Radioligand binding evidence implicates the brain 5-HT2 receptor as a site of action for LSD and phenylisopropylamine hallucinogens

Alterations in brain serotonergic function have been implicated in the mechanism of action of LSD, mescaline, and other similarly acting hallucinogenic drugs of abuse such as STP (2,5-dimethoxyphenylisopropylamine; DOM). In order to test the hypothesis that the mechanism of action of LSD and phenylisopropylamine hallucinogens is through stimulation of a specific brain serotonin receptor sub-type, the affinities of these compounds for radiolabelled 5-HT₂, 5-HT_1A, 5-HT_1B, and 5-HT_1C receptors have been determined using recently developed in vitro radioligand binding methodologies. The 5-HT₂ receptor was labelled with the agonist/hallucinogen radioligand ³H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine). The 5-HT_1A, 5-HT_1B, and 5-HT_1C receptors were labelled with ³H-OH-DPAT, ³H-5-HT, and ³H-mesulergine, respectively. In general, the phenylisopropylamines displayed 10–100 fold higher affinities for the 5-HT₂ receptor than for the 5-HT_1C receptor and 100–1000 fold higher affinities for the 5-HT₂ receptor than for the 5-HT_1A or 5-HT_1B receptor. There was a strong correlation between hallucinogenic potencies and 5-HT₂ receptor affinities of the phenylisopropylamines (r=0.90); the correlation coefficients for the 5-HT_1A, 5-HT_1B, and 5-HT_1C were 0.73, 0.85, and 0.78, respectively. Because there is no evidence that 5-HT_1A-selective or 5-HT_1B-selective agonists are hallucinogenic and because the phenylisopropylamines are potent hallucinogens, a 5-HT₂ receptor interaction is implicated and supports our previous suggestions to this effect. A secondary role for 5-HT_1C receptors cannot be discounted at this time. These results, when integrated with other receptor pharmacological information, indicate that an important component of the mechanism of action of LSD and the phenylisopropylamine hallucinogens is through stimulation of brain 5-HT₂ receptors. Offprint requests to: M. Titeler     

Hallucinogenic drug interactions with neurotransmitter receptor binding sites in human cortex

The binding affinities of four hallucinogenic agents were analyzed at nine neurotransmitter binding sites in human cortex. d-Lysergic acid diethylamide (d-LSD), N,N-dimethyltryptamine (DMT), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane (DOB) display highest affinity for the recently identified DOB binding site labeled by ⁷⁷Br-R(-)DOB. The phenalkylamines, DOI and DOB, display subnanomolar affinity for the ⁷⁷Br-R(-)DOB-labeled site, whereas the indolealkylamines, d-LSD and DMT, display nanomolar affinity for this site. d-LSD was the most potent of the four hallucinogens at six of the other eight sites analyzed in this study. All four hallucinogens also display high affinity for the 5-hydroxytryptamine₂ (5-HT₂) receptor subtype, with potencies ranging from 4 to 360 nM. Marked differences in relative affinities were observed between the indolealkylamines and the phenalkylamines at the 5-HT_1A, 5-HT_1D, and DOB binding sites. These rank-order differences in affinities are likely to account for the differing effects of these agents in various biochemical and physiological assays.     

某些四氢异喹啉类生物碱对大鼠脑内多巴胺和5-羟色胺受体的作用

本文报道十二种四氢异喹啉类生物碱对大鼠脑内D-2,5-HT₁和5-HT₂受体的结合特性。其中l-千金藤碱(l-STP)对这三种受体均有较高的亲和力,其Ki值分别为1.7×10^-7,9.4×10^-8和1.8×10^-7mol。l-莲碱(l-REM)对5-HT₂受体的亲和力与Z-STP相似(Ki=1.7×10^-7mol)。THB,THC和THJ对D-2受体的亲和力介于l-SPD和l-THP之间。本文报道的多数生物碱能同时影响两种或两种以上受体部位的结合特性,提示它们对单胺神经系统可能有复杂的相互作用。     

Complex discriminative stimulus properties of (+)lysergic acid diethylamide (LSD) in C57Bl/6J mice

Rationale The drug discrimination procedure is the most frequently used in vivo model of hallucinogen activity. Historically, most drug discrimination studies have been conducted in the rat. With the development of genetically modified mice, a powerful new tool has become available for investigating the mechanisms of drug-induced behavior. The current paper is part of an ongoing effort to determine the utility of the drug discrimination technique for evaluating hallucinogenic drugs in mice.Objective To establish the training procedures and characterize the stimulus properties of (+)lysergic acid diethylamide (LSD) in mice.Methods Using a two-lever drug discrimination procedure, C57Bl/6J mice were trained to discriminate 0.45 mg/kg LSD vs saline on a VI30 sec schedule of reinforcement, with vanilla-flavored Ensure serving as the reinforcer.Results As in rats, acquisition was orderly, but the training dose was nearly five-fold higher for mice than rats. LSD lever selection was dose-dependent. Time-course studies revealed a rapid loss of the LSD stimulus effects. The 5-HT_2A/2C receptor agonist, 2,5-dimethoxy-4-bromoamphetamine [(–)DOB] (1.0 mg/kg), substituted fully for LSD and the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) (1.6 mg/kg), substituted partially for LSD. Pretreatment with the 5-HT_2A receptor-selective antagonist, MDL 100907, or the 5-HT_1A-selective antagonist WAY 100635, showed that each antagonist only partially blocked LSD discrimination. Substitution of 1.0 mg/kg (–)DOB for LSD was fully blocked by pretreatment with MDL 100907 but unaltered by WAY 100635 pretreatment.Conclusions These data suggest that in mice the stimulus effects of LSD have both a 5-HT_2A receptor and a 5-HT_1A receptor component.     

Cross-tolerance studies of serotonin receptors involved in behavioral effects of LSD in rats

Like hallucinogenic 5-HT₂ agonists, LSD (d-lysergic acid diethylamide) produces characteristic decreases in locomotor activity and investigatory behaviors of rats tested in a novel environment. Because LSD is an agonist at both 5-HT_1A and 5-HT₂ receptors, however, the respective influences of these different receptors in the behavioral effects of LSD remain unclear. In particular, the paucity of selective 5-HT_1A antagonists has made it difficult to assess the specific contribution of 5-HT_1A receptors to the effects of LSD. An alternative approach to the delineation of receptor-specific effects is the use of cross-tolerance regimens. In the present studies, rats were pretreated with saline, 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/kg SC), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (1.0 mg/kg SC), or LSD (60 µg/kg SC), every 12 h for 5 or 8 days. Thirty-six hours later, rats were tested in a behavioral pattern monitor 10 min after injection of saline, 0.5 mg/kg 8-OH-DPAT, 1.0 mg/kg DOI, or 60 µg/kg LSD. As expected, tolerance to the decreases in locomotor activity produced by acute administrations of 8-OH-DPAT, DOI, or LSD occurred when rats were pretreated chronically with 8-OH-DPAT, DOI, or LSD, respectively. Furthermore, pretreatment with either 8-OH-DPAT or DOI produced cross-tolerance to LSD. These results support the hypothesis that the effects of LSD in this model reflect a combination of 5-HT_1A and 5-HT₂ effects and support the view that there is an interaction between 5-HT_1A and 5-HT₂ receptors.     

Serotonin receptor subtype mediation of the interoceptive discriminative stimuli induced by 5-methoxy-N,N-dimethyltryptamine

Male Wistar rats were trained to discriminate the interoceptive effects of 5-methoxy-N,N-dimethyltryptamine (5-OMe-DMT; 1.25 mg/kg, IP) from saline in a two-lever operant chamber. Following discrimination learning, the following drugs (with ED₅₀ dose in mg/kg IP) dose-dependently generalized: lysergic acid diethylamide (LSD, 0.04), 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.11), 6-methoxy-4-(dipropyl-amino)-1,3,4,5-tetrahydrobenz(c,d)indole hydrochloride (BAY R 1531, 0.15), 5-OMe-DMT itself (0.63), ipsapirone (TVX Q 7821, 2.7), and buspirone (3.8). The potencies of these drugs in generalization tests were best correlated with their binding affinities for the 5-HT_1A serotonin receptor subtype (as measured by displacement of ³H-ipsapirone in the hippocampus). Drugs not, or only partially generalizing included quipazine, bufotenin, m-trifluoromethylphenylpiperazine (TFMPP), 5-methoxy-3(1,2,3,6-tetrahydropyridine-4-yl)-1H-indole succinate (RU 24969), citalopram, clomipramine, 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), the buspirone metabolite 1-pyrimidinyl-piperazine (1-PP), methysergide, metergoline, and metitepine. Of the last three compounds with antagonistic activity at 5-HT receptors, as well as ketanserin, pizotifen, and ritanserin, only metitepine and pindolol could fully block the 5-OMe-DMT stimulus. Pizotifen blocked the generalization of quipazine fully, that of 5-OMe-DMT only partially, and that of ipsapirone not at all. These data indicate that the 5-HT_1A receptor subtype is strongly involved in the transduction of the interoceptive discriminative stimuli induced by 5-OMe-DMT, with 5-HT₂ agonism also playing a possible role.     

Interaction of the novel antipsychotic aripiprazole with 5-HT1A and 5-HT2A receptors: functional receptor-binding and in vivo electrophysiological studies

Background Aripiprazole (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butoxy}-3,4-dihydro-2(1H)-quinolinone) is a novel antipsychotic with a mechanism of action that differs from current typical and atypical antipsychotics. Aripiprazole interacts with a range of receptors, including serotonin [5-hydroxytryptamine (5-HT)] and dopamine receptors. Materials and methods This study examined aripiprazole’s interactions with 5-HT systems in vitro and in vivo to further clarify its pharmacologic properties. Results Aripiprazole produced increases in [³⁵S]GTPγS binding to rat hippocampal membranes. Its potency (pEC₅₀ = 7.2) was similar to that of ziprasidone (7.1) and greater than that of 5-HT (6.7) and buspirone (6.4), a 5-HT_1A-receptor partial agonist, whereas its intrinsic activity was similar to that of ziprasidone and buspirone. The stimulatory effect of aripiprazole was blocked by WAY-100635, a 5-HT_1A-receptor antagonist. In in vivo electrophysiology studies, aripiprazole produced a dose-related reduction in the firing rate of 5-HT-containing dorsal raphe neurons in rats, which was both prevented and reversed by WAY-100635 administration. Aripiprazole showed a high affinity for human 5-HT_1A receptors (K _i = 4.2 nM) using parietal cortex membrane preparations. In membranes from cells expressing human recombinant receptors, aripiprazole bound with high affinity to 5-HT_2A receptors (K _i = 3.4 nM), moderate affinity to 5-HT_2C (K _i = 15 nM) and 5-HT₇ (K _i = 39 nM) receptors, and low affinity to 5-HT₆ receptors (K _i = 214 nM) and 5-HT transporter (K _i = 98 nM). In addition, aripiprazole potently blocked 5-HT_2A-receptor-mediated increases in intracellular Ca²⁺ levels in a rat pituitary cell line (IC₅₀ = 11 nM). Discussion These results support a partial agonist activity for aripiprazole at 5-HT_1A receptors in vitro and in vivo, and suggest important interactions with other 5-HT-receptor subtypes. This receptor activity profile may contribute to the antipsychotic activity of aripiprazole in humans.     

3-(ω-Aminoalkyl)-5,5-dialkyl(or spirocycloalkyl-1′,5-)hydantoins as New 5-HT1A and 5-HT2A Receptor Ligands

A series of new 3-(ω-aminoalkyl)-5,5-disubstituted hydantoins, containing 1-phenylpiperazine, 1-(o-methoxyphenyl)piperazine or 1,2,3,4-tetrahydroisoquinoline fragments, were synthesized by standard alkylation procedures and their 5-HT_1A and 5-HT_2A receptor affinities were determined. It has been shown that the investigated derivatives are recognized by 5-HT_1A and 5-HT_2A receptors due to the presence of a 1-arylpiperazine fragment; however, the terminal hydantoin moiety plays an important role in stabilization of the receptor-ligand complex. It has also been found that the two 1-phenylpiperazine derivatives 32 and 36 are new, selective 5-HT_2A receptor ligands (K_i = 34 and 37 nM, respectively), whereas the derivative of 1-(o-methoxyphenyl)piperazine ( 38 ) is a new, highly potent 5-HT_1A receptor ligand (K_i = 0.51 nM) with a moderate affinity for 5-HT_2A receptors (K_i = 213 nM).     

Interactions of selective serotonin reuptake inhibitors with the serotonin 5-HT2C receptor

Interactions of the selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine and its main metabolite norfluoxetine, and the tricyclic anti-depressant (TCA) imipramine with the rat serotonin 5-HT_2C receptor in a clonal cell line and in the rat choroid plexus were investigated by radioligand binding and phosphoinositide (PI) hydrolysis assays. For comparison, the affinities of a variety of other antidepressants of different chemical classes for the cloned rat 5-HT_2C and 5-HT_2A receptors were also determined by radioligand binding assays. Fluoxetine displayed relatively high affinity for the 5-HT_2C receptor in the choroid plexus, with a K_i value for inhibition of [³H]mesulergine binding of 55.4 nM. The K_i values for imipramine, norfluoxetine and citalopram were 136 nM, 203 nM, and 298 nM, respectively. Similar rank order of potency was detected in PI hydrolysis assays, which showed that these drugs are antagonists at the 5-HT_2C receptor without exhibiting inverse agonist activity. [³H]Ketanserin (5-HT_2A) binding assays revealed that the SSRIs fluoxetine, norfluoxetine and citalopram show 10- to 23-fold selectivity for the 5-HT_2C receptor in vitro, whereas the TCA imipramine does not. Many other TCAs also had high to intermediate affinity for both 5-HT_2A and 5-HT_2C receptors. The present data provide evidence that fluoxetine, norfluoxetine and citalopram, along with many other antidepressant compounds, interact directly with the 5-HT_2C receptor.     

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

Summary The agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [³H]5-hydroxytryptamine ([³H]5-HT) binding sites and the affinities of the antagonists for [¹²⁵Iodo]LSD ([¹²⁵I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the K _D values published for displacement of [³H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT₂ binding sites labelled by both [¹²⁵I]LSD and [³H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT₁ binding sites labelled by [³H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT₂-like receptors.     

Potent agonist activity of DOB at 5-HT2 receptors in guinea pig trachea

DOB, 4-bromo-2,5-dimethyloxyamphetamine, a potent hallucinogen, displays high affinity for the 5-HT₂ receptor in binding assays. Since 5-HT contractile responses in guinea pig trachea are mediated by 5-HT₂ receptors, we assessed the activity of DOB and related analogs in this preparation. DOB acts as a partial 5-HT agonist with an EC₅₀=25 nM and is blocked by the potent selective 5-HT₂ receptor antagonists ketanserin, pirenperone and LY 53857, as is 5-HT. These findings indicate that DOB may be useful agent in characterizing 5-HT₂ receptor responses.     

The role of the 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs I: Antagonist correlation analysis

Investigations conducted over the past 3 decades have demonstrated that serotonergic receptors, specifically the 5-HT_2A and 5-HT_2C subtypes, play an important role in the behavioral effects of hallucinogenic compounds. The present study was designed to determine the respective significance of these two receptors in the stimulus effects of LSD and (–)DOM in the rat. Specifically, the interactions of a series of serotonergic antagonists (risperidone, pirenpirone, metergoline, ketanserin, loxapine, LY53857, pizotyline, spiperone, cyproheptadine, mesulergine, promethazine, and thioridazine) with the LSD stimulus and the (–)DOM stimulus in LSD-trained subjects was defined. From these data, IC₅₀ values were determined for the inhibition of the LSD-appropriate responding elicited by either 0.1 mg/kg LSD (15-min pretreatment time) or 0.4 mg/kg (–)DOM (75-min pretreatment). In addition, the affinities of these antagonists for 5-HT_2A and 5-HT_2C receptors were determined in radioligand competition studies. 5-HT_2A affinity correlated significantly with IC₅₀ values for the blockade of the LSD (r=+0.75,P<0.05) and (–) DOM (r=+0.95,P<0.001) stimuli in the LSD trained subjects. 5-HT_2C affinity did not correlate significantly with either series of IC₅₀ values. These data indicate that (1) the stimulus effects of LSD, and (2) the substitution of (–)DOM for the LSD stimulus are mediated by agonist activity at 5-HT_2A receptors.This study was supported in part by US Public Health Service grant DA 03385 (J.C.W., R.A.R.), by National Research Service Award MH 10567 (D.F.), and by a fellowship from Schering-Plough Research Institute (D.F.). Animals used in these studies were maintained in accordance with the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council.     

LSD and structural analogs: pharmacological evaluation at D1 dopamine receptors

The hallucinogenic effects of lysergic acid diethylamide (LSD) have been attributed primarily to actions at serotonin receptors. A number of studies conducted in the 1970s indicated that LSD also has activity at dopamine (DA) receptors. These latter studies are difficult to interpret, however, because they were completed before the recognition of two pharmacologically distinct DA receptor subtypes, D₁ and D₂. The availability of subtype-selective ligands (e.g., the D₁ antagonist SCH23390) and clonal cell lines expressing a homogeneous receptor population now permits an assessment of the contributions of DA receptor subtypes to the DA-mediated effects of LSD. The present study investigated the binding and functional properties of LSD and several lysergamide analogs at dopamine D₁ and D₂ receptors. Several of these compounds have been reported previously to bind with high affinity to serotonin 5HT₂ (i.e.,³H-ketanserin) sites in the rat frontal cortex (K_0.5 5–30 nM). All tested compounds also competed for both D₁-like (³H-SCH 23390) and D₂-like (³H-spiperone plus unlabeled ketanserin) DA receptors in rat striatum, with profiles indicative of agonists (n _H<1.0). The affinity of LSD and analogs for D₂ like receptors was similar to their affinity for 5HT₂ sites. The affinity for D₁ like receptors was slightly lower (2- to 3-fold), although LSD and several analogs bound to D₁ receptors with affinity similar to the prototypical D₁ partial agonist SKF38393 (K_0.5 ca. 25 nM). A second series of experiments tested the binding and functional properties of LSD and selected analogs in C-6 glioma cells expressing the rhesus macaque D_1A receptor. LSD and the analogs tested bound to C-6 mD_1A cells with affinity and kinetics similar to those obtained in rat straitum. Additionally, LSD and selected analogs were able to increase cAMP accumulation, albeit only as partial agonists. Similar to the actions of SKF38393, they could stimulate, as well as block, DA-stimulated cAMP synthesis. These results represent the first clear demonstration of the interaction of LSD with DA D₁ receptors, and provide a basis for evaluating the contribution of D₁ receptors to the biobehavioral actions of LSD.     

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT_1A (pK _i = 7.72) and 5-HT_2A (pK _i = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT_2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC₅₀ = 6.09) and in the hippocampus (pEC₅₀ = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pK _i = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT_1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT_1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT_1A receptors and antagonism at 5-HT_2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).     

A Search for New 5-HT1A/5-HT2A Receptor Ligands. In Vitro and in vivo Studies of 1-[ω-(4-Aryl-1-piperazinyl)alkyl]indolin-2(1H)-ones

A series of 1-[ω-(4-aryl-1-piperazinyl)alkyl]indolin-2(1H)-one derivatives 2–14 was synthesized in order to obtain ligands with a dual 5-HT_1A/5-HT_2A activity. The majority of those compounds ( 2–5, 7, 10–13 ) exhibited a high 5-HT_1A (K_i = 2 – 44 nM) and/or 5-HT_2A affinity (K_i = 51 and 39 for 5 and 7 , respectively). Induction of lower lip retraction (LLR) and behavioral syndrome and inhibition of these efects evoked by 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) were used for determination the agonistic and antagonistic activity, respectively, at 5-HT_1A receptors. The 5-HT_2A antagonistic activity was assessed by the blocking effect on the head twitches induced by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in mice. Two of the tested compounds, 1-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-6-fluoroindolin-2(1H)-one ( 5 ) and 1-{3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl}indolin-2(1H)-one ( 7 ), demonstrated a high 5-HT_1A/5-HT_2A affinity and an in vivo antagonistic activity towards both receptor subtypes.     

Investigation of hallucinogenic and related β-carbolines

Certain β-carbolines are known to be hallucinogenic in humans, and several produce stimulus effects in animals similar to those of the classical hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). Classical hallucinogens bind at 5-HT₂ serotonin receptors and these receptors are thought to play a role in their mechanism of action. In the present study, we examined the binding of 15 β-carbolines at rat 5-HT_2A and 5-HT_2C receptors. Affinities (K_i values) of the β-carbolines ranged from about 100 nM to greater than 10 000 nM depending upon the degree of saturation of the pyridyl ring, and upon the presence and location of methoxy substituents in the benzenoid ring. In a further study, six rats were trained to discriminate the hallucinogenic β-carboline harmaline (3.0 mg/kg, i.p.) from vehicle using a VI-15s schedule of reinforcement. This represents the first time a hallucinogenic β-carboline has been used as a training drug in a drug discrimination study. Administration of DOM to the harmaline-trained animals resulted in 76% harmaline-appropriate responding at 1.25 mg/kg DOM and disruption of behavior at a higher dose. Taken together, the results of the present investigation demonstrate that: (a) certain β-carbolines bind at 5-HT₂ receptors; (b) that harmaline serves as a training drug at 3.0 mg/kg in drug discrimination studies with rats as subjects; and that (c) there is some similarity between the stimulus effects produced by harmaline and DOM.     

Complex stimulus properties of LSD: a drug discrimination study with α2-adrenoceptor agonists and antagonists

The influence of several ₂-adrenergic agents on the discriminative stimulus (DS) properties of lysergic acid diethylamide (LSD) was studied in rats trained to discriminate 0.08 mg/kg (186 nmol/kg) of LSD from saline in a two-lever operant paradigm. Only yohimbine fully mimicked LSD with an ED₅₀ of 2.05 mg/kg (5.24 µmol/kg). Yohimbine's 5-HT_1A agonist properties may be responsible for this substitution. Other ₂-adrenoceptor antagonists, idazoxan with an agonist/antagonist profile at 5-HT_1A receptors and RS 26026-197, a highly selective ₂-adrenoceptor antagonist, failed to produce substitution. Clonidine, an ₂-adrenoceptor agonist, did not substitute for LSD but the response rate was dose-dependently reduced. None of the ₂-adrenergic agents used for pretreatment before LSD inhibited the response to the LSD training dose. Coadministration of clonidine with LSD produced a leftward shift of the dose-response relationship of LSD without a significant change in the slope of the dose-response line. Simultaneous administration of ₂-adrenergic agents with LSD shifted the dose-response curve to the left only when the adrenergic agent also possessed at least moderate affinity for the 5-HT_1A receptor. In addition, radioligand competition experiments were performed that showed LSD to have relatively high affinity (K_i=37 nM) for [³H]clonidine-labeled sites in rat cortex with lower affinity for [³H]yohimbine labeled sites. While previous studies have suggested that the nature of the LSD cue may be essentially expressed by 5-HT₂ receptor activation, the present data show that this cue can be modulated by effects of LSD at 5-HT_1A and at other monoamine neurotransmitter receptors.     

R(−)-2,5-Dimethoxy-4-77bromoamphetamine [77Br-R(−)DOB]: a novel radioligand which labels a 5-HT binding site subtype

R(–)-2,5-Dimethoxy-4-⁷⁷Br-amphetamine [⁷⁷Br-R(–)DOB], a radioligand of high specific activity (1500±200 Ci/mmol), was used to label membrane-associated recognition sites in rat brain. ⁷⁷Br-R(–)DOB sites were of high affinity (K _D=0.19 nM) but low density (B _max=0.32 pmol/g tissue) in rat brain preparations. Competition experiments show that both 5-hydroxytryptamine (5-HT) and 5-HT₂ antagonists display nanomolar potency for these sites. We conclude that ⁷⁷Br-R(–)DOB labels 5-HT recognition sites in rat brain which do not fit into current classifications of 5-HT binding subtypes. This finding may be of aid in deciphering the mechanism of action of hallucinogens in man.     

Actions of α2 adrenoceptor ligands at α2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for α2A adrenoceptors

This study examined the activity of chemically diverse α₂ adrenoceptor ligands at recombinant human (h) and native rat (r) α_2A adrenoceptors as compared with 5-HT_1A receptors. First, in competition binding experiments at hα_2A and h5-HT_1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (±)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for hα_2A versus h5-HT_1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pK _i values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for hα_2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for α₂ adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT_1A receptor-mediated G-protein activation, as indicated by the stimulation of [³⁵S]GTPγS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT_1A receptors. Relative efficacies (E_max values) were 25–35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50–65% for 1-PP, (±)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT =100%). Yohimbine-induced [³⁵S]GTPγS binding was inhibited by the selective 5-HT_1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT_1A receptors, inhibiting 5-HT-stimulated [³⁵S]GTPγS binding. Atipamezole, which exhibited negligeable affinity for 5-HT_1A receptors, was inactive. Third, the affinities for rα_2A differed considerably from the affinities for hα_2A receptors whereas the affinities for r5-HT_1A differed much less from the affinities for h5-HT_1A receptors. This affected markedly the affinity ratios of certain compounds. For example, (±)-idazoxan was only 3.6-fold selective for hα_2A versus h5-HT_1A but 51-fold selective for rα_2A versus r5-HT_1A receptors. Conversely, yohimbine was tenfold selective for hα_2A versus h5-HT_1A adrenoceptors but 4.2-fold selective for rα_2A versus r5-HT_1A receptors. Nevertheless, both atipamezole and DMT were highly selective for both rat and human α_2A versus rat or human 5-HT_1A receptors. In conclusion, these data indicate that: (1) the agonist DMT and the antagonist atipamezole are the ligands of choice to distinguish α₂-mediated from 5-HT_1A-mediated actions, whilst several of the other compounds show only low or modest selectivity for α_2A over 5-HT_1A receptors; (2) caution should be exercised in experimental and clinical interpretation of the actions of traditionally employed α₂ ligands, such as clonidine, yohimbine and (±)-idazoxan, which exhibit marked agonist activity at 5-HT_1A receptors. Received: 2 March 1998 / Accepted: 11 May 1998     

WAY-100635 is a potent dopamine D4 receptor agonist

Rationale and objectives WAY-100635 is a prototypical 5-HT_1A receptor antagonist and has been used widely as a pharmacological probe to investigate the distribution and function of 5-HT_1A receptors. Results from our studies suggested that WAY-100635 was potently inducing effects unrelated to its 5-HT_1A receptor affinity. In the present work, we evaluated the in vitro pharmacology of this compound at two D₂-like receptor subtypes.Method The functional properties and binding affinities of WAY-100635 were evaluated in HEK 293 cells stably expressing dopamine D_2L or D_4.4 receptors.Results Initial screens performed by the NIMH Psychoactive Drug Screening Program indicated that WAY-100635 displayed 940, 370, and 16 nM binding affinities at D_2L, D₃, and D_4.2 receptors, respectively. Subsequent saturation analyses demonstrated that the K _d of [³H]WAY-100635 at D_4.2 receptors was 2.4 nM, only tenfold higher than 5-HT_1A. WAY-100635 and its major metabolite, WAY-100634, were potent agonists in HEK-D_4.4 cells (EC₅₀=9.7±2.2 and 0.65±0.2 nM, respectively). WAY-100635 behaved as a full agonist, and WAY-100634 was a nearly full agonist. In HEK-D_2L cells, WAY-100635 weakly antagonized the effects of 300 nM quinpirole. Subsequent radioligand binding studies confirmed that WAY-100635 possesses high affinity for D_4.4 receptors but binds weakly to D_2L receptors (3.3±0.6 and 420±11 nM, respectively).Conclusions This study demonstrates that WAY-100635 is not a “selective” 5-HT_1A receptor antagonist, as previously reported, and conclusions drawn from studies that employed WAY-100635 as a selective 5-HT_1A antagonist may need to be reevaluated.