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CELLULAR AND MOLECULAR

Differential Effects of 5-Methyl-1-[[2-[(2-methyl-3-pyridyl)oxyl]-5-pyridyl]carbamoyl]-6-trifluoromethylindone (SB 243213) on 5-Hydroxytryptamine_2C Receptor-Mediated Responses

Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas (K.A.B., T.A.S., Y.M.S., W.P.C.); Unité Mixte de Recherche-Centre National de la Recherche Scientifique 5541, Université Victor Segalen Bordeaux 2, Bordeaux Cedex, France (S.N., U.S.); and Neuropharmacology Research, Psychiatry CEDD, GlaxoSmithKline, Harlow, United Kingdom (M.D.W.)

Received March 15, 2006; accepted June 23, 2006.

	Abstract

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5-Methyl-1-[[2-[(2-methyl-3-pyridyl)oxyl]-5-pyridyl]carbamoyl]-6-trifluoromethylindone (SB 243213) is a selective, high-affinity 5-hydroxytryptamine (serotonin)_2C receptor ligand that has been previously characterized as a competitive 5-HT_2C receptor antagonist that has a long duration of activity in vivo. It is active in two preclinical models of anxiety and has an improved anxiolytic profile compared with benzodiazepines. In this study, we further characterized the pharmacological properties of SB 243213 by measuring its effects on each of multiple responses coupled to the 5-HT_2C receptor. In Chinese hamster ovary cells, SB 243213 was an inverse agonist for the phospholipase A₂ response, for guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding, for reduction of constitutive desensitization, and for enhancement of dopamine release in the rat nucleus accumbens, with relative efficacies of 0.6, 1, 1, and 0.6, respectively. However, for the phospholipase C (PLC) signaling cascade, SB 243213 behaved as an antagonist. Although SB 243213 was previously characterized as a competitive antagonist for the PLC response, the magnitude of the dextral shift of the 5-HT concentration-response curve was time-dependent, and the maximal PLC response to 5-HT was decreased, probably as a result of the slow dissociation rate of SB 243213 (initial dissociation rate was 3.2 times slower than SB206553, a prototypical 5-HT_2C receptor inverse agonist). Taken together, these data show that the pharmacological characteristics of SB 243213 at the 5-HT_2C receptor differ depending upon the response measured, and they support the hypothesis that different drugs, acting at the same receptor subtype, can differentially regulate multiple cellular signaling systems.

Like many, if not all, 7-TMS receptors, 5-HT_2C receptors couple to multiple cellular effector systems. Perhaps the best-studied effector system is the phospholipase C (PLC) pathway. Stimulation of PLC by 5-HT_2C receptors leads to the production of inositol phosphates (IP) and diacylglycerol. Another effector pathway that couples to 5-HT_2C receptors, but that is somewhat less well studied, is the phospholipase A₂ (PLA₂) signaling cascade, which leads to the liberation of arachidonic acid (AA) and the subsequent production of a myriad of AA metabolites (Leysen, 2004).

In addition to the classical signaling mechanisms described above, 5-HT_2C receptors couple to several other signaling systems. In addition to the PLC and PLA₂ pathways, 5-HT_2C receptors also activate desensitization mechanisms, such as G protein-coupled receptor kinase (Westphal et al., 1995; Saucier et al., 1998; Backstrom et al., 2000; Berg et al., 2001b; Porter et al., 2001; Schlag et al., 2004). Furthermore, it has been demonstrated that 5-HT_2C receptors can couple to phospholipase D via G₁₃ (McGrew et al., 2002), to pertussis toxin-sensitive G proteins (e.g., G_i/o) (Lucaites et al., 1996), and to PDZ domain-containing proteins (Backstrom et al., 2000). Consequently, the net cellular effect of activation of 5-HT_2C receptors is a coalescence brought about by the concurrent activation of several effector pathways within cells.

Recently, evidence has begun to accumulate from studies with 7-TMS receptor systems that indicates that ligand pharmacology may be more complex than that specified by traditional receptor theory. For example, rather than being a system-independent constant for a particular receptor, it seems that ligand intrinsic efficacy is a variable that changes with cell phenotype and physiological state and is dependent upon cellular signaling machinery (i.e., differs with the response measured) (Clarke and Bond, 1998). This newly discovered ligand behavior has been given many names by different groups: agonist-directed trafficking of receptor stimulus, functional selectivity, stimulus trafficking, and biased agonism to name a few (see references within Kenakin, 1995; Mailman and Gay, 2004; Clarke, 2005).

5-HT_2C receptors seem to be capable of subserving agonist-directed trafficking of receptor stimulus. Werry et al. (2005) found that the relative efficacy of DOI and quipazine was reversed when measured for PLC-IP and calcium responses versus phosphorylation of extracellular signal-regulated kinase1/2 via the 5-HT_2C receptor expressed in CHO cells. In NIH-3T3 cells expressing the 5-HT_2C receptor, it was reported that although lysergic acid diethylamide is equally efficacious as 5-HT in stimulating PLC-IP, it does not elicit increases in intracellular calcium nor does it promote phosphorylation of the 5-HT_2C receptor (Backstrom et al., 1999). Studies from our group have also shown different ligand pharmacological properties depending upon the response measured from activation of 5-HT_2C receptors (Berg et al., 1998, 2001a; Stout et al., 2002; De Deurwaerdere et al., 2004). One of the important implications of agonist-directed trafficking of receptor stimulus is that drugs may have a richer pharmacology than previously thought. Ligands may have more selectivity than that afforded by differential affinity for different receptor subtypes. By developing drugs that are not only receptor subtype-selective but also signal pathway-selective, it may be possible to enhance therapeutic selectivity.

SB 243213 is a selective, high-affinity 5-HT_2C receptor ligand. SB 243213 has been previously characterized as a competitive 5-HT_2C receptor antagonist that has a long duration of activity in vivo, is active in two preclinical models of anxiety, and has an improved anxiolytic profile compared with benzodiazepines (Wood et al., 2001; Blackburn et al., 2002). In this study, we further characterized the pharmacological properties of SB 243213 by measuring its relative efficacy on each of multiple responses coupled to the 5-HT_2C receptor with respect to effects elicited by the 5-HT_2C inverse agonists SB206553 (Kennett et al., 1996) and clozapine (Herrick-Davis et al., 2000). In addition, the pharmacological profile of SB 243213 was also studied in vivo by assessing its influence on the effects induced by the inverse agonist SB206553 and the agonist Ro 60-0175 (Martin et al., 1998) on the release of DA measured in the rat nucleus accumbens (NAc) (De Deurwaerdere et al., 2004).

	Materials and Methods

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Materials
The following materials were purchased from commercial sources: myo-[³H]inositol, [³H]arachidonic acid, and [³⁵S]GTPS were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). 5-HT HCl, DOI, SB206553 HCl, and 8-chloro-11-(4-methyl-1-piperazinyl)-5H6-dibenzo[b,e][1,4]-diazepine (clozapine) were purchased from Sigma/RBI (Natick, MA). Ro 60-0175 HCl was kindly donated by Dr. P. Weber (F. Hoffman-La Roche, Basel, Switzerland). SB 242084 and SB 243213 were generously provided by Dr. M. Wood (Psychiatry CEDD, GlaxoSmithKline, Harlow, UK). Fetal bovine serum was from Gemini Bioproducts (Calabasas, CA). All other tissue culture reagents were purchased from Invitrogen (Carlsbad, CA). All other drugs and chemicals (reagent grade) were purchased from Sigma-Aldrich (St. Louis, MO, and Illkirch, France) and VWR (Strasbourg, France).

In Vitro Methods
Cell Culture. CHO-1C19 and CHO-1C7 cell lines are CHO-K1 cells which stably express nonedited 5-HT_2C-INI receptor at densities of 250 fmol/mg protein and 20 pmol/mg protein, respectively (Berg et al., 1994, 1999).

Cells were maintained in -minimum Eagle's medium supplemented with 5% fetal bovine serum and seeded into 12- or 24-well tissue culture vessels for functional studies or 15-cm dishes for radioligand binding studies at a density of 4 x 10⁴ cells/cm². Following a 24-h plating period, cells were washed with Hanks' balanced salt solution and placed into Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) with 5 µg/ml insulin, 5 µg/ml transferrin, 30 nM selenium, 20 nM progesterone, and 100 µM putrescine (serum-free media) and cultured for an additional 24 h before experimentation. All prolonged ligand pretreatments were done during the serum-free culture period.

Radioligand Binding. CHO-1C7 membranes were prepared from cell pellets by trituration through a 26-gauge needle, followed by centrifugation at 39,000g and sonication at 4°C. Estimates of ligand dissociation were made as described by Christopoulos et al. (1999). Membranes were incubated with 1 µM SB206553, 100 nM SB 243213, or 0.1% vehicle (dimethyl sulfoxide) for 60 min at 37°C, washed three times with ice-cold wash buffer (50 mM HEPES, 2.5 mM MgCl₂, and 2 mM EGTA, pH 7.4, at 23°C), and finally resuspended in assay buffer (wash buffer plus 0.1% ascorbic acid) at a protein concentration of 20 to 40 µg/ml. After washing, 2 nM [³H]mesulergine was added to tubes containing 5 to 10 µg of membrane protein and incubated for various periods up to 1 h. Binding was terminated by addition of ice-cold assay buffer and filtration through 0.5% polyethylenimine-coated GF/C glass fiber filters. This method of assessing the initial association rate of [³H]mesulergine with the 5-HT_2C receptor previously occupied with ligand provides an indirect measure of the relative rates of ligand dissociation.

IP Accumulation and AA Release Measurements. CHO-1C19 or CHO-1C7 cells were labeled in serum-free medium with 1 µCi/ml [³H]myo-inositol for 24 h and with 0.1 to 0.2 µCi/ml [³H]arachidonic acid in the presence of 1 µM unlabeled AA for 4 h at 37°C before experiments. Before initiation of the assay, cells were washed three times with 5-min incubation intervals between each wash. For agonist-stimulation experiments, agonists were added for 10 min in the presence of 20 mM LiCl as described previously (Berg et al., 1998, 1999). Antagonists, when present, were added simultaneously with, or 15 min before, agonists. Basal IP accumulation (i.e., ligand-independent 5-HT_2C receptor activity) was measured following a 25-min incubation (37°C) with 20 mM LiCl as described previously (Berg et al., 1998, 1999). For homologous sensitization studies (Berg et al., 1999), cells were treated for 24 h with ligand and washed three times with 5-min incubation intervals at 37°C. IP accumulation and AA release were measured from cells stimulated with the 5-HT_2C agonist DOI (1 µM) or vehicle for 10 min. For experiments in which both PLC-PI and PLA₂-AA pathways were measured, total [³H]IP accumulation and [³H]AA release were measured from the same multiwell (simultaneously) as described previously (Berg et al., 1998, 1999).

[³⁵S]GTPS Binding. GTPS binding to G_i/o was done in membranes prepared from CHO-1C7 cells as described previously (Evans et al., 2001; De Deurwaerdere et al., 2004). Membranes were prepared by repeated trituration of cells through a 1-ml pipette in ice-cold wash buffer (20 mM HEPES, 3 mM MgCl₂, 0.2 mM EGTA, and 100 mM NaCl, pH 7.4, at 23°C). The homogenate was centrifuged (39,000g; 4°C; 10 min), and the pellet was washed two times by resuspension in 40 volumes of the same buffer and then centrifugation. Membranes were resuspended in assay buffer (wash buffer plus 10 µM GDP, 100 nM okadaic acid, and 10 nM cypermethrin) at a protein concentration of 100 µg/ml. Aliquots (100 µl) of the membrane suspension were preincubated with ligand for 20 min, followed by the addition of [³⁵S]GTPS (0.3 nM, final concentration) for 30 min at 37°C in 96-well Multiscreen filtration plates. Binding was stopped by the addition of ice-cold buffer and rapid filtration, followed by several washes. Nonspecific binding was defined in the presence of 100 µM guanosine 5'-(,-imido)triphosphate. With this method, all [³⁵S]GTPS binding was blocked by pretreatment of cells with pertussis toxin and therefore represents binding to G_i/o and not G_q/11.

Data Analysis. For analysis of concentration-response curves, data were fit with nonlinear regression to the following model:

(1)

where R is the measured response at a given ligand concentration [A], R₀ is the response in the absence of ligand, R_m is response at maximal ligand concentration, EC₅₀ is the concentration of ligand that produces a half-maximal response, and n is the slope factor. Maximal response was calculated as R_m - R₀.

For analysis of initial association rate, data points for the first 15 min of [³H]mesulergine binding were fit to the following equation:

(2)

where B is the measured amount of [³H]mesulergine bound at time t, B_max is the maximal amount of [³H]mesulergine bound at t = , and k is the apparent rate constant.

For signal transduction and binding assays, Student's t test (paired) was used for statistical comparisons. Asterisks denote p values <0.05 (*), <0.01 (**), or <0.001 (***).

In Vivo Methods
Animals. Sprague-Dawley rats (Iffa Credo, Lyon, France) weighing 330 to 380 g were used. Animals were kept at constant room temperature (21 ± 2°C) and relative humidity (60%) with a 12-h light/dark cycle (dark from 8:00 PM) and had free access to water and food. All animals use procedures conformed to International European Ethical Standards (86/609-EEC) and the French National Committee (décret 87/848) for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.

In Vivo Microdialysis. Surgery and perfusion procedures were performed as described previously, with minor modifications (De Deurwaerdere et al., 2004). In brief, rats were anesthetized with a mixture of halothane and nitrous oxide-oxygen [2%; 2:1 (v/v)]. After tracheotomy for artificial ventilation, the animals were placed in a stereotaxic frame, and their rectal temperature was monitored and maintained at 37.3 ± 0.1°C with a heating pad. One microdialysis probe, 2 mm in length, (CMA/11, 240-µm outer diameter, Cuprophan; Carnegie Medicin, Phymep, Paris, France) was implanted in the right NAc (coordinates from interaural point: AP, 11; L, 1.3; and V, 2) according to the atlas of Paxinos and Watson (1998). The probe was perfused at a constant flow rate of 2 µl/min by means of a microperfusion pump (CMA 111; Carnegie Medicin, Phymep) with artificial cerebrospinal fluid containing 154.1 mM Cl^-, 147 mM Na⁺, 2.7 mM K⁺, 1 mM Mg²⁺, and 1.2 mM Ca²⁺, adjusted to pH 7.4 with 2 mM sodium phosphate buffer. Dialysates (30 µl) were collected on ice every 15 min. The in vitro recovery of the probe was about 10% for DA. At the end of each experiment, the brain was removed and fixed in 0.9% NaCl/10% paraformaldehyde solution. The location of the probe was determined histologically on serial coronal sections (60 µm) stained with cresyl violet, and only data obtained from rats with correctly implanted probes were included in the results.

Chromatographic Analysis. Dialysate samples were immediately analyzed by reverse-phase high-performance liquid chromatography coupled with electrochemical detection, as described previously (Bonhomme et al., 1995). The mobile phase (containing 70 mM NaH₂PO₄, 0.1 mM Na₂EDTA, 0.7 mM triethylamine, and 0.1 mM octylsulfonic acid plus 10% methanol, adjusted to pH 4.8 with orthophosphoric acid) was delivered at 1 ml/min flow rate (Shimadzu system LC-10AD-VP; Shimadzu, Croissy Beaubourg, France) through a Hypersyl column (C18; 4.6 x 150 mm, particle size 5 µm; Touzard and Matignon, Paris, France). Detection of DA was carried out with a coulometric detector (Coulochem II; ESA, Paris, France) coupled to a dual-electrode analytical cell (model 5014; ESA). The potential of the electrodes was set at -175 and +175 mV. Output signals were recorded on a computer (Shimadzu system class VP-4). Under these conditions, the sensitivity for DA was 0.5 pg/30 µl with a signal-to-noise ratio of 3:1.

Pharmacological Treatments. Pharmacological treatments were performed after the stabilization of DA levels in the perfusate. A stable baseline, defined as three consecutive samples in which DA contents varied by less than 10% in the NAc, was generally obtained 135 min after the beginning of the perfusion (stabilization period). The selective 5-HT_2C ligand SB 243213 was dissolved in a mixture of 0.9% NaCl containing hydroxypropyl--cyclodextrin (8% by weight) plus 25 mM citric acid and was administered intraperitoneally at 1, 3, and 10 mg/kg in a volume of 2 ml/kg. The 5-HT_2C agonist Ro 60-0175 was dissolved in 0.9% NaCl and administered intraperitoneally at 3 mg/kg in a volume of 1 ml/kg. The 5-HT_2C inverse agonist SB206553 was diluted in a 99:1 (v/v) mixture of apyrogenic water plus lactic acid and administered intraperitoneally at 5 mg/kg in a volume of 2 ml/kg. SB 243213 was administered intraperitoneally at 1 mg/kg 30 min before Ro 60-0175 and 30 or 60 min before SB206553. The doses and administration times of the different 5-HT compounds used were chosen on the basis of previous studies to keep both selectivity and efficiency toward the targeted sites (Kennett et al., 1996; Gobert et al., 2000; Blackburn et al., 2002; De Deurwaerdere et al., 2004). All drug doses were calculated as the free base. In each experimental group, animals received either drugs or their appropriate vehicle.

Data Analysis. DA content in each sample was expressed as the percentage of the average baseline level calculated from the three fractions preceding any treatment. Data correspond to the mean ± S.E.M. values of the percentage obtained in each experimental group. Drug overall effect was calculated as the average of DA content from dialysates collected after their administration.

The statistical analysis of the effect of the 5-HT_2C ligand SB 243213 on basal DA outflow was assessed by a one-way ANOVA (using group as the main factor) with time as repeated measures, performed for the 10 samples that followed its administration. When significant (p < 0.05), the one-way ANOVA was followed by the Fisher's protected least significance difference test (PLSD) to determine the effect of each dose of SB 243213.

The interaction of SB 243213 (pretreatment) with Ro 60-0175 or SB206553 (treatment) was studied by a two-way ANOVA (pretreatment x treatment) with time as repeated measures, performed for the eight samples that followed the treatment administration. When significant (p < 0.05), the ANOVA was followed by the post hoc Fisher's PLSD test to allow multiple comparison between groups.

	Results

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Effect of SB 243213 on PLC, PLA₂, and [³⁵S]GTPS Binding. Figure 1 shows that SB 243213 behaved as a strong inverse agonist for the PLA₂-AA and [³⁵S]GTPS binding responses coupled to the 5-HT_2C receptor. In comparison with the full inverse agonist SB206553, SB 243213 was a partial inverse agonist with a relative efficacy of 0.6 for AA release and a pIC₅₀ of 8.77 ± 0.12 (1.7 nM). For [³⁵S]GTPS binding the relative efficacy of SB 243213 was 1.0 and the pIC₅₀ was 8.89 ± 0.31 (1.3 nM). In contrast to the AA release and basal [³⁵S]GTPS binding responses, SB 243213 did not alter basal activity of the PLC-IP pathway (Figs. 1 and 2A) at concentrations expected to produce full receptor occupancy (1 µM; 1000 x K_i). Thus, SB 243213 seems to be an antagonist for this response. To further define the antagonist property for the PLC-IP response, we tested the ability of SB 243213 to block both inverse agonist and agonist effects on IP accumulation. As shown in Fig. 2, SB 243213 blocked the effects of the inverse agonists, clozapine and SB206553, and of the agonist Ro 60-0175. SB 243213 had no effect in CHO cells that did not express the 5-HT_2C receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 1. A to E, effect of SB 243213 on basal AA release (A and B), activation of Gi (C and D), and IP accumulation (E). CHO-1C7 cells were incubated with either 1 µM clozapine (100 x KB), 300 nM SB206553 (100 x KB), the indicated concentrations of SB 243213 (1 µM in A), or vehicle (0.01% DMSO). The amount of [3H]AA released, [3H]IP accumulated, or [35S]GTPS bound was determined after 25 min of incubation with ligands. Basal levels of AA release (A and B) were 1222 ± 42 dpm, for [35S]GTPS binding (C and D) were 89 ± 7 pmol/mg protein, and for IP accumulation (E) were 6577 ± 407 dpm. The data shown represent the mean ± S.E.M. of three to five independent experiments. *, p < 0.05 compared with effect of SB206553, Student's paired t test.

View larger version (14K): [in this window] [in a new window]   Fig. 2. A, time course of constitutive 5-HT2C receptor activity. SB 243213 does not alter basal IP accumulation, but it blocks the inverse agonist effect of clozapine. CHO-1C7 cells were incubated with 20 mM LiCl in the absence (closed squares) and presence of 1 µM SB 243213 (1000 x Ki; open squares), 1 µM clozapine (100 x KB; closed circles), and both SB 243213 and clozapine (open triangles) for the indicated times. Data represent the mean ± S.E.M. of three experiments. For some data points, the S.E.M. is within the size of the symbol. (B) SB 243213 blocked SB206553-mediated inhibition of PLC. Cells were incubated with 30 nM SB 243213 or vehicle for 15 min, before a 25-min incubation with 100 nM SB206553. Columns represent the mean ± S.E.M. of three experiments. ***, p < 0.001 compared with vehicle. C, SB 243213 blocked agonistmediated stimulation of PLC. Cells were incubated with 30 nM SB 243213 or vehicle for 15 min, before a 10-min stimulation with 100 nM 5-HT or 300 nM Ro 60-0175. Bars represent the mean ± S.E.M. of three experiments. ***, p < 0.001 compared with vehicle, Student's paired t test.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Concentration-response curves for 5-HT in the absence (vehicle) and presence of SB 243213. CHO-1C19 cells were pretreated (15 min) or coincubated with 30 nM SB 243213 and the indicated concentrations of 5-HT. Total IP accumulation for 10 min was measured. Data shown are mean dpm ± S.E.M. of three experiments. The KB calculated from the equation [B]/dr - 1 is 2.4 nM with coincubation and 0.12 nM with pretreatment conditions.

Effect of SB 243213 on Extracellular DA Levels in the NAc. Absolute basal levels of DA in dialysates collected from the NAc did not differ across the different experimental groups throughout the course of the study and were 5.3 ± 0.3 pg/30 µl (mean ± S.E.M., without adjusting for probe recovery; n = 102 animals). Intraperitoneal administration SB 243213 produced a progressive and long-lasting increase in DA extracellular levels in the NAc reaching approximately +30% of basal values at the end of the experiment at the three doses used (one-way ANOVA, F_3,33 = 7.1, p < 0.001; Fig. 4A).

View larger version (28K): [in this window] [in a new window]   Fig. 4. A, effect of the i.p. administration of the 5-HT2C ligand SB 243213 (1, 3, and 10 mg/kg i.p.) on DA extracellular levels in the NAc. SB 243213 was injected at time 0. B, effect of SB 243213 (1 mg/kg i.p.) on Ro 60-0175-induced decrease in DA extracellular levels in the NAc. SB 243213 was injected (vertical arrow) 30 min before the i.p. administration of 3 mg/kg Ro 60-0175 (time 0). Data, obtained from five to seven animals per group, represent mean ± S.E.M. percentages of baseline in each sample (time courses), or averaged (insets) over 2.5 (A) or 2 h (B). **, p < 0.01; and ***, p < 0.001 versus the vehicle group; +++, p < 0.001 versus the Ro 60-0175 group (Fisher's PLSD test).

Figure 5 illustrates the effect of the administration of 1 mg/kg SB 243213 (i.p.) on the increase in DA extracellular levels induced by SB206553 (5 mg/kg i.p.). As reported previously (De Deurwaerdere et al., 2004), SB206553 elicited a significant overall increase in DA outflow, reaching about 50% over basal. SB 243213 (1 mg/kg i.p.) significantly blocked the increase produced by SB206553 when it was administered 30 min before (p < 0.05; Fisher's PLSD test after a two-way ANOVA F_1,18 = 12.9, p < 0.01; Fig. 5A) or 60 min before SB206553 (p < 0.01; Fisher's PLSD test after a two-way ANOVA F_1,22 = 19.4, p < 0.001; Fig. 5B).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effect of the i.p. administration of the 5-HT2C ligand SB 243213 (1 mg/kg i.p.) on SB206553-induced increase in DA extracellular levels in the NAc. SB 243213 was injected (vertical arrows) 30 min (A) and 60 min (B) before the i.p. administration of 5 mg/kg SB206553 (time 0). Data, obtained from five to eight animals per group, represent mean ± S.E.M. percentages of baseline in each sample (time courses) or averaged over 2 h (insets). *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus the vehicle group; +, p < 0.05; ++, p < 0.01 versus the SB206553 group (Fisher's PLSD test).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effect of 24-h treatment with clozapine or SB 243213 on basal (A) and agonist-stimulated (B) 5-HT2C constitutive receptor activity. CHO-1C7 cells were treated for 24 h with vehicle, 1 µM clozapine, or 1 µM SB 243213 followed by extensive washing. A, IP was measured for 25 min. Basal IP accumulation was 1971 ± 88 dpm. Data shown represent mean percentage of basal accumulation ± S.E.M. of four experiments. ***, p < 0.001 compared with vehicle, Student's paired t test. B, accumulation of IP with or without the 5-HT2C agonist DOI (1 µM) for 25 min was measured. DOI-stimulated IP accumulation in vehicle-treated cells was 210 ± 12% above basal. Data shown represent mean percentage of DOI-stimulated IP accumulation ± S.E.M. of four experiments. ***, p < 0.001; **, p < 0.01 compared with vehicle, Student's paired t test.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Mianserin-sensitive 5-HT2C constitutive receptor activity following 24-h treatment with clozapine or SB 243213. CHO-1C7 cells were incubated with 1 µM SB 243213, 1 µM clozapine, or vehicle for 24 h. Following extensive washing to remove ligand, total IP for 25 min with or without mianserin (100 x KB) was measured. Basal IP accumulation was 1942 ± 167 dpm. Data shown are mean percentage of basal activity ± S.E.M. of four experiments. **, p < 0.01 compared with vehicle, Student's paired t test.

Dissociation rate of SB 243213 Binding to the 5-HT_2C Receptor. To determine whether slow dissociation of SB 243213 from the 5-HT_2C receptor could be responsible for the time-dependent shift in the 5-HT concentration-response curve (Fig. 3) and the different effects of prolonged SB 243213 treatment with respect to clozapine (Figs. 6B and 7), we measured the relative rates of dissociation of SB 243213 compared with SB206553 by measuring the association rate of [³H]mesulergine binding after pretreatment of membranes with maximal concentrations of the ligands (with respect to receptor occupancy). The association rate of [³H]mesulergine was markedly slower in membranes pretreated with SB 243213 than with SB206553 (Fig. 8). The initial association rate constants for [³H]mesulergine binding were 0.423 ± 0.08, 0.249 ± 0.11, and 0.078 ± 0.01 min^-1 for vehicle-, SB206553-, and SB 243213-pretreated membranes, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Association of [3H]mesulergine with the 5-HT2C receptor in CHO-1C7 cell membranes that were pretreated with 100 nM SB 243213, 1 µM SB206553, or vehicle (0.1% DMSO) for 60 min at 37°C. After treatment, membranes were washed extensively at 4°C and then incubated for various times with 2 nM [3H]mesulergine at 37°C. Data are normalized to the maximal [3H]mesulergine binding in vehicle-treated membranes and represent mean ± S.E.M. of three experiments.

	Discussion

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The data presented here demonstrate that SB 243213 has different pharmacological properties at the 5-HT_2C receptor, depending upon the response measured. For the PLA₂-AA and G_i pathways, SB 243213 is an inverse agonist (negative efficacy), reducing basal AA release to about 60% of that of the reference inverse agonist SB206553, and reducing basal [³⁵S]GTPS binding to an equivalent extent as SB206553. Likewise, for inactivation of desensitization mechanisms upon prolonged treatment, SB 243213 was also a strong inverse agonist, producing sensitization of the 5-HT_2C-PLC-IP system that was equal to that produced by the reference inverse agonist clozapine. However, SB 243213 behaved as an antagonist (zero efficacy) for acute activation of the 5-HT_2C-PLC-IP pathway. At concentrations that should produce full receptor occupancy (1000 x K_i), SB 243213 did not alter basal IP accumulation and blocked inverse agonist (clozapine and SB206553)-induced decreases and agonist (5-HT and Ro 60-0175)-stimulated increases in IP accumulation.

In vivo, SB 243213 seemed to behave as a partial inverse agonist for 5-HT_2C receptor-mediated regulation of DA release. SB 243213 alone produced a small increase in DA release and antagonized both agonist-(Ro 60-0175) and inverse agonist (SB206553)-induced changes in DA release in the NAc measured with microdialysis. These actions of SB 243213 were similar to those of SB 242084 on DA release in the NAc (De Deurwaerdere et al., 2004); however, the antagonism of the SB206553-induced increase in DA release by SB 243213 had a longer latency than that of SB 242084, requiring approximately 100 min to occur. This is consistent with the in vitro results that showed increased potency of SB 243213 to antagonize 5-HT-stimulated IP accumulation with incubation time, likely as a result of the slow dissociation of SB 243213 from the receptor (see below). Considering that ligands that activate the 5-HT_2C-PLC pathway decrease DA release, ligands that reduce 5-HT_2C-PLC signaling increase DA release and ligands with weak efficacy for the PLC response antagonize effects of stronger agonists and inverse agonists, it seems likely that the 5-HT_2C-PLC pathway plays a predominant role in regulating DA release in vivo, although extrapolation of results from in vitro cell culture systems to in vivo physiological processes should be done with caution.

Taken together, these data demonstrate that the relative efficacy of SB 243213 differs depending upon the response measured. Such ligand action is not consistent with traditional receptor theory that requires that ligand relative efficacy, as a measure of intrinsic efficacy, be response-independent. Although somewhat sporadic, strong evidence has been accumulating for the 5-HT_2C receptor system, and others (for reviews, see Kenakin, 2002; Perez and Karnik, 2005), which suggest that traditional receptor theory requires modification. Previously, we showed that some 5-HT_2C agonists preferentially activate the PLC over the PLA₂ pathway, whereas for other agonists the reverse was true (Berg et al., 1998). In addition, 5-HT_2C agonists differ in their relative efficacy to elicit desensitization of the PLC and the PLA₂ responses (Stout et al., 2002). Werry et al. (2005) reported that 5-HT_2C agonists differentially regulate PLC versus extracellular signal-regulated kinase1/2 signaling, and Backstrom et al. (1999) showed that the relative efficacy of LSD differs for PLC versus calcium mobilization and 5-HT_2C receptor phosphorylation. Differential relative efficacy toward different cellular signaling cascades coupled to the same receptor suggests that drugs may have more specificity than that afforded by differential affinity for receptor subtypes. Such selectivity may be exploited for improved therapeutic efficacy.

We have reported previously that prolonged reduction of constitutive 5-HT_2C receptor activity (with inverse agonists) toward desensitization mechanisms results in enhanced responsiveness of the 5-HT_2C-PLC signaling pathway (Berg et al., 1999). This enhanced responsiveness occurs as a result of increased expression of G_q/11 in the absence of a change in 5-HT_2C receptor density. Here, we found that prolonged treatment with SB 243213 or clozapine enhanced constitutive 5-HT_2C signaling to the PLC pathway. However, surprisingly and unlike the clozapine effect, the enhanced basal IP response was not sensitive to reduction by acute treatment with the inverse agonist mianserin. Furthermore, prolonged SB 243213 treatment did not enhance 5-HT_2C agonist stimulation of IP accumulation, as does clozapine and SB206553 (Berg et al., 1999), but instead reduced it.

One explanation for the opposite effects of prolonged SB 243213 treatment on basal versus agonist-stimulated IP accumulation is perhaps due to response-dependent differences in the pharmacological properties of SB 243213 combined with a slow rate of dissociation from the receptor. As an inverse agonist toward constitutive desensitization, prolonged treatment with SB 243213 would increase basal IP accumulation; however, with a slow rate of dissociation, SB 243213 would remain bound to the receptor after a washout procedure that is effective at removing clozapine and SB206553. Our data suggest that the dissociation of SB 243213 from the 5-HT_2C receptor is markedly slower than that of SB206553. Continued occupancy of the receptor by SB 243213 after prolonged treatment would not alter the enhanced basal IP response [SB 243213 is an antagonist (zero efficacy) for this response], but would block the action of agonists (DOI) to stimulate IP accumulation and would prevent inverse agonists (mianserin) from decreasing the enhanced basal response, as we observed here. It should be noted that although slow dissociation of SB 243213 from the 5-HT_2C receptor is the most parsimonious explanation for our data, which showed that pretreatment of membranes containing the 5-HT_2C receptor with SB 243213, but not SB206553, reduced the rate of association of [³H]mesulergine, there are other interpretations. For example, it is possible that SB 243213, but not SB206553, pretreatment produces a change in the 5-HT_2C receptor conformation (e.g., association with an accessory protein, ligand-induced changes in receptor dimerization, phosphorylation, differential receptor distribution into membrane microdomains) that persists after dissociation of SB 243213 such that affinity of [³H]mesulergine is reduced.

Slow dissociation of SB 243213 would also be expected to alter the kinetics of antagonism of the PLC response. We found that the magnitude of the dextral shift of the 5-HT concentration-response curve for IP accumulation was time-dependent. Furthermore, even though SB 243213 was previously characterized as a competitive antagonist (Wood et al., 2001), we found that SB 243213 reduced the maximal 5-HT response. Although insurmountable antagonism is generally associated with uncompetitive or irreversible antagonism, maximal response depression also occurs for competitive, but slowly dissociating, antagonists when used in experiments in which agonists are applied for short periods. In these cases, the agonist cannot bind until the antagonist dissociates from the receptor. This "hemiequilibrium" can result in pharmacological characteristics that seem to be time-dependent; insurmountability when measured under nonequilibrium (short-term) conditions versus surmountablility in equilibrium (long-term) assays (Christopoulos et al., 1999). Consistent with this hypothesis, equilibrium binding assays indicate that SB 243213 binding to the 5-HT_2C receptor is reversible and surmountable (Wood et al., 2001); however, indirect measurement of the dissociation rate of SB 243213 suggested that it dissociates very slowly from the receptor. Slow receptor dissociation could explain the long duration of action of SB 243213 seen in some behavioral assays (Wood et al., 2001).

In summary, our data demonstrate that SB 243213 displays different pharmacological properties depending upon the response measured. For 5-HT_2C receptor-mediated activation of the PLA₂-AA cascade, [³⁵S]GTPS binding, reduction of constitutive desensitization of PLC signaling and regulation of DA release in vivo, SB 243213 is an inverse agonist with different relative efficacy depending upon the response. However, for stimulation of PLC, SB 243213 behaves as an antagonist. Compared with SB206553, SB 243213 has a relatively slow dissociation rate from the 5-HT_2C receptor, which likely explains the time-dependent increase in antagonist activity (increase potency and reduction of maximal response) of the 5-HT_2C receptor-mediated PLC response. These results support the hypothesis that ligands can traffic the receptor stimulus differentially to cellular signaling cascades and suggest that pharmacological labeling of drugs (as agonists, antagonists, and inverse agonists) should be done with reference to the response measured.

	Acknowledgements

 
We are grateful to Dr. P. Weber for the gift of Ro 60-0175. We thank D. Moison and P. Moya for technical support and Dr. Arthur Christopoulos for helpful discussions.

	Footnotes

 
This work was supported by National Institutes of Health Grant U.S. Public Health Service GM 56852 and grants from the National Alliance for Research on Schizophrenia and Depression, the Centre National de la Recherche Scientifique, and Bordeaux 2 University. S.N. was a fellowship recipient from the Ministère de la Recherche et de l'Enseignement Supérieur during the course of this study.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104448.

ABBREVIATIONS: 5-HT, 5,hydroxytryptamine, serotonin; 7-TMS, 7-transmembrane spanning; PLC, phospholipase C; IP, inositol phosphate; PLA₂, phospholipase A₂; AA, arachidonic acid; DOI, (±)-2,5-dimethoxy-4-iodoamphetamine hydrobromide; CHO, Chinese hamster ovary; SB 243213, 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxyl]-5-pyridyl]carbamoyl]-6-trifluoromethylindone; SB206553, 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole; Ro 60-0175, S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine; SB 242084, 6-chloro-5-methyl-1-[6-(2-methylpiridin-3-yloxy)pyridin-3-yl carbamoyl] indoline; DA, dopamine; NAc, nucleus accumbens; GTPS, guanosine 5'-O-(3-thio)triphosphate; ANOVA, analysis of variance; PLSD, protected least significant difference; i.p., intraperitoneal.

Address correspondence to: Dr. Kelly A. Berg, Department of Pharmacology, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. E-mail: berg{at}uthscsa.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Backstrom JR, Chang MS, Chu H, Niswender CM, and Sanders-Bush E (1999) Agonist-directed signaling of serotonin 5-HT_2C receptors: differences between serotonin and lysergic acid diethylamide (LSD). Neuropsychopharmacology 21: S77-S81.[Medline]

Backstrom JR, Price RD, Reasoner DT, and Sanders-Bush E (2000) Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses. J Biol Chem 275: 23620-23626.[Abstract/Free Full Text]

Berg KA, Clarke WP, Sailstad C, Saltzman A, and Maayani S (1994) Signal transduction differences between 5-hydroxytryptamine type 2A and type 2C receptor systems. Mol Pharmacol 46: 477-485.[Abstract]

Berg KA, Cropper JD, Niswender CM, Sanders-Bush E, Emeson RB, and Clarke WP (2001a) RNA-editing of the 5-HT_2C receptor alters agonist-receptor-effector coupling specificity. Br J Pharmacol 134: 386-392.[CrossRef][Medline]

Berg KA, Maayani S, Goldfarb J, Scaramellini C, Leff P, and Clarke WP (1998) Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Mol Pharmacol 54: 94-104.[Abstract/Free Full Text]

Berg KA, Stout BD, Cropper JD, Maayani S, and Clarke WP (1999) Novel actions of inverse agonists on 5-HT_2C receptor systems. Mol Pharmacol 55: 863-872.[Abstract/Free Full Text]

Berg KA, Stout BD, Maayani S, and Clarke WP (2001b) Differences in rapid desensitization of 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptor-mediated phospholipase C activation. J Pharmacol Exp Ther 299: 593-602.[Abstract/Free Full Text]

Blackburn TP, Minabe Y, Middlemiss DN, Shirayama Y, Hashimoto K, and Ashby CR Jr (2002) Effect of acute and chronic administration of the selective 5-HT_2C receptor antagonist SB-243213 on midbrain dopamine neurons in the rat: an in vivo extracellular single cell study. Synapse 46: 129-139.[CrossRef][Medline]

Bonhomme N, De Deurwaerdere P, Le Moal M, and Spampinato U (1995) Evidence for 5-HT₄ receptor subtype involvement in the enhancement of striatal dopamine release induced by serotonin: a microdialysis study in the halothane-anesthetized rat. Neuropharmacology 34: 269-279.[CrossRef][Medline]

Christopoulos A, Parsons AM, Lew MJ, and El-Fakahany EE (1999) The assessment of antagonist potency under conditions of transient response kinetics. Eur J Pharmacol 382: 217-227.[CrossRef][Medline]

Clarke WP (2005) What's for lunch at the conformational cafeteria? Mol Pharmacol 67: 1819-1821.[Abstract/Free Full Text]

Clarke WP and Bond RA (1998) The elusive nature of intrinsic efficacy. Trends Pharmacol Sci 19: 270-276.[CrossRef][Medline]

De Deurwaerdere P, Navailles S, Berg KA, Clarke WP, and Spampinato U (2004) Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum and nucleus accumbens. J Neurosci 24: 3235-3241.[Abstract/Free Full Text]

Evans KL, Cropper JD, Berg KA, and Clarke WP (2001) Mechanisms of regulation of agonist efficacy at the 5-HT_1A receptor by phospholipid-derived signaling components. J Pharmacol Exp Ther 297: 1025-1035.[Abstract/Free Full Text]

Gobert A, Rivet JM, Lejeune F, Newman-Tancredi A, Adhumeau-Auclair A, Nicolas JP, Cistarelli L, Melon C, and Millan MJ (2000) Serotonin(2C) receptors tonically suppress the activity of mesocortical dopaminergic and adrenergic, but not serotonergic, pathways: a combined dialysis and electrophysiological analysis in the rat. Synapse 36: 205-221.[CrossRef][Medline]

Herrick-Davis K, Grinde E, and Teitler M (2000) Inverse agonist activity of atypical antipsychotic drugs at human 5-hydroxytryptamine2C receptors. J Pharmacol Exp Ther 295: 226-232.[Abstract/Free Full Text]

Jones BJ and Blackburn TP (2002) The medical benefit of 5-HT research. Pharmacol Biochem Behav 71: 555-568.[CrossRef][Medline]

Kenakin T (1995) Agonist-receptor efficacy II: agonist trafficking of receptor signals. Trends Pharmacol Sci 16: 232-238.[CrossRef][Medline]

Kenakin T (2002) Efficacy at G-protein-coupled receptors. Nat Rev Drug Discov 1: 103-110.[CrossRef][Medline]

Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, Thomas D, Baxter GS, Forbes IT, Ham P, et al. (1996) In vitro and in vivo profile of SB 206553, a potent 5-HT_2C/5-HT_2B receptor antagonist with anxiolytic-like properties. Br J Pharmacol 117: 427-434.[Medline]

Leysen JE (2004) 5-HT2 receptors. Curr Drug Targets CNS Neurol Disord 3: 11-26.[CrossRef][Medline]

Lucaites VL, Nelson DL, Wainscott DB, and Baez M (1996) Receptor subtype and density determine the coupling repertoire of the 5-HT₂ receptor subfamily. Life Sci 59: 1081-1095.[CrossRef][Medline]

Mailman RB and Gay EA (2004) Novel mechanisms of drug action: functional selectivity at D-2 dopamine receptors (a lesson for drug discovery). Med Chem Res 13: 115-126.[CrossRef]

Martin JR, Bos M, Jenck F, Moreau J, Mutel V, Sleight AJ, Wichmann J, Andrews JS, Berendsen HH, Broekkamp CL, et al. (1998) 5-HT_2C receptor agonists: pharmacological characteristics and therapeutic potential. J Pharmacol Exp Ther 286: 913-924.[Abstract/Free Full Text]

McGrew L, Chang MS, and Sanders-Bush E (2002) Phospholipase D activation by endogenous 5-hydroxytryptamine 2C receptors is mediated by G₁₃ and pertussis toxin-insensitive G subunits. Mol Pharmacol 62: 1339-1343.[Abstract/Free Full Text]

Paxinos G and Watson C (1998) The Rat Brain in Sterotaxic Coordinates, Academic Press, New York.

Perez DM and Karnik SS (2005) Multiple signaling states of G-protein-coupled receptors. Pharmacol Rev 57: 147-161.[Abstract/Free Full Text]

Porter RH, Malcolm CS, Allen NH, Lamb H, Revell DF, and Sheardown MJ (2001) Agonist-induced functional desensitization of recombinant human 5-HT₂ receptors expressed in CHO-K1 cells. Biochem Pharmacol 62: 431-438.[CrossRef][Medline]

Roth BL and Shapiro DA (2001) Insights into the structure and function of 5-HT₂ family serotonin receptors reveal novel strategies for therapeutic target development. Expert Opin Ther Targets 5: 685-695.[CrossRef][Medline]

Roth BL, Willins DL, Kristiansen K, and Kroeze WK (1998) 5-Hydroxytryptamine2-family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-hydroxytryptamine2C): where structure meets function. Pharmacol Ther 79: 231-257.[CrossRef][Medline]

Saucier C, Morris SJ, and Albert PR (1998) Endogenous serotonin-2A and -2C receptors in Balb/c-3T3 cells revealed in serotonin-free medium: desensitization and down-regulation by serotonin. Biochem Pharmacol 56: 1347-1357.[CrossRef][Medline]

Schlag BD, Lou Z, Fennell M, and Dunlop J (2004) Ligand dependency of 5-hydroxytryptamine 2C receptor internalization. J Pharmacol Exp Ther 310: 865-870.[Abstract/Free Full Text]

Stout BD, Clarke WP, and Berg KA (2002) Rapid desensitization of the serotonin(2C) receptor system: effector pathway and agonist dependence. J Pharmacol Exp Ther 302: 957-962.[Abstract/Free Full Text]

Werry T, Gregory K, Sexton P, and Christopoulos A (2005) Characterization of serotonin 5-HT2C signaling to extracellular signal related kinases 1 and 2. J Neurochem 93: 1603-1615.[CrossRef][Medline]

Westphal RS, Backstrom JR, and Sanders-Bush E (1995) Increased basal phosphorylation of the constitutively active serotonin 2C receptor accompanies agonist-mediated desensitization. Mol Pharmacol 48: 200-205.[Abstract]

Wilbanks AM, Laporte SA, Bohn LM, Barak LS, and Caron MG (2002) Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors. Biochemistry 41: 11981-11989.[CrossRef][Medline]

Wood MD, Reavill C, Trail B, Wilson A, Stean T, Kennett GA, Lightowler S, Blackburn TP, Thomas D, Gager TL, et al. (2001) SB-243213; a selective 5-HT_2C receptor inverse agonist with improved anxiolytic profile: lack of tolerance and withdrawal anxiety. Neuropharmacology 41: 186-199.[CrossRef][Medline]

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