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TPN672 [7-(2-(4-(benzothiophen-4-yl) piperazin-1-yl)ethyl)quinolin-2(1H)-one maleate] is a novel antipsychotic candidate with high affinity for serotonin and dopamine receptors that is currently in clinical trial for the treatment of psychiatric disorders. In vitro binding study showed that TPN672 exhibited extremely high affinity for serotonin 1A receptor (5-HT 1A R) ( K i = 0.23 nM) and 5-HT 2A R ( K i = 2.58 nM) as well as moderate affinity for D 3 R ( K i = 11.55 nM) and D 2 R ( K i = 17.91 nM). In vitro functional assays demonstrated that TPN672 acted as a potent 5-HT 1A R agonist, D 2 R/D 3 R partial agonist, and 5-HT 2A R antagonist. TPN672 displayed robust antipsychotic efficacy in rodent models (e.g., blocking phencyclidine-induced hyperactivity), significantly better than aripiprazole, and ameliorated negative symptoms and cognitive deficits in the sociability test, dark avoidance response, Morris water maze test, and novel object recognition test. The results of electrophysiological experiments showed that TPN672 might inhibit the excitability of the glutamate system through activating 5-HT 1A R in medial prefrontal cortex, thereby improving cognitive and negative symptoms. Moreover, the safety margin (the ratio of minimum catalepsy-inducing dose to minimum effective dose) of TPN672 was about 10-fold, which was superior to aripiprazole. In conclusion, TPN672 is a promising new drug candidate for the treatment of schizophrenia and has been shown to be more effective in attenuating negative symptoms and cognitive deficits while having lower risk of extrapyramidal symptoms and hyperprolactinemia. SIGNIFICANCE STATEMENT: TPN672 is a promising new drug candidate for the treatment of schizophrenia and has been shown to be more effective in attenuating negative symptoms and cognitive deficits while having a lower risk of extrapyramidal symptoms and hyperprolactinemia. A phase I clinical trial is now under way to test its tolerance, pharmacokinetics, and pharmacodynamic effects in human volunteers. Accordingly, the present results will have significant impact on the development of new antischizophrenia drugs., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Radiotracers suitable for positron emission tomography studies often serve as preclinical tools for in vivo receptor occupancy. The serotonin 1B receptor (5-HT(1B)) subtype is a pharmacological target used to discover treatments for various psychiatric and neurological disorders. In psychiatry, 5-HT(1B) antagonists may provide novel therapeutics for depression and anxiety. We report on the in vitro and in vivo evaluation of tritiated 5-methyl-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylicacid (4-morpholin-4-yl-phenyl)-amide ([N-methyl-(3)H(3)]AZ10419369), a potent 5-HT(1B) radiotracer. [N-methyl-(3)H(3)]-AZ10419369 showed saturable single-site high-affinity in vitro binding (guinea pig, K(d) = 0.38 and human, K(d) = 0.37) to guinea pig or human 5-HT(1B) receptors in recombinant membranes and high-affinity (K(d) = 1.9 nM) saturable (B(max) = 0.099 pmol/mg protein) binding in membranes from guinea pig striatum. When [N-methyl-(3)H(3)]AZ10419369 was administered to guinea pigs by intravenous bolus, the measured radioactivity was up to 5-fold higher in brain areas containing the 5-HT(1B) receptor (striatum/globus pallidus, midbrain, hypothalamus, and frontal cortex) compared with the cerebellum, the nonspecific binding region. Specific uptake peaked 30 min after injection with slow dissociation from target regions, as suggested by the in vitro binding kinetic profile. Pretreatment with 6-fluoro-8-(4-methyl-piperazin-1-yl)-4-oxo-4H-chromene-2-carboxylic acid [4-(4-propionyl-piperazin-1-yl)-phenyl]-amide (AZD1134) and 2-aminotetralin (AR-A000002), 5-HT(1B)-selective ligands, inhibited [N-methyl-(3)H(3)]AZ10419369-specific binding in a dose-dependent manner. In the guinea pig striatum, AZD1134 (ED(50) = 0.017 mg/kg) occupies a greater percentage of the 5-HT(1B) receptors at a lower administered dose than AR-A000002 (ED(50) = 2.5 mg/kg). In vivo receptor occupancy is an essential component to build binding-efficacy-exposure relationships and compare novel compound pharmacology. [N-methyl-(3)H(3)]AZ10419369 is a useful preclinical tool for investigating 5-HT(1B) receptor occupancy for novel compounds targeting this receptor.

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT)2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT2C receptor agonists serotonin, Ro 60-0175 [(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine], and WAY-161503 [(4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one]; partial agonists mCPP [1-(m-chlorophenyl)piperazine] and DOI [(+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]; inverse agonists SB-206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide] and mianserin; and neutral antagonists SB-242084 [6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline] and 5-methoxygramine on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca2+ measurements (flourometric imaging plate reader). The 5-HT2C-GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in approximately 50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.

There is increasing evidence for a role of 5-hydroxytrypta-mine-6 (5-HT6) receptors in cognitive function. In the rat and human brain, 5-HT6 receptors are widely expressed and highly enriched in the basal ganglia. However, in the mouse brain, only very low levels of 5-HT6 receptor mRNA and receptor protein, measured by TaqMan reverse transcriptase-polymerase chain reaction and selective radioligand binding, could be detected, with no evidence of enrichment in the basal ganglia. The mouse receptor was cloned and transiently expressed in human embryonic kidney 293 cells to characterize its pharmacological profile. Despite significant sequence homology between human, rat, and mouse 5-HT6 receptors, the pharmacological profile of the mouse receptor was significantly different from the rat and human receptors. Four amino acid residues, conserved in rat and human and divergent in mouse receptors, were identified, and various mutant receptors were generated and their pharmacologies studied. Residues 188 (tyrosine in mouse, phenylalanine in rat and human) in transmembrane region 5 and 290 (serine in mouse, asparagine in rat and human) in transmembrane region 6 were identified as key amino acids responsible for the different pharmacological profiles. Molecular modeling of the receptor and docking of selective and nonselective compounds was undertaken to elucidate the ligand receptor interactions. The binding pocket was predicted to be different in the mouse compared with rat and human 5-HT6 receptors, and the models were in excellent agreement with the observed mutation results and have been used extensively in the design of further selective 5-HT6 antagonists.

5-Hydroxytryptamine(1A) (5-HT(1A)) receptors have been implicated in the pathophysiology and treatment of anxiety and depression, and are a target for novel drug development. This is the first study examining the human brain in vivo occupancy by a novel, selective, silent 5-HT(1A) antagonist. 2-[4-[4-(7-Chloro-2,3-dihydro-1,4-benzdioxyn-5-yl)-1-piperazinyl]butyl]-1,2-benzisothiazol-3-(2H)-one-1,1-dioxide (DU 125530), a compound in clinical development, has potential applications in the treatment of anxiety and mood disorders. Positron emission tomography (PET) and [(11)C][O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY-100635), were used to assess 5-HT(1A) autoreceptor and postsynaptic receptor occupancy in 12 healthy male volunteers. Over a 10- to 40-mg daily dose range, DU 125530 was well tolerated, and exhibited a dose-dependent occupancy from 0 to 72% at 2 h post the last dose. Occupancy correlated significantly with plasma levels of DU 125530, and a fitting of the data to a standard single-site binding model gave a maximum occupancy of approximately 80%, and a half-saturation concentration (ED(50)) of approximately 7 ng/ml. At 24 h after the last dose 5-HT(1A) occupancy was approximately 50% of that achieved at 2 h. This study demonstrates that high occupancy of the human brain 5-HT(1A) receptor can be achieved at doses producing minimal acute side effects.

We have examined the cellular processes underlying the desensitization of the 5-hydroxytryptamine (5-HT)(2A) receptor induced by agonist or antagonist exposure. Treatment of C6 glioma cells with either 5-HT or the 5-HT(2A) receptor antagonist ketanserin resulted in an attenuation in 5-HT(2A) receptor function, specifically the accumulation of inositol phosphates stimulated by the partial agonist quipazine. 5-HT-induced desensitization of the 5-HT(2A) receptor involved receptor internalization through a clathrin- and dynamin-dependent process because it was prevented by concanavalin A, monodansylcadaverine, and by expression of the dominant negative mutants beta-arrestin (319-418) and dynamin K44A. Although short-term (i.e., 10 min) 5-HT and ketanserin exposure resulted in the same degree of desensitization, ketanserin-induced desensitization was not prevented by these agents and did not involve receptor internalization. In contrast, prolonged ketanserin exposure (i.e., 2 h) resulted in 5-HT(2A) receptor internalization through a clathrin- and dynamin-dependent process, as was observed after agonist treatment. Inhibitors of protein kinase C or calcium-calmodulin kinase II did not attenuate or prevent 5-HT-induced desensitization of the receptor. 5-HT(2A) receptor desensitization induced by 5-HT and prolonged ketanserin treatment, but not by short-term ketanserin treatment, was prevented by the expression of the dominant negative mutant of G protein-coupled receptor kinase (GRK)2, GRK2-K220R, and by an anti-GRK2/3 antibody. Our data indicate a dual mechanism of early and late desensitization by the antagonist ketanserin. Short-term ketanserin treatment reduced the specific binding of the agonist radioligand [(125)I](+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([(125)I]DOI) and the ability of 5'-guanylylimidodiphosphate to attenuate this binding, suggesting that at the early stage of antagonist-induced desensitization the capacity of the 5-HT(2A) receptor to couple to G protein is impaired.

The 5-hydroxytryptamine(7) (5-HT(7)) receptor was originally defined by molecular biology techniques. The 5-HT(7) receptor protein and mRNA are found in brain areas, such as the CA3 subfield of the hippocampus, that are involved in various neuropsychiatric disease states. No functional response has previously been attributed to activation of the 5-HT(7) receptor in any of these brain areas. Calcium spike-induced slow afterhyperpolarizations (sAHP) were recorded from CA3 hippocampal pyramidal cells using intracellular recording techniques in a brain slice preparation maintained in vitro. A concentration-dependent inhibition of the sAHP amplitude was obtained when 5-HT was used as the agonist. To identify whether the 5-HT(7) receptor was one of the receptors mediating the inhibition of the sAHP amplitude, 5-HT agonists and antagonists were tested in the presence of WAY-100635 and GR-113808 to block 5-HT(1A) and 5-HT(4) receptor activation, respectively. The rank order potency of the agonists was 5-carboxyamidotryptamine (5-CT) > 5-HT > 5-methoxytryptamine (5-MeOT). Other agonists with high affinity at 5-HT(2), 5-HT(3), 5-HT(1B), 5-HT(1D), or 5-HT(6) receptors did not produce any response when tested at 10 microM. Ritanserin, mesulergine, and SB-269770 were competitive antagonists of the 5-CT inhibition of sAHP amplitude, with affinity (pA(2)) values of 6.8, 7. 9, and 8.8, respectively. Methiothepin was also an effective antagonist but was insurmountable. Other antagonists with affinity for the 5-HT(2), 5-HT(3), or 5-HT(6) receptor had no effect. Based on the rank order potency of the agonists and antagonists, one of the receptors that mediates the decrease in sAHP amplitude in CA3 hippocampal pyramidal cells was concluded to be the 5-HT(7) receptor.

LY426965 [(2S)-(+)-1-cyclohexyl-4-[4-(2-methoxyphenyl)-1-piperazinyl]2-methyl- 2-phenyl-1-butanone monohydrochloride] is a novel compound with high affinity for the cloned human 5-hydroxytryptamine (HT)(1A) receptor (K(i) = 4.66 nM) and 20-fold or greater selectivity over other serotonin and nonserotonin receptor subtypes. Both in vitro and in vivo studies indicate that LY426965 is a full antagonist and has no partial agonist properties. LY426965 did not stimulate [(35)S]guanosine-5'-O-(3-thio) triphosphate (GTPgammaS) binding to homogenates of cells expressing the cloned human 5-HT(1A) receptor in vitro but did inhibit 300 nM 5-HT-stimulated [(35)S]GTPgammaS binding with a K(i) value of 3.07 nM. After both p.o. and s.c. administration, LY426965 blocked the lower lip retraction, flat body posture, hypothermia, and increase in rat serum corticosterone induced by the 5-HT(1A) agonist 8-OH-DPAT (8-hydroxy-2-dipropylaminotetralin). In pigeons, LY426965 dose-dependently blocked the stimulus cue induced by 8-OH-DPAT but had no 8-OH-DPAT-like discriminative properties. LY426965 completely reversed the effects of nicotine withdrawal on the auditory startle reflex in rats. In microdialysis experiments, LY426965 administered together with fluoxetine significantly increased extracellular levels of serotonin above those achievable with fluoxetine alone. In electrophysiological studies, the administration of LY426965 produced a slight elevation of the firing rate of 5-HT neurons in the dorsal raphe nucleus of anesthetized rats and both blocked and reversed the effects of fluoxetine on 5-HT neuronal activity. These preclinical results indicate that LY426965 is a selective, full 5-HT(1A) antagonist that may have clinical use as pharmacotherapy for smoking cessation and depression and related disorders.

Clozapine, an atypical antipsychotic used in the treatment of refractory schizophrenia, causes neutropenia and agranulocytosis in 3 and 0.8% of patients, respectively. Clozapine undergoes bioactivation to a chemically reactive nitrenium ion, which has been shown to cause neutrophil cytotoxicity. To define further the mechanism of cell death, we have investigated the toxicity of clozapine, its stable metabolites, and its chemically reactive nitrenium ion to neutrophils and lymphocytes. Clozapine was able to induce neutrophil apoptosis at therapeutic concentrations (1-3 microM) only when it was bioactivated to the nitrenium ion. The parent drug caused apoptosis at supratherapeutic concentrations (100-300 microM) only. Neutrophil apoptosis induced by the nitrenium ion, but not by the parent drug itself, was inhibited by antioxidants and genistein and was accompanied by cell surface haptenation (assessed by flow cytometry) and glutathione depletion. Dual-color flow cytometry showed that neutrophils that were haptenated were the same cells that underwent apoptosis. No apoptosis of lymphocytes was evident with the nitrenium ion or the parent drug, despite the fact that the former caused cell surface haptenation, glutathione depletion, and loss of membrane integrity. Demethylclozapine, the major stable metabolite in vivo, showed a profile that was similar to, although less marked than that observed with clozapine. N-oxidation of clozapine or replacement of the nitrogen (at position 5) by sulfur produced compounds that were entirely nontoxic to neutrophils. In conclusion, the findings of the study expand on potential mechanisms of clozapine-induced cytotoxicity, which may be of relevance to the major forms of toxicity encountered in patients taking this drug.

Several novel N-arylpiperazine derivatives were synthesized and tested for their 1) affinity and functional activity on 5-hydroxytryptamine(1A) (5-HT(1A)) receptors in vitro; 2) activity in models predictive of antagonism at somatodendritic and postsynaptic 5-HT(1A) receptors; and 3) effects on the micturition reflex in anesthetized and conscious rats. These studies also included 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN 190), 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4, 5]decane-7,9-dione dihydrochloride (BMY 7378), and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl-N-(2-pyridinyl)cyclohex anecarboxamide (WAY 100635). Almost all compounds were found to be potent and selective for the human recombinant 5-HT(1A) receptor, with K(i) values in the nanomolar range. [(35)S]GTPgammaS binding in HeLa cells expressing the recombinant human 5-HT(1A) receptor allowed classification of the compounds into neutral antagonists and partial agonists. Almost all neutral antagonists were active in blocking 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT)-induced forepaw treading in rats (postsynaptic model) and hypothermia in mice (somatodendritic model) with the same potency, whereas compounds showing partial agonistic activity were active in the postsynaptic model but were inactive, or poorly active, in the somatodendritic model. Neutral antagonists potently inhibited volume-induced bladder-voiding contractions in anesthetized rats. Contractions were completely blocked, and the disappearance of bladder contractions lasted 7 to 13 min after the highest doses tested. Furthermore, neutral antagonists increased bladder volume capacity in conscious rats during continuous transvesical cystometry, whereas micturition pressure was only slightly, and not dose-dependently, reduced. Partial agonists were inactive or poorly active, inducing a disappearance time of bladder contractions that did not exceed 6 min in anesthetized rats, and failing to increase bladder volume capacity in conscious rats. These findings indicate that only neutral 5-HT(1A) receptor antagonists are endowed with inhibitory effects on the bladder.

Ro 63-0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [3H]Ro 63-0563, the first selective 5-HT6 receptor radioligand. Specific binding of [3H]Ro 63-0563 (nonspecific binding defined in the presence of 10 microM methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nM and 4.96 nM, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [3H]Ro 63-0563 (methiothepin > D-lysergic acid diethylamide > clozapine approximately Ro 63-0563 > lisuride > ergotamine approximately Ro 04-6790 > 5-HT > amitriptyline approximately metergoline approximately mianserin approximately ritanserin > methysergide > mesulergine) was similar to that obtained by using either [3H]D-lysergic acide diethylamide or [3H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [3H]Ro 63-0563 labeled a single binding site with Kd and Bmax values of 11. 7 nM and 175 fmol/mg protein, respectively. In porcine striatal membranes, [3H]Ro 63-0563 also labeled a single binding site with Kd and Bmax values of 8.0 nM and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6 receptor, which suggested the presence of 5-HT6 receptors in porcine striatum.

Acute pancreatitis was induced in mice by feeding with a choline-deficient ethionine-supplemented diet. All the mice developed acute pancreatitis, and approximately 80% of them died within 4 days. Stereomicroscopic and light microscopic examinations revealed that pancreatic necrosis and circulatory disturbance that were not apparent on day 1 were increased markedly on days 2 and 3. Serum levels of pancreatic enzymes were normal or reduced on day 1 but then increased to peak on day 3. Plasma 5-hydroxyindoleacetic acid levels, which may indicate serotonin release, were significantly increased on days 1 through 3. Pretreatment with D, L-p-chlorophenylalanine methylester hydrochloride (200-400 mg/kg) significantly attenuated the mortality of the mice with pancreatitis. Dose-dependent attenuation was also obtained with ketaserin (0. 01-10 mg/kg), cyproheptadine (0.01-10 mg/kg), pindolol (0.1-100 mg/kg) and NAN-190 (0.1-100 mg/kg), but not with 0.01 to 10 mg/kg of ICS205-930 or M-840, and the activities were significantly correlated with the binding affinities for serotonin2 receptor on the rat cerebral cortex. In addition, ketanserin or cyproheptadine attenuated the morphologic changes in the choline-deficient ethionine-supplemented diet mice at a dose (3.2 mg/kg) that hardly affected the serum enzyme levels. We propose that serotonin2 receptor activation plays an important role in the aggravation of diet-induced acute pancreatitis.

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.

(R)-(+)-2-Amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl]+ ++pyrrolidin-3-yl]thiazole (NRA0045), a novel thiazole derivative, has high affinities for the human cloned dopamine D4.2, D4.4 and D4.7 receptors, with Ki values of 2.54, 0.55 and 0.54 nM, respectively. NRA0045 is approximately 91-fold more potent at the dopamine D4.2 receptor, compared with human cloned dopamine D2L receptor. NRA0045 also has high affinities for the serotonin (5-HT)2A receptor (Ki = 1.92 nM) and alpha-1 adrenoceptor (Ki = 1.40 nM) but weak affinities (IC50 values are approximately 1 microM) for six other neurotransmitter receptors (adenosine1, 5-HT1A, 5-HT1C, dopamine transporter, alpha2A and alpha2A) and negligible affinities (IC50 values are over 10(-5) M) for 42 other receptors, including neurotransmitters and hormones, ion channels and second messenger systems. Locomotor hyperactivity induced by methamphetamine (1 mg/kg i.p.) in mice was dose-dependently antagonized by NRA0045 (ED50 = 0.5 mg/kg i.p. and 1.9 mg/kg p.o., respectively). Methamphetamine (10 mg/kg i.p.)-induced stereotyped behavior in mice was dose-dependently antagonized by NRA0045, whereas NRA0045 did not exceed 50% inhibition even at the highest dose given (30 mg/kg i.p.). Catalepsy was dose-dependently and significantly induced by NRA0045 in rats, whereas NRA0045 did not exceed 50% induction even at the highest dose given (30 mg/kg i.p.). Thus NRA0045 blocks behaviors associated with activation of the mesolimbic/mesocortical dopaminergic neurons more selectively than behaviors associated with nigrostriatal dopaminergic neurons. In rats, tryptamine-induced clonic seizure, a 5-HT2 receptor-mediated behavior, was also dose-dependently inhibited by NRA0045 (ED50 = 1.7 mg/kg i.p.). Norepinephrine-induced lethality is regarded as being induced through the alpha-1 adrenoceptor. NRA0045 dose-dependently antagonized norepinephrine-induced lethality in rats (ED50 = 0.2 mg/kg i.p.). Thus NRA0045 may have a unique antipsychotic activity with regard to dopamine D4 and 5-HT2A receptors and alpha-1 adrenoceptor antagonistic activities, without producing the extrapyramidal side effects.

We assayed [3H]5-hydroxytryptamine ([3H]5-HT) binding in rat hypothalamic membranes to confirm the possible of measuring 5-HT7 receptors. Binding was tested in the presence of 3 microM (+/-)-pindolol, a concentration higher than previously suggested for the same purpose (0.1 micron). This higher concentration was, however, needed to fully saturate 5-HT1A and 5-HT1B receptors without interaction with 5-HT7 receptors. Under these conditions, [3H]5-HT binding could be further inhibited with methiothepin (used to determine nonspecific binding) and with 5-HT, with an IC50 of 1.4 nM and a slope of 1. The inhibition curves of (+/-)-8-hydroxy-dipropylaminotetralin, ritanserin, and mianserin were shallow (slopes, 0.35-0.58) and could be better analyzed with the two-site model, indicating that the pindolol-insensitive [3H]5-HT binding sites in rat hypothalamic membranes are heterogeneous. Although the IC50 of the compounds tested suggests that one population of sites is actually associated with 5-HT7 receptors, our data clearly indicate that this binding assay does not selectively label 5-HT7 receptors in native tissues. These results challenge a previous report and suggest that the proposed down-regulation of 5-HT7 receptors after fluoxetine treatment should be considered with caution. The development of more selective and sensitive binding assays will probably offer significant advantage.

Ziprasidone (CP-88,059) is a combined 5-HT (serotonin) and dopamine receptor antagonist which exhibits potent effects in preclinical assays predictive of antipsychotic activity. Whereas the compound is a dopamine antagonist in vitro and in vivo, its most potent action is antagonism of 5-HT2A receptors, where its affinity is an order of magnitude greater than that observed for dopamine D2 sites. Laboratory and clinical findings have led to a hypothesis that antagonism of 5-HT2A receptors in the brain limits the undesirable motor side effects associated with dopamine receptor blockade and improves efficacy against the negative symptoms of schizophrenia. Ziprasidone possesses an in vitro 5-HT2A/dopamine D2 receptor affinity ratio higher than any clinically available antipsychotic agent. In vivo, ziprasidone antagonizes 5-HT2A receptor-induced head twitch with 6-fold higher potency than for blockade of d-amphetamine-induced hyperactivity, a measure of central dopamine D2 receptor antagonism. Ziprasidone also has high affinity for the 5-HT1A, 5-HT1D and 5-HT2C receptor subtypes, which may further enhance its therapeutic potential. The prediction of antipsychotic efficacy without severe motor side effects is supported by the relatively weak potency of ziprasidone to produce catalepsy in animals, contrasted with its potent antagonism of conditioned avoidance responding and dopamine agonist-induced locomotor activation and stereotypy. The compound is well tolerated in animals at doses producing effective dopamine antagonism in the brain. Ziprasidone should be a valuable addition to the treatment of psychotic disorders.

The present study assessed compounds displaying affinity for 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C receptors for their ability to substitute for, enhance or antagonize the discriminative stimulus effects of cocaine (10 mg/kg) in rats. In substitution tests, the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT; 0.2-1.6 mg/kg), the 5-HT1A/B receptor agonists RU 24969 (0.25-2 mg/kg) and CGS 12066B (2-16 mg/kg), the 5-HT1B/2C receptor agonists m-chlorophenylpiperazine (mCPP; 0.25-2 mg/kg) and m-trifluoromethylphenylpiperazine (TFMPP; 0.125-2 mg/kg), the 5-HT2A/2C receptor agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane ([+/-]-DOB; 0.0625-0.5 mg/kg), the 5-HT2C receptor agonist MK 212 (0.25-1 mg/kg) and the nonselective 5-HT receptor agonist quipazine (1-8 mg/kg) engendered 30% to 70% cocaine-appropriate responding. The DA receptor antagonists SCH 23390 (0.025 or 0.05 mg/kg) and haloperidol (0.125 or 0.25 mg/kg) failed to block the partial substitution of RU 24969 (1 mg/kg) for cocaine. In combination tests, a fixed dose of either quipazine (4 mg/kg), RU 24969 (0.25, 0.5 or 1 mg/kg) or TFMPP (0.5 mg/kg) but not 8-OH-DPAT (0.4 mg/kg) or CGS 12066B (16 mg/kg) produced a leftward shift in the cocaine dose-response curve (0.625-5 mg/kg). In contrast, coadministration of either mCPP (0.25-2 mg/kg) or MK 212 (0.125-2 mg/kg) plus a dose of cocaine (5 mg/kg) that produced > 85% cocaine-appropriate responding when given alone partially antagonized cocaine; mCPP (1 mg/kg) also produced a rightward shift in the cocaine dose-response curve. Neither 8-OH-DPAT (0.2-1.6 mg/kg), (+/-)-DOB (0.125-0.5 mg/kg) nor quipazine (2-8 mg/kg) blocked the cocaine stimulus. The 5-HT1A receptor antagonist NAN 190 (0.2-0.8 mg/kg), the 5-HT2A/2C receptor antagonist LY 53857 (0.5-4 mg/kg) and the 5-HT/DA receptor antagonist pirenperone (0.5-4 mg/kg) neither substituted for nor enhanced cocaine; however, pirenperone but not NAN 190 or LY 53857 partially blocked the cocaine (10 mg/kg) response. Although 5-HT receptor compounds do not substitute for cocaine, several 5-HT receptor agonists (i.e., the indole derivative RU 24969 and the arylpiperazines mCPP, MK 212, TFMPP and quipazine), but not antagonists, differentially modulate the stimulus effects of cocaine.(ABSTRACT TRUNCATED AT 400 WORDS)

Iloperidone (1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone) demonstrated a potent antipsychotic profile in several in vitro and in vivo animal models. Iloperidone displaced ligand binding at D2 dopamine receptors (IC50 = 0.11 microM) and displayed a high affinity for serotonin (5-HT2) receptors (IC50 = 0.011 microM) and alpha-1 receptors (IC50 = 0.00037 microM). In vivo, iloperidone antagonized apomorphine-induced climbing behavior in mice at low doses with good oral bioavailability, prevented 5-HT-induced head twitch in rats at low doses, and inhibited self-stimulation behavior in rats, pole climb avoidance in rats and continuous Sidman avoidance responding in monkeys. The latter assay also demonstrated a good duration of action. Iloperidone was substantially less active in models of extrapyramidal side effect (EPS) liability, such as preventing apomorphine-induced stereotypy and causing catalepsy in rats. In single dopamine neuron sampling studies, iloperidone demonstrated clozapine-like effects on the number of active midbrain dopamine neurons. Based on the significant increase in the open arm time seen after iloperidone treatment in the elevated plus maze assay and increased interaction score in social interaction, iloperidone may also have favorable effects in the clinic on anxiety and, possibly, negative symptoms. Clinical trials are under way of the use of iloperidone for the treatment of schizophrenia.

Recently discovered serotonin3 (5-HT3) receptor antagonists are potent antiemetics in cytotoxic drug-induced vomiting. The specific site where 5-HT3 receptor antagonists act to abolish emesis is controversial. The major objective of this study was to determine whether the antiemetic effect of 5-HT3 receptor antagonists is exerted in the brain areas that reside inside or outside of the blood-brain barrier. Tropisetron, zatosetron (LY277359 maleate) and its quaternary analog zatosetron-QUAT were used in this study. Zatosetron and zatosetron-QUAT showed high affinity and selectivity for 5-HT3 receptors in radioligand binding studies. Both compounds antagonized 5-HT-induced bradycardia in rats with an approximate ID50 of 0.7 and 0.2 microgram/kg i.v., respectively. Zatosetron and tropisetron significantly inhibited cisplatin-evoked emesis in dogs (estimated ID50 values of 34.4 +/- 2.3 micrograms/kg and 108.3 +/- 4.8 micrograms/kg i.v., respectively). Zatosetron-QUAT (0.01-1.0 mg/kg i.v.) had no effect. [14C]-zatosetron-QUAT (100 micrograms/kg) was not detected in the brain after i.v. administration to rats, consistent with the inability of charged compounds to achieve significant brain concentrations. However, i.c.v. administration (100 ng/kg) of zatosetron-QUAT reduced emetic episodes significantly (11.6 +/- 1.6 vs. 2.8 +/- 1.2). These studies suggest that, in dogs, antagonism of 5-HT3 receptors located within the blood-brain barrier is important to block cisplatin-induced emesis.

Expression of the 5-hydroxytryptamine type 2C (5-HT 2C) receptor in NIH/3T3 fibroblasts results in agonist-independent 5-HT2C receptor activation. Some 5-HT2c receptor antagonists decrease this activation and are termed inverse agonists. The present study uses this system to evaluate functional and receptor binding properties of other 5-HT2C receptor antagonists. A number of inverse agonists, including clozapine, and a neutral antagonist (methysergide) were identified in a functional assay. Guanine nucleotides increased the affinity of a radiolabeled inverse agonist ([3H]mesulergine), suggesting that inverse agonists bind the G protein-uncoupled form of the 5-HT2C receptor with high affinity. Competition binding was performed using conditions that separately labeled the G protein-coupled and -uncoupled forms of the receptor. These studies demonstrated that inverse agonists bound the uncoupled form of the 5-HT2C receptor with higher affinity, compared with the G protein-coupled form. Agonists, on the other hand, had higher affinity for the coupled form whereas neutral antagonists had equal affinity for both forms of the receptor. Thus, 5-HT2C receptor neutral antagonists exhibited functional and receptor binding properties consistent with those of classical receptor antagonists. However, 5-HT2C receptor inverse agonists displayed functional and receptor binding properties that were opposite those of agonists.

Recent findings have suggested a relationship between 5-hydroxytryptamine (5-HT)4 receptors and sigma binding sites. To test this idea, the affinity of 5-HT4 receptor ligands for sigma binding sites was examined. In contrast to the 5-HT4 receptor ligands BIMU-1 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3- dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride] and BIMU-8 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3- yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-benzamidazole-1-carbox ami de hydrochloride], DAU 6215 ]N-(endo-8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-2,3-dihydro-2-oxo-1H- benzimidazole-1-carboxamide hydrochloride], 5-HT and 5-methoxytryptamine had low affinity for sigma binding sites (pKi < 6). Conversely, the sigma ligands haloperidol and pentazocine had low affinity for 5-HT4 receptors. Thus, no relationship was found between the affinity of ligands at 5-HT4 receptors and sigma binding sites. However, one potent 5-HT4 receptor antagonist, RS-23597-190 [3-(piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride], had high affinity for sigma-1 (pKi = 8.4) but not sigma-2 (pKi = 6.2) binding sites. [3H]RS-23597-190 bound to a saturable site with the pharmacology of a sigma-1 binding site: (pIC50) haloperidol (9.0) > (+)-pentazocine (8.8) > (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine (8.2) > 1,3-di-o-tolyl-guanidine (8.0) > (-)-pentazocine (7.8) = (+)-SKF 10,047 [N-allylnormetazocine] > (-)-SKF 10,047 (6.2) > BIMU-1 (5.3) > 5-HT and 5-methoxytryptamine. The distribution of [3H]RS-23597-190 binding sites was similar to that described for other sigma radioligands, with the greatest binding densities in cranial nerve nuclei, the tegmental nucleus and in the mamillary nucleus. In contrast to (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine, [3H]RS-23597-190 binding was not allosterically modulated by phenytoin. These studies do not support the notion of an obvious relationship between sigma and 5-HT4 receptors, but they provide additional insight into the structure/affinity relationship of ligands at specific sigma binding sites, and they uncover a novel sigma-1 receptor ligand whose binding is insensitive to the action of phenytoin.

1-(Cyclopropylmethyl)-4-(2-(4-fluorophenyl)-2-oxoethyl) piperidine HBr (DuP 734) is a novel sigma receptor ligand which exhibits promise in preclinical animal models as an antipsychotic agent without motor side effects. In vitro and in vivo receptor binding profiles of DuP 734 in mouse brain using [3H]DuP 734 and [3H]N-allylnormetazocine ((+)-SKF 10,047) were studied. The pharmacology and stereospecificity of [3H]DuP 734-labeled sites in mouse brain, both in vitro and in vivo, was consistent with sigma receptor pharmacology. Specific in vivo binding of [3H]DuP 734 in brain peaked 1 hr after i.v. injection and this level of binding was maintained up to 4 hr. On the other hand, plasma concentration of [3H]DuP 734 decreased rapidly within 20 min after injection, indicating different pharmacokinetics between brain and plasma levels. Nonspecific binding, defined using 1 mg/kg (2.66 mumol/kg) of haloperidol, was approximately 30% of total binding 1 hr after injection of radiotracer. Administration of DuP 734 potently antagonized the binding of [3H]DuP 734 and [3H](+)-SKF 10,047 to brain sigma receptors in vivo with ID50 values of 0.02 and 0.07 mg/kg (0.07 and 0.25 mumol/kg), respectively. However, (+)-SKF 10,047, which possess a high affinity (IC50 = 22.5 nM) for [3H]DuP 734 binding in vitro, failed to displace [3H]DuP 734 binding in vivo. Thus in vitro receptor binding data may not predict in vivo receptor occupancy. Overall, the data suggest [3H] DuP 734 is a good ligand for in vivo imaging of sigma receptors.

Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.

The molecular processes by which agonists and antagonists bind to serotonin2 [5-hydroxytryptamine (5-HT2)] receptors are currently unknown. Three molecular models have proposed that conserved aromatic residues help to anchor the phenyl ring of 5-HT via stacking or pi-pi-type interactions with the 5-HT2 receptor. To test these models we made single point mutations (Phe339-->Leu339 and Phe340-->Leu340) of two aromatic residues that are conserved among all guanine nucleotide-binding protein-coupled 5-HT receptors and a single point mutation (Phe125-->Leu125) that exchanges a 5-HT2 for a 5-HT1c sequence. [3H]Mesulergine binding was abolished by Phe340-->Leu340 and unchanged with the Phe339-->Leu339 and Phe125-->Leu125 mutations, whereas [3H]ketanserin binding affinity was diminished by the Phe339-->Leu339 mutation and unchanged by Phe340-->Leu340 and Phe125-->Leu125. We also found that the affinities of three ergot derivatives (mesulergine, methysergide, and lisuride) were decreased by 88-1079-fold with only the Phe340-->Leu340 mutation. We also discovered that 4-[125I]iodo-2,5-(dimethoxy)phenylisopropylamine (DOI) binding was abolished in COS-7 cells expressing 5-HT2 (Phe340-->Leu340) receptors but maintained in cells expressing the Phe339-->Leu339 and Phe125-->Leu125 mutations. Additionally, the Ki values for several agonists and partial agonists (5-HT, DOI, m-chlorophenylpiperazine, trifluoromethylphenylpiperazine, bufotenine, and MK-212) were greatly diminished (26-14,000-fold decrease) only with the Phe340-->Leu340 receptor mutation. Finally, the Phe340-->Leu340 mutant receptors displayed an attenuated or abolished ability to augment phosphoinositide hydrolysis in COS-7 cells with four separate agonists (5-HT, MK-212, bufotenine, and quipazine). Taken together, these results are consistent with the idea that agonists and certain ergot derivatives anchor to 5-HT2 receptors, in part, via specific interactions with aromatic residue Phe340 located in transmembrane region VI.

The rat 5-hydroxytryptamine (5-HT)2F (serotonin2F) receptor is a newly cloned member of the 5-HT2/1C receptor family. The pharmacology of the 5-HT2F receptor was explored using a variety of structurally different compounds in a radioligand binding assay. In addition, the 5-HT2F receptor was shown to stimulate production of inositol 1,4,5-trisphosphate in the transformed cells. Based on the affinities of the compounds tested, their known affinities for certain of the other 5-HT receptors, and the fact that activation of the cloned 5-HT2F receptor stimulates inositol 1,4,5-trisphosphate production, the 5-HT2F receptor was determined to be a novel receptor and a member of the 5-HT2/1C receptor family. In addition, several agonists and partial agonists were evaluated for contractile activity in the rat stomach fundus, and these activities were correlated with their binding affinities at the 5-HT2F receptor. A highly significant correlation was found, providing additional evidence that is consistent with the 5-HT2F receptor being the stomach fundal contractile receptor. [3H]5-HT had high affinity for this receptor both at 37 degrees and at 0 degree (Kd = 7.87 +/- 0.55 and 0.12 +/- 0.02 nM, respectively). The difference in affinity for [3H]5-HT at the two temperatures prompted an investigation of potential temperature-dependent differences in the binding affinities of agonists versus antagonists. Agonists such as 5-HT, 5-methoxytryptamine, etc., showed higher affinity for the 5-HT2F receptor at 0 degree than at 37 degrees, whereas antagonists such as methysergide, 1-naphthylpiperazine, etc., showed no difference in affinity for this receptor at the two different temperatures. Therefore, the affinity of a compound for the 5-HT2F receptor at 37 degrees versus 0 degree was shown to be useful for predicting agonist or antagonist activity. Additionally, information is provided about some of the structural requirements for the affinity of certain tryptamines at the 5-HT2F receptor.

A new potent, selective and p.o. active serotonergic [5-hydroxytryptamine (5-HT2)] receptor antagonist, SR 46349B [trans, 4-([3Z)3-(2-dimethylaminoethyl)oxyimino-3(2-flurophenyl++ +)propen-1-yl]phenol hemifumarate) has been characterized by a series of "in vitro" and "in vivo" methods. Based upon binding studies with 5-HT2 receptors in rat brain cortical membranes and blockade of 5-HT-induced contractions in isolated tissues (rabbit thoracic aorta, rat jugular vein, rat caudal artery, rat uterus and guinea pig trachea), SR 46349B showed high affinity for 5-HT2 receptors. Furthermore, SR 46349B displayed moderate affinity for the 5-HT1C receptor and had no affinity for the other 5-HT1 subclass (5-HT1A, 5-HT1B or 5-HT1D), dopamine (D1 or D2), "alpha" adrenergic (alpha-1 or alpha-2), sodium and calcium channel and histamine (H1) receptors. It did not interact with histamine (H1), alpha-1 adrenergic and 5-HT3 receptors in smooth muscle preparations. No inhibition of the uptake of norepinephrine, dopamine or 5-HT was seen. Based upon blockade of pressor responses to 5-HT in pithed rats and in vivo binding studies in mice, SR 46349B was found to be a potent and p.o. active 5-HT2 receptor antagonist with a relatively long duration of action. Behavioral experiments, including mescaline- and 5-hydroxytryptophan-induced head twitches and learned helplessness, as well as sleep-waking cycle and EEG spectral parameter studies, indicated that SR 46349B has a classical 5-HT2 psychopharmacological antagonist profile.

5-Hydroxytryptamine (5HT)1C and 5HT2 receptors appear to be closely related, from a molecular viewpoint, displaying similar second messenger systems and a high degree of sequence homology. However, there are striking differences in the interactions of 5HT with 5HT1C and 5HT2 receptors; 5HT is generally more potent in stimulating responses mediated through 5HT1C receptors than responses mediated through 5HT2 receptors. Also [3H]5HT labels 5HT1C receptors and not 5HT2 receptors. In order to explore more fully the molecular rationale for these differences, radioligand binding studies were performed in rat, human, and porcine brain and choroid plexus tissues and in mammalian cells transfected with rat 5HT1C or 5HT2 receptors; second messenger studies (inositol phosphate accumulation) were performed in the transfected cells. The second messenger studies confirmed the approximately 10-fold higher potency of 5HT in stimulating intracellular responses through 5HT1C receptors (EC50 = 8.3 nM) than in stimulating intracellular responses through 5HT2 receptors (EC50 = 101 nM). An agonist radioligand selective for the 5HT1C and 5HT2 receptors, 2,5-dimethoxy-(4-[125I]iodo)phenylisopropylamine, was used, as well as [3H]5HT, [3H]mesulergine (antagonist radioligand for 5HT1C receptors), and [3H]ketanserin (antagonist radioligand for 5HT2 receptors). Computer-assisted analyses of the binding data revealed two agonist affinity states for the 5HT1C receptor. The agonist high affinity state of the receptor was modifiable by guanyl nucleotides. The proportion of agonist high affinity states, relative to the total receptor population, was approximately 10% for both receptors. The apparent higher affinity of 5HT for the radiolabeled 5HT1C receptors was due to the higher affinity 5HT displayed for the agonist low affinity state of the 5HT1C receptor, compared with the affinity of 5HT for the agonist low affinity state of the 5HT2 receptor. The correspondence between the higher affinity of 5HT for the agonist low affinity state of the 5HT1C receptor, relative to the 5HT2 receptor, and the higher potency of 5HT in stimulating 5HT1C responses indicates that 5HT interacts with the agonist low affinity state of the 5HT1C and 5HT2 receptors in initiating its biological effects. These observations indicate that guanine nucleotide-binding protein (G protein)-coupled receptors can exhibit high affinity for neurotransmitters in both the free receptor and the G protein-coupled states and that receptors exhibiting this property may represent a novel subfamily of G protein-coupled receptors.

The purpose of this study was to assess the duration of (+-)-3,4-methylenedioxymethamphetamine's (MDMA's) effects on serotonin containing neurons in nonhuman primates. Fifteen squirrel monkeys were used: three served as controls, 12 received MDMA s.c. at a dose of 5 mg/kg twice daily for 4 consecutive days. Two weeks, 10 weeks, 8 months and 18 months after drug treatment, groups (n = 3) of MDMA-treated monkeys, along with controls, were examined for regional brain content of serotonin and 5-hydroxyindoleacetic acid, and for the number of [3H] paroxetine-labeled serotonin uptake sites. Two weeks after MDMA treatment, monkeys showed profound reductions in all three serotonergic presynaptic markers. By 10 weeks, there was evidence of partial recovery in some brain regions (e.g., hippocampus, caudate nucleus, frontal cortex). However, by 18 months, it was evident that recovery did not continue, as serotonergic deficits returned to the level of severity observed 2 weeks after MDMA treatment. This was the case in all brain regions examined except the thalamus and hypothalamus. In the thalamus, the level of serotonin increased to 63% of control, whereas that of 5-hydroxyindoleacetic acid recovered completely. In the hypothalamus, concentrations of serotonin and 5-hydroxyindoleacetic acid were 140 and 187% of control, respectively. These results suggest that MDMA produces lasting effects on serotonergic neurons in nonhuman primates, with most brain regions showing evidence of persistent denervation and some showing signs of reinnervation (thalamus) or possibly even hyperinnervation (hypothalamus). The morphological and functional correlates of these enduring neurochemical changes in the MDMA-treated primate remain to be delineated.

8B-N-cyclohexyl-6-methyl-1(1-methylethyl)ergoline-8-carboxamide (LY237733) is an ergoline with potent and highly selective 5-hydroxytryptamine (5-HT) antagonist activity. The in vitro radioligand displacement studies showed that LY237733 has a preferential affinity for 5-HT1c and 5-HT2 receptors compared to other monoaminergic receptors. This characteristic is shared with other previously described ergoline 5-HT antagonists, such as LY53857 and sergolexole. In parallel ligand displacement assays, LY237733 had a similar potency to sergolexole. LY237733 was equipotent to sergolexole, but slightly less potent than LY53857 in the antagonism of 5-HT-induced elevation in blood pressure and quipazine-induced elevation in corticosterone levels, which are considered to be measures of 5-HT2 and possibly 5-HT1c antagonist activity. LY237733 failed to antagonize pergolide or 8-hydroxy-2-(di-n-propylamino)tetralin-induced elevations in serum corticosterone levels, indicating selectivity for the 5-HT1c/2 receptor, relative to 5-HT1a and D2 dopaminergic receptors. The only in vivo response that could be detected after administration of LY237733 alone in doses less than 1 mg/kg was the amplification of male rat sexual behavior. LY237733 was 10 to 100 times more potent than LY53857 or sergolexole in augmenting sexual responses of male rats with different levels of sexual response capacity. LY237733 has a much longer serum half-life than sergolexole. These studies have provided the pre-clinical rationale to evaluate the effects of this compound in the treatment of sexual disorders such as psychogenic erectile dysfunction, and other therapeutic indications for a 5-HT2 antagonist, including depression, anxiety, schizophrenia and migraine.

The affinity of a new serotonin (S) derivative, serotonin-O-carboxymethyl-glycyl-tyrosinamide (S-CM-GTNH2), for the various 5-hydroxytryptamine (5-HT)1 receptor subtypes was tested using quantitative autoradiography on rat and guinea pig brain sections. In the rat, S-CM-GTNH2 is 57 and 24 times more potent at 5-HT1B sites (IC50 = 28 nM) than at 5-HT1A (IC50 = 1600 nM) and 5-HT1C sites (IC50 = 670 nM), respectively. In the guinea pig, the affinity of S-CM-GTNH2 for 5-HT1D sites (IC50 = 67 nM) is 21 times higher than at 5-HT1A sites (IC50 = 1400 nM). S-CM-GTNH2 shows a low affinity (less than 10 microM) for 5-HT2 and 5-HT3 binding sites. This new ligand is therefore highly specific for 5-HT1B and 5-HT1D binding sites and can be used to further characterize the involvement of these subtypes in physiological studies focusing particularly on behavioral effects.

The presence of 5-hydroxytryptamine (5-HT)1C/2 binding sites in autonomic regions of the thoracic spinal cord and their role in the regulation of sympathetic outflow to the cardiovascular system were examined. Light microscopic receptor autoradiography was used to visualize the binding of the 5-HT1C/2 ligand, [125I]-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [( 125I]DOI). In thoracic spinal cord, a discrete and preferential localization of specific [125I]DOI binding sites was found in the intermediolateral cell column. To determine the mean arterial pressure (MAP) and heart rate (HR) effects resulting from activation of 5-HT1C/2 receptors in spinal cord, DOI and alpha-methyl-5-HT were administered intrathecally (i.t.) to anesthetized, artificially ventilated rats. DOI (1-100 micrograms) caused initial decreases followed by increases in MAP and HR, whereas alpha-methyl-5-HT (1-30 micrograms) only decreased MAP and HR. The distribution of [125I]DOI after i.t. administration and the effects of a peripherally administered 5-HT1C/2 antagonist 6-methyl-1-(1-methylethyl)-ergoline-8 beta-carboxylic acid, 2-hydroxyl-1-methyl propyl ester and maleate salt (LY53857) showed that the pressor effects of i.t. DOI were due to peripheral leakage and suggested that the depressor effects were due to a spinal site of action. The depressor effects of DOI were prevented by peripheral administration of phentolamine. Pretreatment with i.t. administration of 5-HT1C/2 antagonists (LY53857, ketanserin and mianserin) did not block the depressor or bradycardic effects of i.t. administration of DOI. Only LY53857 was effective in blocking the depressor effects of i.t. administration of alpha-methyl-5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5-HT2 (serotonin) receptor has traditionally been labeled with antagonist radioligands such as [3H]ketanserin and [3H]spiperone, which label both agonist high-affinity (guanyl nucleotide-sensitive) and agonist low-affinity (guanyl nucleotide-insensitive) states of this receptor. The hallucinogen 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) is an agonist which labels the high-affinity guanyl nucleotide-sensitive state of brain 5-HT2 receptors selectively. In the present study, conditions for autoradiographic visualization of (+/-)-[125I]DOI-labeled 5-HT2 receptors were optimized and binding to slide-mounted sections was characterized with respect to pharmacology, guanyl nucleotide sensitivity and anatomical distribution. In slide-mounted rat brain sections (+/-)-[125I]DOI binding was saturable, of high affinity (KD approximately 4 nM) and displayed a pharmacologic profile typical of 5-HT2 receptors. Consistent with coupling of 5-HT2 receptors in the high-affinity state to a guanyl nucleotide regulatory protein, [125I]DOI binding was inhibited by guanyl nucleotides but not by adenosine triphosphate. Patterns of autoradiographic distribution of [125I]DOI binding to 5-HT2 receptors were similar to those seen with [3H]ketanserin- and [125I]-lysergic acid diethylamide-labeled 5-HT2 receptors. However, the density of 5-HT2 receptors labeled by the agonist [125I]DOI was markedly lower (30-50%) than that labeled by the antagonist [3H]ketanserin. High densities of [125I]DOI labeling were present in olfactory bulb, anterior regions of cerebral cortex (layer IV), claustrum, caudate putamen, globus pallidus, ventral pallidum, islands of Calleja, mammillary nuclei and inferior olive. Binding in hippocampus, thalamus and hypothalamus was generally sparse. Of note, choroid plexus, a site rich in 5-HT1c receptors had a high density of [125I]DOI binding sites but [3H]ketanserin binding in this region was low. Studies in which [125I]DOI binding to 5-HT2 receptors was blocked with spiperone revealed persisting robust [125I]DOI binding in choroid plexus, which was guanyl nucleotide-sensitive and displayed a pharmacologic profile consistent with its binding to 5-HT1c receptors. These studies suggest that [125I]DOI may be useful as a radiolabel for visualizing the agonist high-affinity state of 5-HT2 receptors and for visualizing 5-HT1c receptors.

6-Nitroquipazine (DU 24565; 6-nitro 2-piperazinylquinoline) is a very potent 5-hydroxytryptamine (5-HT; serotonin) uptake inhibitor. It has been demonstrated very recently that [3H]-6-nitroquipazine is a suitable radioligand for studying 5-HT uptake sites. The present study evaluates [3H]6-nitroquipazine as a radioligand for in vivo labeling of 5-HT uptake sites in mouse brain. Very high uptake of radioactivity in the brain after i.v. administration of [3H]-6-nitroquipazine was shown. Regional distribution of the radioactivity in mouse brain 3 hr after injection of [3H]-6-nitroquipazine was in the order (highest to lowest) hypothalamus greater than midbrain greater than striatum greater than hippocampus greater than cerebral cortex greater than medulla oblongata greater than cerebellum. The regional distribution of in vivo [3H]-6-nitroquipazine binding in mouse brain was highly correlated with that in rat brain obtained from previous in vitro binding studies. Coadministration of carrier 6-nitroquipazine (5 mg/kg) significantly decreased the radioactivity in the hypothalamus, whereas that in the cerebellum and cerebral cortex was increased. Because the cerebellum has very low density of [3H]-6-nitroquipazine binding sites, the radioactivity in the cerebellum could, therefore, reflect the amount on nonspecific binding and free ligand. Kinetic studies showed highest in vivo specific binding 1 hr after injection of [3H]-6-nitroquipazine and slow clearance of specific binding. Specific binding in the hypothalamus was inhibited in a stereoselective manner by the stereoisomers of norzimelidine. Furthermore, specific binding in the hypothalamus was reduced by several 5-HT uptake inhibitors, in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo receptor-binding properties of the new serotonin antagonist, ritanserin, are reported. In in vitro binding assays, ritanserin shows high affinity binding to serotonin-S2 sites in rat frontal cortex tissue: IC50 = 0.9 nM without drug preincubation and 0.3 nM with 30-min drug preincubation; IC50 values for histamine-H1, dopamine-D2, and adrenergic-alpha 1 and -alpha 2 sites were 39-, 77-, 107-, and 166-fold higher, and at up to 1 microM, the drug did not bind to serotonin-S1 sites. In in vitro assays, ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Arylpiperazines (MK-212, quipazine, m-chlorophenylpiperazine and m-trifluoromethylphenylpiperazine) caused a serotonin (5-HT) receptor-mediated contraction of rat fundic strips. m-chlorophenylpiperazine and m-trifluoromethylphenylpiperazine had high affinity for the receptor but little efficacy, whereas quipazine and MK-212 had lesser affinity and much greater efficacy. 5-HT itself was the most potent (EC50 = 6-9 nM) agonist and possessed the greatest affinity (KA = 9.7 nM). Assessment of receptor occupancy vs. functional response (as well as receptor alkylation studies) demonstrated a very small, if any, receptor reserve in this tissue. Several arylquinolizines were found to be competitive antagonists of 5-HT-induced contraction, the most potent being L-653,267 and rauwolscine (KB values = 1.9 and 3.8 nM). Clozapine, trazodone and propranolol were identified as less potent, competitive antagonists, whereas various ergolines (including LY 53857), L-646,462 (cyproheptadine analog) and mianserin were noncompetitive. Potent 5-HT2 receptor antagonists (pirenpirone and ketanserin) antagonized only weakly or were without effect against 5-HT, indicating that the fundic 5-HT receptor is not of the 5-HT2 subtype. Because the fundic receptor has high affinity for 5-HT (as does the 5-HT1 binding site in brain tissue), the possible correspondence of the fundic 5-HT receptor with the 5-HT1 recognition site in rat brain cortex was considered. 5-HT, the nonindole agonists (1-arylpiperazines) and the competitive antagonists all competed with [3H]-5-HT for the 5-HT1 site. However, all compounds except 5-HT had Hill slopes significantly less than 1.0, precluding a valid comparison with dissociation constants derived pharmacologically in the fundus. With respect to having a high affinity for 5-HT, the 5-HT receptor mediating contraction of fundic smooth muscle resembles the 5-HT1 recognition site (as defined in brain tissue by radioligand binding), but identity remains unproven.

Recent studies indicate that there are multiple subtypes of the 5-hydroxytryptamine 1 (5-HT1) receptor. Previously, we provided evidence consistent with the finding that multiple states of the 5-HT1 receptor are present when the binding of [3H]-5-HT is measured in the absence of guanine nucleotides. When 1 mM GTP was present in the [3H]-5-HT receptor binding assay, the high affinity state was eliminated. As the presence of multiple states of a receptor complicates the interpretation of the inhibition of [3H]-5-HT binding caused by serotonin agonists and antagonists, we examined the ability of a series of these drugs to compete for 15 nM [3H]-5-HT binding in the presence of 1 mM GTP in the rat frontal cortex. Eight agonists and five antagonists showed selectivity for the two subtypes of the 5-HT1 receptor, whereas three agonists and four antagonists showed the same affinity for these two receptors subtypes. Most of the compounds examined exhibited only a modest 10- to 30-fold degree of selectivity. However, 1-(m-trifluoromethylphenyl) piperazine and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)indole were about 65-fold selective and spiperone was over 100-fold selective for one of the receptor subtypes. The subtype specificity of the selective compounds was determined using either spiperone, a selective 5-HT 1A compound, or 1-(m-trifluoromethylphenyl)piperazine, a selective 5-HT 1B compound, to preferentially inhibit one of the receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examines the effects of repeated systemic administration (20 mg/kg s.c., twice daily for 4 days) of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on levels of brain monoamines, their metabolites and on the density of monoamine uptake sites in various regions of rat brain. Marked reductions (30-60%) in the concentration of 5-hydroxyindoleacetic acid were observed in cerebral cortex, hippocampus, striatum, hypothalamus and midbrain at 2 weeks after a 4-day treatment regimen of MDMA or MDA; less consistent reductions in serotonin (5-HT) content were observed in these brain regions. In addition, both MDMA and MDA caused comparable and substantial reductions (50-75%) in the density of [3H]paroxetine-labeled 5-HT uptake sites in all brain regions examined. In contrast, neither MDMA nor MDA caused any widespread or long-term changes in the content of the catecholaminergic markers (i.e., norepinephrine, dopamine, 3,4 dihydroxyphenylacetic acid and homovanillic acid) or in the number of [3H]mazindol-labeled norepinephrine or dopamine uptake sites in the brain regions examined. These data demonstrate that MDMA and MDA cause long-lasting neurotoxic effects with respect to both the functional and structural integrity of serotonergic neurons in brain. Furthermore, our measurement of reductions in the density of 5-HT uptake sites provides a means for quantification of the neurodegenerative effects of MDMA and MDA on presynaptic 5-HT terminals.

3H-(+/-)-4-Bromo-2,5-dimethoxyphenylisopropylamine (3H-DOB), a putative agonist radioligand, was synthesized and used to label 5-HT2 receptors in a particulate fraction prepared from rat frontal cortex tissue homogenates. The specific binding (defined by the difference in 3H-DOB binding in the presence and absence of 10(-6) M cinanserin, a potent and specific 5-HT2 antagonist) displayed high affinity (KD = 4.1 X 10(-10) M) and saturability with a Bmax of 17.9 fmol/mg of protein. The distribution of specific 3H-DOB binding in nine brain regions correlated closely with the distribution of 3H-ketanserin (an antagonist radioligand)-labeled 5-HT2 receptors. Competition studies in frontal cortex homogenates using a variety of compounds revealed a distinct 5-HT2 receptor pharmacology. A series of 5-HT2 antagonists exhibited high affinities in competition studies for specific 3H-DOB binding. The absolute potencies of these antagonists as well as their order of potencies closely correlated with their potencies in competing for 3H-ketanserin-labeled brain 5-HT2 receptors. A series of 5-HT2 agonists also exhibited high affinities in competition studies for specific 3H-DOB binding. Although the order of potencies of these agonists was similar to their order in competing for 3H-ketanserin-labeled brain 5-HT2 receptors, the agonists displayed 10-100-fold higher affinities for the 3H-DOB-labeled sites than for the 3H-ketanserin-labeled sites. The level of specific 3H-DOB binding in the frontal cortex homogenates was approximately 5% of the levels of 3H-ketanserin-labeled 5-HT2 receptors (358 fmol/mg of protein). Taken together, these results indicate that 3H-DOB labels a subset of brain 5-HT2 receptors that has high affinity for agonists as well as antagonists); 3H-ketanserin appears to label both subsets of brain 5-HT2 receptors. Antagonists apparently do not discriminate between these two subsets of 5-HT2 receptors. 3H-DOB specific binding to 5-HT2 receptors was potently inhibited by guanosine 5'-(beta, gamma-imido)triphosphate and guanosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of GTP) with IC50 values of 42 and 21 nM, respectively, whereas adenosine 5'-(beta, gamma-imido)triphosphate and adenosine 5'-O-(3-thio)triphosphate (nonhydrolyzable derivatives of ATP) had no effect. In summary, 3H-DOB specific binding displays the pharmacological characteristics of a 5-HT2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

We investigated the mode of action of the potent antagonist ICI 170,809 in the 5-hydroxytryptamine (5-HT)2 receptor system of arterial smooth muscle. We used isolated preparations from rat tail artery and calf coronary artery with the endothelium rubbed off. In tail artery ICI 170,809 (0.3-30 nM) antagonized surmountably and nearly competitively the contractile effects of 5-HT (pKB = 10.0) and partially prevented the depression of 5-HT-induced contractions caused by methysergide. Increasing methysergide concentrations gradually prevented the protective effect of ICI 170,809. The combination of 30 nM ICI 170,809 with 300 nM of its demethylated analog ICI 169,369 (pKB = 8.8) caused surmountable blockade of the effects of 5-HT as expected from competition of the three drugs for the same receptor. In calf coronary artery ICI 170,809 (1-100 nM) reduced the maximum contractile response to 5-HT by 35% and caused competitive antagonism (pKB = 10.4) of the remaining 65% of the responses to 5-HT. ICI 169,369 (100 nM) completely prevented the depression of the maximum response to 5-HT caused by ICI 170,809. Methysergide (3 nM) depressed the maximum response to 5-HT by 65 and 30% in the absence and presence of ICI 170,809. The results are consistent with the existence of two interconvertible states R in equilibrium R' of the 5-HT2 receptor. The equilibrium of R in equilibrium R' is shifted toward R' by methysergide greater than ICI 170,809 much greater than ICI 169,369.

5-Hydroxytryptamine3 (5-HT3) receptors are present in both central and peripheral neuronal tissues but radioligand binding studies have thus far been limited to crude membranes from brain and vagus nerve. The present studies describe the isolation and characterization from the rabbit small bowel of neuronal membranes enriched in binding sites for the potent 5-HT3 ligand, [3H]zacopride. The number of specific [3H]zacopride binding sites per milligram of protein was increased 6-fold in a 10,000 to 100,000 x g membrane fraction as compared to the homogenate. [3H]Zacopride bound to these membranes with high specificity (greater than 90%), exhibited high affinity for a homogeneous population of binding sites (Kd = 0.3 nM) and its binding was inhibited competitively by other 5-HT3 compounds with the following rank order of potency: ICS 205-930 greater than GR 38032F greater than or equal to quipazine greater than BRL 24924 approximately MDL 72222 much greater than metoclopramide greater than 2-CH3-5-HT3. On a discontinuous sucrose gradient, specific [3H]zacopride binding was increased an additional 3.5-fold and copurified with three plasma membrane markers. Fractionation on a continuous sucrose gradient demonstrated that specific [3H]zacopride binding was associated with the enteric neuronal plasma membranes. Comparative studies in rabbit vagus nerve also demonstrated a large number (maximum binding = 148 fmol/mg of protein) of high affinity [3H]zacopride binding sites (Kd = 0.4 nM), in membranes that exhibited a density and binding characteristics similar to those from enteric neurons. Thus, membranes enriched in 5-HT3 binding sites can be isolated from both enteric and vagus neurons and [3H]zacopride is a potent ligand useful for characterization of these sites.

[3H]ICS 205-930 recognition sites were analyzed in membranes prepared from murine neuroblastoma N1E-115 cells. [3H]ICS 205-930 bound rapidly, reversibly, and stereoselectively to a homogeneous population of high affinity recognition sites: Bmax = 40 +/- 5 fmol/mg of protein, pKD = 9.20 +/- 0.05 (n = 11). Nonlinear regression and Scatchard analysis of saturation data suggested the existence of a single class of [3H]ICS 205-930 recognition sites on N1E-115 cells. The affinity of [3H]ICS 205-930 determined in kinetic studies was in agreement with that obtained under equilibrium conditions. Competition studies carried out with a large variety of agonists and antagonists also suggested the presence of a homogeneous population of [3H]ICS 205-930 recognition sites. [3H]ICS 205-930-binding sites displayed the pharmacological profile of a 5-HT3 receptor. Potent 5-HT3 receptor antagonists showed nM affinities for [3H]ICS 205-930-binding sites with the following rank order of potency: SDZ 206-830 greater than SDZ 206-792 greater than ICS 205-930 greater than BRL 43694 greater than quipazine greater than BRL 24924 greater than MDL 72222 greater than GR 38032F. Methiothepine, mCPP, and metoclopramide showed sub-microM affinity. The rank order of potency of agonists was: 5-HT greater than phenylbiguanide = 2-methyl-5-HT much greater than 5-methoxytryptamine = 5-carboxamidotryptamine. All antagonist competition curves were steep (pseudo-Hill coefficients not lower than 1), monophasic, and best fit for a one-site model; 5-HT and 2-methyl-5-HT produced pseudo-Hill coefficients of 1.2-1.4. Drugs acting at 5-HT1, 5-HT2, alpha- and beta-adrenergic, dopaminergic, and histaminergic receptors (methysergide, ketanserin, propranolol, phentolamine, sulpiride, SCH 23390, cimetidine) were essentially inactive at 10 mumol/liter. The binding of [3H]ICS 205-930 was not affected by guanine and adenine nucleotides (GTP, GppNHp, and ATP) at 1 mmol/liter. These nucleotides did not affect the binding of agonists, suggesting that 5-HT3 recognition sites are not coupled to G-proteins. The interactions of agonists and antagonists with [3H]ICS 205-930 recognition sites were competitive in nature, as demonstrated by saturation experiments carried out with [3H]ICS 205-930 in the presence and the absence of unlabeled compounds: apparent Bmax values were not reduced, whereas apparent KD values were increased in the presence of competing ligands.(ABSTRACT TRUNCATED AT 400 WORDS)