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5HT-receptor antagonist properties of SCH 23390 in vascular smooth muscle and brain
European Journal of Pharmacology. 105 (1984) 339- 342
339
Elsevier Short communication 5 H T - R E C E P T O R A N T A G O N I S T P R O P E R T I E S O F SCH 23390 IN VASCULAR S M O O T H M U S C L E AND BRAIN PETE:R E. IIICKS. tlANS SCHOEMAKERand SALOMON Z. LANGER * Department of Biology. Laboratoires d'Etudes et de Recherches ,~ynthblabo. 58. rue de la Glacibre. 75013 Parts, France
Received 30 July 1984, accepted 13 August 1984
P.E. HICKS, H. SCHOEMAKER and S.Z. LANGER, 5HT-receptor antagonist properties of S C H 23390 in vascular smooth muscle and brain. European J. Pharmacol. 105 (1984)339-342. The dopamine D~-receptor antagonist SCH 23390 was a potent competitive antagonist of 5HT-induced vasoconstriction in the isolated perfused rat tail artery preparation (pA 2 8.17) but a very weak antagonist of phenylephrine-induced responses (pA 2 5.94). In rat brain cerebral cortex, SCH 23390 inhibited 5-HT2-sensitive [3H]spiperone binding with an ICs0 of 112 nM. Binding of [3H]5HT to 5HT 1 receptors in the cortex was inhibited by SCH 23390 with an IC5o of 2.49 p.M. SCH 23390 has significant affinity for 5HT receptors in addition to the reported selective dopamine D~-receptor antagonist properties. SCH 23390
5-HT receptor antagonists
I. Introduction
2. Materials and methods
SCH 23390 [(R-(+ )-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine] has been reported to be a potent antagonist of dopamine D~-receptors in the central nervous system (lorio et al., 1983; Hyttel, 1984) and has recently been shown to be a very selective antagonist at the smooth muscle D~-receptor subtype which mediates vasodilatation in dog renal (Goldberg et al., 1984) or mesenteric vasculature (Hilditch et al., 1984). On the other hand, it was shown that SCH 23390 has affinity for the 5HT2-sensitive [aH]spiperone binding site in rat brain (Hyttel, 1983). We now report on a potent 5HT receptor antagonist effect of SCH 23390 in the isolated perfused tail artery preparation of the rat (Hicks and Langer, 1983). We also studied the inhibition by SCH 23390 of radioligand binding to 5HT receptor subtypes in the rat cerebral cortex.
2.1. R a t tail artery preparation
• To whom all correspondenceshould be addressed, 0014-2999/84/$03.00 :'~'1984 Elsevier Science Publishers B.V.
Male Sprague-Dawley rats (250-300 g) were anaesthetised with pentobarbitone (60 m g / k g i.p.). The proximal tail artery was removed, cannulated and perfused and superfused at a flow rate of 4 m l / m i n with Krebs bicarbonate medium at 37°C, bubbled with 95% O 2 and 5% CO 2 (according to the method of Hicks and Langer, 1983). Vasoconstrictor responses were evoked by different concentrations of 5HT, or phenylephrine, and were measured as increases in back perfusion pressure in mmHg. The Krebs medium routinely contained propranoiol (1 p,M), indomethacin (2.6 p,M) and pargyline (10 #M) in order to block/3-adrenoceptors, prostaglandin synthesis and monoamine oxidase activity. Preparations were first sensitised (for 1 h) to repeated challenges with phenylephrine (1 p,M) until stable responses were obtained. Subsequently, concentration-response curves for phenylephrine (0.1-3 p,M) or 5HT (0.03-1 p,M) were determined. The concentration-response curves for these agonists were repeated after incubation of
34O
the tissue for 30 min with increasing concentrations of SCH 23390 (10 nM, 100 nM and 1000 nM) in the perfusing medium,
2.2. Receptor binding Male Sprague-Dawley rats weighing 150-200 g were used. Following decapitation, the brain was excised and the cerebral cortex dissected. Binding of [3H]5HT was measured essentially as described by Schnellmann et al. (1984). Briefly, the cerebral cortex was homogenized in 50 mM Tris-HCl buffer (pH 7.4 at 25°C) using a polytron. A twice washed membrane preparation was prepared by repeated centrifugation for 10 min at 50000 × g and resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM Tris buffer (pH 7.4) containing 0.1%, ascorbic acid, 4 mM CaCI z and 10 ttM pargyline and preincubated for 10 min at 37°C. An aliquot (750 p,1) of this membrane preparation (10 mg tissue/ml) was then incubated for 10 min at 37°C with 5 nM [3H]5HT in a final volume of 1 ml. Following incubation, the membranes were collected by filtration over Whatman G F / B filters that were washed subsequently using three 5 ml aliquots of Tris buffer. Nonspecific binding was defined by using 10 ~tM unlabelled 5HT and amounted to 40% of the total binding at the concentration of 5 nM [~H]5HT. To evaluate the binding of [3H]spiperone to 5HT 2 receptors in the rat cerebral cortex, a thrice washed homogenate was prepared as described above in 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCI and 5 mM KCI. The final membrane suspension (30 mg tissue/ml) was preincubated for 5 min at 37°C with 0.1% ascorbic acid and 10 ~M pargyline. After incubation of the membranes with 0.3 nM [ 3H]spiperone for 20 rain at 37 oc, 1 mi aliquots were filtered over Whatman G F / B filters, and washed twice with 10 ml cold buffer. Specific binding was defined as that displaced by 100 ~M 5HT and represented 50%. of the total binding at 0.3 nM [3H]spiperone. In each case, the filters were dried after filtration, and the retained radioactivity then quantified by liquid scintillation spectrometry. Drug inhibition of binding was studied using 5
to 8 concentrations of the concentration required to specific binding (ICsu) was ventional graphic and least sion analysis.
test compound. The inhibit 50% of the calculated using consquares linear regres-
2.3. Drugs (-)-Phenylephrine hydrochloride (Koch-Eight): 5-hydroxytryptamine creatinine sulphate (Sigma): ( + )-propranolol hydrochloride (I.C.I.); indomethacin (Sigma): pargyline hydrochloride (Sigma): SCH 23390 ([R-(+)-8-chloro-2,3,4,5-tetrahydro3-methyl-5-phenyl-lH-3-benzazepine] (Schering): metcrgoline (Farmitalia); [3H]spiperone (23.8 C i / m m o l ) (New England Nuclear): [3H]5-hydroxytryptamine creatinine sulphate (13.5 C i / mmol) (Amersham).
3. Results In the perfused-superfused rat tail artery preparation vasoconstrictor concentration-response curves evoked by either 5HT, or phenylephrine were readily reproducible in four consecutive concentration-response curves. Incubation of the preparation with increasing concentrations of SCH 23390 in the perfusing medium caused progressive parallel rightward displacement of the 5HT concentration-response curve (fig. 1) with no significant depression of the maximum response to 5HT. Schild analysis of these data is shown in table 1. I'ABI_L1 Antagonist potency of SCIt 23390 against 5tiT- or phenylephrine-induced contractions in the perfused rat tail artery preparation, p A 2 and slope were deri','ed from Schild analysis, of the antagonism of responsecurves for 5HT or phenylephrinc by SCH 23390 tested at three concentrations (30 min incuhation). "/he results were calculated at the level of ECho values [or the agonists. Values in parentheses are 95% confidence intervals. n: number of experimenls. Agonist
n
5HT
4 4
Phenylephrine
pA 2 8.17 (8.12-8.23) 5.94 (5.79-6.26)
Slope __ 1.06 (0.93-1.21) 1.26 (1.0 -1.42)
341
,00_
/
Control
,,0aM, 7-
I1 ~MI '
--
--
1
_L
E= E
a2
¢...3
0
003
0.1
D3
1
3
10
30
]
)
100
300
5HI (I,M I Fig. I. Blockade by SCH 23390 of the vasoconstrictor responses to 5-hydroxytryptamine in the perfused and superfused rat tail artery in vitro. Ordinate: V a s o c o n s t r i c t o r effect e x p r e s s e d as p e r c e n t o f the m a x i m u m c o n t r o l r e s p o n s e to 5 l I T . Ahsctssa: L o g c o n c e n t r a t i o n o f 5 H T ( # M ) . S h o w n a r e m e a n + S.E.M.. n = 4 for e a c h c o n c e n t r a t i o n of S C H 23390. V a s o c o n s t r i c t o r r e s p o n s e s to 5 H T ( e b e f o r e o r a f t e r S C H 233911 ( • • 10 n M : 13 LI 100 n M ; or • • 1 IuM).
The vasoconstrictor effects of phenylephrine were also antagonised by SCH 23390 but only at high concentrations resulting in pA 2 values which were at least 2 log units lower than that against 5HT (table 1). Binding studies using membranes from the rat cerebral cortex showed that SCH 23390 inhibits [3H]spiperone binding to 5HT 2 receptors with an IC50 of 112 _+ 25 nM. SCH 23390 was less potent as an inhibitor of [3HI5HT binding to 5HT) receptors in this tissue (IC50 = 2.49 + 0.73 p.M).
4. Discussion SCH 23390 is a potent antagonist of dopamine D~-receptors in both the central nervous system (lorio et al., 1983; Hyttel, 1983) and vascular smooth muscle (Goldberg et al., 1984: Hilditch et al., 1984). It was also reported that SCH 23390 is either weak or inactive at blocking dopamine D 2receptors (lorio et al., 1983). The present results indicate that SCH 23390 possesses potent 5HT receptor antagonist properties in vascular smooth muscle in vitro but is devoid of at-receptor blocking properties except at high concentrations, The receptor binding data indicate that SCH
O)
23390 has a higher affinity for the [3H]spiperone recognition site (5HT2) than for the [3H]5HT site (5HTt) in rat cerebral cortex. Similarly, Hyttel (1983) reported a relatively high affinity (K i = 30 nM) for SCH 23390 to inhibit [3H]spiperone binding to 5HT 2 receptors. These data are not incompatible with the 5HT receptor antagonist properties of SCH 23390 in tail arteries being mediated through 5HT 2 receptors. The 5HT receptors mediating vasoconstriction in the rat tail artery are however not easy to characterize in terms of 5HT receptor subtype because methysergide, methergoline and ketanserin are all potent antagonists of 5HT-induced contraction in this tissue (Hicks and Langer, 1983: Bradley et al., 1983). The significance of the 5HT receptor antagonist properties of SCH 23390 in relation to the potential neuroleptic action of this drug, and its behavioural and pharmacological profile in the central nervous system remain to be clarified, since many of the classical neuroleptic drugs have very high affinity for 5HT, recognition sites in receptor binding assays (Hyttel, 1983; Barone et al., 1984). However these findings emphasise the need to carefully examine the activity of new compounds on a wide range of receptor sites before they can
3,42
be used as selective agents in the classification of receptor subtypes, preferably using both functional tests and receptor binding assays.
References Barone, D., A. Condo, U. Piazza and A. Glasser, 1984, Zetidoline: a new antidopaminergic agent unable to interact in vitro with S2-serotonergic receptors of the rat brain, IRCS Med. Sci. 12, 204. Bradley, P.B., P.P.A. Humphrey and R.H. Williams, 1983, Arc vascular "I)' and '5HT2'-receptors for 5-hydroxytryptamine the same?. Br. J. Pharmacol. 79, 295P. Goldherg, L.I., D. Glock, J.D. Kohli and A. Barnett, 1984, Separation of peripheral dopamine receptors by a selective
DA I antagonist S('H 23390, Hs,pertension 6 (Suppl. 11,
1-25. Hicks, P.t-. and S.Z. l,anger, 1983, Antagonism by tetrahvdrofl-carboline of the vasoconstrictor responses to trypt~iminc in rat tail arteries, European J. Pharmacol. 96, 145. Hilditch, A., G.M. Drew and R.J. Naylor, 1984, S('1t 23390 is a very potent and selective antagonist at vascular dopamine receptors, European J. Pharmacol. 97, 333. Hyttel, J., 1983, SCH 23390 - the first selccti~,'e dopamine I) I antagonist, European J. Pharmacol. 91, 153. loric, L.C., A. Barnett, I-:.tt. Leitz, P. Hower and C.A. Korduha, 1983, S('H 23390, a potential benzazepinc anlispychotic with unique interactions on dopaminergic systems, J. Pharmacol. Exp. Ther. 226, 462. Schnellmann, R.G., S.J. Waters and D.L. Nelson. 1984, [ ~lt]5Hydroxytryptamine binding sites: species and tissue ,,~riation. J. Neurochem. 42, 65.
339
Elsevier Short communication 5 H T - R E C E P T O R A N T A G O N I S T P R O P E R T I E S O F SCH 23390 IN VASCULAR S M O O T H M U S C L E AND BRAIN PETE:R E. IIICKS. tlANS SCHOEMAKERand SALOMON Z. LANGER * Department of Biology. Laboratoires d'Etudes et de Recherches ,~ynthblabo. 58. rue de la Glacibre. 75013 Parts, France
Received 30 July 1984, accepted 13 August 1984
P.E. HICKS, H. SCHOEMAKER and S.Z. LANGER, 5HT-receptor antagonist properties of S C H 23390 in vascular smooth muscle and brain. European J. Pharmacol. 105 (1984)339-342. The dopamine D~-receptor antagonist SCH 23390 was a potent competitive antagonist of 5HT-induced vasoconstriction in the isolated perfused rat tail artery preparation (pA 2 8.17) but a very weak antagonist of phenylephrine-induced responses (pA 2 5.94). In rat brain cerebral cortex, SCH 23390 inhibited 5-HT2-sensitive [3H]spiperone binding with an ICs0 of 112 nM. Binding of [3H]5HT to 5HT 1 receptors in the cortex was inhibited by SCH 23390 with an IC5o of 2.49 p.M. SCH 23390 has significant affinity for 5HT receptors in addition to the reported selective dopamine D~-receptor antagonist properties. SCH 23390
5-HT receptor antagonists
I. Introduction
2. Materials and methods
SCH 23390 [(R-(+ )-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine] has been reported to be a potent antagonist of dopamine D~-receptors in the central nervous system (lorio et al., 1983; Hyttel, 1984) and has recently been shown to be a very selective antagonist at the smooth muscle D~-receptor subtype which mediates vasodilatation in dog renal (Goldberg et al., 1984) or mesenteric vasculature (Hilditch et al., 1984). On the other hand, it was shown that SCH 23390 has affinity for the 5HT2-sensitive [aH]spiperone binding site in rat brain (Hyttel, 1983). We now report on a potent 5HT receptor antagonist effect of SCH 23390 in the isolated perfused tail artery preparation of the rat (Hicks and Langer, 1983). We also studied the inhibition by SCH 23390 of radioligand binding to 5HT receptor subtypes in the rat cerebral cortex.
2.1. R a t tail artery preparation
• To whom all correspondenceshould be addressed, 0014-2999/84/$03.00 :'~'1984 Elsevier Science Publishers B.V.
Male Sprague-Dawley rats (250-300 g) were anaesthetised with pentobarbitone (60 m g / k g i.p.). The proximal tail artery was removed, cannulated and perfused and superfused at a flow rate of 4 m l / m i n with Krebs bicarbonate medium at 37°C, bubbled with 95% O 2 and 5% CO 2 (according to the method of Hicks and Langer, 1983). Vasoconstrictor responses were evoked by different concentrations of 5HT, or phenylephrine, and were measured as increases in back perfusion pressure in mmHg. The Krebs medium routinely contained propranoiol (1 p,M), indomethacin (2.6 p,M) and pargyline (10 #M) in order to block/3-adrenoceptors, prostaglandin synthesis and monoamine oxidase activity. Preparations were first sensitised (for 1 h) to repeated challenges with phenylephrine (1 p,M) until stable responses were obtained. Subsequently, concentration-response curves for phenylephrine (0.1-3 p,M) or 5HT (0.03-1 p,M) were determined. The concentration-response curves for these agonists were repeated after incubation of
34O
the tissue for 30 min with increasing concentrations of SCH 23390 (10 nM, 100 nM and 1000 nM) in the perfusing medium,
2.2. Receptor binding Male Sprague-Dawley rats weighing 150-200 g were used. Following decapitation, the brain was excised and the cerebral cortex dissected. Binding of [3H]5HT was measured essentially as described by Schnellmann et al. (1984). Briefly, the cerebral cortex was homogenized in 50 mM Tris-HCl buffer (pH 7.4 at 25°C) using a polytron. A twice washed membrane preparation was prepared by repeated centrifugation for 10 min at 50000 × g and resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM Tris buffer (pH 7.4) containing 0.1%, ascorbic acid, 4 mM CaCI z and 10 ttM pargyline and preincubated for 10 min at 37°C. An aliquot (750 p,1) of this membrane preparation (10 mg tissue/ml) was then incubated for 10 min at 37°C with 5 nM [3H]5HT in a final volume of 1 ml. Following incubation, the membranes were collected by filtration over Whatman G F / B filters that were washed subsequently using three 5 ml aliquots of Tris buffer. Nonspecific binding was defined by using 10 ~tM unlabelled 5HT and amounted to 40% of the total binding at the concentration of 5 nM [~H]5HT. To evaluate the binding of [3H]spiperone to 5HT 2 receptors in the rat cerebral cortex, a thrice washed homogenate was prepared as described above in 50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCI and 5 mM KCI. The final membrane suspension (30 mg tissue/ml) was preincubated for 5 min at 37°C with 0.1% ascorbic acid and 10 ~M pargyline. After incubation of the membranes with 0.3 nM [ 3H]spiperone for 20 rain at 37 oc, 1 mi aliquots were filtered over Whatman G F / B filters, and washed twice with 10 ml cold buffer. Specific binding was defined as that displaced by 100 ~M 5HT and represented 50%. of the total binding at 0.3 nM [3H]spiperone. In each case, the filters were dried after filtration, and the retained radioactivity then quantified by liquid scintillation spectrometry. Drug inhibition of binding was studied using 5
to 8 concentrations of the concentration required to specific binding (ICsu) was ventional graphic and least sion analysis.
test compound. The inhibit 50% of the calculated using consquares linear regres-
2.3. Drugs (-)-Phenylephrine hydrochloride (Koch-Eight): 5-hydroxytryptamine creatinine sulphate (Sigma): ( + )-propranolol hydrochloride (I.C.I.); indomethacin (Sigma): pargyline hydrochloride (Sigma): SCH 23390 ([R-(+)-8-chloro-2,3,4,5-tetrahydro3-methyl-5-phenyl-lH-3-benzazepine] (Schering): metcrgoline (Farmitalia); [3H]spiperone (23.8 C i / m m o l ) (New England Nuclear): [3H]5-hydroxytryptamine creatinine sulphate (13.5 C i / mmol) (Amersham).
3. Results In the perfused-superfused rat tail artery preparation vasoconstrictor concentration-response curves evoked by either 5HT, or phenylephrine were readily reproducible in four consecutive concentration-response curves. Incubation of the preparation with increasing concentrations of SCH 23390 in the perfusing medium caused progressive parallel rightward displacement of the 5HT concentration-response curve (fig. 1) with no significant depression of the maximum response to 5HT. Schild analysis of these data is shown in table 1. I'ABI_L1 Antagonist potency of SCIt 23390 against 5tiT- or phenylephrine-induced contractions in the perfused rat tail artery preparation, p A 2 and slope were deri','ed from Schild analysis, of the antagonism of responsecurves for 5HT or phenylephrinc by SCH 23390 tested at three concentrations (30 min incuhation). "/he results were calculated at the level of ECho values [or the agonists. Values in parentheses are 95% confidence intervals. n: number of experimenls. Agonist
n
5HT
4 4
Phenylephrine
pA 2 8.17 (8.12-8.23) 5.94 (5.79-6.26)
Slope __ 1.06 (0.93-1.21) 1.26 (1.0 -1.42)
341
,00_
/
Control
,,0aM, 7-
I1 ~MI '
--
--
1
_L
E= E
a2
¢...3
0
003
0.1
D3
1
3
10
30
]
)
100
300
5HI (I,M I Fig. I. Blockade by SCH 23390 of the vasoconstrictor responses to 5-hydroxytryptamine in the perfused and superfused rat tail artery in vitro. Ordinate: V a s o c o n s t r i c t o r effect e x p r e s s e d as p e r c e n t o f the m a x i m u m c o n t r o l r e s p o n s e to 5 l I T . Ahsctssa: L o g c o n c e n t r a t i o n o f 5 H T ( # M ) . S h o w n a r e m e a n + S.E.M.. n = 4 for e a c h c o n c e n t r a t i o n of S C H 23390. V a s o c o n s t r i c t o r r e s p o n s e s to 5 H T ( e b e f o r e o r a f t e r S C H 233911 ( • • 10 n M : 13 LI 100 n M ; or • • 1 IuM).
The vasoconstrictor effects of phenylephrine were also antagonised by SCH 23390 but only at high concentrations resulting in pA 2 values which were at least 2 log units lower than that against 5HT (table 1). Binding studies using membranes from the rat cerebral cortex showed that SCH 23390 inhibits [3H]spiperone binding to 5HT 2 receptors with an IC50 of 112 _+ 25 nM. SCH 23390 was less potent as an inhibitor of [3HI5HT binding to 5HT) receptors in this tissue (IC50 = 2.49 + 0.73 p.M).
4. Discussion SCH 23390 is a potent antagonist of dopamine D~-receptors in both the central nervous system (lorio et al., 1983; Hyttel, 1983) and vascular smooth muscle (Goldberg et al., 1984: Hilditch et al., 1984). It was also reported that SCH 23390 is either weak or inactive at blocking dopamine D 2receptors (lorio et al., 1983). The present results indicate that SCH 23390 possesses potent 5HT receptor antagonist properties in vascular smooth muscle in vitro but is devoid of at-receptor blocking properties except at high concentrations, The receptor binding data indicate that SCH
O)
23390 has a higher affinity for the [3H]spiperone recognition site (5HT2) than for the [3H]5HT site (5HTt) in rat cerebral cortex. Similarly, Hyttel (1983) reported a relatively high affinity (K i = 30 nM) for SCH 23390 to inhibit [3H]spiperone binding to 5HT 2 receptors. These data are not incompatible with the 5HT receptor antagonist properties of SCH 23390 in tail arteries being mediated through 5HT 2 receptors. The 5HT receptors mediating vasoconstriction in the rat tail artery are however not easy to characterize in terms of 5HT receptor subtype because methysergide, methergoline and ketanserin are all potent antagonists of 5HT-induced contraction in this tissue (Hicks and Langer, 1983: Bradley et al., 1983). The significance of the 5HT receptor antagonist properties of SCH 23390 in relation to the potential neuroleptic action of this drug, and its behavioural and pharmacological profile in the central nervous system remain to be clarified, since many of the classical neuroleptic drugs have very high affinity for 5HT, recognition sites in receptor binding assays (Hyttel, 1983; Barone et al., 1984). However these findings emphasise the need to carefully examine the activity of new compounds on a wide range of receptor sites before they can
3,42
be used as selective agents in the classification of receptor subtypes, preferably using both functional tests and receptor binding assays.
References Barone, D., A. Condo, U. Piazza and A. Glasser, 1984, Zetidoline: a new antidopaminergic agent unable to interact in vitro with S2-serotonergic receptors of the rat brain, IRCS Med. Sci. 12, 204. Bradley, P.B., P.P.A. Humphrey and R.H. Williams, 1983, Arc vascular "I)' and '5HT2'-receptors for 5-hydroxytryptamine the same?. Br. J. Pharmacol. 79, 295P. Goldherg, L.I., D. Glock, J.D. Kohli and A. Barnett, 1984, Separation of peripheral dopamine receptors by a selective
DA I antagonist S('H 23390, Hs,pertension 6 (Suppl. 11,
1-25. Hicks, P.t-. and S.Z. l,anger, 1983, Antagonism by tetrahvdrofl-carboline of the vasoconstrictor responses to trypt~iminc in rat tail arteries, European J. Pharmacol. 96, 145. Hilditch, A., G.M. Drew and R.J. Naylor, 1984, S('1t 23390 is a very potent and selective antagonist at vascular dopamine receptors, European J. Pharmacol. 97, 333. Hyttel, J., 1983, SCH 23390 - the first selccti~,'e dopamine I) I antagonist, European J. Pharmacol. 91, 153. loric, L.C., A. Barnett, I-:.tt. Leitz, P. Hower and C.A. Korduha, 1983, S('H 23390, a potential benzazepinc anlispychotic with unique interactions on dopaminergic systems, J. Pharmacol. Exp. Ther. 226, 462. Schnellmann, R.G., S.J. Waters and D.L. Nelson. 1984, [ ~lt]5Hydroxytryptamine binding sites: species and tissue ,,~riation. J. Neurochem. 42, 65.