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Solubilization of serotonin S2-receptors from human brain
European Journal of Pharmacology, 100 (1984) 329-333
329
Elsevier
S O L U B I L I Z A T I O N OF S E R O T O N I N S2-RECEPTORS F R O M H U M A N BRAIN A. S C H O T T E *, J.M. M A L O T E A U X *'** and P.M. L A D U R O N ***
* Laboratoire de Chimie Physiologique, International Institute of Cellular and Molecular Pathology, B 1200 Brussels, Belgium, and *** Department of Biochemical Pharmacology, Janssen Pharmaceutica Research Laboratories, B 2340 Beerse, Belgium Received 26 January 1984, accepted 7 February 1984
A. SCHOTTE, J.M. MALOTEAUX and P.M. LADURON, Solubilization of serotonin S2-receptors from human brain, European J. Pharmacol. 100 (1984) 329-333. Serotonin S2-receptors were solubilized from human brain by means of the mild detergent, lysolecithin. Previous studies have shown that the serotonin S2-receptors in human brain are mainly enriched in the cortex. A total particulate fraction from human cortex was treated with 0.25% lysolecithin. [3H]Ketanserin binding sites from the soluble extract showed the binding characteristics of S2-receptors: potent 5HT antagonists like pirenperone, methysergide and pipamperone competed for [3 H]ketanserin binding at nanomolar concentrations. The agonists bufotenin and serotonin themselves were more active than the potent dopamine agonist tetraline. Binding was saturable with a low K D (1.07 nM) and reversible. There was a good correlation between the drug potencies in both soluble and membrane preparations and also with the IC50 values previously obtained in membrane preparations and soluble extract from rat brain. Therefore, lysolecithin allows serotonin S2-receptors from human brain to be obtained in a molecularly dispersed form with the same high affinity properties as in the original membranes. Serotonin
[ 3H]Ketanserin
Soluble receptors
1. Introduction Various ligands were found to label serotonin receptors in in vitro binding assays but all do not bind to the same site. [3H]Serotonin for instance is known to label serotonin S 1 binding sites (Peroutka et al., 1981), which are recognition sites for indole derivatives without physiological relevance (Laduron and Ilien, 1982). In contrast, in the frontal cortex [3H]spiperone labels serotonin receptors (Leysen and Laduron, 1977; Leysen et al., 1978) which were called the S2-receptor sites (Peroutka et al., 1981). Recently [3H]ketanserin was reported to be a more selective ligand for serotonin S2-receptors in all brain regions (Leysen et al.,
** To whom all correspondence should be addressed: Dr J.M. Maloteaux, Laboratoire Chimie Physiol., I.C.P., U C L 7539, 75 Av. Hippocrate, 1200 Bruxelles, Belgium. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Human cortex
1982). There is no doubt about their physiological role since they are directly involved in the antagonism of various serotonergic effects measured both in vitro and in vivo (Peroutka et al., 1981; Leysen et al., 1978; 1982). Solubilization of serotonin S2-receptors was first achieved from rat frontal cortex by using lysolecithin (Ilien et al., 1980). The macromolecular complex so obtained and labelled with [3H]spiperone retained the high affinity characteristics of the serotonin S2-receptors in the original membrane (Ilien et al., 1982a). Recent work from our laboratory showed that [3H]ketanserin is a more appropriate ligand for labelling the S2-receptors -solubilized from rat and dog brain (Ilien et al., 1982b). After having described the regional distribution of S2 sites in human brain (Schotte et al., 1983) we now report on their solubilization from human cortex.
330
2. Materials and methods Human brains were obtained 6-10 h after the patient's death and immediately dissected on ice; tissue samples were then stored at - 8 0 ° C until the time of assay. Since the serotonin S2-receptors are mainly enriched in the cortex of the human brain (Schotte et al., 1983) only this brain region was used for the solubilization assays. After thawing, tissue samples were homogenized in 50 mM Tris-HCl using a Potter Elvehjem homogenizer with a conical glass tube and a motor-driven teflon pestle (4 strokes at 120 rpm). A total particulate fraction was then prei~ared as previously described. This membrane preparation was treated at 0°C (15 min at 16000 rpm) with 0.25% lysolecithin (L-a-phosphatidyl choline from egg yolk, type I, No. L-4129, Sigma) suspended in 10 mM sodium phosphate pH 7.2, 1 mM EDTA, 0.02% NaN 3 and 0.25 M sucrose. After centrifugation at 182000 x g for 1 h (rotor SW65, Spinco centrifuge), 0.4 ml aliquots of the supernatant (which was taken as soluble extract) were incubated for 20 min at 30°C with 1 nM [3H]ketanserin (87 Ci/mmol, NEN, Boston, Mass.) in presence of various concentrations of unlabelled drugs, unless states otherwise. After incubation the ligand-receptor complex was separated from the free ligand, using the charcoal procedure (Ilien et al., 1982a,b). To absorb the free ligand, 50 ~l of an activated charcoal suspension (10% charcoal and 2% BSA in water)
80
T etraline
60. e~
40 ae 8
7
6
5
4
Fig. 2. Inhibition curves of [~H]ketanserin binding by various agonist drugs in a soluble extract from human brain cortex. Non-specific binding (blank value) ( . . . . . . ) was defined by inhibition with 1 0 - 6 M methysergide.
were added to the incubation medium which was then rapidly centrifuged in a microfuge. Finally a 0.2 ml aliquot of the last supernatant was placed in a scintillation vial and its radioactivity counted (Packard scintillation spectrometer). Specific binding was defined as the difference between total binding and binding in the presence of 10 -6 M unlabelled methysergide (blank).
3. Results Agonists and antagonists from various chemical and pharmacological classes were tested for [3H]ketanserin binding using the lysolecithin ex-
2.4 ~.
I0O
.~
80
-~
60
3
- log C ( M )
r
2.0
s /
-~ 1.6 t
i
i
1.2
40
!
0.6
~:
0.4
? 2 20 "G ae
o 98
76
9876
9876
9876
109876
KETANSERIN PIPAMPERONE SPtPERONEMETHYSERGIDE PIRENPERONE
0 i
2
3
4
nM
3H-KETANSERLN
- l o g C (M)
Fig. 1. Inhibition curves for [3H]ketanserin binding by various drugs in soluble extracts from human brain cortex. Non-specific binding ( . . . . . . ) was defined by inhibition with 10 -6 M methysergide.
Fig. 3. Binding in a soluble extract from human brain cortex with increasing concentrations of [ 3 H]ketanserin. Displaceable binding (O) was defined as the difference of total binding (©) and non-displaceable (A) [3 H]ketanserin binding in presence of 10 -6 M methysergide.
331
tract from human cortex. Fig. 1 shows [3H]ketanserin displacement curves obtained with the serotonergic antagonists ketanserin, pipamperone, spiperone, methysergide and pirenperone. All these drugs showed IC50 values lower than 9 nM; methysergide, ketanserin and pipamperone gave rise to the same plateau value at maximal inhibition. In contrast pirenperone and spiperone displaced more binding sites than did the other drugs. Domperidone and prazosin, a dopaminergic and an al-adrenergic antagonist respectively did not compete with [3H]ketanserin binding at nanomolar concentrations. Specific [3 H]ketanserin binding of soluble extract represents 45% of the total binding. The serotonergic agonists serotonin and bufotenin inhibited [3H]ketanserin binding at concentrations 500 fold higher than the serotonergic antagonists but were still more potent than 2-(N,N-dipropyl)-amino-5,6-dihydroxytetraline, a potent dopamine agonist (fig. 2). Table 1 gives the IC50 values of [3H]ketanserin binding for various drugs using lysolecithin extracts from human, dog and rat brain; the values obtained for the different species are very similar. The soluble extract of human cortex was also incubated with concentrations of [3H]ketanserin
TABLE 1 Comparison of 1Cso values for [3H]ketanserin binding in soluble extracts from human, dog and rat brain. Drugs
bound/free
0.014
IN 0,012 0.010 0.008 0.006 0.004 0,002
0.2
0.4
0.6
0.8
bound
fmol/mg solubilized tissue
Fig. 4. Scatchard plots of specific [3H]ketanserin binding to solubilized serotonin S2-receptors.
increasing from 0.35 nM to 4 nM (fig. 3); nonspecific binding was assayed in the presence of 10 -6 M methysergide. Specific binding reached a plateau value at [3H]ketanserin concentrations higher than 1.5 nM, while non-specific binding increased linearly. A single slope was observed in the Scatchard analysis and allowed the calculation of a Bm,x value of 0.79 fmol/mg wet tissue and a K D of 1.07 nM which is quite comparable to the value of 0.69 nM previously reported for membrane preparations (Schotte et al., 1983).
IC5o (nM) Human
Dog
Rat
0.9 3.5 5.6 7.0 8.9 42. 247 5 012 26 600
1.4 4.2 7.5 " 7.3 " 10 a 178 a 281 28 900 a -
1.1 7.9 15 " 6.3 a 18 ~ 331 a 1000 > 10000 " -
Antagonists Pirenperone Ketanserin Pipamperone Spiperone Methysergide ( + )-Butaclamol Domperidone Prazosin ( - )-Butaclamol
•
4 IE
5
Prazosm •T • Tetrallne Ser°t ° n l n ~• i ol
6 _o
7
Domperidone ~ethyser9ide• :etanserine Spiperone •
8
• (+)-Butadarnol
Agonists Bufotenin Serotonin N,N-(Dipropyl)-amino5,6-dihydroxytetraline "
501 1995
1580 4 220
860 5 957
66 800
19 900 "
44 700 ~
Taken from Ilien et al. (1982a).
10
9
8
7
6
5
4
Soluble ( p IC50 .M )
Fig. 5. Correlation between drug affinities for [3H]ketanserin binding sites in soluble and membrane preparations of human brain cortex.
332
As shown in fig. 5 a good correlation (Spearman rank correlation coefficient: r = 0.92 n = 10) was obtained between the IC50 values of various drugs in membrane preparations and in soluble extracts. Most of the solubilization criteria were fulfilled: lack of sedimentation even after a second centrifugation (100000 × g × 60 min) and no retention on small pore filters (Gelman acrodisc 0.2 /~m pores).
4. Discussion The first attempts to solubilize serotonin S2-receptors involved rat frontal cortex using lysolecithin and [3H]spiperone as ligand (Ilien et al., 1980; Ilien et al., 1982a). One of the greatest difficulties encountered at that time was the relatively high non-specific binding; this problem was solved when [3H]ketanserin became available for labelling serotonin S2-receptors (Leysen et al., 1982). Its use enabled us to easily label solubilized serotonin S2-receptors from dog and rat brain and now those from human brain. The fact that the highest content of S2-receptor sites was in the human cortex has made this region the material of choice to undertake solubilization experiments. Lysolecithin appeared to be the most convenient detergent as it was for rat brain. As already noted (Laduron and Ilien, 1982) numerous criteria must be fulfilled in order to assess the specificity of binding sites as well as their true solubilization. Drug displacement experiments reveal that the potent serotonin antagonists like pirenperone, pipamperone, methysergide and spiperone competed with [3H]ketanserin binding in the nanomolar range. Fig. 1 shows clearly that methysergide and pipamperone displaced fewer sites than did pirenperone or spiperone; this means that the two latter drugs most probably also partly displace non-specific sites. Therefore, methysergide and pipamperone should be considered as the most appropriate drugs for getting a blank value. Since it is preferable to determine a blank value with a drug of different chemical nature from the ligand, unlabelled ketanserin should not be used for this purpose. The serotonergic agonists, bufotenin and
serotonin displaced at lower concentrations than e.g. tetralin which is a potent dopamine agonist (IC50 value in human striatum: 43 nM, unpublished results). The affinity of dopaminergic or adrenergic drugs was much less marked than that of serotonin antagonists. Therefore, one m a y conclude that the [3 H]ketanserin binding in lysolecithin human brain extract was of a serotonergic nature. The binding was reversible and saturable. The low K D value (1.07 nM) was quite compatible with the values obtained in rat frontal cortex (1.9 nM) and in membrane preparations of human cortex (0.69 nM) (Schotte et al., 1983). The straight line in the Scatchard analysis is noteworthy; it suggests a homogenous population of [3H]ketanserin binding sites. Although the K D are very similar for serotonin S2-receptors in both membranes and soluble extract, the n u m b e r of sites labelled with [3H]ketanserin recovered in soluble extracts was not so high; about 6% of the original membrane sites could still be labelled after solubilization. Such a feature seems to be a general rule for the solubilization of brain neurotransmitter receptors, such a low yield has been reported for dopamine and serotonin receptors from rat brain (Laduron and Ilien, 1982; Ilien et al., 1982a,b). The two main solubilization criteria (cf. Laduron and Ilien, 1982) were fulfilled: the absence of sedimentation of [3H]ketanserin binding sites in the soluble extract and the lack of retention on Gelman small pore filter indicate that the S2 serotonin receptors were truly dispersed in a molecular form.
Acknowledgements Part of this work was supported by a grant from IWONL. J.M. Maloteaux is an aspirant of the Belgian FNRS.
References llien, B., H. Gorissen and P.M. Laduron, 1980, Sohibilization of serotonin receptors from rat frontal cortex, Biochem. Pharmacol. 29, 3341.
333 Ilien, B., H. Gorissen and P.M. Laduron, 1982a, Characterization of solubilized serotonin ($2) receptors in rat brain, Mol. Pharmacol. 22, 243. Ilien, B., A. Schotte and P.M. Laduron, 1982b, Solubilized serotonin receptors from rat and dog brain. Selective labelling with [3H]-ketanserin, FEBS Lett. 138, 311. Laduron, P. and B. Ilien, 1982, Solubilization of brain muscarinic, dopaminergic and serotonergic receptors: a critical analysis, Biochem. Pharmacol. 31,445. Leysen, J.E. and P.M. Laduron, 1977, A serotonergic component of neuroleptic receptors, Arch. Int. Pharmacodyn. Ther. 230, 337. Leysen, J.E., C.J.E. Niemegeers, J.P. Tollenaere and P.M.
Laduron, 1978, Serotonergic component of neuroleptic receptors, Nature 272, 168. Leysen, J.E., C.J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3 H]-Ketanserin (R41467), a selective [3 H]ligand for serotonin (S2) receptors: binding properties, brain distribution and functional role, Mol. Pharmacol. 21,301. Peroutka, S.J., R.M. Lebovitz and S.H. Snyder, 1981, Two distinct central serotonin receptors with different physiological functions, Science 212, 827. Schotte, A., J.M. Maloteaux and P.M. Laduron, 1983, Characterization and regional distribution of serotonin S2 receptors in human brain, Brain Res. 276, 231.
329
Elsevier
S O L U B I L I Z A T I O N OF S E R O T O N I N S2-RECEPTORS F R O M H U M A N BRAIN A. S C H O T T E *, J.M. M A L O T E A U X *'** and P.M. L A D U R O N ***
* Laboratoire de Chimie Physiologique, International Institute of Cellular and Molecular Pathology, B 1200 Brussels, Belgium, and *** Department of Biochemical Pharmacology, Janssen Pharmaceutica Research Laboratories, B 2340 Beerse, Belgium Received 26 January 1984, accepted 7 February 1984
A. SCHOTTE, J.M. MALOTEAUX and P.M. LADURON, Solubilization of serotonin S2-receptors from human brain, European J. Pharmacol. 100 (1984) 329-333. Serotonin S2-receptors were solubilized from human brain by means of the mild detergent, lysolecithin. Previous studies have shown that the serotonin S2-receptors in human brain are mainly enriched in the cortex. A total particulate fraction from human cortex was treated with 0.25% lysolecithin. [3H]Ketanserin binding sites from the soluble extract showed the binding characteristics of S2-receptors: potent 5HT antagonists like pirenperone, methysergide and pipamperone competed for [3 H]ketanserin binding at nanomolar concentrations. The agonists bufotenin and serotonin themselves were more active than the potent dopamine agonist tetraline. Binding was saturable with a low K D (1.07 nM) and reversible. There was a good correlation between the drug potencies in both soluble and membrane preparations and also with the IC50 values previously obtained in membrane preparations and soluble extract from rat brain. Therefore, lysolecithin allows serotonin S2-receptors from human brain to be obtained in a molecularly dispersed form with the same high affinity properties as in the original membranes. Serotonin
[ 3H]Ketanserin
Soluble receptors
1. Introduction Various ligands were found to label serotonin receptors in in vitro binding assays but all do not bind to the same site. [3H]Serotonin for instance is known to label serotonin S 1 binding sites (Peroutka et al., 1981), which are recognition sites for indole derivatives without physiological relevance (Laduron and Ilien, 1982). In contrast, in the frontal cortex [3H]spiperone labels serotonin receptors (Leysen and Laduron, 1977; Leysen et al., 1978) which were called the S2-receptor sites (Peroutka et al., 1981). Recently [3H]ketanserin was reported to be a more selective ligand for serotonin S2-receptors in all brain regions (Leysen et al.,
** To whom all correspondence should be addressed: Dr J.M. Maloteaux, Laboratoire Chimie Physiol., I.C.P., U C L 7539, 75 Av. Hippocrate, 1200 Bruxelles, Belgium. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Human cortex
1982). There is no doubt about their physiological role since they are directly involved in the antagonism of various serotonergic effects measured both in vitro and in vivo (Peroutka et al., 1981; Leysen et al., 1978; 1982). Solubilization of serotonin S2-receptors was first achieved from rat frontal cortex by using lysolecithin (Ilien et al., 1980). The macromolecular complex so obtained and labelled with [3H]spiperone retained the high affinity characteristics of the serotonin S2-receptors in the original membrane (Ilien et al., 1982a). Recent work from our laboratory showed that [3H]ketanserin is a more appropriate ligand for labelling the S2-receptors -solubilized from rat and dog brain (Ilien et al., 1982b). After having described the regional distribution of S2 sites in human brain (Schotte et al., 1983) we now report on their solubilization from human cortex.
330
2. Materials and methods Human brains were obtained 6-10 h after the patient's death and immediately dissected on ice; tissue samples were then stored at - 8 0 ° C until the time of assay. Since the serotonin S2-receptors are mainly enriched in the cortex of the human brain (Schotte et al., 1983) only this brain region was used for the solubilization assays. After thawing, tissue samples were homogenized in 50 mM Tris-HCl using a Potter Elvehjem homogenizer with a conical glass tube and a motor-driven teflon pestle (4 strokes at 120 rpm). A total particulate fraction was then prei~ared as previously described. This membrane preparation was treated at 0°C (15 min at 16000 rpm) with 0.25% lysolecithin (L-a-phosphatidyl choline from egg yolk, type I, No. L-4129, Sigma) suspended in 10 mM sodium phosphate pH 7.2, 1 mM EDTA, 0.02% NaN 3 and 0.25 M sucrose. After centrifugation at 182000 x g for 1 h (rotor SW65, Spinco centrifuge), 0.4 ml aliquots of the supernatant (which was taken as soluble extract) were incubated for 20 min at 30°C with 1 nM [3H]ketanserin (87 Ci/mmol, NEN, Boston, Mass.) in presence of various concentrations of unlabelled drugs, unless states otherwise. After incubation the ligand-receptor complex was separated from the free ligand, using the charcoal procedure (Ilien et al., 1982a,b). To absorb the free ligand, 50 ~l of an activated charcoal suspension (10% charcoal and 2% BSA in water)
80
T etraline
60. e~
40 ae 8
7
6
5
4
Fig. 2. Inhibition curves of [~H]ketanserin binding by various agonist drugs in a soluble extract from human brain cortex. Non-specific binding (blank value) ( . . . . . . ) was defined by inhibition with 1 0 - 6 M methysergide.
were added to the incubation medium which was then rapidly centrifuged in a microfuge. Finally a 0.2 ml aliquot of the last supernatant was placed in a scintillation vial and its radioactivity counted (Packard scintillation spectrometer). Specific binding was defined as the difference between total binding and binding in the presence of 10 -6 M unlabelled methysergide (blank).
3. Results Agonists and antagonists from various chemical and pharmacological classes were tested for [3H]ketanserin binding using the lysolecithin ex-
2.4 ~.
I0O
.~
80
-~
60
3
- log C ( M )
r
2.0
s /
-~ 1.6 t
i
i
1.2
40
!
0.6
~:
0.4
? 2 20 "G ae
o 98
76
9876
9876
9876
109876
KETANSERIN PIPAMPERONE SPtPERONEMETHYSERGIDE PIRENPERONE
0 i
2
3
4
nM
3H-KETANSERLN
- l o g C (M)
Fig. 1. Inhibition curves for [3H]ketanserin binding by various drugs in soluble extracts from human brain cortex. Non-specific binding ( . . . . . . ) was defined by inhibition with 10 -6 M methysergide.
Fig. 3. Binding in a soluble extract from human brain cortex with increasing concentrations of [ 3 H]ketanserin. Displaceable binding (O) was defined as the difference of total binding (©) and non-displaceable (A) [3 H]ketanserin binding in presence of 10 -6 M methysergide.
331
tract from human cortex. Fig. 1 shows [3H]ketanserin displacement curves obtained with the serotonergic antagonists ketanserin, pipamperone, spiperone, methysergide and pirenperone. All these drugs showed IC50 values lower than 9 nM; methysergide, ketanserin and pipamperone gave rise to the same plateau value at maximal inhibition. In contrast pirenperone and spiperone displaced more binding sites than did the other drugs. Domperidone and prazosin, a dopaminergic and an al-adrenergic antagonist respectively did not compete with [3H]ketanserin binding at nanomolar concentrations. Specific [3 H]ketanserin binding of soluble extract represents 45% of the total binding. The serotonergic agonists serotonin and bufotenin inhibited [3H]ketanserin binding at concentrations 500 fold higher than the serotonergic antagonists but were still more potent than 2-(N,N-dipropyl)-amino-5,6-dihydroxytetraline, a potent dopamine agonist (fig. 2). Table 1 gives the IC50 values of [3H]ketanserin binding for various drugs using lysolecithin extracts from human, dog and rat brain; the values obtained for the different species are very similar. The soluble extract of human cortex was also incubated with concentrations of [3H]ketanserin
TABLE 1 Comparison of 1Cso values for [3H]ketanserin binding in soluble extracts from human, dog and rat brain. Drugs
bound/free
0.014
IN 0,012 0.010 0.008 0.006 0.004 0,002
0.2
0.4
0.6
0.8
bound
fmol/mg solubilized tissue
Fig. 4. Scatchard plots of specific [3H]ketanserin binding to solubilized serotonin S2-receptors.
increasing from 0.35 nM to 4 nM (fig. 3); nonspecific binding was assayed in the presence of 10 -6 M methysergide. Specific binding reached a plateau value at [3H]ketanserin concentrations higher than 1.5 nM, while non-specific binding increased linearly. A single slope was observed in the Scatchard analysis and allowed the calculation of a Bm,x value of 0.79 fmol/mg wet tissue and a K D of 1.07 nM which is quite comparable to the value of 0.69 nM previously reported for membrane preparations (Schotte et al., 1983).
IC5o (nM) Human
Dog
Rat
0.9 3.5 5.6 7.0 8.9 42. 247 5 012 26 600
1.4 4.2 7.5 " 7.3 " 10 a 178 a 281 28 900 a -
1.1 7.9 15 " 6.3 a 18 ~ 331 a 1000 > 10000 " -
Antagonists Pirenperone Ketanserin Pipamperone Spiperone Methysergide ( + )-Butaclamol Domperidone Prazosin ( - )-Butaclamol
•
4 IE
5
Prazosm •T • Tetrallne Ser°t ° n l n ~• i ol
6 _o
7
Domperidone ~ethyser9ide• :etanserine Spiperone •
8
• (+)-Butadarnol
Agonists Bufotenin Serotonin N,N-(Dipropyl)-amino5,6-dihydroxytetraline "
501 1995
1580 4 220
860 5 957
66 800
19 900 "
44 700 ~
Taken from Ilien et al. (1982a).
10
9
8
7
6
5
4
Soluble ( p IC50 .M )
Fig. 5. Correlation between drug affinities for [3H]ketanserin binding sites in soluble and membrane preparations of human brain cortex.
332
As shown in fig. 5 a good correlation (Spearman rank correlation coefficient: r = 0.92 n = 10) was obtained between the IC50 values of various drugs in membrane preparations and in soluble extracts. Most of the solubilization criteria were fulfilled: lack of sedimentation even after a second centrifugation (100000 × g × 60 min) and no retention on small pore filters (Gelman acrodisc 0.2 /~m pores).
4. Discussion The first attempts to solubilize serotonin S2-receptors involved rat frontal cortex using lysolecithin and [3H]spiperone as ligand (Ilien et al., 1980; Ilien et al., 1982a). One of the greatest difficulties encountered at that time was the relatively high non-specific binding; this problem was solved when [3H]ketanserin became available for labelling serotonin S2-receptors (Leysen et al., 1982). Its use enabled us to easily label solubilized serotonin S2-receptors from dog and rat brain and now those from human brain. The fact that the highest content of S2-receptor sites was in the human cortex has made this region the material of choice to undertake solubilization experiments. Lysolecithin appeared to be the most convenient detergent as it was for rat brain. As already noted (Laduron and Ilien, 1982) numerous criteria must be fulfilled in order to assess the specificity of binding sites as well as their true solubilization. Drug displacement experiments reveal that the potent serotonin antagonists like pirenperone, pipamperone, methysergide and spiperone competed with [3H]ketanserin binding in the nanomolar range. Fig. 1 shows clearly that methysergide and pipamperone displaced fewer sites than did pirenperone or spiperone; this means that the two latter drugs most probably also partly displace non-specific sites. Therefore, methysergide and pipamperone should be considered as the most appropriate drugs for getting a blank value. Since it is preferable to determine a blank value with a drug of different chemical nature from the ligand, unlabelled ketanserin should not be used for this purpose. The serotonergic agonists, bufotenin and
serotonin displaced at lower concentrations than e.g. tetralin which is a potent dopamine agonist (IC50 value in human striatum: 43 nM, unpublished results). The affinity of dopaminergic or adrenergic drugs was much less marked than that of serotonin antagonists. Therefore, one m a y conclude that the [3 H]ketanserin binding in lysolecithin human brain extract was of a serotonergic nature. The binding was reversible and saturable. The low K D value (1.07 nM) was quite compatible with the values obtained in rat frontal cortex (1.9 nM) and in membrane preparations of human cortex (0.69 nM) (Schotte et al., 1983). The straight line in the Scatchard analysis is noteworthy; it suggests a homogenous population of [3H]ketanserin binding sites. Although the K D are very similar for serotonin S2-receptors in both membranes and soluble extract, the n u m b e r of sites labelled with [3H]ketanserin recovered in soluble extracts was not so high; about 6% of the original membrane sites could still be labelled after solubilization. Such a feature seems to be a general rule for the solubilization of brain neurotransmitter receptors, such a low yield has been reported for dopamine and serotonin receptors from rat brain (Laduron and Ilien, 1982; Ilien et al., 1982a,b). The two main solubilization criteria (cf. Laduron and Ilien, 1982) were fulfilled: the absence of sedimentation of [3H]ketanserin binding sites in the soluble extract and the lack of retention on Gelman small pore filter indicate that the S2 serotonin receptors were truly dispersed in a molecular form.
Acknowledgements Part of this work was supported by a grant from IWONL. J.M. Maloteaux is an aspirant of the Belgian FNRS.
References llien, B., H. Gorissen and P.M. Laduron, 1980, Sohibilization of serotonin receptors from rat frontal cortex, Biochem. Pharmacol. 29, 3341.
333 Ilien, B., H. Gorissen and P.M. Laduron, 1982a, Characterization of solubilized serotonin ($2) receptors in rat brain, Mol. Pharmacol. 22, 243. Ilien, B., A. Schotte and P.M. Laduron, 1982b, Solubilized serotonin receptors from rat and dog brain. Selective labelling with [3H]-ketanserin, FEBS Lett. 138, 311. Laduron, P. and B. Ilien, 1982, Solubilization of brain muscarinic, dopaminergic and serotonergic receptors: a critical analysis, Biochem. Pharmacol. 31,445. Leysen, J.E. and P.M. Laduron, 1977, A serotonergic component of neuroleptic receptors, Arch. Int. Pharmacodyn. Ther. 230, 337. Leysen, J.E., C.J.E. Niemegeers, J.P. Tollenaere and P.M.
Laduron, 1978, Serotonergic component of neuroleptic receptors, Nature 272, 168. Leysen, J.E., C.J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3 H]-Ketanserin (R41467), a selective [3 H]ligand for serotonin (S2) receptors: binding properties, brain distribution and functional role, Mol. Pharmacol. 21,301. Peroutka, S.J., R.M. Lebovitz and S.H. Snyder, 1981, Two distinct central serotonin receptors with different physiological functions, Science 212, 827. Schotte, A., J.M. Maloteaux and P.M. Laduron, 1983, Characterization and regional distribution of serotonin S2 receptors in human brain, Brain Res. 276, 231.