Basic Pharmacology, eds. G.L. Gessa and G.U. Corsini (Raven Press, New York) p. 41. Domesick, V.B., 1981, The anatomical basis for feedback and feed-forward in the striatonigral system, in: Apomorphine and Other Dopaminomimetics, Vol. 1, Basic Pharmacology, eds. G.L. Gessa and G.U. Corsini (Raven Press, New York) p. 27. Frey, U., S. Hartmann and H. Matthies, 1989, Domperidone, an inhibitor of the D2-receptor, blocks a late phase of an electrically induced LTP in the CA1 region in rats, Biomed. Biochim. Acta 48, 473. Hanbauer, I. and W. Phyall, 1980, Involvement of calmodulin in the modulation of dopamine receptor function, Adv. Biochem. Psychopharmacol. 24, 133. Hanbauer, I., S. Pradhan and H.Y.T. Yang, 1980, Role of calmodulin in dopaminergic transmission, Ann. N.Y. Acad. Sci. 356, 292. Haselhorst, U., 1984, Das Amphetaminmodell - ein potentiell psychoserelevantes Tiermodell (Promotion A, Medizinische Akademie Magdeburg). Honza, R., 1983, Wirkung dopaminerger Pharmaka auf die Retention einer erworbenen Verhaltensweise bei Ratten und Glykosilierung yon Hirnproteinen in vitro bei Dopamin-Supersensitivit~it (Promotion A, Medizinische Akademie Magdeburg). Horn, A.S., A.C. Cuello and R.R. Miller, 1974, Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase activity, J. Neurochem. 22, 265. Jork, R. and H. Matthies, 1985, Zellul~ire Mechanismen von Lern- und Ged~ichtnisprozessen, Funkt. Biol. Med. 4, 146. Lowry, O.H., H.J. Rosenbrough, A.L. Farr and R.J. Randall,
210 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Matthies, H., 1989, Neurobiological aspects of learning and memory, Annu. Rev. Psychol. 40, 381. Popov, N., W. Pohle, B. L/Sssner, S. Schulzeck, S. Schrnidt, T. Ott and H. Matthies, 1973, Regional distribution of RNA and protein radioactivity in the rat after intraventricular apphcation of labeled precursors, Acta Biol. Med. Germ. 31, 51. Popov, N.S., K.G. Reymann, K. Schulzeck, S. Schulzeck and H. Matthies, 1988, Alterations in calmoduhn content in fractions of rat hippocampal slices during tetanic- and calcium-induced long-term potentiation, Brain Res. Bull. 21,201. Schulzeck, S., H. Struy, N. Popov and H. Matthies, 1983, Hirn-Calmodulin. Isolierung und radiochemische Bestimmung, Zentralbl. Pharm. 122, 180. Stoclet, J.C., D. Gerard, M.C. Kilhoffer, C. Lugnier, R. Miller
and P. Schaffer, 1987, Calmodulin and its role in intracellular calcium regulation, Prog. Neurobiol. 29, 321. Struy, H., 1983, Grundlegende Untersuchungen zur Entwicklung und Anwendung yon Radioimmuntesten fiir die mikrobiologische und immunologische Diagnostik (Promotion B, Medizinische Akademie Magdeburg). Tarsy, D. and R.J. Baldessarini, 1974, Behavioural supersensitivity to apomorphine following chronic treatment with drugs which interfere with the synaptic functions of catecholamines, Neuropharmacology 13, 927. Toru, M., 1982, studies on pathophysiology of schizophrenia using experimental animals, Folia Psychiat. Neurol. Jpn. 36, 442. Van Eldik, L.J. and D.M. Watterson, 1981, Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site, J. Biol. Chem. 256, 4205.
205
Elsevier EJPMOL 90019
Influence of dopamine receptor agonists and antagonists on calmodulin translocation in different brain regions Nikolai Popov * and Hansjiirgen Matthies Institute of Pharmacology and Toxicology, Medical Academy, 3090 Magdeburg, G.D.R.
Received 21 February 1989, accepted 14 March 1989
Acute and chronic experiments were performed to study the effects of intraperitoneally administered dopamine receptor agonists and antagonists on the translocation of cytosolic and membrane-bound calmodulin in the striatum and hippocampus of the rat brain. Single doses of apomorphine and a low-dose amphetamine (1.25 mg/kg) resulted in a translocation of calmodulin, as measured by a decrease in membrane-bound and increase in cytosolic calmodulin in the striatum, whereas bromocryptine was ineffective. Amphetamine exerted a similar effect in the hippocampus and striatum. However, a high-dose amphetamine (5 mg/kg) had an opposite effect on translocation, in that there was an increase in membrane-bound calmodulin. Chronically applied amphetamine (5 mg/kg) and haloperidol (1 mg/kg), i.e. under conditions of dopamine receptor supersensitivity, tended to decrease cytosolic calmodulin in the striatum and hippocampus, and to increase membrane-bound calmodulin. The findings and, especially, the chronic effects of haloperidol and the high dose of amphetamine are interpreted in the fight of the current concept which suggests that transmitters and intraneuronal signalling systems converge, thereby influencing the more complex processes of neuronal plasticity, rather than receptor sensitivity only. Amphetamine; Haloperidol; Dopamine; Striatum; Hippocampus; Calmodulin translocation
1. Introduction
Calmodulin, a calcium-binding protein, has been found to play an essential role in various systems of intracellular signalling by mediating transmitter-induced changes in Ca 2+ concentrations (for review see Stoclet et al., 1987). The translocation of calmodulin from the cytosol to membranes and vice versa seems to be of crucial significance in these processes (Hanbauer et al., 1980; Costa, 1981). This involvement of transmitter signals in intraneuronal transduction suggests that the action of specific neurotropic drugs could also induce characteristic changes in the distribution a n d / o r content of calmodulin. Eluci-
dation of these events would contribute to a better understanding of the interplay between messenger systems in the intracellular control of neuronal functions and the mechanisms of action of pharmacologically active substances. It was our aim to study whether calmodulin translocates from the cytosolic c o m p a r t m e n t to membrane and vice versa in brain structures after acute or repeated intraperitoneal (i.p.) administration of dopamine receptor agonists and antagonists. The chronic treatment was applied under conditions chosen to attain dopamine receptor supersensitivity.
2. Materials and methods 2.1. A n i m a l s a n d t r e a t m e n t
* To whom all correspondence should be addressed: Institute of Pharmacology and Toxicology, Medical Academy, Leipzigerstrasse 44, 3090 Magdeburg, G.D.R.
Male rats of Wistar origin from our own breeding stock were used. Eight-week-old animals were
0922-4106/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
206 used for the acute experiments, while 5-week-old rats were used for the chronic treatment. The animals were provided with food and water ad libitum. All drugs used were injected i.p.: apomorphine hydrochloride (SPOFA, Prague, Czechoslovakia), bromocryptine mesylate (Sigma, Deisenhofen, F.R.G.), amphetamine racemate (VEB FahlbergList, Magdeburg, G.D.R.), haloperidol (Gedeon Richter, Budapest, Hungary) and clozapine (Leponex*, Sandoz, Basel, Switzerland). The animals in the acute experiments were killed 30-120 min after drug administration as detailed below (the number of animals per group is indicated in parentheses): 1 mg/kg apomorphine, 30 min (5) and 60 min (5); 10 mg/kg bromocryptine, 40 min (5) and 120 min (5); 1.25 mg/kg amphetamine, 40 min (10); 5 mg/kg amphetamine, 40 min (5); 1 mg/kg haloperidol, 40 min (10); 20 mg/kg clozapine, 40 min (5). The control animals (at least 5) received saline; these animals were killed after the corresponding periods following drug administration. The chronic series involved 3 groups of 8 rats each; the animals were treated daily with 1 mg/kg haloperidol, 5 mg/kg amphetamine or saline for 3 weeks. The rats were killed 2 days after withdrawal and the brains were dissected out as described elsewhere (Popov et al., 1973). The latter procedure was also used for the acute experiments, and the caudate putamen, n, accumbens and substantia nigra were dissected additionally in the chronic experiments (Horn et al., 1974). The brain regions were frozen on dry ice and stored at - 20 ° C until use.
2.2. Biochemical analysis Brain tissue from the individual regions was homogenized with a microhomogenizer in a solution containing 0.32 M sucrose and 1 mM disodium EDTA and, using a microtube adaptor, was centrifuged in a refrigerated ultracentrifuge at 100000 × g for 60 min. The resulting supernatant was heated (90 ° C, 1 min) and re-centrifuged at 100000 x g for 30 min. The supernatant obtained was designated as the water-soluble (cytosolic) fraction. The water-insoluble pellet from the first
centrifugation step was homogenized in 0.05 M Tris-HC1 buffer, pH 7.4, containing 0.1% Lubrol, heated (90°C, 1 min), sonicated (3 times for 15 s each) and kept at 4°C for 16 h. After centrifugation at 100 000 x g for 30 min, the resulting supernatant was designated the Lubrol-soluble fraction and contained membrane-bound calmodulin. The protein content was determined in aliquots of each fraction by the method of Lowry et al. (1951). The content of calmodulin was measured in triplicate by a modified, competitive, solid-phase radioimmunoassay (Schulzeck et al., 1983; Struy, 1983) with antiserum raised against performic acid-oxidized calmodulin from bovine brain (Van Eldik and Watterson, 1981). Briefly, the samples were pretreated with 20 mM chloramine T (to increase the sensitivity of the antigen-antibody reaction), and after 2 min the reaction was stopped by the addition of 40 mM potassium metabisulphite (Biber et al., 1984). Thereafter, each sample was incubated in the presence of a defined amount of rabbit antiserum to calmodulin. The amount of non-bound antibody was then detected by the addition of a25I-labelled anti-rabbit sheep globulin to the complex which consisted of PVC blister as solid phase covered by adsorbed calmodulin (antigen) on the inner surface, and calmodulin antibodies (unbound during the preceding step). This approach of labelling a third component (detector) in addition to the unlabelled antigen and antibody was adopted to preclude iodination-induced alterations of calmodulin and the calmodulin antibodies. The content was calculated with the aid of associated calibration curves, and is reported as big protein. The two-tailed Student's t-test in groups was used to compute the statistical significance of differences between means obtained for the control and drug-treated groups.
3. Results
3.1. Acute effects of dopaminergic drugs Single doses of the substances used did not influence the calmodulin content of the cytosolic and membrane-bound fractions in the frontal
207
51
cytosolic
4 3 2 1
DQ
striatum L
5 t membrane-t
hippocamp.
4
J
3 2 1
apo
mg/kg 5 rain 30 60
brcr
amph
5
1.2s 5
40 120
40
halo cloz 1 20 40
amph
1.2s 5 40
Fig. l. Influence of dopamine receptor agonists and antagonists on the translocation of calmodulin from cytosolic to membrane-bound forms, and vice versa, in the rat striatum and hippocampus. The ordinate indicates the calmodulin values ( n g / # g protein). M e a n s + S . E . M . of 10 saline controls are represented by horizontal lines: cytosolic calmodulin 2.92 + 0.38 and 2.52 for the striatum and hippocampus, respectively; membrane-bound calmodulin 3.20+0.21 and 2.90+0.35, respectively, l.p. doses and time of treatment are shown in the bottom part of the figure; a p o = apomorphine, brcr = bromocryptine, a m p h = amphetamine, halo = haloperidol, cloz = clozapine. The means of drug-treated animal determinations + S.E.M. are represented as bars. Empty circle, one filled circle and two filled circles stand for P < 0.05, P < 0.02 and P < 0.01, respectively, revealing statistical significance of differences between drug-treated animals and saline controls.
cortex and cerebellum. Figure 1 depicts the results obtained (a) in the dopamine-rich striatum, which is frequently used to study biochemical alterations related to the action of dopaminergic drugs and to changes in dopamine receptor responsiveness, and (b) in the hippocampus, which contains little dopamine and which is assumed to play a role in learning and memory formation (for review see Matthies, 1989) and which exhibits phenomena of neuronal plasticity, such as post-tetanic long-term
potentiation. As demonstrated in fig. 1, the D1/D-2 dopamine receptor agonist, apomorphine, and the preferential D-2 receptor agonist, bromocryptine, had different effects: apomorphine caused a significant decrease in membrane-bound calmodulin accompanied by a tendency to an increased content in the cytosolic fraction in the striatum, while bromocryptine did not influence the calmodulin content in either of the fractions from this region. After application of a low dose (1.25 mg/kg) of the indirect agonist, amphetamine, the distribution of calmodulin changed in a manner similar to that noted after apomorphine: a significant increase in the cytosolic fraction was accompanied by a significant decrease in membrane-bound calmodulin. In contrast, a relatively high dose of amphetamine (5.0 mg/kg) resulted in an opposite effect: a significant increase in membrane-bound calmodulin occurred with a simultaneous distinct, even though insignificant, decrease in cytosolic calmodulin. The dopamine receptor antagonists, haloperidol (1.0 mg/kg) and clozapine (20.0 mg/kg), had no effect on the distribution of calmodulin in either striatal fraction. In the hippocampus, only the low dose of amphetamine caused detectable changes: the cytosolic fraction of calmodulin was significantly enhanced and the membrane-bound fraction tended to decrease, thus exhibiting qualitative alterations identical to those occurring after the low-dose amphetamine in the striatum.
3.2. Effects of chronic treatment with haloperidol and amphetamine Chronic treatment with haloperidol and a high dose of amphetamine was given in the manner used in our laboratories to produce supersensitivity in rats as determined by the behavioural effects of single doses of apomorphine (Tarsy and Baldessarini, 1974; Honza, 1983) and amphetamine (Toru, 1982; Haselhorst, 1984). Table 1 shows the results obtained in the rat striatum, nucleus accumbens, substantia nigra and hippocampus. Two days after a 3 week treatment with a daily dose of 1.0 mg/kg haloperidol or 5.0 mg/kg amphetamine, cytosolic calmodulin was found to be diminished or not altered, while membrane-
208 TABLE 1 Alterations in the cytosolic and membrane-bound content of caimodulin in rat brain regions by chronically applied drugs. The animals received daily i.p. injections of saline, 1 m g / k g haloperidol or 5 m g / k g amphetamine for 21 days. Two days after withdrawal, the rats were killed and tissue fractions were prepared for biochemical analysis. In all cases, n = 8 per group. Drugs
Calmodulin content (ng//~g protein ± S.E.M.) Cytosolic
Membranebound
Striatum (caudate putaraen) Saline Haloperidol Amphetamine
2.66 ± 0.42 2.89 ± 0.22 2.12 ± 0.46
3.14±0.26 6.03±0.87 c 4.85±0.60 a
2.46 ± 0.25 2.10 + 0.35 2.33 5:0.60
3.11 5:0.22 4.32+0.98 3.73 + 0.57
2.58 ± 0.23 2.50 ± 0.33 3.39 ± 0.40
3.04 + 0.21 3.62 + 0.39 4.71 + 0.50 b
2.69 ± 0.22 1.71 ± 0.37 a 1.66 ± 0.20 a
2.94 + 0.27 3.43 + 0.29 3.95+0.32 a
Nucleus accumbens Saline controls Haloperidol Amphetamine
Substantia nigra Saline controls Haloperidol Amphetamine
H ippocampus Saline controls Haloperidol Amphetamine
a p < 0.05, b p < 0.02, c p < 0.01, controls vs. drug-treated rats.
bound calmodulin increased. The haloperidol-induced increase in membrane-bound calmodulin was highly significant in the striatum. A significant increase in membrane-bound calmodulin after amphetamine occurred in the striatum, substantia nigra and hippocampus. The changes in calmodulin in the cytosolic fractions were less pronounced; the decrease of calmodulin was significant in the hippocampus after both haloperidol and amphetamine treatment.
4. Discussion
The present findings suggest that calmodulin is involved in the dopamine receptor-mediated actions of drugs. Occupation of dopamine receptors
by a dopamine receptor agonist, given as a single dose, and activation of dopamine release seem to induce the translocation of calmodulin from the membrane-bound to the cytosolic fraction. In view of the significance of the different receptor subtypes for dopamine (for review see Clark and White, 1987; Chiodo, 1988), the ineffectiveness of bromocryptine suggests that D-1 receptors are predominantly involved in calmodulin-related intracellular events. The low amphetamine dose induced the same extent of translocation as apomorphine did and the changes were similar in the nigrostriatal system and the hippocampus. The results obtained after apomorphine and the low dose of amphetamine were consistent with previous data reporting an increase in cytosolic and a decrease in membrane-bound calmodulin (Hanbauer and Phyall, 1980; Hanbauer et al., 1980). However, an opposite effect was observed in the striatum after a high dose of amphetamine (5.0 mg/kg) which increased membrane-bound calmodulin considerably and reduced cytosolic calmodulin. Even though such an inverse effect would be expected to occur after application of dopamine receptor antagonists, single doses of haloperidol or clozapine, which do not cause noticeable behavioural changes, did not influence the calmodulin content in either fraction of the striatum and hippocampus. Membrane-bound calmodulin, however, did increase and cytosolic calmodulin decreased after a 3 week treatment with the antagonist, haloperidol, just as it did after repeated high doses of amphetamine. Under the present experimental conditions, dopamine receptor supersensitivity (Honza, 1983), a particular form of neuronal plasticity, developed after the long-lasting blockade of dopamine receptors or the repeated strong activation of dopaminergic transmission. The occurrence of this supersensitivity is well documented for the caudate putamen which is amply provided with dopaminergic terminals originating from the cell bodies located in distinct structures of substantia nigra (Domesick, 1981; Di Chiara et al., 1981). Supersensitivity to the actions of dopamine can also be obtained in the hippocampus, as demonstrated by the effects of local microinjection of dopaminergic drugs into the hippocampal structure on the retention of a
209
learned behaviour. Furthermore, dopaminergic drugs induce an increased fucosylation of hippocampal glycoproteins (Honza, 1983; Jork and Matthies, 1985). The existence of a particular mesohippocampal dopamine system has been evidenced (for review see Bischoff, 1986), and recent studies have revealed the relevance of dopaminergic influences for the longer-term maintenance of hippocampal post-tetanic potentiation (Frey et al., 1989). In view of these results, dopaminergic activation, when coinciding with other specific transmitter-mediated signals, is very likely to produce the effects outlined above in the hippocampus. Such an assumption could explain the opposing effects of low and high doses of amphetamine: a low dose of amphetamine only stimulates dopamine release and causes the same translocation of calmodulin from membranes to the cytosol as that observed after direct activation of the dopamine D-1 receptor by apomorphine. Inversely, high doses of amphetamine also stimulate other transmitter systems and functional structures which, by postsynaptic convergence, induce other intraneuronal signalling systems related to the more complex processes of neuronal plasticity, such as those that occur in the course of chronic treatment and the development of receptor supersensitivity. This suggestion is consistent with the initial translocation of calmodulin from the cytosolic compartment to membranes observed after the induction of hippocampal long-term potentiation (Popov et al., 1988). Therefore, high doses of amphetamine result in similar increases in membrane-bound calmodulin to those caused by repeated administration of either haloperidol or amphetamine, thus producing changes in receptor responsiveness. Further investigations are needed to elucidate which subtypes of dopamine receptors are involved in the different effects of low and high doses of amphetamine.
Acknowledgements The skilful technical assistance of Mrs. Dora Ntiss and Mrs. Helga Tischrneyer is gratefully acknowledged.
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