European Journal of Pharmacology, 197 (1991) 221-223 0 1991 Elsevier Science Pubhshers B.V. 0014-2999/91/$03,50
221
ADONIS 001429999100380K
EJP 20825
Short communication
ter
arizi Caterina
Department of Neuroscience
Arnbrosio
‘B.B. Brodie
and Ennio Stefanini
‘, UniLprsity of Cagliari, Via Porcell4, 09124 Cagliari,
Received 12 March 1991,
Italy
accepted 19 March 1991
Flunarizine dose dependently inhibits the binding of both [‘Hlspiperone and [3HlSCH 23390 with Ki values of 112 + 9 and 532 + 39 nM, respectively. The inhibition of [3’ Qpiperone binding by flunarizine was competitive as revealed by the Schild plot. The results indicate that flunarizine is a fairly potent dopamine D, receptor antagonist and offer a possible explanation for the extrapyramidal side-effects observed in patients treated with the drug.
Flunarizine; [3H]Spiperone; i3HlSCH 23390; Dopamine receptors
1. Introduction A number of Ca2+ antagonists, in addition to their inhibitory effect on transmembrane Ca2+ influx, have been shown to modify dopaminergic transmission (Greeb, 1986; Greeb et al., 1987). In particular flunarizine has been shown to share with neuroleptics the ability to increase concentrations of the dopamine (DA) metabolite 3,4_dihydroxyphenylacetic acid (DOPAC) in the striatum (Stefanini et al., 1987) and to potentiate cocaine-induced release of DA in the striatum (Pani et al., 1990). Clinical observations have consistently shown that flunarizine produces extrapyramidal side-effects (Chousa et al., 1986). These observations suggest that flunarizine might block the D, type DA receptors considered to be involved in the above responses. To test this hypothesis we studied the interaction of flunarizine with the binding of [3H]spiperone to DA D, receptors and of [3H]SCH 23390 to D, receptors in S!Iiatal membranes.
2. Materials and methods [3H]Spiperone (23.3 Ci/mmol) and [3H]SCH 23390 (60.4 Ci/mmol) were obtained from NEN DuPont (Boston, MA, U.S.A.); ( - )-sulpiride from Ravizza S.p.A. (Muggio, Italy); ketanserine from RBI (Natick, MA, U.S.A); SCH 23390 from Schering Corp. (Kenil-
Correspondence to: E. Stefanini, Department of Neuroscience, Via Porcell 4, 09124 Cagliari, Italy.
worth, NJ, U.S.A.); flunarizine from Sigma Chemie (Deisenhofen, WGermany). Male Sprague-Dawley rats (Morini, S.Polo D’Enza, Italy), weighing 180-225 g, were killed by decapitation. The brains were rapidly removed, placed in ice-cold saline for 2 min, and the striatum was dissected out. Tissues were processed as described previously (Stefanini et al., 1987). Briefly, tissues were homogenized in 100 volumes of ice-cold 50 mM Tris-HCl, pH 7.7, by using a tight teflon/g!ass Potter homogenizer, and centrifuged at 50000 x g for 20 min at 4°C. The supernatant was discarded, the pellet was resuspended as above in 20 volumes of 50 Mm Tris-HCl pH 7.4, aliquoted and stored at -80°C until processed. Tissue suspensions were thawed, diluted with Tris-HCl 50 mM, NaCl 120 mM, KCI 5 mM, EDTA 1 mM, ascorbic acid O.l%, pH 7.4, and re-homogenized in buffer to obtain about 100 pg protein 100 ~1 of buffer. One hundred and fifty microlitres of tissue preparation were added to triplicate glass tubes containing incubation buffer, which contained 0.2-0.5 nM [3H]spiperone (0.025-1.0 nM for saturation experiments) or 0.4-0.8 nM [ 3H]SCH 23390 (0.01-1.0 nM for saturation experiments), and unlabelled drugs in a final volume of 2.0 or 0.5 ml, respectively. The incubation was carried out at 37°C for 45 min for [3H]spiperone binding and at room temperature for 45 min for 13H]SCH 23390 binding. Membrane-bound radioactivity was trapped on GF/C Whatman glass fiber filters by using a Brandell manifold apparatus (Biomedical Research and Development Laboratories Inc., Gaithesburg, MA, U.S.A.). Ah the assays were carried out in the presence of 30 nM ketanserine. Specific binding of [3Hlspiperone or
olsplacera
cone.
-log
CM1
Fig. 1. inhibition of [‘H]spiperone (0.1X nM) specific binding by flunarizine tclose circles) and ( - kulpiride (open circles) in striatal krmugrnatrs. The data are ihe means of three experiments performed in triplicate.
[“H]SCH 23390 were defined as the total binding minus the non-specific counts obtained in the presence of 50 PM (- )-sulpir& and 1 PM SCH 23390 respectively.
[‘H]Spiperone (0.2-0.5 nM) bound to striatal membranes with a specific binding ranging from 81 f 7% (0.2 nM) to 51.8 f 5% (0.5 nM) of total binding, a K, of 57 f 7.8 pM and a B,,, of 281 f 29 fmol/mg protein [‘HISCH 23390 bound with a specific binding of S2-89% (0.8-0.4 nM) of total binding, a K, of 0.3 + 0.04 nM and a B,, of 1089 fmol/mg protein. Fhmarizine inhibited both ]“H]SCH 23390 and [ “Hlspiperone bind.12
r
Fig. 2. Eauilibrium measurements cf [“Hlspiperone binding in the presence of 10 nM (A ).30 nM ( ), l&l nM (o), 1OCjO nM (0) and in the absence (e) of flunarizine, respectively. The Schild plot of the data is shown in the inset. The dose ratio (DR) is the ratio of dissociation constants of [“fllspiperone obtained in the presence and absence of flunarizine. The data are the means of two experiments. each carried out in triplicate.
ing in a concentration-dependent manner. Antagonism against [ ‘H]SCH 23390 and [“Hlspiperone binding was characterized by a Ki of 539 _+39 nM and 112 + 12 nM, respectively; the Ki value of fiunarizine againsl [‘Hlspiperone binding was about half that of (- )sulpiride (K, 55 + 4 nM, fig. 1). In order to characterize the nature of the displacement of [“Hlspiperone by flunarizine, the inhibition of radioactive ligand binding by fhtnarizine was measured in striatal membranes to obtain equilibrium binding isotherms for [“Hlspiperone in the presence of increasing concentrations of flunarizine. In fig. 2 the results of these experiments are reported as Scatchard and Schild plots. It can be seen that the apparent dissociation constant of [3H]spiperone increased with increasing concentratios of flunarizine, whereas the B,,, values were not statistically different from the value of the [3H]spiperone control binding isotherm. These results are also shown in the form of Schild plot (inset, fig. 2). The data represented are consistent with competitive inhibition based on the linearity of the Schild plot (r = 0.98).
The present results show that flunarizine inhibits [“Hlspiperone and [“H]SCH 23390 binding to D, and D, striatal receptors, respectively. The affinity of flunarizine for Dz receptors was much higher than its affinity for D, receptors, being in the order of magnitude of that of (-)-sulpiride, a classic D2 receptor antagonist. Moreover, similarly to ( - I-sulpiride, flunarizine interacts with D, receptors in a competitive manner. Other Ca2+ antagonists, such as bepridil D600 and verapamil, also displace [“Hlspiperone binding. Vice versa, different neuroleptics, such as pimozide and thioridazine, have been shown to block D, receptors and also to be potent Ca2+ channel antagonists (Gould et al., 1983). These findings question whether D, receptors and L-type Ca2+ channels, with which the above compounds interact, might share some structural similarity. The finding that Ca2+ antagonists block D, receptors might explain some experimental findings, such as the inhibition by these drugs of amphetamine-induced motor stimulation in mice (Greeb, 19861, and the increase of DA metabolism in rats (Fadda et al., 1989), as well as some clinical observations, such as the ability to produce parkinsonian sideeffects (Chousa et al., 1986) and hyperprolactinemia (Bonucceiii et al., 1988). The property to block both D, receptors and, to a lesser extent D, receptors, is consistent with the suggestion that Ca’+ antagonists might be effective antipsychotic agents in mania (HBschl et al., 1986). However, this issue is complicated by recent results from our laboratory showing that flunarizine is a potert inhibitor of DA uptake by striatal synapto-
223
somes in vitro (Devote et al., submitted), an effect which should eventually result in enhanced extraneuronal DA conce9ltrations. Moreover we have found recently that flunarizine increases DA release and potentiates that induced by cocaine in the striatum, as measured by brain microdialysis (Pani et al., 1990). In conclusion, the overall observations indicate that flunarizine has multiple actions in the brain. The net effect on dopaminergic transmission might depend on the affinity of flunarizine for the different receptors and on the concentration that is reached in the brain after systemic administration of the drug.
Acknowledgements I would like to thank G.L. Gessa for his suggestions and constructive criticism during the preparation of the manuscript and Ravizza S.p.A. for supporting this research.
References Bonuccelli, U.. P. Piccini, A.M. Paoletti, A. Nuti, A. Colzi. G.B. Melis and A. Muratorio, 1988, Flunarizine increses PRL in normal and migraineous women, .I. Neural Transm. 74, 43. Chousa. C., A. Scaramelli, J.L. Caamano, 0. De Medina. R. Aljanati and S. Romero, 1986, Parkinsonism, tardive dyskinesia, akathisia and depression induced by flunarizine, Lancet i. 1303.
Dewar, M.K. and T.A. Reader,
1989, Specific (‘HJSCH23390 binding to dopamine D, receptors in cerebral cortex and neostriatum: role of disultide and sulphydryl groups, J. Neurochem. 472. Fadda, F., G.L. Cessa, E. Mosca and E. Stefanini, 1989, Different effects of the Ca’+ antagonists nimodipine and flunarizine on dopamine metabolism in the rat brain, J. Neural Transm. 75, 195. Gould, R.J., K.M.M. Murphy, I.J. Reinolds and S.H. Snyder, 1983. Antischizophrenic drugs of diphenylbutylpiperidine type act as Caz+ channel antagonists, Proc. Natl. Acad. Sci. U.S.A. 83.7513. Greeb, A.J., 1986, Nifedipine and flunarizine block amphetamine-induced behavioural stimulation in mice, Life Sci. 38, 2375. Greeb, A.J.. R.C. Shelton and W.J. Freed, 1987, diltiazem or verapamil prevents haloperidol-induced apomorphine supersensitivity in mice, J. Neural Transm. 68, 241. Hiisghl, C., J. Blahos and J. Kabes, 1986, The use of Caz’ channel blockers in psychiatry, in: Biological Psychiatry, eds. C. Shagass et al. (Elsevier) p. 329. Pani, L., A. Kuzmin. E. Stefanini, G.L. Gessa and Z.L. Rossetti. 1990, Flunarizine potentiates cocaine-induced dopamine release and motor stimulation in rats, European J. Pharmacol. 190, 223. Pileblad, E. and A. Carlsson, 1986, In vivo effects of the calcium antagonists nimodipine on dopamine metabolism in mouse brain, J. Neural Transm. 66, 171. Pani, L.. A. Kuzmin. E. Stefanini, G.L. Gessa and Z.L. Rossetti, 1990, Flunarizine potentiates cocaine-induced dopamine release and motor stimulation in rats, European J. Pharmacol. 190, 223. Stefanini. E., A.M. Ortu, F. Vernaleone and G.L. Gessa, 1987, ["Hjf - JSulpiride binding in rat striatum, cortex and anterior pituitary: an improved assay. Pharmacol. Res. Commun. 19, 11. 777.
221
ADONIS 001429999100380K
EJP 20825
Short communication
ter
arizi Caterina
Department of Neuroscience
Arnbrosio
‘B.B. Brodie
and Ennio Stefanini
‘, UniLprsity of Cagliari, Via Porcell4, 09124 Cagliari,
Received 12 March 1991,
Italy
accepted 19 March 1991
Flunarizine dose dependently inhibits the binding of both [‘Hlspiperone and [3HlSCH 23390 with Ki values of 112 + 9 and 532 + 39 nM, respectively. The inhibition of [3’ Qpiperone binding by flunarizine was competitive as revealed by the Schild plot. The results indicate that flunarizine is a fairly potent dopamine D, receptor antagonist and offer a possible explanation for the extrapyramidal side-effects observed in patients treated with the drug.
Flunarizine; [3H]Spiperone; i3HlSCH 23390; Dopamine receptors
1. Introduction A number of Ca2+ antagonists, in addition to their inhibitory effect on transmembrane Ca2+ influx, have been shown to modify dopaminergic transmission (Greeb, 1986; Greeb et al., 1987). In particular flunarizine has been shown to share with neuroleptics the ability to increase concentrations of the dopamine (DA) metabolite 3,4_dihydroxyphenylacetic acid (DOPAC) in the striatum (Stefanini et al., 1987) and to potentiate cocaine-induced release of DA in the striatum (Pani et al., 1990). Clinical observations have consistently shown that flunarizine produces extrapyramidal side-effects (Chousa et al., 1986). These observations suggest that flunarizine might block the D, type DA receptors considered to be involved in the above responses. To test this hypothesis we studied the interaction of flunarizine with the binding of [3H]spiperone to DA D, receptors and of [3H]SCH 23390 to D, receptors in S!Iiatal membranes.
2. Materials and methods [3H]Spiperone (23.3 Ci/mmol) and [3H]SCH 23390 (60.4 Ci/mmol) were obtained from NEN DuPont (Boston, MA, U.S.A.); ( - )-sulpiride from Ravizza S.p.A. (Muggio, Italy); ketanserine from RBI (Natick, MA, U.S.A); SCH 23390 from Schering Corp. (Kenil-
Correspondence to: E. Stefanini, Department of Neuroscience, Via Porcell 4, 09124 Cagliari, Italy.
worth, NJ, U.S.A.); flunarizine from Sigma Chemie (Deisenhofen, WGermany). Male Sprague-Dawley rats (Morini, S.Polo D’Enza, Italy), weighing 180-225 g, were killed by decapitation. The brains were rapidly removed, placed in ice-cold saline for 2 min, and the striatum was dissected out. Tissues were processed as described previously (Stefanini et al., 1987). Briefly, tissues were homogenized in 100 volumes of ice-cold 50 mM Tris-HCl, pH 7.7, by using a tight teflon/g!ass Potter homogenizer, and centrifuged at 50000 x g for 20 min at 4°C. The supernatant was discarded, the pellet was resuspended as above in 20 volumes of 50 Mm Tris-HCl pH 7.4, aliquoted and stored at -80°C until processed. Tissue suspensions were thawed, diluted with Tris-HCl 50 mM, NaCl 120 mM, KCI 5 mM, EDTA 1 mM, ascorbic acid O.l%, pH 7.4, and re-homogenized in buffer to obtain about 100 pg protein 100 ~1 of buffer. One hundred and fifty microlitres of tissue preparation were added to triplicate glass tubes containing incubation buffer, which contained 0.2-0.5 nM [3H]spiperone (0.025-1.0 nM for saturation experiments) or 0.4-0.8 nM [ 3H]SCH 23390 (0.01-1.0 nM for saturation experiments), and unlabelled drugs in a final volume of 2.0 or 0.5 ml, respectively. The incubation was carried out at 37°C for 45 min for [3H]spiperone binding and at room temperature for 45 min for 13H]SCH 23390 binding. Membrane-bound radioactivity was trapped on GF/C Whatman glass fiber filters by using a Brandell manifold apparatus (Biomedical Research and Development Laboratories Inc., Gaithesburg, MA, U.S.A.). Ah the assays were carried out in the presence of 30 nM ketanserine. Specific binding of [3Hlspiperone or
olsplacera
cone.
-log
CM1
Fig. 1. inhibition of [‘H]spiperone (0.1X nM) specific binding by flunarizine tclose circles) and ( - kulpiride (open circles) in striatal krmugrnatrs. The data are ihe means of three experiments performed in triplicate.
[“H]SCH 23390 were defined as the total binding minus the non-specific counts obtained in the presence of 50 PM (- )-sulpir& and 1 PM SCH 23390 respectively.
[‘H]Spiperone (0.2-0.5 nM) bound to striatal membranes with a specific binding ranging from 81 f 7% (0.2 nM) to 51.8 f 5% (0.5 nM) of total binding, a K, of 57 f 7.8 pM and a B,,, of 281 f 29 fmol/mg protein [‘HISCH 23390 bound with a specific binding of S2-89% (0.8-0.4 nM) of total binding, a K, of 0.3 + 0.04 nM and a B,, of 1089 fmol/mg protein. Fhmarizine inhibited both ]“H]SCH 23390 and [ “Hlspiperone bind.12
r
Fig. 2. Eauilibrium measurements cf [“Hlspiperone binding in the presence of 10 nM (A ).30 nM ( ), l&l nM (o), 1OCjO nM (0) and in the absence (e) of flunarizine, respectively. The Schild plot of the data is shown in the inset. The dose ratio (DR) is the ratio of dissociation constants of [“fllspiperone obtained in the presence and absence of flunarizine. The data are the means of two experiments. each carried out in triplicate.
ing in a concentration-dependent manner. Antagonism against [ ‘H]SCH 23390 and [“Hlspiperone binding was characterized by a Ki of 539 _+39 nM and 112 + 12 nM, respectively; the Ki value of fiunarizine againsl [‘Hlspiperone binding was about half that of (- )sulpiride (K, 55 + 4 nM, fig. 1). In order to characterize the nature of the displacement of [“Hlspiperone by flunarizine, the inhibition of radioactive ligand binding by fhtnarizine was measured in striatal membranes to obtain equilibrium binding isotherms for [“Hlspiperone in the presence of increasing concentrations of flunarizine. In fig. 2 the results of these experiments are reported as Scatchard and Schild plots. It can be seen that the apparent dissociation constant of [3H]spiperone increased with increasing concentratios of flunarizine, whereas the B,,, values were not statistically different from the value of the [3H]spiperone control binding isotherm. These results are also shown in the form of Schild plot (inset, fig. 2). The data represented are consistent with competitive inhibition based on the linearity of the Schild plot (r = 0.98).
The present results show that flunarizine inhibits [“Hlspiperone and [“H]SCH 23390 binding to D, and D, striatal receptors, respectively. The affinity of flunarizine for Dz receptors was much higher than its affinity for D, receptors, being in the order of magnitude of that of (-)-sulpiride, a classic D2 receptor antagonist. Moreover, similarly to ( - I-sulpiride, flunarizine interacts with D, receptors in a competitive manner. Other Ca2+ antagonists, such as bepridil D600 and verapamil, also displace [“Hlspiperone binding. Vice versa, different neuroleptics, such as pimozide and thioridazine, have been shown to block D, receptors and also to be potent Ca2+ channel antagonists (Gould et al., 1983). These findings question whether D, receptors and L-type Ca2+ channels, with which the above compounds interact, might share some structural similarity. The finding that Ca2+ antagonists block D, receptors might explain some experimental findings, such as the inhibition by these drugs of amphetamine-induced motor stimulation in mice (Greeb, 19861, and the increase of DA metabolism in rats (Fadda et al., 1989), as well as some clinical observations, such as the ability to produce parkinsonian sideeffects (Chousa et al., 1986) and hyperprolactinemia (Bonucceiii et al., 1988). The property to block both D, receptors and, to a lesser extent D, receptors, is consistent with the suggestion that Ca’+ antagonists might be effective antipsychotic agents in mania (HBschl et al., 1986). However, this issue is complicated by recent results from our laboratory showing that flunarizine is a potert inhibitor of DA uptake by striatal synapto-
223
somes in vitro (Devote et al., submitted), an effect which should eventually result in enhanced extraneuronal DA conce9ltrations. Moreover we have found recently that flunarizine increases DA release and potentiates that induced by cocaine in the striatum, as measured by brain microdialysis (Pani et al., 1990). In conclusion, the overall observations indicate that flunarizine has multiple actions in the brain. The net effect on dopaminergic transmission might depend on the affinity of flunarizine for the different receptors and on the concentration that is reached in the brain after systemic administration of the drug.
Acknowledgements I would like to thank G.L. Gessa for his suggestions and constructive criticism during the preparation of the manuscript and Ravizza S.p.A. for supporting this research.
References Bonuccelli, U.. P. Piccini, A.M. Paoletti, A. Nuti, A. Colzi. G.B. Melis and A. Muratorio, 1988, Flunarizine increses PRL in normal and migraineous women, .I. Neural Transm. 74, 43. Chousa. C., A. Scaramelli, J.L. Caamano, 0. De Medina. R. Aljanati and S. Romero, 1986, Parkinsonism, tardive dyskinesia, akathisia and depression induced by flunarizine, Lancet i. 1303.
Dewar, M.K. and T.A. Reader,
1989, Specific (‘HJSCH23390 binding to dopamine D, receptors in cerebral cortex and neostriatum: role of disultide and sulphydryl groups, J. Neurochem. 472. Fadda, F., G.L. Cessa, E. Mosca and E. Stefanini, 1989, Different effects of the Ca’+ antagonists nimodipine and flunarizine on dopamine metabolism in the rat brain, J. Neural Transm. 75, 195. Gould, R.J., K.M.M. Murphy, I.J. Reinolds and S.H. Snyder, 1983. Antischizophrenic drugs of diphenylbutylpiperidine type act as Caz+ channel antagonists, Proc. Natl. Acad. Sci. U.S.A. 83.7513. Greeb, A.J., 1986, Nifedipine and flunarizine block amphetamine-induced behavioural stimulation in mice, Life Sci. 38, 2375. Greeb, A.J.. R.C. Shelton and W.J. Freed, 1987, diltiazem or verapamil prevents haloperidol-induced apomorphine supersensitivity in mice, J. Neural Transm. 68, 241. Hiisghl, C., J. Blahos and J. Kabes, 1986, The use of Caz’ channel blockers in psychiatry, in: Biological Psychiatry, eds. C. Shagass et al. (Elsevier) p. 329. Pani, L., A. Kuzmin. E. Stefanini, G.L. Gessa and Z.L. Rossetti. 1990, Flunarizine potentiates cocaine-induced dopamine release and motor stimulation in rats, European J. Pharmacol. 190, 223. Pileblad, E. and A. Carlsson, 1986, In vivo effects of the calcium antagonists nimodipine on dopamine metabolism in mouse brain, J. Neural Transm. 66, 171. Pani, L.. A. Kuzmin. E. Stefanini, G.L. Gessa and Z.L. Rossetti, 1990, Flunarizine potentiates cocaine-induced dopamine release and motor stimulation in rats, European J. Pharmacol. 190, 223. Stefanini. E., A.M. Ortu, F. Vernaleone and G.L. Gessa, 1987, ["Hjf - JSulpiride binding in rat striatum, cortex and anterior pituitary: an improved assay. Pharmacol. Res. Commun. 19, 11. 777.
