- Email: [email protected]
Characterization of binding sites for two classes of calcium channel antagonists in human forebrain
European Journal of Pharmacology, 117 (1985) 139-142
139
Elsevier Short communication C H A R A C T E R I Z A T I O N OF B I N D I N G S I T E S F O R T W O C L A S S E S OF C A L C I U M C H A N N E L A N T A G O N I S T S IN H U M A N F O R E B R A I N REMI QUIR1ON Douglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, 6875 Blvd. Lasalle, Verdun, Qubbec H4H lR3, Canada
Received 13 August 1985, accepted 3 September 1985
R. QUIRION, Characterization of binding sites for two classes of calcium channel antagonists in human forebrain, European J. Pharmacol. 117 (1985) 139-142. 1,4-Dihydropyridine ([3H]PN200-110) and phenylalkylamine ([3H](-)D-888) bound with high affinity to human brain cortex homogenates. The ligand selectivity pattern indicates that [3H]PN200-110 binding was inhibited by related 1A-dihydropyridine analogues but not by phenylalkylamines. On the other hand, phenylalkylamines were potent inhibitors of [3H](- )D-888 binding in human brain. The radioautographic distribution of [3 H]PN200-110 and [3 H](-)D-888 binding sites demonstrates that 1,4-dihydropyridine and phenylalkylamine sites were similarly distributed in human forebrain. Calcium channel antagonist
1,4-Dihydropyridine
1. Introduction Over the last few years, various groups have reported on the existence of specific binding sites for calcium channel antagonists in peripheral as well as brain tissues (for reviews, Glossmann et ai., 1982; Janis and Triggle, 1984; Spedding, 1985). The physiological relevance of these sites in the central nervous system remains to be established. However, recent data have demonstrated that calcium channel blockers such as nifedipine and verapamil are potent inhibitors of certain calcium channels in various brain preparations (Spedding, 1985). Thus, it appears that under appropriate conditions, calcium channel antagonists could act as powerful blockers of certain types of brain calcium channels. Binding sites for calcium channel antagonists are discretely distributed in rat brain. Various groups have shown that 1,4-dihydropyridine binding sites were highly localized in areas enriched in synaptic zones such as the hippocampus and the cortex (Cortes et al., 1984; Murphy et al., 1982; Quirion, 1983). Moreover, Ferry et al. (1984) have 0014-2999/85/$03.30 ~ 1985 Elsevier Science Publishers B.V.
Phenylalkylamine
Human brain
Autoradiography
recently demonstrated that the distribution of 1,4dihydropyridine and phenylalkylamine binding sites was identical in guinea-pig brain. This suggests that these two classes of calcium channel antagonists bind to the same protein complex to induce their effects. I now report on the comparative binding properties and distribution of sites for these two classes of calcium channel blockers in human forebrain studied in membrane binding assays as well as by in vitro receptor autoradiography. The data indicate that 1,4-dihydropyridine and phenylalkylamine binding sites are similarly distributed in human brain. Thus, these two classes of calcium channel blockers are likely to act through the same population of sites in human brain.
2. Materials and methods Membrane binding assays were done with 'control' human brains obtained at autopsy (mean delay of 17 _+ 5 h, n = 6), and rapidly frozen on dry ice then kept at - 8 0 ° C until used. Subse-
140 quently, punches (500-750 rag) were taken from various areas of the cortex and the basal ganglia and homogenized in Krebs-Ringer buffer using a Brinkmann polytron at setting 5 for 20 s. The homogenates were then centrifuged three times for 15 min at 39000 × g and the final pellets were resuspended in Krebs-Ringer buffer to yield a protein concentration of 2.0-2.5 mg/ml. For the 1,4-dihydropyridine experiments, 200 >1 of this preparation was then incubated in near total darkness for 60 min in Krebs-Ringer buffer at 37°C with various concentrations of [3H]PN200-110 (84 C i / m m o l ; New England Nuclear) ranging between 0.03-5.0 nM and in presence or absence of 1.0/,M nimodipine to reach the specifically bound ligand. For the phenylalkylamine experiments, 200 ,tl of membrane preparations were incubated in near total darkness for 60 min in 50 mM Tris • HC1 buffer, pH 7.4 at 25°C with various concentrations of [3H](-)D-888 (83 Ci/mmol; gift from Knoll AG, West Germany) ranging between 0.05-5.0 nM and in presence or absence of 1.0/,M verapamil to reach the specifically bound ligand. Incubations were terminated by rapid filtration (Cell Harvester, Brandel Co. USA) through G F / B filter strips followed by two 4 ml rinses using cold incubation buffer. The filter strips were presoaked in 0.01% polyethyleneimine to decrease binding of ligands to filters. Binding of ligands was quantitated by counting filters in 6 ml Scinti-Verse II (Fisher Scientific Ltd., Canada) scintillation cocktail. For 0.5 nM [3H]PN200-110, the specific binding represented 65-70% of totally bound ligand while it was 45-50% for 0.5 nM [3H](-)D-888. All binding data were analyzed by computerized curve-fitting analysis. For receptor autoradiography, slide-mounted sections of human brain were prepared as described before for other mammalian brain (Quirion et al., 1981). Briefly, control brains were cut at autopsy (mean delay of 21 _+ 3 h, n = 5) into 4 mm thick whole hemisphere sections that were rapidly frozen in 2-methyl butane at - 4 0 ° C , mounted on cryostats chucks and cut into 30 >m sections using a large stage LKB cryomicrotome kept at - 1 7 ° C . Sections were thaw-mounted on precleaned gelatin-coated slides, dessicated overnight at 4°C than stored at - 8 0 ° C until used.
Using such a protocol made it possible to avoid most ice crystal formation (fig. 1). All brains were obtained from patients who died of cardiovascular diseases or melanomas, without evident neurological and psychiatric complications. For the 1,4-dihydropyridine experiments, frozen slide-mounted sections were incubated for 60 rain at room temperature in near total darkness in Krebs buffer solution containing 0.5 nM [ 3H]PN200-110. For phenylalkylamine binding experiments, sections were incubated for 60 rain at room temperature in near total darkness in 50 mM Tris- HCI, pH 7.4 containing 0.5 hM [ 3 H ] ( - ) D 888. At the end of incubations, slides were rinsed for 4 min in cold incubation buffer, dipped in cold distilled water to remove salts, rapidly dried then juxtaposed tightly against tritium-sensitive film (Ultrofilm, LKB Instruments). The films were developed after 12 weeks as previously described (Quirion et al., 1981). Specific binding was defined as described above for membrane binding assays.
3. Results
[3H]PN200-110 bound with high affinity to a saturable population of sites in human brain homogenates, The affinity (Kd) in all the forebrain regions investigated (43) ranged between 0.10-1.41 nM and the number of sites (Bmax) between 60.1208 f m o l / m g protein. Supavilai and Karobath (1984) have recently reported similar data for rat brain. The highest density of sites was found in the frontotemporal cortex (Brodmann's area 24) while the lowest concentration was in the globus pallidus. The caudate, putamen, amygdala and hippocampus had intermediate densities of 1,4-dihydropyridine binding sites. Similarly, [3H](-)D-888 appeated to bind to a saturable class of sites in human brain homogenates. However, the affinity of [3H](-)D-888 for its binding sites was not as high as observed with [3H]PN200-110. In Brodmann's area 24, the K d of [3HI(-)D-888 was 4.3 nM while it was 0.33 nM for [ 3H]PN200-110. Their respective B.... were in the same ranghe (235 vs. 204 f m o l / m g protein, respectively). Other brain regions were not investigated for [ 3 H I ( - )D-888 binding parameters.
141 TABLE 1
~a
Potencies of various calcium channel antagonists in inhibiting 0.5 nM [3H]PN200-110 and 1.5 nM [3H](-)D-888 binding in human brain cortex membrane preparations. The values are the means_+S.E.M. of three determinations, each in triplicate. IC5o represents the concentration of drugs needed to displace 50% of specifically bound ligands. Drug
[3H]PN200-110 IC5o (nM)
1,4-Dihydropyridine Nisoldipine 0.41 _+0.09 Nimodipine 0.41 +0.10 Nitrendipine 1.02 + 0.30 Nifedipine 3.0 + 0.5 Phenylalkylamine ( - )D-888 > 10,000 ( + )D-888 > 20,000 ( - )D-600 > 20,000 Verapamil > 20,000
[3H]( -)D-888 IC5o (nM)
3.9 _+ 0.6 54.8 _+ 8.4 28.5_+ 1.7 202 _+36
As shown in table 1, various 1,4-dihydropyridines were potent competitors of [3H]PN200-110 binding in human brain cortex, while phenylalkylamines were very weak. On the other hand, phenylalkylamines were very potent on [3 H]D-888 binding with ICs0 in the low nanomolar range (table 1). The stereoselectivity of [3H]D-888 binding sites is shown with the enantiomers of D-888. The ( - ) isomer was at least ten times more active than the ( + ) isomer in inhibiting [ 3 H ] ( - ) D - 8 8 8 binding (table 1). As shown in fig. 1, 1,4-dihydropyridine (A) and phenylalkylamine (B) binding sites are discretely distributed in human brain: High densities of both classes of sites are present in cortex, especially in layers I and III (fig. 1). High densities of sites were also seen in the hippocampus, especially in the granule cell layer of the dentate gyrus (not shown). Moderate to high densities of sites were found in caudate, putamen and claustrum (fig. 1). Lower densities are present in most thalamic (fig. 1) and hypothalamic nuclei and amygdala (not shown). Very low densities of sites were found in white matter areas (fig. 1). The global distribution of calcium channel antagonist binding sites in human brain is very similar to the one already reported for the rat brain (Cortes et al., 1984; Ferry et al., 1984; Murphy et al., 1982; Quirion, 1983). Moreover, the autoradiographic distribution of 1,4-di-
Fig. 1. Photomicrographsof the distribution of [ 3HIPN200-110 (A) and [3H](- )D-888 (B) binding sites in coronal sections of human brain at the level of the striatum. Abbreviations used: C, caudate; CL, claustrum; G, globus pallidus; P, putamen and T, thalamus. hydropyridine and of phenylalkylamine calcium channel antagonist binding sites is almost identical in human forebrain (fig. 1). Similar resuks have been reported for rat brain (Ferry et al., 1984).
4. Discussion This is the first report of the existence in human brain of specific binding sites for two different classes of calcium channel blockers. Peroutka and Allen (1983), who used [3H]nimodipine, have demonstrated the existence of 1,4-dihydropyridine binding sites in human brain homogenates. These sites had an affinity in the nanomolar range and a ligand selectivity pattern very similar to the one observed in the present study. Thus, both [3H]nimodipine and [3H]PN200-110 could be used as radiolabelled probes to characterize 1,4-dihydropyridine binding sites in human brain. There is another class of calcium channel antagonists with specific binding sites in human brain preparations. [3H](-)D-888, a phenylalkylamine, binds with high affinity to an apparently single class of saturable sites in human brain cortex. The ligand selectivity pattern for
142 these sites is very different from the one observed for the 1,4-dihydropyridine b i n d i n g sites. For example, while various p h e n y l a l k y l a m i n e s are potent competitors on their own sites, they are almost inactive on the 1,4-dihydropyridine sites. Similar data have recently been reported for guinea-pig b r a i n (Ferry et al., 1984). However, there are m a n y reports suggesting that both classes of b i n d i n g sites are allosterically linked together (for review see, G l o s s m a n n et al., 1982: Janis a n d Triggle, 1984). The respective radioautographic d i s t r i b u t i o n of [3H]PN200-110 a n d [ 3 H ] ( - ) D - 8 8 8 b i n d i n g sites in h u m a n foreb r a i n also supports this hypothesis. In this tissue, 1,4-dihydropyridine a n d p h e n y l a l k y l a m i n e b i n d ing sites are distributed identically with high densities of sites in cortical areas, caudate and putamen. Similar results have been reported for guinea-pig b r a i n (Ferry et al., 1984). Thus, various biochemical as well as radioautographic data strongly support the hypothesis that 1,4-dihydrop y r i d i n e a n d p h e n y l a l k y l a m i n e b i n d i n g sites are linked together in b r a i n tissues. The f u n c t i o n a l relevance of the presence of high densities of calcium c h a n n e l a n t a g o n i s t b i n d i n g sites in several h u m a n forebrain regions remains to be established. However, the observation that b i n d i n g sites for calcium c h a n n e l blockers are m a i n l y located in areas enriched in synaptic contacts ( M u r p h y et al., 1982) suggests that these sites could be associated with dendritic fields. Thus, it is possible that calcium c h a n n e l a n t a g o n i s t b i n d ing could be used as a marker of synaptic integrity in various neurological a n d psychiatric diseases. F u r t h e r investigations are needed to s u b s t a n t i a t e this interesting hypothesis.
Acknowledgements This project has been financed by the Medical Research Council of Canada and the Douglas Hospital Research Centre. Knoll, AG (West Germany) and Miles Laboratories (New
Haven, CT) are gratefully acknowledged for their gifts of verapamil, D-600, ( )D-888, (+)D-888, [~H]D-888 and nimodipine, nisoldipine and 'nitrendipine, respectively. The expert secretarial assistance of Mrs. Joan Currie is acknowledged.
References Cortes, R., P. Supavilai, M. Karobath and J.M. Palacios, 1984, Calcium antagonist binding sites in the rat brain: quantitative autoradiographic mapping using the 1,4-dihydropyridines [~H]PN200-110 and [3H]PY108-068, J. Neural Transm. 60, 169. Ferry, D.R., A. Goll, C. Gadow and H. Glossmann, 1984, ( )-3 H-Desmethoxyverapamillabelling of putative calcium channels in brain: autoradiographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazen binding sites, Naunyn-Schmiedeb. Arch. Pharmacol. 327, 183. Glossmann, H., D.R. Ferry, F. Lubbecke, R. Mewes and F. Hofmann, 1982, Calcium channels: direct identification with radioligand binding studies, Trends Pharmacol. Sci. 3, 43l. Janis, R.A. and D.J. Triggle, 1984, 1,4-Dihydropyridine Ca2" channel antagonists and activators: a comparison of binding characteristics with pharmacology, Drug Dev. Res. 4, 257. Murphy, K.M.M., R.J. Gould and S.H. Snyder. 1982. Autoradiographic visualization of [~H]nitrendipine binding sites in rat brain: localization to synaptic zones, European J. Pharmacol. 81, 517. Peroutka, S.J. and G.S. Allen, 1983, Calcium channel antagonist binding sites labeled by ~H-nimodipine in human brain, J. Neurosurg. 59, 933. Quirion, R., 1983, Autoradiographic localization of a calcium channel antagonist, 3H-nitrendipine binding sites in rat brain, Neurosci. Lett. 36, 267. Quirion, R., R.P. Hammer, Jr., M. Herkenham and C.B. Pert, 1981, Phencyclidine (angel dust)/sigma *opiate' receptor: visualization by tritium-sensitive film, Proc. Natl. Acad. Sci. U.S.A. 78, 5881. Spedding, M., 1985, Calcium antagonist subgroups, Trends Pharmacol. Sci. 6, 109. Supavilai, P. and M. Karobath, 1984, The interaction of [:~H]PY108-068 and of [)H]PN200-110 with calcium channel binding sites in rat brain, J. Neural Transm. 60, 149.
139
Elsevier Short communication C H A R A C T E R I Z A T I O N OF B I N D I N G S I T E S F O R T W O C L A S S E S OF C A L C I U M C H A N N E L A N T A G O N I S T S IN H U M A N F O R E B R A I N REMI QUIR1ON Douglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, 6875 Blvd. Lasalle, Verdun, Qubbec H4H lR3, Canada
Received 13 August 1985, accepted 3 September 1985
R. QUIRION, Characterization of binding sites for two classes of calcium channel antagonists in human forebrain, European J. Pharmacol. 117 (1985) 139-142. 1,4-Dihydropyridine ([3H]PN200-110) and phenylalkylamine ([3H](-)D-888) bound with high affinity to human brain cortex homogenates. The ligand selectivity pattern indicates that [3H]PN200-110 binding was inhibited by related 1A-dihydropyridine analogues but not by phenylalkylamines. On the other hand, phenylalkylamines were potent inhibitors of [3H](- )D-888 binding in human brain. The radioautographic distribution of [3 H]PN200-110 and [3 H](-)D-888 binding sites demonstrates that 1,4-dihydropyridine and phenylalkylamine sites were similarly distributed in human forebrain. Calcium channel antagonist
1,4-Dihydropyridine
1. Introduction Over the last few years, various groups have reported on the existence of specific binding sites for calcium channel antagonists in peripheral as well as brain tissues (for reviews, Glossmann et ai., 1982; Janis and Triggle, 1984; Spedding, 1985). The physiological relevance of these sites in the central nervous system remains to be established. However, recent data have demonstrated that calcium channel blockers such as nifedipine and verapamil are potent inhibitors of certain calcium channels in various brain preparations (Spedding, 1985). Thus, it appears that under appropriate conditions, calcium channel antagonists could act as powerful blockers of certain types of brain calcium channels. Binding sites for calcium channel antagonists are discretely distributed in rat brain. Various groups have shown that 1,4-dihydropyridine binding sites were highly localized in areas enriched in synaptic zones such as the hippocampus and the cortex (Cortes et al., 1984; Murphy et al., 1982; Quirion, 1983). Moreover, Ferry et al. (1984) have 0014-2999/85/$03.30 ~ 1985 Elsevier Science Publishers B.V.
Phenylalkylamine
Human brain
Autoradiography
recently demonstrated that the distribution of 1,4dihydropyridine and phenylalkylamine binding sites was identical in guinea-pig brain. This suggests that these two classes of calcium channel antagonists bind to the same protein complex to induce their effects. I now report on the comparative binding properties and distribution of sites for these two classes of calcium channel blockers in human forebrain studied in membrane binding assays as well as by in vitro receptor autoradiography. The data indicate that 1,4-dihydropyridine and phenylalkylamine binding sites are similarly distributed in human brain. Thus, these two classes of calcium channel blockers are likely to act through the same population of sites in human brain.
2. Materials and methods Membrane binding assays were done with 'control' human brains obtained at autopsy (mean delay of 17 _+ 5 h, n = 6), and rapidly frozen on dry ice then kept at - 8 0 ° C until used. Subse-
140 quently, punches (500-750 rag) were taken from various areas of the cortex and the basal ganglia and homogenized in Krebs-Ringer buffer using a Brinkmann polytron at setting 5 for 20 s. The homogenates were then centrifuged three times for 15 min at 39000 × g and the final pellets were resuspended in Krebs-Ringer buffer to yield a protein concentration of 2.0-2.5 mg/ml. For the 1,4-dihydropyridine experiments, 200 >1 of this preparation was then incubated in near total darkness for 60 min in Krebs-Ringer buffer at 37°C with various concentrations of [3H]PN200-110 (84 C i / m m o l ; New England Nuclear) ranging between 0.03-5.0 nM and in presence or absence of 1.0/,M nimodipine to reach the specifically bound ligand. For the phenylalkylamine experiments, 200 ,tl of membrane preparations were incubated in near total darkness for 60 min in 50 mM Tris • HC1 buffer, pH 7.4 at 25°C with various concentrations of [3H](-)D-888 (83 Ci/mmol; gift from Knoll AG, West Germany) ranging between 0.05-5.0 nM and in presence or absence of 1.0/,M verapamil to reach the specifically bound ligand. Incubations were terminated by rapid filtration (Cell Harvester, Brandel Co. USA) through G F / B filter strips followed by two 4 ml rinses using cold incubation buffer. The filter strips were presoaked in 0.01% polyethyleneimine to decrease binding of ligands to filters. Binding of ligands was quantitated by counting filters in 6 ml Scinti-Verse II (Fisher Scientific Ltd., Canada) scintillation cocktail. For 0.5 nM [3H]PN200-110, the specific binding represented 65-70% of totally bound ligand while it was 45-50% for 0.5 nM [3H](-)D-888. All binding data were analyzed by computerized curve-fitting analysis. For receptor autoradiography, slide-mounted sections of human brain were prepared as described before for other mammalian brain (Quirion et al., 1981). Briefly, control brains were cut at autopsy (mean delay of 21 _+ 3 h, n = 5) into 4 mm thick whole hemisphere sections that were rapidly frozen in 2-methyl butane at - 4 0 ° C , mounted on cryostats chucks and cut into 30 >m sections using a large stage LKB cryomicrotome kept at - 1 7 ° C . Sections were thaw-mounted on precleaned gelatin-coated slides, dessicated overnight at 4°C than stored at - 8 0 ° C until used.
Using such a protocol made it possible to avoid most ice crystal formation (fig. 1). All brains were obtained from patients who died of cardiovascular diseases or melanomas, without evident neurological and psychiatric complications. For the 1,4-dihydropyridine experiments, frozen slide-mounted sections were incubated for 60 rain at room temperature in near total darkness in Krebs buffer solution containing 0.5 nM [ 3H]PN200-110. For phenylalkylamine binding experiments, sections were incubated for 60 rain at room temperature in near total darkness in 50 mM Tris- HCI, pH 7.4 containing 0.5 hM [ 3 H ] ( - ) D 888. At the end of incubations, slides were rinsed for 4 min in cold incubation buffer, dipped in cold distilled water to remove salts, rapidly dried then juxtaposed tightly against tritium-sensitive film (Ultrofilm, LKB Instruments). The films were developed after 12 weeks as previously described (Quirion et al., 1981). Specific binding was defined as described above for membrane binding assays.
3. Results
[3H]PN200-110 bound with high affinity to a saturable population of sites in human brain homogenates, The affinity (Kd) in all the forebrain regions investigated (43) ranged between 0.10-1.41 nM and the number of sites (Bmax) between 60.1208 f m o l / m g protein. Supavilai and Karobath (1984) have recently reported similar data for rat brain. The highest density of sites was found in the frontotemporal cortex (Brodmann's area 24) while the lowest concentration was in the globus pallidus. The caudate, putamen, amygdala and hippocampus had intermediate densities of 1,4-dihydropyridine binding sites. Similarly, [3H](-)D-888 appeated to bind to a saturable class of sites in human brain homogenates. However, the affinity of [3H](-)D-888 for its binding sites was not as high as observed with [3H]PN200-110. In Brodmann's area 24, the K d of [3HI(-)D-888 was 4.3 nM while it was 0.33 nM for [ 3H]PN200-110. Their respective B.... were in the same ranghe (235 vs. 204 f m o l / m g protein, respectively). Other brain regions were not investigated for [ 3 H I ( - )D-888 binding parameters.
141 TABLE 1
~a
Potencies of various calcium channel antagonists in inhibiting 0.5 nM [3H]PN200-110 and 1.5 nM [3H](-)D-888 binding in human brain cortex membrane preparations. The values are the means_+S.E.M. of three determinations, each in triplicate. IC5o represents the concentration of drugs needed to displace 50% of specifically bound ligands. Drug
[3H]PN200-110 IC5o (nM)
1,4-Dihydropyridine Nisoldipine 0.41 _+0.09 Nimodipine 0.41 +0.10 Nitrendipine 1.02 + 0.30 Nifedipine 3.0 + 0.5 Phenylalkylamine ( - )D-888 > 10,000 ( + )D-888 > 20,000 ( - )D-600 > 20,000 Verapamil > 20,000
[3H]( -)D-888 IC5o (nM)
3.9 _+ 0.6 54.8 _+ 8.4 28.5_+ 1.7 202 _+36
As shown in table 1, various 1,4-dihydropyridines were potent competitors of [3H]PN200-110 binding in human brain cortex, while phenylalkylamines were very weak. On the other hand, phenylalkylamines were very potent on [3 H]D-888 binding with ICs0 in the low nanomolar range (table 1). The stereoselectivity of [3H]D-888 binding sites is shown with the enantiomers of D-888. The ( - ) isomer was at least ten times more active than the ( + ) isomer in inhibiting [ 3 H ] ( - ) D - 8 8 8 binding (table 1). As shown in fig. 1, 1,4-dihydropyridine (A) and phenylalkylamine (B) binding sites are discretely distributed in human brain: High densities of both classes of sites are present in cortex, especially in layers I and III (fig. 1). High densities of sites were also seen in the hippocampus, especially in the granule cell layer of the dentate gyrus (not shown). Moderate to high densities of sites were found in caudate, putamen and claustrum (fig. 1). Lower densities are present in most thalamic (fig. 1) and hypothalamic nuclei and amygdala (not shown). Very low densities of sites were found in white matter areas (fig. 1). The global distribution of calcium channel antagonist binding sites in human brain is very similar to the one already reported for the rat brain (Cortes et al., 1984; Ferry et al., 1984; Murphy et al., 1982; Quirion, 1983). Moreover, the autoradiographic distribution of 1,4-di-
Fig. 1. Photomicrographsof the distribution of [ 3HIPN200-110 (A) and [3H](- )D-888 (B) binding sites in coronal sections of human brain at the level of the striatum. Abbreviations used: C, caudate; CL, claustrum; G, globus pallidus; P, putamen and T, thalamus. hydropyridine and of phenylalkylamine calcium channel antagonist binding sites is almost identical in human forebrain (fig. 1). Similar resuks have been reported for rat brain (Ferry et al., 1984).
4. Discussion This is the first report of the existence in human brain of specific binding sites for two different classes of calcium channel blockers. Peroutka and Allen (1983), who used [3H]nimodipine, have demonstrated the existence of 1,4-dihydropyridine binding sites in human brain homogenates. These sites had an affinity in the nanomolar range and a ligand selectivity pattern very similar to the one observed in the present study. Thus, both [3H]nimodipine and [3H]PN200-110 could be used as radiolabelled probes to characterize 1,4-dihydropyridine binding sites in human brain. There is another class of calcium channel antagonists with specific binding sites in human brain preparations. [3H](-)D-888, a phenylalkylamine, binds with high affinity to an apparently single class of saturable sites in human brain cortex. The ligand selectivity pattern for
142 these sites is very different from the one observed for the 1,4-dihydropyridine b i n d i n g sites. For example, while various p h e n y l a l k y l a m i n e s are potent competitors on their own sites, they are almost inactive on the 1,4-dihydropyridine sites. Similar data have recently been reported for guinea-pig b r a i n (Ferry et al., 1984). However, there are m a n y reports suggesting that both classes of b i n d i n g sites are allosterically linked together (for review see, G l o s s m a n n et al., 1982: Janis a n d Triggle, 1984). The respective radioautographic d i s t r i b u t i o n of [3H]PN200-110 a n d [ 3 H ] ( - ) D - 8 8 8 b i n d i n g sites in h u m a n foreb r a i n also supports this hypothesis. In this tissue, 1,4-dihydropyridine a n d p h e n y l a l k y l a m i n e b i n d ing sites are distributed identically with high densities of sites in cortical areas, caudate and putamen. Similar results have been reported for guinea-pig b r a i n (Ferry et al., 1984). Thus, various biochemical as well as radioautographic data strongly support the hypothesis that 1,4-dihydrop y r i d i n e a n d p h e n y l a l k y l a m i n e b i n d i n g sites are linked together in b r a i n tissues. The f u n c t i o n a l relevance of the presence of high densities of calcium c h a n n e l a n t a g o n i s t b i n d i n g sites in several h u m a n forebrain regions remains to be established. However, the observation that b i n d i n g sites for calcium c h a n n e l blockers are m a i n l y located in areas enriched in synaptic contacts ( M u r p h y et al., 1982) suggests that these sites could be associated with dendritic fields. Thus, it is possible that calcium c h a n n e l a n t a g o n i s t b i n d ing could be used as a marker of synaptic integrity in various neurological a n d psychiatric diseases. F u r t h e r investigations are needed to s u b s t a n t i a t e this interesting hypothesis.
Acknowledgements This project has been financed by the Medical Research Council of Canada and the Douglas Hospital Research Centre. Knoll, AG (West Germany) and Miles Laboratories (New
Haven, CT) are gratefully acknowledged for their gifts of verapamil, D-600, ( )D-888, (+)D-888, [~H]D-888 and nimodipine, nisoldipine and 'nitrendipine, respectively. The expert secretarial assistance of Mrs. Joan Currie is acknowledged.
References Cortes, R., P. Supavilai, M. Karobath and J.M. Palacios, 1984, Calcium antagonist binding sites in the rat brain: quantitative autoradiographic mapping using the 1,4-dihydropyridines [~H]PN200-110 and [3H]PY108-068, J. Neural Transm. 60, 169. Ferry, D.R., A. Goll, C. Gadow and H. Glossmann, 1984, ( )-3 H-Desmethoxyverapamillabelling of putative calcium channels in brain: autoradiographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazen binding sites, Naunyn-Schmiedeb. Arch. Pharmacol. 327, 183. Glossmann, H., D.R. Ferry, F. Lubbecke, R. Mewes and F. Hofmann, 1982, Calcium channels: direct identification with radioligand binding studies, Trends Pharmacol. Sci. 3, 43l. Janis, R.A. and D.J. Triggle, 1984, 1,4-Dihydropyridine Ca2" channel antagonists and activators: a comparison of binding characteristics with pharmacology, Drug Dev. Res. 4, 257. Murphy, K.M.M., R.J. Gould and S.H. Snyder. 1982. Autoradiographic visualization of [~H]nitrendipine binding sites in rat brain: localization to synaptic zones, European J. Pharmacol. 81, 517. Peroutka, S.J. and G.S. Allen, 1983, Calcium channel antagonist binding sites labeled by ~H-nimodipine in human brain, J. Neurosurg. 59, 933. Quirion, R., 1983, Autoradiographic localization of a calcium channel antagonist, 3H-nitrendipine binding sites in rat brain, Neurosci. Lett. 36, 267. Quirion, R., R.P. Hammer, Jr., M. Herkenham and C.B. Pert, 1981, Phencyclidine (angel dust)/sigma *opiate' receptor: visualization by tritium-sensitive film, Proc. Natl. Acad. Sci. U.S.A. 78, 5881. Spedding, M., 1985, Calcium antagonist subgroups, Trends Pharmacol. Sci. 6, 109. Supavilai, P. and M. Karobath, 1984, The interaction of [:~H]PY108-068 and of [)H]PN200-110 with calcium channel binding sites in rat brain, J. Neural Transm. 60, 149.