Introduction

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Although neuroleptics such as haloperidol moderate the positive symptoms of schizophrenia via blockade of mesolimbic D₂ receptors, they are not effective in all patients and poorly alleviate negative-cognitive symptoms (Kinon and Lieberman, 1996). Furthermore, concomitant blockade of striatal and hypophyseal populations of D₂ sites provokes motor and endocrine extrapyramidal side effects, respectively (Cunningham-Owens, 1996). In the design of improved antipsychotic agents (Meltzer, 1995), many recent studies have focused on the following conceptual developments of this classic, "mesolimbic dopaminergic/D₂ hyperactivity" theory of schizophrenia (Brunello et al., 1995; Kinon and Lieberman, 1996). First, a reduced activity of dopaminergic pathways innervating frontal cortex (FCX) may contribute to largely intractable, negative and cognitive symptoms (Knable and Weinberger, 1997; Parellada et al., 1998). Second, D₃ and D₄ receptors, which are closely related to their D₂ counterparts, may be involved in the pathogenesis and management of schizophrenia (Levant, 1997; Wilson et al., 1998). Third, in addition to dopaminergic transmission, a dysfunction of serotonergic and adrenergic networks may be implicated in psychotic states and their management (Baldessarini et al., 1992; Nutt, 1994; Roth and Meltzer, 1995).

Within this framework, the dibenzodiazepine clozapine has attracted considerable interest because it preferentially reinforces mesocortical versus subcortical dopaminergic transmission, possesses marked affinity for D₄ receptors, and interacts with a diversity of serotonergic and adrenergic receptors (ARs; Brunello et al., 1995; Meltzer, 1995; Josselin et al., 1997; Millan et al., 1998b; Wilson et al., 1998). These distinctive actions of clozapine likely underlie its superior ("atypical") clinical profile (Brunello et al., 1995). Clozapine is, thus, efficacious in a proportion of patients refractory to neuroleptics, exerts antipsychotic activity in the absence of extrapyramidal side effects, and may control negative-cognitive symptoms, although its efficacy against primary negative symptoms is still debated (Brunello et al., 1995). Apart from the potential importance of non-D₂ dopaminergic receptors, several serotonergic and AR subtypes have been implicated in the actions of clozapine. For example, the pronounced antagonist actions of clozapine at 5-hydroxytryptamine (5-HT)_2A receptors may permit antipsychotic efficacy in the relative absence of extrapyramidal side effects (Roth and Meltzer, 1995; Schmidt et al., 1995).

Adrenergic mechanisms, via actions in the FCX and other corticolimbic loci, play an important role in the control of mood and cognition (Arnsten, 1997; Coull et al., 1997). Furthermore, a perturbation of adrenergic transmission has been related to positive crises, intensification of negative symptoms, and the risk of relapse after the discontinuation of treatment (Maes et al., 1993). Notably, clozapine displays antagonist properties at ₁- and ₂-ARs (Baldessarini et al., 1992; Nutt, 1994; Blake et al., 1998; Elman et al., 1999). In this respect, the following points deserve emphasis. First, psychostimulants, such as cocaine and amphetamine, increase extracellular levels of norepinephrine (NE); correspondingly, activation of ₁-ARs in both mesolimbic structures and the FCX may be involved in their excitatory actions (Baldessarini et al., 1992; Darracq et al., 1998). Second, blockade of ₁-ARs preferentially suppresses mesolimbic versus nigrostriatal dopaminergic transmission (Lane et al., 1990; Svensson et al., 1995). Third, antagonism of ₁-ARs facilitates thalamic gating of sensory input to the cortex, a process compromised in psychotic patients (McCormick and Pape, 1995; Bakshi and Geyer, 1997). Fourth, blockade of inhibitory ₂-AR heteroceptors on terminals of dopaminergic fibers in FCX enhances mesocortical DA release (Gobert et al., 1998). Fifth, combined treatment with the ₂-AR antagonist idazoxan and the neuroleptic fluphenazine affords a "clozapine-like" profile of antipsychotic activity (Litman et al., 1996), and ₂-AR antagonist actions are implicated in the functional actions of clozapine in humans (Nutt, 1994; Elman et al., 1999). Finally, ₂-AR antagonist properties limit extrapyramidal side effects (Kalkman et al., 1998) and contribute to an improvement in mood (Nutt, 1994).

From these considerations, the potential interest of antipsychotics sharing the adrenergic receptorial profile of clozapine appears evident. However, the potent histaminic and muscarinic actions of clozapine are accompanied by autonomic/cardiovascular side effects, and its conversion to a "netrenium" metabolite can provoke agranulocytosis (Cunningham-Owens, 1996). In this light, the present (and accompanying) papers describe a novel, potential antipsychotic, S18327 (1-{2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)piperid-1-yl]ethyl}3-phenyl imidazolin-2-one), which is chemically distinct from clozapine. Thus, the latter is a tricyclic, dibenzodiazepine derivative, whereas S18327 (Fig. 1) is a benzoisoxazolepiperidine coupled to a phenylimidazoline via an ethylene linker. In contrast to clozapine, S18327 possesses only weak affinity for muscarinic and histaminic receptors. Nevertheless, S18327 mimics both the monoaminergic profile of clozapine and its distinctive pattern of antipsychotic versus extrapyramidal activity in vivo, actions to which its marked ₁- and ₂-AR antagonist properties make an important contribution.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Chemical structure of S18327.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Affinity of S18327 for Multiple Monoaminergic Receptors. The affinities of S18327 for multiple, native and cloned, human dopaminergic, adrenergic, and serotonergic receptors were determined using standard protocols described in detail previously (Millan et al., 1998b) and summarized (Tables 1-3). Nonlinear, regression analysis of isotherms was performed by use of the program Prism (GraphPAD Software, San Diego, CA), which yielded IC values. These were transformed into K_i values according to the Cheng-Prusoff equation: K_i = IC₅₀/(1 + L/K_d), where L corresponds to the radioligand concentration and K_d to its dissociation constant (Kenakin, 1997).

View this table: [in this window] [in a new window]   TABLE 1 Affinities of S18327 at multiple dopaminergic receptors and DA reuptake sites Data are means ± S.E. of 3 determinations performed in triplicate. For details of methods, see Millan et al., 1998b.

View this table: [in this window] [in a new window]   TABLE 2 Affinities of S18327 at multiple adrenergic receptors and NE reuptake sites Data are means ± S.E. of 3 determinations performed in triplicate. For details of methods, see Millan et al., 1998b.

View this table: [in this window] [in a new window]   TABLE 3 Affinities of S18327 at multiple serotonergic receptors and 5-HT reuptake sites Data are means ± S.E. of 3 determinations performed in triplicate. For details of methods, see Millan et al., 1998b.

Antagonist Properties of S18327 at Specific Monoaminergic Receptors: [³⁵S]GTPS binding. As described in detail previously (Newman-Tancredi et al., 1997, 1999; Millan et al., 1998b), and summarized (Table 4), the binding of [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTPS; 1000 Ci/mmol; NEN, Les Ulis, France) at human (h)D₂, hD₃, hD₄, h_2A-AR, h5-HT_1A, h5-HT_1B, and h5-HT_1D receptors was used as a measure of the efficacy with which S18327 interacts at these sites. S18327 was evaluated both alone (agonist activity) and, where appropriate, in the presence of a fixed concentration of dopamine (DA), NE, or 5-HT (antagonist activity). Agonist efficacy was expressed relative to that of a maximally effective concentration of DA, NE, or 5-HT (defined as 100%). For the antagonist studies, concentration-response curves of the blocking properties of S18327 were analyzed as described in Newman-Tancredi et al. (1999) to yield K_b values.

View this table: [in this window] [in a new window]   TABLE 4 Determination of the influence of S18327 on [35S]GTPS binding at cloned, human receptors transfected into CHO cells Summary of experimental conditions for [35S]GTPS binding at various dopaminergic and serotonergic receptor types. For more detailed protocols, see Newman-Tancredi et al., 1997 and 1999.

Antagonist Properties of S18327 at h_1A-ARs and h5-HT_2A Receptors: Intracellular Ca²⁺ Concentration ([Ca²⁺]_i). Chinese hamster ovary (CHO) cells expressing h5-HT_2A receptors or h_1A-ARs were harvested and incubated for 60 min at room temperature with 5 µM Fura-2 acetoxymethyl ester. The cells were pelleted by centrifugation (1500g, 10 min, 10°C), resuspended in Hanks' medium, and incubated for 15 min at 37°C to allow hydrolysis of Fura-2 acetoxymethyl ester to Fura-2. The cells were then recentrifuged as above and resuspended in Hanks' medium with 5-HT or NE and/or S18327. After incubation for 12 min, assays were terminated by the addition of Triton X-100 (0.1%) and EGTA (10 mM). [Ca²⁺]_i levels were calculated from the fluorescence detected at a wavelength of 510 nm after excitation at wavelengths of 340 and 380 nm. The latter correspond to the absorbance peaks of Fura-2 in its Ca²⁺-complexed and noncomplexed forms, respectively.

Antagonist Properties of S18327 at hD₃ Receptors: Mitogen-Activated Protein (MAP) Kinase. CHO cells expressing hD₃ receptors were grown in 6-well plates until 90% confluent. The cells were then washed with serum-free medium and incubated overnight in this medium. Drugs were diluted in serum-free medium and added to cells to obtain the appropriate, final concentration. Cells were preincubated for 5 min with antagonists and then stimulated with DA (100 nM) for 5 min. At the end of the incubation period, 0.25 ml/well of Laemmi's sample buffer containing 200 mM dithiotreitol was added. Whole-cell lysates were boiled for 3 min at 95°C. Then, 14 µl of cell extract was loaded onto 15-well, 10% polyacrylamide gels and "fully" activated MAP kinase was revealed using a monoclonal antibody specifically raised against the phosphorylated pp42^mapk (ERK 2) and pp44^mapk (ERK 1) forms on both threonine and tyrosine residues (NanoTools, Denzlingen, Germany), followed by enhanced chemiluminescence detection with horseradish peroxidase as a secondary antibody (Amersham, Les Ulis, France).

Studies In Vivo. All studies in vivo used male Wistar rats weighing 220 to 240 g obtained from Iffa Credo (L'Arbresle, France). They were maintained in sawdust-lined cages with unlimited access to water and food. Laboratory humidity was 60 ± 5%, and temperature was 21 ± 1°C. Lights were on from 7:30 AM to 7:30 PM. All animals were adapted for at least 1 week to laboratory conditions before use.

Antagonist Properties of S18327 at Cerebral Populations of ₂-ARs: Autoradiographic Evaluation of [³⁵S]GTPS Binding In Situ. The potential activity of S18327 at cerebral populations of ₂-ARs, visualized by [³⁵S]GTPS in the amygdala, was determined as follows. Slides with three or four brain sections were incubated for 60 min in 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.2 mM EGTA, 0.2 mM dithiothreitol, 2.5 mM GDP, 10 mM MgCl₂, and 0.05 nM [³⁵S]GTPS plus NE and/or S18327. After incubation, sections were washed with ice-cold buffer and then dipped into ice-cold, deionized distilled water. The slides were dessicated under air flow and placed in x-ray cassettes apposed to ³⁵S-sensitive film for 4.5 days at 20°C. Binding densities were measured by computerized densitometry using a Leica Q600 image analyzer (Leica, Paris, France) and ¹⁴C-standard microscales. Data are expressed as a percentage increase in [³⁵S]GTPS binding induced by drug treatment relative to that observed under basal (non-drug-treated) conditions (defined as 0%). The amygdala was selected for this study inasmuch as this central nervous system region shows a robust [³⁵S]GTPS response to NE, which is fully blocked by selective ₂-AR antagonists (Newman-Tancredi et al., in press).

Influence of S18327 on Hypothermia Elicited by Preferential D₃ versus D₂ Agonist, PD128,907. Core (rectal) temperature was measured in lightly restrained rats by the use of a digital thermisoprobe as described previously (Millan et al., 1995). Basal core temperature was determined; 30 min later, vehicle or S18327 was administered, and then 30 min later, vehicle or PD128,907 (0.63 mg/kg s.c.) was injected. Again, 30 min later, core temperature was redetermined, and the difference from pretreatment values calculated. Data were analyzed by ANOVA, and the ID₅₀ plus 95% confidence limit (CL) values were calculated.

Influence of S18327 on Rotation in Unilateral, Substantia Nigra-Lesioned Rats. The procedure used has been described previously (Millan et al., 1998b). Separate groups of rats with a unilateral 6-hydroxydopamine (8 µg/4 µl) lesion of the left or right substantia nigra, pars compacta, were trained with either the D₁ agonist SKF38393 (0.63 mg/kg s.c.) or the D₂ agonist quinpirole (0.02 mg/kg s.c.). The induction of rotation was measured 45 to 60 min and 20 to 50 min after the administration of quinpirole and SKF38393, respectively. Rotation was evaluated automatically using a harness coupled to a Rotacount 8 (Columbus Instruments, Columbus, OH) apparatus. In alternate sessions, rats were treated 25 min before SKF38393 or quinpirole with either S18327 or vehicle. This permitted expression of the action of S18327 as a function of the mean of the preceding and subsequent vehicle sessions (defined as 100%). Data were analyzed by a paired Student's t test, and the ID₅₀ (95% CL) was calculated.

Influence of S18327 on 5-HT_2A Receptor-Mediated Secretion of Corticosterone (CS). The antagonist properties of S18327 at 5-HT_2A receptors in vivo were evaluated by its ability to inhibit the increase in plasma levels of CS elicited by the 5-HT_2A agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Vehicle or S18327 and vehicle or DOI (2.5 mg/kg s.c.) were administered, respectively, 60 and 30 min before the determination of CS levels, performed with a CS radioimmunoassay (RPA548; Amersham). Cross-reactivity with other steroids was <0.1%. Levels are expressed in ng/ml of plasma. Data were analyzed by two-way ANOVA followed by Dunnett's test.

Modulation by S18327 of Electrical Activity of Dopaminergic, Adrenergic, and Serotonergic Neurons in Anesthetized Rats. Procedures described in detail previously (Aghajanian et al., 1977; Lejeune et al., 1997) were used for evaluation of the influence of S18327 on the electrical activity of dopaminergic, adrenergic, and serotonergic perikarya localized in the ventrotegmental area (VTA), locus ceruleus (LC), and dorsal raphe nucleus (DRN), respectively. Separate groups were used for each nucleus. Rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed in a stereotaxic apparatus, after which a tungsten microelectrode was lowered into the VTA, LC, or DRN. Coordinates were as follows: VTA, AP = 5.5 from bregma, L = 0.7, and H = 7/8.5 from dura; LC, AP = 1.1 from 0, L = 1.2, and H = 5.5/6.5; and DRN, AP = 7/8 from bregma, L = 0.0, and H = 5/6.5. Specific populations of dopaminergic, adrenergic, and serotonergic neurons were identified according to their waveform as described previously (above citations) and baseline recording performed during 5 min. The influence of S18327 (dissolved in sterile water and injected i.v. in a volume of 0.5 ml/kg) on firing rate was evaluated after its administration in cumulative doses at intervals of 2 to 3 min. For examination of the antagonist properties of S18327 on dopaminergic neurons of the VTA, it was administered 3 min after a single dose of the dopaminergic agonist apomorphine (0.031 mg/kg i.v.). To examine mechanisms underlying the inhibitory influence of S18327 on serotonergic neurons of the DRN, a single dose of S18327 was injected (0.25 mg/kg i.v.) followed, 3 min later, by an injection of the 5-HT_1A antagonist WAY100,635 (0.031 mg/kg i.v.). In an additional experiment, as detailed in Lejeune et al. (1994), a single dose of the ₁-AR agonist cirazoline (0.005 mg/kg i.v.) was injected 3 min before a single dose of S18327 (0.25 mg/kg i.v.). All drug effects were determined during 60 s at the time of peak drug action. Data acquisition and analysis were performed with Spike 2 software (CED, Cambridge, England). Data are percent change from preinjection, basal values (defined as 0%). Data were analyzed by ANOVA followed by Newman-Keuls test.

Influence of S18327 on Cerebral Synthesis of DA, NE, and 5-HT. The modulation by S18327 of cerebral turnover of DA, NE, and 5-HT was evaluated as described previously (Millan et al., 1998b). DA and 5-HT turnover was determined in the striatum (rich in DA, but not NE), and NE and 5-HT turnover was evaluated in the hippocampus (rich in NE, but not DA). The actions of S18327 were measured 60 min after its administration and 30 min after injection of the decarboxylase inhibitor NSD1015 (100 mg/kg s.c.). HPLC analysis followed by electrochemical detection was used for determination of tissue levels of L-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP) as described previously (Millan et al., 1998b). Levels of L-DOPA and 5-HTP were expressed relative to those of vehicle values (defined as 0%). Data were analyzed by ANOVA followed by Dunnett's test.

Influence of S18327 on Cerebral DA Turnover. As detailed previously (Millan et al., 1998b), the ratio of levels of the DA metabolite dihydroxyphenylalaninecarboxylic acid (DOPAC) to those of DA itself were determined in projection regions of the nigrostriatal pathway (striatum), the mesolimbic pathway (nucleus accumbens and olfactory tubercles), and the mesocortical pathway (FCX) 30 min after the administration of S18327. Levels of DOPAC and DA were determined by HPLC and electrochemical detection. DOPAC/DA ratios were expressed relative to those of vehicle values (defined as 0%). Data were analyzed by ANOVA followed by Dunnett's test.

Influence of S18327 on Dialysate Levels of DA, NE, and 5-HT in FCX, Nucleus Accumbens, Striatum, and Hippocampus of Freely Moving Rats. The procedures used for quantification of DA, NE, and 5-HT levels in single dialysate samples of the FCX, accumbens and striatum of freely moving rats have been extensively documented elsewhere (Gobert et al., 1998; Millan et al., 1998b). Using essentially identical procedures, we also examined levels of NE and 5-HT in single dialysates of the hippocampus of freely moving rats with a guide cannula (CMA 11) implanted under pentobarbital anesthesia (60.0 mg/kg i.p.) at the coordinates AP = 3.6, L = ±1.2, and DV = 2.3. A cuprophane CMA/11 probe, 2 mm length, 0.24 mm diameter, was used for this structure. Experiments were performed 5 days after the placement of guide cannulas. Samples were taken every 20 min, and after three basal samples, S18327 or vehicle was injected s.c. and sampling continued for an additional 3 h. The influence of S18327 and vehicle was expressed relative to basal values (defined as 0%). In the interaction experiments performed in the FCX, WAY100,635 (0.16 mg/kg s.c.) or S18616 (0.00063 mg/kg s.c.) was injected 20 min before S18327 (1.25 mg/kg s.c.) or vehicle. The assay sensitivity was 0.1 to 0.2 pg/sample for DA, NE, and 5-HT in each case. Data were analyzed by ANOVA with sampling time as the repeated within-subject factor.

Drugs. S18327 and other drugs were dissolved in sterile water, if required with the addition of a few drops of lactic acid. In this case, the pH was readjusted to as close to neutrality as possible (>5.0). Unless otherwise indicated, drugs were administered s.c. Cirazoline HCl was obtained from Synthélabo (Bagneux, France). Apomorphine HCl was obtained from Sigma (Chesnes, France). SKF38393 [(±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol HCl], quinpirole HCl, DOI (1-[2,5-dimethoxy-4-iodophenyl]2-aminopropane HCl), and PD128,907 [(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]1,4-oxazin-9-ol) HCl] were obtained from Research Biochemicals (Natick, MA). S18327, WAY100,635 [(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclo-hexanecarboxamide fumarate], and S18616 ([7,8](2-chlorobenzo)-2-amino-1-aza-3-oxa-[4,5]spirodeca-1,7-diene HCl) were synthetized by Servier chemists (J.-L. Peglion and A. Cordi).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Binding Profile of S18327 at Multiple Dopaminergic Receptors. S18327 displayed affinities of about 70-90 nM for native and cloned rat and human D₂ receptors (Table 1). Its affinity for cloned rat and human D₃ receptors was similar. In contrast, the affinity of S18327 for hD₄ receptors was 10-fold greater than that at hD₂ sites. At native rat D₁, cloned hD₁, and cloned hD₅ receptors, S18327 manifested affinities of 60 to 160 nM. S18327 had negligible affinity for DA reuptake sites (>1000 nM).

Binding Profile of S18327 at Multiple ARs. S18327 showed high affinities (1.0 nM) for rat frontocortical ₁-ARs as well as for native rat _1A- and _1B-ARs (Table 2). Similarly, the affinities of S18327 for cloned h_1A-, h_1B-, and h_1D-ARs were high (1.0 nM). At cortical ₂-ARs and cloned human h_2A-, h_2B-, and h_2C-ARs, the affinities of S18327 were 40 to 200 nM. The affinity of S18327 at -ARs and NE reuptake sites was negligible (>1000 nM).

Binding Profile of S18327 at Multiple Serotonergic Receptors. S18327 displayed affinities of 63 and 45 nM for native rat 5-HT_1A and cloned h5-HT_1A receptors, respectively, yet low (>1000 nM) affinity for native and cloned 5-HT_1B receptors (Table 3). Its affinity for h5-HT_1D receptors was 374 nM. The affinity of S18327 for both native and cloned 5-HT_2A receptors was high (4.0 nM). On the other hand, it showed comparatively low affinities of 50 to 500 nM for cloned h5-HT_2B receptors and for native 5-HT_2C and cloned h5-HT_2C sites. S18327 manifested negligible (1000 nM) affinity for 5-HT₃, 5-HT₄, 5-HT_5A, and 5-HT₆ receptors and 5-HT reuptake sites. However, the affinity of S18327 at 5-HT₇ receptors was 15 nM.

Binding Profile of S18327 for Other, Nonmonoaminergic Receptors and Enzymes. S18327 displayed affinity of 80 nM for native, rat ₁ receptors. Its affinity for native, rat ₂ sites was 530 nM. S18327 had negligible affinity (>10 µM) for all other receptor types examined: nicotinic, acetylcholine uptake, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, N-methyl-D-aspartate, glycine, -aminobutyric acid_A, -aminobutyric acid_B, benzodiazepine, adenosine₁, adenosine₂, imidazoline₁, imidazoline₂, µ-opioid, cannabinoid₁, neuropeptide Y, neurokinin₁, neurokinin₂, Ca²⁺ channels, site 2 Na⁺ channels, K⁺ channels, monoamine oxidase A, thromboxane₂, estrogen, progesterone, testosterone, bradykinin₂, endothelin_A, endothelin_B, nitric oxide synthase, phospholipase A₂, phosphodiesterase, 5-lipoxygenase, acetylcholinesterase, and cyclooxygenase. The interaction of S18327 at histaminic and muscarinic sites is described in detail in the accompanying paper; however, it should be noted here that its affinity was 17.5 nM at native, histamine₁ receptors and 600 nM at cloned, human muscarine₁ receptors.

Antagonist Properties of S18327 at hD₂, hD₃, and hD₄ Receptors: Blockade of DA-Stimulated [³⁵S]GTPS Binding and MAP Kinase. As described previously (Newman-Tancredi et al., 1997, 1999), DA markedly enhanced [³⁵S]GTPS binding at hD₂, hD₃, and hD₄ receptors with EC₅₀ values of 364.0 ± 42.0, 13.2 ± 2.0, and 102.0 ± 13.0 nM, respectively (Fig. 2, A-C). These actions of DA (defined as 100%) were concentration-dependently inhibited by S18327, which did not itself modify [³⁵S]GTPS binding. K_b values for S18327 at hD₂, hD₃, and hD₄ sites were 34.0 ± 7.4 (Fig. 2A), 119.0 ± 28.0 (Fig. 2B), and 12.5 ± 3.2 nM (Fig. 2C), respectively. DA also markedly stimulated the activity of MAP kinase in CHO cells transfected with hD₃ receptors (Fig. 2D); this action was similarly abolished by S18327, which did not itself influence MAP kinase activity.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Antagonist actions of S18327 at cloned hD2, hD3, and hD4 receptors as determined by inhibition of DA-stimulated [35S]GTPS binding and MAP kinase activity. A, inhibition of [35S]GTPS binding at hD2 receptors. B, inhibition of [35S]GTPS binding at hD3 receptors. C, inhibition of [35S]GTPS binding at hD4 receptors. D, inhibition of hD3 receptor-coupled MAP kinase stimulation. Values are representative of at least three independent determinations performed in triplicate.

Antagonist Properties of S18327 at D₃/D₂ Receptors In Vivo: Inhibition of Hypothermia Elicited by PD128,907. The preferential D₃ versus D₂ agonist, PD128,907 (0.63 mg/kg s.c.), elicited hypothermia in rats (PD128,907, 2.0 ± 0.2°C; vehicle, +0.5 ± 0.1°C; P < .05. Its action was dose-dependently inhibited by S18327 with an ID₅₀ (95% CL) value of 6.1 (2.4-15.1) mg/kg s.c.

Antagonist Actions of S18327 at D₁ and D₂ Receptors: Inhibition of SKF38393- and Quinpirole-Elicited Rotation in Rats. The preferential agonists at D₁ and D₂ receptors, SKF38393, and quinpirole, respectively, each provoked contralateral rotation in rats sustaining unilateral lesions of the substantia nigra, pars compacta. S18327 dose-dependently and fully blocked their actions with ID₅₀ (95% CL) values of 1.4 (0.4-3.6) and 0.5 (0.2-1.2) mg/kg s.c., respectively.

Antagonist Actions of S18327 at h_1A-ARs: Inhibition of Increases in [Ca²⁺]_i Levels Elicited by NE. At cloned h_1A-ARs, NE elicited a pronounced elevation in [Ca²⁺]_i levels with an EC₅₀ value of 95.0 ± 32.0 nM and a maximal effect of 1119 ± 37 nM (Fig. 3). Typical basal levels were 140 to 200 nM (Fig. 3A). In analogy to the prototypical ₁-AR antagonist prazosin, which blocked the action of NE with a K_b value of 0.4 ± 0.1 nM, S18327 concentration-dependently abolished the action of NE with a K_b value of 3.6 ± 1.3 nM (Fig. 3B). Applied alone, at concentrations up to 10 µM, S18327 was inactive.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Antagonist actions of S18327 at h1A-ARs as determined by inhibition of NE-induced increases in [Ca2+]i levels. A, influence of S18327 alone. B, blockade of the actions of NE by S18327, which is expressed relative to NE (NAD) alone (100%). Values are representative of at least three independent determinations performed in triplicate.

Antagonist Actions of S18327 at h_2A-ARs: Inhibition of Activation of [³⁵S]GTPS Binding by NE. At cloned h_2A-ARs transfected into CHO cells, NE enhanced [³⁵S]GTPS binding with an EC₅₀ value of 230.0 ± 26.0 nM and a maximal effect of 2.7 ± 0.4-fold stimulation relative to basal values (Fig. 4A). This effect was abolished by the prototypical ₂-AR antagonist yohimbine with a K_b value of 2.1 ± 0.5 nM. S18327 also concentration-dependently abolished the action of NE with a K_b value of 162.0 ± 46.0 nM (Fig. 4B). Moreover, in the presence of fixed concentrations of S18327, the concentration-response curve for NE was progressively displaced in parallel to the right without any loss of maximal effect (Fig. 4C). The pA₂ acquired from Schild analysis (Fig. 4D), 6.9, corresponded well to its pK_i at these sites of 7.0. Furthermore, the slope of 1.00 ± 0.07 (r = 0.99) is consistent with competitive antagonist properties of S18327 at h₂-ARs. Alone, S18327 (10 µM) did not affect [³⁵S]GTPS binding.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Antagonist actions of S18327 at h2A-ARs as determined by inhibition of NE (NAD)-induced [35S]GTPS binding. A, influence of S18327 alone. B, concentration-dependent blockade of the action of NE by S18327. C, displacement by S18327 of the concentration-response curve to the right. D, Schild transformation of data in C. Values are representative of at least three independent determinations performed in triplicate.

Antagonist Actions of S18327 at h_2A-ARs: Inhibition of Activation of [³⁵S]GTPS Binding by NE at ₂-ARs in Rat Amygdala In Situ. In analogy to these studies of cloned h_2A-ARs, NE was shown to markedly activate [³⁵S]GTPS binding in rat amygdala (Fig. 5B). S18327 (10 µM), which did not itself modify [³⁵S]GTPS binding (Fig. 5C), abolished the action of NE (Fig. 5D).

View larger version (86K): [in this window] [in a new window]   Fig. 5.   Inhibition by S18327 of NE-induced [35S]GTPS binding at 2-ARs in rat amygdala (Amyg). A, basal binding of [35S]GTPS in the absence of NE. B, binding of [35S]GTPS in the presence of NE (10 µM). C, binding of [35S]GTPS in the presence of S18327 (10 µM). D, binding of [35S]GTPS in the presence of NE and S18327. Data shown are representative of four independent experiments. Basal levels of [35S]GTPS activity (A) were 384 ± 85 nCi/g tissue equivalent; these were defined as 0%, and the influence of drugs is expressed relative to these (B, +32 ± 10%; C, 5.5 ± 7.6%; and D, 7.5 ± 9.6). The differences of NE from matched basal values and of S18327/NE from control/NE values were significant (P < .05) in each case (Student's two-tailed paired t test).

Antagonist and "Inverse Agonist" Actions of S18327 at h5-HT_1A, h5-HT_1B, and h5-HT_1D Receptors: Blockade of 5-HT-Stimulated [³⁵S]GTPS Binding. 5-HT markedly stimulated [³⁵S]GTPS binding at h5-HT_1A, h5-HT_1B, and h5-HT_1D receptors with EC₅₀ values of 17.5 ± 2.0, 8.9 ± 1.4, and 1.3 ± 0.2 nM, respectively. At h5-HT_1A sites, S18327 elicited a very mild stimulation, attaining 14.2 ± 0.8% of the maximal influence of 5-HT (100%) and with an EC₅₀ value of 97.1 ± 30.1 nM. Furthermore, it exerted antagonist properties at these sites in concentration-dependently inhibiting the stimulation elicited by the 5-HT_1A receptor agonist (±)-8-hydroxy-2-dipropylaminotetralin (100 nM), with a K_b value of 129.0 ± 13.0 nM. At 5-HT_1B and 5-HT_1D receptors, S18327, applied alone, inhibited basal binding by 27.0 ± 6.1% and 51.2 ± 4.5%, respectively, suggesting "inverse agonist" properties. The inhibition was concentration dependent at both h5-HT_1B and h5-HT_1D receptors with IC₅₀ values of 4074 ± 566 and 70 ± 18 nM, respectively. In view of the low affinity of S18327 at h5-HT_1B and h5-HT_1D sites and its inverse agonist actions, interaction studies with 5-HT were not undertaken.

Antagonist Properties of S18327 at h5-HT_2A Receptors: Inhibition of Increases in [Ca²⁺]_i Levels Elicited by 5-HT. At cloned h5-HT_2A receptors, 5-HT evoked an increase in [Ca²⁺]_i levels with an EC₅₀ value of 124 ± 14 nM and a maximal effect of 728 ± 45 nM. Typical basal levels were 300 to 350 nM (Fig. 6A). This action of 5-HT was blocked by the prototypical antagonist mianserin with a K_b value of 3.9 ± 1.9 nM. In analogy, S18327 concentration-dependently blocked the action of 5-HT with a K_b value of 1.4 ± 0.5 nM (Fig. 6B). S18327 was inactive alone at concentrations up to 10 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Antagonist actions of S18327 at 5-HT2A receptors as determined by inhibition of 5-HT-stimulated [Ca2+]i levels in CHO cells and by inhibition of DOI-induced CS secretion in rats. A, influence of S18327 alone. B, blockade by S18327 of the actions of 5-HT. C, inhibition by S18327 of DOI-induced CS secretion. For A and B, values are representative of three independent determinations performed in triplicate. For C, data are mean ± S.E. (n = 5-8 per value). On two-way ANOVA: interaction, F4,86 = 7.6, P < .05; influence of S18327 alone, F4,38 = 2.1, P > .05; influence of S18327 versus DOI, F4,48 = 8.0, P < .05. *P < .05, significance of differences from respective vehicle values (Dunnett's test).

Antagonist Properties of S18327 at Native 5-HT_2A Receptors: Inhibition of DOI-Induced CS Secretion. The 5-HT_2A agonist DOI increased circulating levels of CS. This effect of DOI was dose-dependently and significantly inhibited by S18327, which, at the highest dose tested, tended itself (nonsignificantly) to elevate CS levels (Fig. 6C). [Notably, clozapine and certain other antipsychotics likewise elicit modest increases in CS levels, but the mechanisms underlying the intrinsic influence of S18327 and these agents on CS secretion remain to be clarified (Rivet et al., 1997, 1999)].

Influence of S18327 on Firing Rate of LC-Localized Adrenergic Neurons. S18327 elicited a dose-dependent increase in the firing rate of adrenergic neurons localized in the LC with an AD₅₀ (95% CL) value of 120 (70-200) µg/kg i.v. (Fig. 7A).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Influence of S18327 on the activity of ascending adrenergic neurons. A, enhancement by S18327 of the electrical activity of adrenergic neurons in the LC. B, increase by S18327 of extracellular levels of NE in the nucleus accumbens of freely moving rats. C, increase by S18327 of extracellular levels of NE in the hippocampus of freely moving rats. D, increase by S18327 of NE synthesis in the hippocampus (L-DOPA precursor accumulation after inhibition of decarboxylase). Dialysate levels of NE (noradrenaline) are expressed as a percentage of basal, preinjection values, which were defined as 0% (1.1 ± 0.1 and 0.9 ± 0.1 pg/20 µl dialysate for accumbens and hippocampus, respectively). Induction of NE synthesis is expressed as a percentage of basal, preinjection values (0.44 ng/mg protein), which were defined as 0%. Data are means ± S.E. (n  5 per value). On ANOVA: A, F6,30 = 25.6, P < .05; B, F1,10 = 20.1, P < .05; C, F1,10 = 37.7, P < .05; and D, F3,46 = 4.5, P < .05. *P < .05, significance of difference from respective vehicle values.

Influence of S18327 on Synthesis and Dialysate Levels of NE in Hippocampus and Accumbens. In freely moving rats, at a dose (10.0 mg/kg s.c.) that increased hippocampal NE turnover, S18327 markedly increased extracellular levels of NE in the nucleus accumbens (Fig. 7B). Similarly, a marked rise in dialysate levels of NE in the hippocampus was seen with S18327 (10.0 mg/kg; Fig. 7C). After suppression of decarboxylase activity with NSD1015, S18327 modestly elevated hippocampal levels of the NE precursor, L-DOPA (Fig. 7D).

Influence of S18327 on Firing Rate of VTA-Localized Dopaminergic Neurons. The electrical activity of dopaminergic neurons in the VTA was markedly inhibited by the dopaminergic agonist, apomorphine (31 µg/kg i.v.; Fig. 8A). S18327 dose-dependently abolished this action of apomorphine with an ID₅₀ (95% CL) value of 300 (100-900) µg/kg i.v. (Fig. 8A). Administered alone, S18327 did not alter firing rate (Fig. 8A). Furthermore, S18327 did not alter the firing pattern (regular or "bursts"; not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Influence of S18327 on the activity of ascending dopaminergic neurons. A, blockade by S18327 of the inhibitory influence of the dopaminergic agonist apomorphine (0.031 mg/kg i.v.) on the electrical activity of dopaminergic neurons in the VTA. B-E, influence of S18327 on the turnover (DOPAC/DA ratios) of DA in various cerebral structures. F, influence of S18327 on DA synthesis in striatum (L-DOPA accumulation after inhibition of decarboxylase). Induction of DA turnover is expressed as a percentage of basal, preinjection DOPAC/DA ratios, which were defined as 0%. Basal levels of DOPAC and DA (in ng/mg protein) were as follows: accumbens, DA = 94.7 ± 11.5, DOPAC = 12.2 ± 1.0; FCX, DA = 1.3 ± 0.1, DOPAC = 0.6 ± 0.1; olfactory tubercle, DA = 41.0 ± 2.9, DOPAC = 7.7 ± 0.9; and striatum, DA = 128.4 ± 9.2, DOPAC = 12.9 ± 0.8. Induction of striatal DA synthesis is expressed as a percentage of basal, preinjection, basal values of L-DOPA (11.4 ± 0.8 ng/mg protein), which were defined as 0%. Data are means ± S.E. (n  5 per value). On ANOVA: A, S18327 alone, F4,18 = 1.1, P > .05; S18327 plus apomorphine, F4,18 = 222.0, P < .05; B, F4,17 = 7.0, P < .05; C, F4,15 = 3.2, P < .05; D, F4,17 = 11.3, P < .05; E, F4,17 = 32.4, P < .05; and F, F4,76 = 29.3, P < .05. *P < .05, significance of difference from respective vehicle values.

Influence of S18327 on Cerebral DA Synthesis. S18327, administered over a broad dose range and up to high doses, modestly enhanced ratios of the DA metabolite DOPAC to DA in the nucleus accumbens (Fig. 8B), the FCX (Fig. 8C), the olfactory tubercles (Fig. 8D), and the striatum (Fig. 8E). Similarly, after inhibition of decarboxylase with NSD1015, S18327 increased levels of the DA precursor L-DOPA in the striatum (Fig. 8F).

Influence of S18327 on Firing Rate of DRN-Localized Serotonergic Neurons. S18327 dose-dependently inhibited the firing rate of DRN-localized serotonergic neurons with an ID₅₀ (95% CL) value of 90 (60-160) µg/kg i.v. (Fig. 9A). The selective 5-HT_1A antagonist WAY100,635 at a dose (31 µg/kg i.v.) that abolishes the inhibitory influence of 5-HT_1A agonists on DRN firing (Lejeune et al., 1997) failed to modify the inhibitory influence of S18327 (250 µg/kg i.v.; Fig. 9B). In distinction, pretreatment with the ₁-AR agonist cirazoline, at a dose (5 µg/kg i.v.) that markedly reduces the inhibitory influence of prazosin and clozapine on DRN firing (Lejeune et al., 1994), significantly attenuated the action of S18327 (Fig. 9B).

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Influence of S18327 on the activity of ascending serotonergic neurons. A, inhibition by S18327 of the electrical activity of serotonergic neurons in the DRN. B, attenuation by the 1-AR agonist cirazoline (0.005 mg/kg i.v.), but not the 5-HT1A antagonist WAY100,635 (0.031 mg/kg i.v.), of the inhibitory influence of S18327 (0.25 mg/kg i.v.) on the electrical activity of serotonergic neurons. C, influence of S18327 on synthesis of 5-HT in striatum (5-HTP accumulation after inhibition of decarboxylase). D, influence of S18327 on extracellular levels of 5-HT in the striatum of freely moving rats. E, influence of S18327 on synthesis of 5-HT in hippocampus. F, influence of S18327 on extracellular levels of 5-HT in the hippocampus of freely moving rats. Dialysate levels of 5-HT are expressed as a percentage of basal, preinjection values, which were defined as 0%; these were 0.55 ± 0.07 and 0.37 ± 0.02 pg/20 µl dialysate for striatum and hippocampus, respectively. Induction of synthesis is also expressed as a percentage of basal, preinjection values of 5-HTP, which were defined as 0%; these were 1.2 ± 0.1 and 1.1 ± 0.1 ng/mg protein for the striatum and hippocampus, respectively (n  5 per value). On ANOVA: A, F5,29 = 147.0, P < .05; B, F2,13 = 25.4, P < .05; C, F4,71 = 4.1, P < .05; D, S18327 (0.16), F1,14 = 1.2, P > .05; S18327 (1.25), F1,14 = 2.8, P > .05; S18327 (2.5), F1,18 = 16.0, P < .05 and S18327 (10.0), F1,14 = 19.3, P < .05; E, F4,65 = 2.0, P > .05; and F, F1,12 = 2.6, P > .05. *P < .05, significance of difference from respective vehicle values.

Influence of S18327 on Cerebral 5-HT Synthesis and Dialysate Levels of 5-HT in Striatum, Hippocampus, and Nucleus Accumbens. After administration of the decarboxylase inhibitor NSD1015, S18327 elicited a modest but significant decrease in levels of the 5-HT precursor 5-HTP in the striatum (innervated by the DRN) but not the dorsal hippocampus (innervated by the median raphe nucleus; Fig. 9, C and E, respectively). Similarly, in the striatum, but not the dorsal hippocampus, S18327 decreased dialysate levels of 5-HT (Fig. 9, D and F, respectively). Dialysate levels of 5-HT were not modified by S18327 (10.0 mg/kg) in nucleus accumbens (not shown).

Influence of S18327 on Dialysate Levels of DA in FCX Compared with Nucleus Accumbens and Striatum. S18327 elicited a dose-dependent and pronounced elevation in dialysate levels of DA in the FCX of freely moving rats (Fig. 10A). In the accumbens, although S18327 elicited a significant increase in DA levels, its magnitude was significantly less marked than that in FCX (Fig. 10, A and B). Similarly, the facilitatory influence of S18327 on extracellular levels of DA in the striatum was significantly less pronounced than that in the FCX (Fig. 10, A and C).

View larger version (33K): [in this window] [in a new window]   Fig. 10.   Influence of S18327 on extracellular levels of DA in the FCX compared with nucleus accumbens and striatum of freely moving rats. A-C, effect of various doses of S18327 over time. D, dose-response relationships for S18327 (area under curve analysis) over the entire period of sampling (20-180 min). Dialysate levels are expressed as a percentage of basal, preinjection values, which were defined as 0%; these were 1.1 ± 0.1, 8.3 ± 1.2, and 13.0 ± 1.5 pg/20 µl dialysate for FCX, nucleus accumbens, and striatum, respectively. Data are means ± S.E. (n  5 per value). ANOVA, with dose as between factor and with time as within factor, was performed over 20 to 180 min: A, S18327 (0.04), F1,18 = 0.1, P > .05; S18327 (0.63), F1,17 = 3.1, P > .05; S18327 (1.25), F1,17 = 33.9, P < .05; S18327 (2.5), F1,18 = 116.1, P < .05 and S18327 (10.0), F1,16 = 68.0, P < .05; B, S18327 (0.16), F1,20 = 2.1, P > .05; S18327 (1.25), F1,21 = 0.1, P > .05; S18327 (2.5), F1,24 = 16.1, P < .05 and S18327 (10.0), F1,22 = 63.3, P < .05 and C, S18327 (0.16), F1,20 = 3.5, P > .05; S18327 (1.25), F1,20 = 67.4, P < .05; and S18327 (2.5), F1,24 = 21.4, P < .05 and S18327 (10.0), F1,21 = 243.0, P < .05. *P < .05, significance of differences from respective vehicle values. D, ANOVA for actions in FCX compared with accumbens and striatum for common doses of S18327 as follows: F2,99 = 26.7, P < .001. *P < .05, significance of differences between nucleus accumbens and striatum dose-response curves versus that for FCX.

Influence of S18327 on Levels of NE and 5-HT in FCX. Levels of 5-HT in the FCX were unaffected by S18327 (Fig. 11A), whereas S18327 dose-dependently increased levels of NE therein (Fig. 11B).

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Influence of S18327 on extracellular levels of 5-HT and NE (NAD) in the FCX of freely moving rats. Dialysate levels are expressed as a percentage of basal, preinjection values, which were defined as 0%; these were 0.47 ± 0.15 and 0.68 ± 0.06 pg/20 µl dialysate for NE and 5-HT, respectively. Data are means ± S.E. (n  5 per value). ANOVA, with dose as between factor and with time as within factor, was performed over 20 to 180 min: 5-HT, S18327 (0.04), F1,17 = 1.7, P > .05; S18327 (0.63), F1,16 = 0.1, P > .05; S18327 (1.25), F1,16 = 0.4, P > .05; S18327 (2.5), F1,16 = 1.9, P > .05 and S18327 (10.0), F1,15 = 2.9, P > .05 and NE, S18327 (0.04), F1,16 = 0.4, P > .05; S18327 (0.63), F1,15 = 15.0, P < .05; S18327 (1.25), F1,15 = 24.9, P < .05; and S18327 (2.5), F1,16 = 10.0, P < .05 and S18327 (10.0), F1,14 = 11.5, P < .05. *P < .05, significance of difference from respective vehicle values.

Influence of the ₂-AR Agonist, S18616, Compared with the 5-HT_1A Antagonist, WAY100,635, on Modulation by S18327 of FCX Levels of DA and NE. The novel and potent ₂-AR agonist, S18616 (0.00063 mg/kg s.c.; Millan, 1998), suppressed FCX levels of DA and NE and abolished their elevation by S18327 (Fig. 12, left). S18616 itself also decreased levels of 5-HT (Fig. 12, left). In contrast, the selective 5-HT_1A receptor antagonist WAY100,635 (0.16 mg/kg s.c.) did not significantly modify the influence of S18327 (1.25 mg/kg s.c.) on FCX levels of DA and NE or 5-HT (Fig. 12, right). Administered alone, WAY100,635 did not itself modify FCX levels of these monoamines (Fig. 12, right).

View larger version (35K): [in this window] [in a new window]   Fig. 12.   Lack of influence of the 5-HT1A antagonist WAY100,635 compared with the 2-AR agonist S18616 on the modulation by S18327 of FCX levels of DA and NE (NAD) in FCX of freely moving rats. Data are means ± S.E. (n  5 per value). They are expressed as a percentage of basal, preinjection values, which were defined as 0% (see legends to Figs. 10 and 11). ANOVA, with drug as between factor and with time as within factor, was performed over 40 to 180 min: DA, influence of S18327 alone, F1,18 = 46.2, P < .05; influence of WAY100,635 alone, F1,19 = 0.9, P > .05, and WAY100,635/S18327, F1,16 = 0.7, P > .05; influence of S18616 alone, F1,17 = 13.7, P < .05 and S18616/S18327, F1,13 = 32.3, P < .05. NE, influence of S18327 alone, F1,17 = 32.3, P < .05; influence of WAY100,635 alone, F1,17 = 0.1, P > .05 and WAY100,635/S18327, F1,15 = 2.2, P > .05; influence of S18616, F1,16 = 14.7, P < .05 and S18616/S18327, F1,14 = 30.6, P < .05. 5-HT, influence of S18327 alone, F1,19 = 0.1, P > .05; influence of WAY100,635 alone, F1,20 = 0.1, P > .05 and WAY100,635/S18327, F1,14 = 0.1, P > .05; and influence of S18616, F1,18 = 34.2, P < .05 and S18616/S18327, F1,13 = 49.9, P < .05. *P < .05, significance of differences of S18616/S18327 compared with vehicle/S18327 values. #P < .05, significance of differences of S18616/vehicle compared with vehicle/vehicle values.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Antagonist Actions at Dopaminergic Receptors. [³⁵S]GTPS binding, which provides a measure of G protein coupling (Missale et al., 1998; Newman-Tancredi et al., 1999), revealed the antagonist activity of S18327 at hD₂ receptors. In vivo, antagonist properties of S18327 at postsynaptic D₂ receptors were reflected by blockade of rotation elicited by quinpirole in unilateral substantia nigra-lesioned rats (see Results), whereas antagonist actions at presynaptic D₂ sites were indicated by reversal of the inhibitory influence of apomorphine on VTA-localized dopaminergic neurons (Fig. 8; Lejeune et al., 1997; Gobert et al., 1998; Koeltzow et al., 1998). The latter action may also involve a (minor) population of colocalized D₃ autoreceptors (Gobert et al., 1995; Tepper et al., 1997). Furthermore, the attenuation by S18327 of hypothermia elicited by the preferential D₃ agonist PD128,907 (see Results) may involve antagonism of (postsynaptic) D₂ and D₃ receptors (Millan et al., 1995; Xu et al., 1999). Indeed, in analogy to hD₂ receptors, S18327 antagonized DA-stimulated [³⁵S]GTPS binding at hD₃ receptors (Fig. 2; Newman-Tancredi et al., 1999). Moreover, S18327 also abolished DA activation of MAP kinase (Fig. 2), a downstream component of the transduction cascade initiated by hD₃ receptor activation (Cussac et al., 1998). Although the importance of D₂ receptor blockade for antipsychotic agents is well established, the significance of D₃ receptor antagonism remains unclear (Gurevich et al., 1997; Levant, 1997). Similarly, the potential role of D₄ receptor blockade awaits clinical confirmation, and a clinical study with the selective D₄ antagonist L745,870 found that it was ineffective in acutely ill, neuroleptic-responsive inpatients (Kramer et al., 1997). However, Gazi et al. (1998) recently questioned the antagonist properties of this ligand at human hD₄ receptors. Furthermore, on the basis of alterations in D₄ receptor density in schizophrenic patients, it has been argued that preferential D₄ versus D₂ receptor blockade may afford an improved antipsychotic profile (Roth et al., 1995; Lahti et al., 1998; Millan et al., 1998c; Wilson et al., 1998). In fact, the "atypical" antipsychotic, clozapine, possesses higher affinity for hD₄ versus hD₂ sites (Millan et al., 1998c; Wilson et al., 1998), and this preference was more pronounced for S18327, which likewise behaved as a D₄ antagonist (Fig. 2), as shown by [³⁵S]GTPS binding (Newman-Tancredi et al., 1997).

Antagonist Action at ₁- and ₂-ARs. S18327 displayed high affinity for various ₁-AR subtypes and potently antagonized the NE-induced increase in [Ca²⁺]_i levels at h_1A-ARs. Pronounced, central ₁-AR antagonist actions may contribute to (improved) antipsychotic properties (see the introduction). However, peripheral ₁-AR antagonism is associated with orthostatic hypotension. Indeed, in analogy to the ₁-AR antagonist prazosin and to clozapine and other antipsychotics possessing pronounced ₁-AR antagonist properties (see accompanying paper; Cunningham-Owens, 1996), S18327 reduces arterial blood pressure in rats (M.J.M. and M.B., unpublished observations). Such cardiovascular actions in humans can complicate drug use, but they generally are moderated by drug titration and subside on repetitive administration (Cunningham-Owens, 1996). We have systematically examined the actions of prazosin in the functional models described in the present and accompanying paper (M.J.M. and M.B., unpublished observations). With the exception of certain, specific responses known to involve central ₁-AR blockade (discussed herein and in the accompanying paper), there is no evidence that a hypotension due to peripheral ₁-AR receptor blockade can account for the influence of S18327 on dopaminergic and adrenergic transmissionnot for its antipsychotic and other functional properties in vivo. The affinity of S18327 for rat/h_2A-ARs was comparable with that at rat/hD₂ sites. A [³⁵S]GTPS protocol revealed antagonist properties of S1