Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.     

Repeated cocaine modifies the neuroendocrine responses to the 5-HT1C/5-HT2 receptor agonist DOI.

Endocrine responses to the serotonin (5-HT) 5-HT1C/5-HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) were utilised to evaluate cocaine-induced alterations in postsynaptic 5-HT receptor function. Rats received cocaine HCl (0, 5 or 15 mg/kg i.p.) twice daily for 7 days. Effects of DOI (0, 0.5, 2 or 10 mg/kg i.p.) on plasma adrenocorticotropic hormone (ACTH), corticosterone, prolactin, oxytocin and renin concentrations were assessed 42 h after the final cocaine injection. DOI dose dependently increased the plasma concentrations of each hormone. Cocaine potentiated the DOI-induced elevations of plasma ACTH, corticosterone and prolactin concentrations. In contrast, the oxytocin response was reduced, and the renin response was unaltered by cocaine exposure. The data suggest that 5-HT2 receptor-mediated responses for ACTH, corticosterone and prolactin secretion become supersensitive following repeated cocaine. In contrast, the 5-HT2 receptor-mediated response for oxytocin secretion is subsensitive. The cocaine-induced changes in postsynaptic 5-HT receptor function are likely a consequence of deficits in the function of 5-HT nerve terminals, that we have documented previously.     

Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT2C receptor isoforms

In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine-serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT2) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT2 receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT2C receptor (VNI isoform) mediated calcium signaling (EC50 1070 nM, IC50 281 nM). It exhibited no appreciable 5-HT2A or 5-HT2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT2A or 5-HT2B receptor expressed in CHO cells (IC50s of 369 and 0.46 nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT2A (IC50, 2.5 nM)>5-HT2B (47 nM)>5-HT2C (69 nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC50s of 571, 1086 and 2099 nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC50s of 1138 and 1000 nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC50 594 nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC50, 79 nM)>VSV (101 nM)>INI (3856 nM). The modest 5-HT2A antagonism and 5-HT2C partial agonism, along with reported D2 and 5-HT1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine-serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT2C agonism may also underlie the minimal weight gain seen with aripiprazole.     

Ketanserin analogues: structure-affinity relationships for 5-HT2 and 5-HT1C serotonin receptor binding.

Ketanserin is the prototypic 5-HT2 serotonin antagonist; although it has been an important tool for the study of serotonin pharmacology, it has had relatively little impact on drug design because remarkably little is known about its structure-affinity relationships. Furthermore, ketanserin also binds at 5-HT1C receptors and even less is known about the influence of its structural features on 5-HT1C receptor affinity. The present study reveals that the fluoro and carbonyl groups of the 4-fluorobenzoyl portion of ketanserin make small contributions to 5-HT2 binding and that the intact benzoylpiperidine moiety is an important feature. Ring-opening of the piperidine ring reduces affinity. Although the quinazoline-2,4-dione moiety also contributes to binding, it appears to play a smaller role and can be structurally simplified with retention of 5-HT2 affinity. N-(4-Phenylbutyl)-4-(4-fluorobenzoyl)piperidine (39), for example, binds with nearly the same affinity (Ki = 5.3 nM) as ketanserin (Ki = 3.5 nM). All of the compounds examined bind at 5-HT1C sites with lower affinity than ketanserin, and some of the simplified analogues bind with nearly 10 times the 5-HT2 versus 5-HT1C selectivity of ketanserin; however, none displays > 120-fold selectivity. Several of the compounds, such as the amide 32 and the urea 33 represent examples of new structural classes of 5-HT2 ligands.     

Mechanisms involved in the hyperglycemic effect of the 5-HT1C/5-HT2 receptor agonist, DOI.

Previous experiments have indicated that 5-HT2 receptors and catecholaminergic systems mediate the rise in plasma glucose levels elicited by acute administration of the 5-HT1c/5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). On this basis, we investigated the location of these serotonin receptors and the nature of this catecholaminergic involvement. Administration of DOI (0.4 mg/kg i.v.) to conscious rats (bearing jugular catheters) elicited a rapid rise in plasma glucose which was associated with a decreased insulin response to a glucose bolus (300 mg/kg i.v.). Pretreatment with the peripherally acting 5-HT1c/5-HT2 receptor antagonist, BW 501C67 (0.5 mg/kg i.v. 10 min beforehand) prevented the rise in plasma glucose triggered by the peripherally acting 5-HT1c/5-HT2 receptor agonist, alpha-methyl-5-HT (0.75 mg/kg i.v.), but amplified the rise elicited by DOI. Pretreatment with chlorisondamine (1 mg/kg i.v. 10 min beforehand) or adrenalectomy 20 h beforehand prevented the DOI-induced hyperglycemia. On the other hand, pretreatment with dexamethasone (0.35 mg/kg s.c. 2 h and 20 min beforehand) did not affect the DOI-induced hyperglycemia. It is concluded that the hyperglycemic effect of DOI administration is mediated by centrally located 5-HT2 receptors and, in turn, adrenal epinephrine release.     

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.     

5-HT3 receptor antagonism by anpirtoline, a mixed 5-HT1 receptor agonist/5-HT3 receptor antagonist.

1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.     

Positive allosteric modulator of the human 5-HT2C receptor

The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.     

Characterisation of 5-HT2 receptor subtypes in the Suncus murinus intestine.

The involvement of 5-HT2 receptor subtypes in mediating a contraction response in the isolated intestine of Suncus murinus was investigated using DOI ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane, a 5-HT2 receptor agonist) which produced a bell-shaped concentration response curve that was significantly (p < 0.05) reduced by methysergide (a 5-HT1/2 receptor antagonist, 1 microM) but not ketanserin (a 5-HT2A receptor antagonist, 1 microM), yohimbine (a 5-HT2B receptor antagonist, 1 microM) or a combination of ondansetron (a 5-HT3 receptor antagonist, 1 microM) plus SB204070 (8-amino-7-chloro(N-butyl-4-piperidyl) methylbenzo-1,4-dioxan-5-carboxylate hydrochloride, a 5-HT4 receptor antagonist, 1 nM). The contraction response to the lower concentrations of DOI (10 nM-0.3 microM) was reduced in the presence of SB206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2 ,3-f]indole, a 5-HT2B/2C receptor antagonist, 1 microM), whilst conversely, the reducing response to the higher concentrations of DOI (1-30 microM) was prevented. A repeated challenge with 3 microM DOI produced a smaller response (desensitisation) and also reduced the response to 5-HT (5-hydroxytryptamine, 0.3 microM) that was inhibited by SB206553 (1 microM). Data indicate that 5-HT2C receptors are likely candidates to mediate the contractile response to DOI and demonstrate desensitisation to repeated challenges.     

Modified 5-HT3A receptor function by co-expression of alternatively spliced human 5-HT3A receptor isoforms

Serotonin (5-HT) exerts fast excitatory responses by activation of 5-HT3 receptors, irrespective of whether they are homomerically composed of 5-HT3A subunits or heteromerically assembled of 5-HT3A and 5-HT3B subunits. Here we describe a short, truncated (h5-HT3AT) and a long (h5-HT3AL) splice variant of the human 5-HT3A (hS-HT3A) receptor subunit. The deduced protein of the short isoform consists of 238 amino acids (aa) with a single transmembrane domain (M1). Compared to the known 5-HT3A receptor, the long isoform contains 32 additional aa in the extracellular loop between M2 and M3. Both splice variants are co-expressed together with the 5-HT3A subunit in the amygdala and hippocampus, whereas in the placenta only the short variant is co-expressed. Both splice variants, when expressed in transfected human embryonic kidney (HEK) 293 cells, are not able to form functional homomeric receptors, but modify 5-HT response at heteromeric h5-HT3A receptors. Co-expression of the short variant considerably decelerates the desensitization of the 5-HT3 receptor; thus, heteromeric assemblies of h5-HT3A and the h5-HT3AT subunit exhibit 5-HT-induced cation fluxes which are much larger than those of homomeric hS-HT3A receptors. In contrast, heteromeric complexes containing the h5-HT3AL subunit display reduced cation fluxes. In conclusion, the splice variants increase the functional diversity of 5-HT3 receptors.     

Deramciclane, a putative anxiolytic drug, is a serotonin 5-HT2C receptor inverse agonist but fails to induce 5-HT2C receptor down-regulation

 Deramciclane (EGIS-3886) is a novel anxiolytic agent that binds with high affinity to 5-HT_2A/2C receptors. The interactions of deramciclane with the serotonin 5-HT_2C receptor were characterized further using receptor phosphoinositide hydrolysis assays and receptor autoradiography. Deramciclane antagonized 5-HT_2C receptor mediated 5-HT-stimulated phosphoinositide hydrolysis with an IC₅₀ value of 168 nM. Deramciclane also decreased basal phosphoinositide hydrolysis by up to 33% (EC₅₀ = 93 nM) in a physiological system in the choroid plexus, suggesting that deramciclane possesses inverse agonist properties at this receptor. Administration of single doses of 0.5 mg/kg and 10 mg/kg resulted in a maximal 5-HT_2C receptor occupancy of up to 45% and 79%, respectively, in the choroid plexus. Chronic (14 days) treatment with 0.5 mg/kg or 10 mg/kg deramciclane did not alter [¹²⁵I]DOI (agonist) or [³H]mesulergine (antagonist) binding to 5-HT_2C receptors in the choroid plexus compared to saline-treated controls, as determined by quantitative receptor autoradiography. In comparison, the effects of deramciclane on 5-HT_2A binding characteristics and receptor occupancy were also studied. Deramciclane treatment resulted in 5-HT_2A receptor occupancy of up to 78%, but no significant effect of chronic treatment on 5-HT_2A receptor agonist binding levels was found. In conclusion, these data indicate that deramciclane is a 5-HT_2C receptor inverse agonist and occupies 5-HT_2C receptors during treatment, and that chronic treatment with deramciclane does not lead to 5-HT_2C receptor down-regulation. Received: 24 October 1996/Final version: 8 August 1997     

Comparisons of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2C receptors

Since the classical hallucinogens were initially reported to produce their behavioral effects via a 5-HT₂ agonist mechanism (i.e., the 5-HT₂ hypothesis of hallucinogen action), 5-HT₂ receptors have been demonstrated to represent a family of receptors that consists of three distinct subpopulations: 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors. Today, there is greater support for 5-HT_2A than for 5-HT_2C receptor involvement in the behavioral effects evoked by these agents. However, with the recent discovery of 5-HT_2B receptors, a new question arises: do classical hallucinogens bind at 5-HT_2B receptors? In the present study we examined and compared the binding of 17 phenylisopropylamines at human 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors. Although there was a notable positive correlation (r>0.9) between the affinities of the agents at all three populations of 5-HT₂ receptors, structural modification resulted only in small differences in 5-HT_2B receptor affinity such that the range of affinities was only about 50-fold. As with 5-HT_2A and 5-HT_2C receptor affinity, there is a significant correlation (r>0.9, n=8) between 5-HT_2B receptor affinity and human hallucinogenic potency. Nevertheless, given that 5-HT_2A and 5-HT_2A/2C antagonists – antagonists with low affinity for 5-HT_2B receptors – have been previously shown to block the stimulus effects of phenylisopropylamine hallucinogens, it is likely that 5-HT_2A receptors play a more prominent role than 5-HT_2B and 5-HT_2C receptors in mediating such effects despite the affinity of these agents for all three 5-HT₂ receptor subpopulations.     

The 5-HT1A receptor agonist flesinoxan shares discriminative stimulus properties with some 5-HT2 receptor antagonists.

Ten homing pigeons were trained to discriminate the selective 5-HT1A receptor agonist flesinoxan (0.25 mg/kg p.o.) from its vehicle in a fixed-ratio (FR) 30 two-key operant drug discrimination procedure. The 5-HT2 receptor antagonist mianserin (ED50 = 4.8 mg/kg) fully substituted for flesinoxan, whereas ketanserin, ritanserin, mesulergine, and SB200646A substituted only partially, suggesting an interaction between 5-HT1A and 5-HT2 receptors. However, the 5-HT2 receptor agonists [DOI (0.6 mg/kg), TFMPP (10 mg/kg), mCPP (4 mg/kg)] were unable to antagonize the flesinoxan cue. The 5-HT1A receptor antagonists DU125530 (0.5-13 mg/kg) and WAY100,635 (0.1-1 mg/kg) partially antagonized the generalization of mianserin to flesinoxan. Taken together, these results are in accordance with the hypothesis that 5-HT1A receptor activation exerts an inhibitory effect on activation of 5-HT2 receptors. These results are in broad agreement with existing theories on 5-HT1A and 5-HT2 receptor interaction. Furthermore, it is argued that the discriminative stimulus properties of a drug may undergo qualitative changes with prolonged training.     

New 5-HT2C receptor agonists

While there is evidence for the involvement of different serotonin (5hydroxytryptamine; 5-HT) receptors in the regulation of food intake and body weight maintenance, the data supporting the role of the 5-HT_2C receptor are especially strong. This information has elevated 5-HT_2C receptor activation into one of the most competitive research areas for antiobesity therapy, a therapeutic area with few (if any) safe, effective pharmacological agents available. Varying amounts of evidence also exist to support the use of 5-HT_2C agonists for the pharmacological treatment of several other conditions, including anxiety, depression, obsessive–compulsive disorder, sexual dysfunction, epilepsy, urinary incontinence and hot flushes. The therapeutic potential of 5-HT_2C receptor modulation has spawned a successful search for 5-HT_2C receptor agonists. This review will focus on recent patent applications through August 2003 that describe compounds that have agonist activity at the 5-HT_2C receptor, and should be complementary to previous Expert Opinion discussions on the 5-HT_2C receptor [1-7].     

Influence of the 5-HT(2C) receptor antagonist SB242,084 on behaviour produced by the 5-HT(2) agonist Ro60-0175 and the indirect 5-HT agonist dexfenfluramine

Ro60-0175 has been described as a selective agonist at the 5-HT(2C) receptor, yet it has only 10- fold higher affinity at the 5-HT(2C) compared to the 5-HT(2A) subtype, and equivalent affinity for the 5-HT(2B) receptor. The selective 5-HT(2C) receptor antagonist SB242,084 (0.5 mg kg(-1) i.p.), blocked the hypoactivity and penile grooming induced by Ro60-0175 (1 mg kg(-1) s.c.). The combination of SB242,084 (0.5 mg kg(-1) i.p.) and Ro60-0175 (3 - 10 mg kg(-1)) produced a completely different pattern of behaviours including wet-dog shakes, hyperactivity and back muscle contractions. These latter effects were blocked by the selective 5-HT(2A) receptor antagonist MDL100,907 (0.5 mg kg(-1) i.p.), but not the 5-HT(2B) receptor antagonist SB215,505 (3 mg kg(-1) p.o.). The indirect 5-HT releaser/reuptake inhibitor dexfenfluramine (1 - 10 mg kg(-1) i.p.) produced a mild increase in locomotor activity, penile grooming, and occasional back muscle contractions and wet-dog shakes. Pre-treatment with SB242,084 (0.5 mg kg(-1)), blocked the incidence of penile grooming, and markedly potentiated both the dexfenfluramine-induced hyperactivity, the incidence of back muscle contractions, and to a lesser extent wet-dog shakes. Some toxicity was also evident in animals treated with dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)), but not in any other treatment groups. The hyperactivity and toxicity produced by the dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)) combination was replicated in a further study, and hyperthermia was also recorded. Both hyperthermia and toxicity were blocked by MDL100,907 (0.5 mg kg(-1)) but not SB215,505 (3 mg kg(-1)). An attenuation of the hyperlocomotor response was also observed following MDL100,907. These findings suggest that 5-HT(2C) receptor activation can inhibit the expression of behaviours mediated through other 5-HT receptor subtypes.     

Aversive stimulus properties of the 5-HT2C receptor agonist WAY 161503 in rats

Serotonin2C (5-HT2C) receptors may influence motivation and reward through effects on the mesocorticolimbic dopamine (DA) system. Previous work from this laboratory indicated that 5-HT2C receptor stimulation does not induce place conditioning when animals are tested in a drug-free state, but does result in decreased locomotor activity and increased frequency thresholds for electrical self-stimulation of the ventral tegmental area (VTA). The present study was conducted to determine whether the 5-HT2C receptor agonist WAY 161503 may induce place conditioning in a state-dependent manner and also whether this compound will induce gustatory avoidance conditioning in the conditioned taste aversion (CTA) paradigm. The effects of the 5-HT2C receptor agonist WAY 161503 in the place conditioning and CTA (two-bottle choice test) paradigms were assessed in male Sprague-Dawley rats. Administration of WAY 161503 (3.0 mg/kg) induced a state-dependent conditioned place aversion and a CTA to saccharin. The differential state dependency of 5-HT2C receptor agonists' effects in place conditioning (state dependent) and CTA (non-state dependent) is consistent with the activation of different brain systems in these two paradigms. The state-dependent effects in place conditioning underscore the need to include controls for state dependency in studies of 5-HT receptor related compounds.     

Chronic mianserin treatment decreases 5-HT2 receptor binding without altering 5-HT2 receptor mRNA levels

Rats were injected with 15 mg/kg (i.p.) mianserin or vehicle (saline) for 4, 10 or 21 days and 5-HT2 receptor binding and mRNA levels measured. Treatment with mianserin induced a substantial decrease in 5-HT2 radioligand binding (44-59% decrease; P less than 0.05 vs. control). No changes in the amount of 5-HT2 or, as a control, beta-actin mRNAs were found (P greater than 0.05 vs. control). These results indicate that mianserin decreases 5-HT2 radioligand binding without altering steady-state 5-HT2 mRNA levels.     

Cloned human 5-HT1A receptor pharmacology determined using agonist binding and measurement of cAMP accumulation

Twenty agonists and nine antagonists were evaluated for their ability to compete for [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) binding to the cloned human serotonin-1A (ch-5-HT1A) receptor expressed in Chinese hamster ovary cells and for their ability to alter adenylyl cyclase activity in the same cells. The most potent full agonists of high affinity included N,N-dipropyl-5-carboxamidotryptamine (pEC50=9.6 +/- 0.1), MDL 73005EF (pEC50=9.3 +/- 0.2), 5-methyl-urapidil (pEC50=9.2 +/- 0.1), 5-carboxamidotryptamine (pEC50=9.1 +/- 0.2), R(+)-8-OH-DPAT (pEC50=8.6 +/- 0.1) and BMY-7378 (pEC50=8.6 +/- 0.1). WB-4101 (pEC50=8.3 +/- 0.2; IA=79%), clozapine (pEC50=8.1 +/- 0.3; IA=29%), (buspirone (pEC50=7.6 +/- 0.2; IA=79%), quipazine (pEC50 <5; IA=45%) and R-DOI (pEC50 < 5; IA=31%) were weaker agonists with partial agonist properties. The most potent antagonists were WAY-100,635 (pKi=10.2 +/- 0.1), methiothepin (pKi=8.8 +/- 0.2), spiperone (pKi=8.7 +/- 0.2) and NAN-190 (pKi=8.5 +/- 0.2). The receptor affinities and functional potencies were well correlated (r=0.88; P <0.0001). Our binding data correlated well with the pharmacology of endogenous 5-HT1A receptors in the rabbit iris-ciliary body (r=0.91; P <0.001) and rat hippocampus (r=0.93, P <0.0001). Our functional cAMP data correlated well with other cAMP accumulation data (r=0.8, P <0.01 vs calf hippocampus) but less so with [35S]-GTPgammaS binding to the ch-5-HT(1A) receptor as a functional activity read-out (r=0.58, P <0.05). The present study provides a detailed pharmacological characterization of the ch-5-HT1A receptor using binding and functional assays.     

Effects of the 5-HT2C receptor agonist Ro60-0175 and the 5-HT2A receptor antagonist M100907 on nicotine self-administration and reinstatement

The reinforcing effects of nicotine are mediated in part by brain dopamine systems. Serotonin, acting via 5-HT(2A) and 5-HT(2C) receptors, modulates dopamine function. In these experiments we examined the effects of the 5-HT(2C) receptor agonist Ro60-0175 and the 5-HT(2A) receptor antagonist (M100907, volinanserin) on nicotine self-administration and reinstatement of nicotine-seeking. Male Long-Evans rats self-administered nicotine (0.03 mg/kg/infusion, IV) on either a FR5 or a progressive ratio schedule of reinforcement. Ro60-0175 reduced responding for nicotine on both schedules. While Ro60-0175 also reduced responding for food reinforcement, response rates under drug treatment were several-fold higher than in animals responding for nicotine. M100907 did not alter responding for nicotine, or food, on either schedule. In tests of reinstatement of nicotine-seeking, rats were first trained to lever press for IV infusions of nicotine; each infusion was also accompanied by a compound cue consisting of a light and tone. This response was then extinguished over multiple sessions. Injecting rats with a nicotine prime (0.15 mg/kg) reinstated responding; reinstatement was also observed when responses were accompanied by the nicotine associated cue. Ro60-0175 attenuated reinstatement of responding induced by nicotine and by the cue. The effects of Ro60-0175 on both forms of reinstatement were blocked by the 5-HT(2C) receptor antagonist SB242084. M100907 also reduced reinstatement induced by either the nicotine prime or by the nicotine associated cue. The results indicate that 5-HT(2C) and 5-HT(2A) receptors may be potential targets for therapies to treat some aspects of nicotine dependence.